Fusosome compositions for CNS delivery

Abstract

To provide fusosome compositions for CNS delivery.SOLUTION: The present disclosure provides, at least in part, methods and compositions for in vivo fusosome delivery. In some embodiments, the fusosome comprises a combination of elements that promote specificity for target cells, e.g., one or more of a fusogen, a positive target cell-specific regulatory element, and a non-target cell-specific regulatory element. In some embodiments, the fusosome compositions comprise one or more modifications that decrease an immune response against the fusosome.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - References to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 767,358, filed on November 14, 2018, entitled "Phososome Compositions for CNS Cell Delivery", and U.S. Patent Application Publication No. 62 / 900,064, filed on September 13, 2019, entitled "Phososome Compositions for CNS Cell Delivery", the contents of which are hereby incorporated by reference in their entirety for all purposes.

[0002] Incorporation by Reference of Sequence Listing This application has been filed with an electronic sequence listing. The sequence listing was created on November 14, 2019, and is provided as a file named 186152003340SeqList.TXT with a size of 819 kilobytes. The electronic form information of the sequence listing is hereby incorporated by reference in its entirety.

Background Art

[0003] Complex biologics are promising therapeutic candidates for various diseases. However, since the plasma membrane functions as a barrier between the cell and the extracellular space, it is difficult to deliver large biological factors into cells. In the art, there is a need for new methods to deliver complex biologics into the cells of a subject.

Summary of the Invention

Means for Solving the Problems

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

[0005] Enumeration of Embodiments 1. The following: a) A lipid bilayer containing a fusogen; and b) The following: (i) A payload gene encoding an exogenous agent, for example, a payload gene encoding an exogenous agent in Table 5 or Table 6, optionally, the exogenous agent is described in any one of SEQ ID NOs: 134 - 154, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence described in any one of SEQ ID NOs: 134 - 154, and includes a functional fragment or a functional variant thereof having an amino acid sequence thereof, a payload gene; and (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, a fusosome, wherein the positive target cell - specific regulatory element increases the expression of the payload gene in the target cell as compared to a 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, and the NTCSRE decreases the expression of the payload gene in non - target cells as compared to a similar fusosome lacking the NTCSRE, optionally, the target cell is a first type of CNS cell, 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, an astrocyte or a microglial cell), or 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, the fusosome according to Embodiment 1.

[0007] 3. The following: a) A lipid bilayer containing a fusogen; and b) The following: (i) An exogenous agent, for example, a payload gene encoding an exogenous agent of Table 5 or Table 6, optionally, the exogenous agent is described in any one of SEQ ID NOs: 134 to 154, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence described in any one of SEQ ID NOs: 134 to 154, a payload gene comprising a functional fragment or a functional variant thereof having an amino acid sequence, (ii) A nucleic acid comprising a promoter operably linked to the payload gene, a fusosome comprising the promoter being selected from the 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, for example, by the sequence of the promoter of 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 comprising a fusogen; and b) The following: (i) A payload gene encoding an exogenous agent, for example, a payload gene encoding an exogenous agent of Table 5 or Table 6, optionally, the exogenous agent is described in any one of SEQ ID NOs: 134 to 154, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence described in any one of SEQ ID NOs: 134 to 154, a payload gene comprising a functional fragment or a functional variant thereof having an amino acid sequence, (ii) 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, and a fusosome comprising the same, wherein the NTCSRE reduces the expression of the payload gene in non-target cells or tissues as compared to a similar fusosome lacking the NTCSRE.

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

[0010] 6. The nucleic acid further comprises a positive target cell-specific regulatory element (e.g., a target cell-specific promoter) operably linked to the payload gene, and the positive target cell-specific regulatory element increases the expression of the payload gene in target cells as compared to a similar fusosome lacking the positive target cell-specific regulatory element, the target cells are the first type of CNS cells, optionally, the non-target cells are the second different type of CNS cells or non-CNS cells, optionally, the target cells are neurons and the non-target cells are glial cells (e.g., oligodendrocytes, astrocytes or microglial cells), or The fusosome according to 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 containing a payload gene encoding an exogenous agent, for example, a payload gene encoding an exogenous agent in Table 5 or Table 6, and optionally, the exogenous agent is described in any one of SEQ ID NOs: 134-154, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence described in any one of SEQ ID NOs: 134-154, a nucleic acid containing a payload gene comprising a functional fragment or a functional variant thereof; 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 a reduced level (e.g., reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to fusosomes generated from otherwise identical unmodified source cells.

[0012] 8. The fusosome of any of the foregoing embodiments, being one or more of the following: i) The fusosome fuses 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, compared to non-target cells; ii) The fusosome fuses 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, compared to another fusosome; iii) the fusosome fuses with the target cells at a rate such that the agent within the fusosome 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 fusosome delivers nucleic acid, such as 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 nucleic acid, such as retroviral nucleic acid, to 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 to another fusosome; or vi) the fusosome delivers nucleic acid, such as retroviral nucleic acid, to target cells at a rate such that the agent in the fusosome 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. Any of the fusosomes of the foregoing embodiments to which one or more of the following apply (e.g., all two or three): the fusosome is a retroviral vector, the lipid bilayer is composed of an envelope, e.g., a viral envelope, and the nucleic acid is retroviral nucleic acid.

[0014] 10. Any of the fusosomes of the foregoing embodiments, wherein the nucleic acid comprises one or more (e.g., all) of the following nucleic acid sequences: 5' LTR (e.g., including U5 and lacking a functional U3 domain), Psi packaging element (Psi), central polypurine tract (cPPT) promoter operably linked to a payload gene, payload gene (optionally including an intron prior to the open reading frame), poly A tail sequence, WPRE, and 3' LTR (e.g., lacking U5 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, such as a 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, and the NTCSRE reduces the expression of the payload gene in non-target cells or tissues as compared to otherwise similar fusosomes lacking the NTCSRE, optionally, the target cells are a first type of CNS cell, the non-target cells are a second different type of CNS cell or non-CNS cell, optionally, the target cells are neurons and the non-target cells are glial cells (e.g., oligodendrocytes, astrocytes or microglial cells), or The fusosome according to any one of embodiments 7 to 15, wherein the target cells are glial cells (e.g., oligodendrocytes, astrocytes or microglial cells) and the non-target cells are neurons.

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

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

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

[0024] 20. The fusosome of any one 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 one of embodiments 7-20, 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 according to any of the foregoing 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 foregoing embodiments, wherein the fusogen comprises VSV-G.

[0028] 24. The fusosome according to 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 nuclear 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 according to embodiment 25, wherein the CNS cell - specific promoter comprises the motif of Table 3.

[0031] 27. The fusosome according to embodiment 25 or 26, wherein the positive CNS cell - specific regulatory element comprises a promoter selected from the 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 - 6, or 16 - 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 - 6, or 16 - 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 negative TCSRE or NTCSRE contains a non-target cell-specific miRNA recognition sequence that is bound by the miRNAs in Table 4, for example, by one or more (e.g., two or more) of miR-338-3p, miR-9, miR-125b-5p, miR-342-3p, or miR-124, and optionally, the miRNA is the sequence described in any one of SEQ ID NOs: 156 to 162, or includes it, and the fusosome according to any one of Embodiments 4 to 6, 16 to 21, or 28 to 30.

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

[0037] 33. The fusosome according to any one of the foregoing embodiments, wherein a nucleic acid, such as a retroviral nucleic acid, contains one or more insulator sequences.

[0038] 34. The fusosome according to Embodiment 33, wherein a nucleic acid, such as a retroviral nucleic acid, contains two insulator sequences, for example, a first insulator sequence upstream of the payload gene and a second insulator sequence downstream of the payload gene, and for example, the first insulator sequence and the second insulator sequence contain the same or different sequences.

[0039] 35. The fusosome according to any one of the foregoing embodiments, which is not genotoxic or does not increase the rate of tumor formation in target cells.

[0040] 36. The fusosome according to any one of the foregoing embodiments, wherein a nucleic acid, such as a retroviral nucleic acid, can be integrated into the genome of target cells.

[0041] 37. The fusosome according to Embodiment 36, wherein the nucleic acid, such as a retroviral nucleic acid, is an integrative lentivirus or a non-integrative lentivirus.

[0042] 38. The fusosome according to any of the foregoing 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 according to any of Embodiments 4 to 6 and 9 to 38, which is one or more of the following: i) Less than 10%, 5%, 4%, 3%, 2%, or 1% of the exogenous agent that is detectably present in the subject is in non-target cells; ii) At least 90%, 95%, 96%, 97%, 98%, or 99% of the cells of the subject that detectably contain the exogenous agent are target cells (e.g., cells of a single cell type); iii) Less than 1,000,000, 500,000, 200,000, 100,000, 50,000, 20,000, or 10,000 cells of the subject that 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 detected in non-target cells of the subject.

[0044] 40. The fusosome according to any of the foregoing embodiments, wherein a nucleic acid, such as a retroviral nucleic acid, encodes a positive TCSRE and / or NTCSRE or a negative TCSRE.

[0045] 41. The fusosome according to any of the foregoing embodiments, wherein a nucleic acid, such as a retroviral nucleic acid, contains a complement of a positive TCSRE and / or NTCSRE or a negative TCSRE.

[0046] 42. The fusosome according to any of embodiments 40 or 41, comprising a target cell-specific promoter in which positive TCSRE 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 according to any of embodiments 40 to 42, comprising an miRNA recognition sequence in which negative TCSRE or NTCSRE reduces gene expression by at least 10%, 25%, 50%, 75%, or 100% in non-target cells compared to target cells.

[0048] 44. The fusosome according to any of the foregoing embodiments, which does not deliver nucleic acids, such as retroviral nucleic acids, to non-target cells, such as neurons, glial cells, antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, non-conventional antigen-presenting cells, macrophages, dendritic cells, myeloid dendritic cells, plasmacytoid dendritic cells, CD11c+ cells, CD11b+ cells, spleen cells, B cells, hepatocytes, endothelial cells, or non-cancerous cells.

[0049] 45. For example, using quantitative PCR, for example, using the assay of Example 1, less than 10%, 5%, 2.5%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, or 0.000001% of non-target cell types (e.g., one or more neurons, glial cells, antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, non-conventional antigen-presenting cells, macrophages, dendritic cells, myeloid dendritic cells, plasmacytoid dendritic cells, CD11c+ cells, CD11b+ cells, spleen cells, B cells, hepatocytes, endothelial cells, or non-cancerous cells) contain nucleic acids, such as retroviral nucleic acids. The fusosome according to any of the foregoing embodiments.

[0050] 46. The target cell contains from 0.00001 to 10, 0.0001 to 10, 0.001 to 10, 0.01 to 10, 0.1 to 10, 0.5 to 5, 1 to 4, 1 to 3, or 1 to 2 copies of a nucleic acid per host genome, such as a retroviral nucleic acid or a part thereof, for example, the copy number of the nucleic acid, for example, the retroviral nucleic acid is evaluated after in vivo administration, a fusosome of any of the foregoing embodiments.

[0051] 47. A fusosome of any of the foregoing embodiments, wherein: less than 10%, 5%, 2.5%, 1%, 0.5%, 0.1%, 0.01% of non-target cells (e.g., neurons, glial cells, antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, non-conventional 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 an exogenous agent; or an exogenous agent (e.g., a 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, non-conventional 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, a fusosome of any of the foregoing embodiments.

[0052] 48. A fusosome according to any of the foregoing embodiments, wherein the fusosome delivers a nucleic acid, such as a retroviral nucleic acid, to a target cell, such as 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. At least 0.00001%, 0.0001%, 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) contain nucleic acids, e.g., retroviral nucleic acids, when using, for example, quantitative PCR, when using, for example, the assay of Example 3, and are fusosomes according to any of the foregoing embodiments.

[0054] 50. At least 0.00001%, 0.0001%, 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) contain exogenous agents and are fusosomes according to any of the foregoing embodiments.

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

[0056] 52. The ratio of the average copy number of a nucleic acid, such as a retroviral nucleic acid or a part thereof, in a target cell to the average copy number of the nucleic acid, such as a retroviral nucleic acid or a part thereof, in a non-target cell is, for example, at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000 according to, for example, the assays of Example 1 and Example 3, according to, for example, a quantitative PCR assay, for any of the aforementioned embodiments of fusosomes.

[0057] 53. The ratio of the median copy number of a nucleic acid, such as a retroviral nucleic acid or a part thereof, in a target cell to the median copy number of the nucleic acid, such as a retroviral nucleic acid or a part thereof, in a non-target cell is, for example, at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000 according to, for example, a quantitative PCR assay, for example, using the assays of Example 1 and Example 3, for any of the aforementioned embodiments of fusosomes.

[0058] 54. The ratio of target cells containing an exogenous RNA agent to non-target cells containing the exogenous RNA agent is, for example, at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000 according to, for example, a reverse transcription quantitative PCR assay, for any of the aforementioned embodiments of fusosomes.

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

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

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

[0062] 58. The ratio of the average exogenous protein effector substance level in target cells to the average exogenous protein effector substance level in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, for example, according to a FACS assay, for example, using the assays of Examples 2 and 4, for any of the aforementioned embodiments of the fusosome.

[0063] 59. The ratio of the median exogenous protein effector substance level in target cells to the median exogenous protein effector substance level in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, for example, according to a FACS assay, for example, using the assays of Examples 2 and 4, for any of the aforementioned embodiments of the fusosome.

[0064] 60. Any of the fusosomes of the aforementioned embodiments comprising one or both of the following: i) An exogenous or overexpressed immunosuppressive protein on the lipid bilayer, for example, an envelope; and ii) An immunostimulatory protein present at a non-existent or reduced level (e.g., reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to fusosomes generated from unmodified source cells that are otherwise similar.

[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. At least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are circulating 2 hours after administration, the fusosomes of any of the foregoing embodiments.

[0070] 66. At least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are circulating 4 hours after administration, the fusosomes of any of the foregoing embodiments.

[0071] 67. At least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are circulating 8 hours after administration, the fusosomes of any of the foregoing embodiments.

[0072] 68. At least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are circulating 12 hours after administration, the fusosomes of any of the foregoing embodiments.

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

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

[0075] 71. A fusosome according to 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 circulating 36 hours after administration.

[0076] 72. A fusosome according to 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 circulating 48 hours after administration.

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

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

[0079] 75. A fusosome according to any of the preceding embodiments, wherein a serum sample from an animal administered the fusosome has a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more decrease in anti-fusosome antibody titer as compared to a serum sample from a subject administered unmodified cells.

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

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

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

[0083] 79. The fusosome generated from NMC-HLA-G cells has a reduced rate of lysis, e.g., PBMC-mediated lysis, NK cell-mediated lysis, and / or CD8+ T cell-mediated lysis, at a specific time point compared to the fusosome generated from NMC or NMC-empty vector, which is the fusosome of any of the foregoing embodiments.

[0084] 80. The modified fusosome is the fusosome of any of the foregoing embodiments that avoids phagocytosis by macrophages.

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

[0086] 82. When the macrophage is incubated with the retroviral vector derived from NMC-CD47 as compared with the vector derived from NMC or the NMC empty vector, the phagocytosis index of the fusosome of any of the foregoing embodiments decreases.

[0087] 83. In macrophage phagocytosis, compared with a reference fusosome, for example, an unmodified fusosome that is otherwise similar to the fusosome, it has a decrease in macrophage phagocytosis of, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, and the decrease in macrophage phagocytosis is determined by assaying the in vitro phagocytosis index as described in, for example, Example 8. The fusosome of any of the foregoing embodiments.

[0088] 84. When the fusosome composition is incubated with macrophages in an in vitro assay of macrophage phagocytosis, it has a phagocytosis index of 0, 1, 10, 100, or more, as measured by the assay of Example 8. The fusosome of any of the foregoing embodiments.

[0089] 85. The fusosome of any of the foregoing embodiments, which is modified and has a reduced complement activity as compared with the unmodified fusosome.

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

[0091] The dose of the fusosome with 87.200 pg / ml of C3a is higher in the case of the modified fusosome (e.g., HEK293-DAF) incubated with the corresponding mouse serum (e.g., HEK-293 DAF mouse serum) than in the case of the reference fusosome (e.g., HEK293 retroviral vector) incubated with the corresponding mouse serum (e.g., HEK293 mouse serum), the fusosome of any of the foregoing embodiments.

[0092] The dose of the fusosome with 88.200 pg / ml of C3a is higher in the case of the modified fusosome (e.g., HEK293-DAF) incubated with naive mouse serum than in the case of the reference fusosome (e.g., HEK293 retroviral vector) incubated with naive mouse serum, the fusosome of any of the foregoing embodiments.

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

[0094] 90. The fusosome of any of the foregoing 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 to 90, wherein the complement regulatory protein comprises one or more of a protein that binds to decay-accelerating factor (DAF, CD55), such as factor H (FH)-like protein-1 (FHL-1), such as C4b-binding protein (C4BP), such as complement receptor 1 (CD35), such as membrane cofactor protein (MCP, CD46), such as protectin (CD59), such as a protein that inhibits classical and alternative complement pathway CD / C5 convertase enzymes, such as a protein that regulates MAC assembly.

[0096] 92. For example, a lysosome produced by the method of Example 10 from cells transfected with DNA encoding an shRNA targeting MHC class I, such as a retroviral vector derived from NMC-shMHC class I, has lower expression of MHC class I compared to NMC and the NMC vector control, of any of the foregoing embodiments.

[0097] 93. A measure of immunogenicity for the lysosome is serum inactivation, such as serum inactivation measured as described in Example 11 and as described herein, of any of the foregoing embodiments of the lysosome.

[0098] 94. The percentage of cells receiving an exogenous agent is the same between lysosome samples incubated with serum from lysosome-naive mice and heat-inactivated serum, of any of the foregoing embodiments of the lysosome.

[0099] 95. The percentage of cells receiving an exogenous agent is the same between a lysosome sample incubated with serum from lysosome-naive mice and a serum-free control incubation, of any of the embodiments of the lysosome.

[0100] 96. The percentage of cells receiving an exogenous agent is less in a lysosome sample incubated with positive control serum than in a lysosome sample incubated with serum from lysosome-naive mice, of any of the foregoing embodiments of the lysosome.

[0101] 97. A modified lysosome, such as one modified by the methods described herein, has a decrease in serum inactivation (e.g., a decrease compared to administration of an unmodified lysosome) following administration of the modified lysosome multiple times (e.g., more than once, e.g., two or more times), of any of the foregoing embodiments of the lysosome.

[0102] 98. Any of the fusosomes described in the foregoing embodiments, wherein the fusosomes described herein are not inactivated by serum after multiple administrations.

[0103] 99. Any of the fusosomes described in the foregoing embodiments, wherein the measure of the immunogenicity of the fusosome is, for example, serum inactivation after multiple administrations, for example, serum inactivation after multiple administrations measured as described herein, for example, as described in Example 12.

[0104] 100. Any of the fusosomes described in the foregoing 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 with modified (e.g., HEK293-HLA-G) fusosomes.

[0105] 101. Any of the fusosomes described in the foregoing 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. Any of the fusosomes described in the foregoing embodiments, wherein the percentage of cells receiving the exogenous agent does not differ between fusosome samples incubated with serum from mice treated with vehicle and serum from mice treated with modified (e.g., HEK293-HLA-G) fusosomes.

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

[0108] 104. Any of the fusosomes described in the foregoing embodiments, wherein the measure of the immunogenicity against the fusosome is an antibody response.

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

[0110] 106. Serum from fusosome-naïve mice shows a greater signal (e.g., fluorescence) than serum from negative controls, such as mice depleted of IgM and IgG, indicating, for example, that immunogenicity has occurred, and any of the fusosomes of the foregoing embodiments.

[0111] 107. Serum from fusosome-naïve mice shows a similar signal (e.g., fluorescence) compared to the negative control, indicating, for example, that immunogenicity has not occurred detectably, and any of the fusosomes of the foregoing embodiments.

[0112] 108. Any of the fusosomes of the foregoing embodiments, which is a modified fusosome, modified, for example, by the method described herein, and when measured as described herein, for example, as described in Example 14, the humoral response is reduced (e.g., reduced compared to administration of unmodified fusosomes) after multiple administrations (e.g., more than once, e.g., two or more times) of the modified fusosome.

[0113] 109. The fusosome is produced by the method of Example 5, 6, 7, or 14 from cells transfected with, for example, HLA-G or HLA-E cDNA, and any of the fusosomes of the foregoing embodiments.

[0114] 110. The humoral response is evaluated by determining the level value of anti-fusosome antibodies (e.g., IgM, IgG1, and / or IgG2 antibodies), and any of the fusosomes of the foregoing embodiments.

[0115] 111. A fusosome (e.g., NMC-HLA-G) that has been modified, when compared to a control, e.g., an NMC fusosome or an NMC-empty fusosome, has a reduced antiviral IgM or IgG1 / 2 antibody titer (e.g., measured by fluorescence intensity by FACS) after injection, and is a fusosome of any of the foregoing embodiments.

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

[0117] 113. A fusosome in which a signal (e.g., mean fluorescence intensity) is similar in recipient cells from mice treated with fusosomes and mice treated with PBS, and is a fusosome of any of the foregoing embodiments.

[0118] 114. A fusosome in which a measure of the immunogenicity of recipient cells is a macrophage response, and is a fusosome of any of the foregoing embodiments.

[0119] 115. A recipient cell that is not targeted by macrophages or is targeted at a level below the reference level, and is a fusosome of any of the foregoing embodiments.

[0120] 116. A fusosome in which a phagocytosis index, measured as described herein, e.g., as described in Example 16, is similar in recipient cells from mice treated with fusosomes and mice treated with PBS, and is a fusosome of any of the foregoing embodiments.

[0121] 117. A fusosome in which a measure of the immunogenicity of recipient cells is a PBMC response, and is a fusosome of any of the foregoing embodiments.

[0122] 118. A fusosome in which recipient cells do not induce a PBMC response, and is a fusosome of any of the foregoing embodiments.

[0123] 119. Any of the fusosomes of the foregoing embodiments, wherein the percentage of CD3+ / CMG+ cells is similar in recipient cells derived from mice treated with fusosomes and mice treated with PBS when measured as described herein, for example, as described in Example 17.

[0124] 120. Any of the fusosomes of the foregoing embodiments, wherein the measure of the immunogenicity of the recipient cells is the natural killer cell response.

[0125] 121. Any of the fusosomes of the foregoing embodiments, wherein the recipient cells do not induce a natural killer cell response or induce a natural killer cell response that is, for example, lower than a reference value.

[0126] 122. Any of the fusosomes of the foregoing embodiments, wherein the percentage of CD3+ / CMG+ cells is similar in recipient cells derived from mice treated with fusosomes and mice treated with PBS when measured as described herein, for example, as described in Example 18.

[0127] 123. Any of the fusosomes of the foregoing embodiments, wherein the measure of the immunogenicity of the recipient cells is the CD8+ T cell response.

[0128] 124. Any of the fusosomes of the foregoing embodiments, wherein the recipient cells do not induce a CD8+ T cell response or induce a CD8+ T cell response that is, for example, lower than a reference value.

[0129] 125. Any of the fusosomes of the foregoing embodiments, wherein the percentage of CD3+ / CMG+ cells is similar in recipient cells derived from mice treated with fusosomes and mice treated with PBS when measured as described herein, for example, as described in Example 19.

[0130] 126. Any of the fusosomes of the foregoing embodiments, wherein the fusogen is a retargeted fusogen.

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

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

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

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

[0135] 131. The method of embodiment 130, wherein the target tissue or target cell is present in the 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 according to any of embodiments 1-127 or the pharmaceutical composition of claim 128.

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

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

[0139] 135. The fusosome according to any one of embodiments 132 to 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 the lysosomal disease or disorder.

[0140] 136. The method according to 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; Friedrich ataxia; Rett syndrome; Canavan disease; pyridoxine-dependent epilepsy; Batten disease, fucosidosis; Krabbe disease; Tay-Sachs disease; Sandhoff disease; beta-mannosidosis; metachromatic leukodystrophy; mucolipidosis IIIa; mucolipidosis IIIb; or mucolipidosis IV.

[0141] 137. The fusosome according to any one 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 gene deletion.

[0142] 138. Use of the fusosome according to any one of embodiments 1 to 127 or the pharmaceutical composition of embodiment 128 for manufacturing an agent for use in treating a subject (e.g., a human subject) having a gene deletion. [[ID=_{18]]

[0143] 139. The fusosome or pharmaceutical composition for use according to embodiment 137, or the use according to embodiment 138, wherein the fusosome comprises a payload gene encoding an exogenous agent for treating a gene deletion.

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

[0145] 141. 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; Friedrich ataxia; Rett syndrome; Canavan disease; pyridoxine-dependent epilepsy; Batten disease, fucosidosis; Krabbe disease; Tay-Sachs disease; Sandhoff disease; beta-mannosidosis; metachromatic leukodystrophy; mucolipidosis IIIa; mucolipidosis IIIb; or mucolipidosis IV, and the fusosome or pharmaceutical composition for the use according to embodiment 137, 139 or 140, or the use according to embodiment 138, 139 or 140.

[0146] 142. The following: a) providing a cell comprising a nucleic acid, such as a retroviral nucleic acid, and a fusogen; b) culturing the cell under conditions that allow for the production of fusosomes, and c) separating, concentrating, or purifying the fusosomes from the cell, thereby producing fusosomes, a method for producing a fusosome according to any one of embodiments 1 to 127.

[0147] Other features, objects, and advantages of the present invention will become 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, in this specification, all GenBank, Unigene, and Entrez sequences mentioned in any table are incorporated by reference. Unless otherwise specified, the sequence accession numbers specified herein, including the tables in this specification, refer to the database entries as of May 15, 2018. When a gene or protein refers to multiple sequence accession numbers, all sequence variants are included. In addition, the materials, methods, and examples are illustrative only 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 explaining the invention, specific embodiments described herein are illustrated herein. However, it should be understood that the invention is not limited to the exact arrangements and means of the embodiments shown in the drawings.

Brief Description of the Drawings

[0150]

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

[0152] I. Definitions The terms used in the claims and the specification are defined as described below, unless otherwise specified.

[0153] As used herein, "detectably present" means that the exogenous agent is detectably present when used in the context of an exogenous agent being 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, "vesicle" refers to an amphiphilic lipid bilayer surrounding a lumen or cavity, and a fusogen that interacts with the amphiphilic lipid bilayer. In embodiments, the vesicle contains nucleic acid. In some embodiments, the vesicle is a membrane-enclosed preparation. In some embodiments, the vesicle is derived from a source cell.

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

[0156] As used herein, "fusogen" refers to an agent or molecule that creates an interaction between two membranes-enclosed lumens. In embodiments, the fusogen promotes membrane fusion. In other embodiments, the fusogen creates a connection, e.g., a pore, between two lumens (e.g., the lumen of a retroviral vector and the cytoplasm of a target cell). In some embodiments, the fusogen comprises a complex of two or more proteins, e.g., none of the proteins have only fusion activity. In some embodiments, the fusogen comprises a targeting domain.

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

[0158] As used herein, the term "effective amount" means an amount of a pharmaceutical composition sufficient to meaningfully and positively modify the condition and / or state being treated (e.g., to provide a positive clinical response). The effective amount of an 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 combination therapy, the particular active ingredient(s) being used, the particular pharmaceutically acceptable excipient and / or carrier being utilized, and like factors based on the knowledge and expertise of the attending physician.

[0159] As used herein, "exogenous agent" with respect to a virus, VLP or fusosome refers to an agent that is not included in the corresponding wild-type virus or the fusogen made from the corresponding wild-type source cell and is not encoded. 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 does not naturally exist in the source cell. In some embodiments, the exogenous agent naturally exists in the source cell but is exogenous to the virus. In some embodiments, the exogenous agent does not naturally exist in the recipient cell. In some embodiments, the exogenous agent naturally exists in the recipient cell but is not present at the desired level or at the desired time. In some embodiments, the exogenous agent comprises RNA or protein.

[0160] As used herein, the term "pharmaceutically acceptable" refers to excipients, compositions and / or dosage forms that are suitable for use in contact with the tissues of humans and animals within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problems or complications, and 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, the promoter functions in concert with one or more enhancers distal to the gene.

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

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

[0164] As used herein, "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, and 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., a miRNA recognition site that causes degradation or inhibition of 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 a miRNA sequence present in an mRNA encoding an exogenous protein agent, such that the mRNA is degraded or inhibited in non-target cells. In some embodiments, the NTCSRE increases the level or activity of a downregulator or inhibitor of the 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), where the target cells are CNS cells. Thus, a non-CNS cell-specific regulatory element refers to a nucleic acid sequence that reduces the level of an exogenous agent in non-CNS cells as compared to CNS cells, and the nucleic acid encoding the exogenous agent is operably linked to the non-CNS cell-specific regulatory element.

[0166] As used herein, "retargeted fusogen" refers to a fusogen that includes a targeting moiety having a sequence that is not part of the naturally occurring form of the fusogen. In embodiments, the fusogen includes a different targeting moiety as 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 as compared to the naturally occurring form of the fusogen, for example, in a transmembrane domain, a fusogenically active domain, or a cytoplasmic domain.

[0167] As used herein, "retroviral nucleic acid" refers to a nucleic acid that alone or in combination with a helper cell, helper virus, or helper plasmid contains at least the minimal sequence requirements for packaging into a retrovirus or retroviral vector. In some embodiments, the retroviral nucleic acid further comprises or encodes an exogenous agent, a positive target cell-specific regulatory element, a non-target cell-specific regulatory element, or a negative TCSRE. In some embodiments, the retroviral nucleic acid comprises one or more of a 5' LTR (e.g., to facilitate integration), U3 (e.g., to activate viral genomic RNA transcription), R (e.g., Tat binding region), U5, 3' LTR (e.g., to facilitate integration), a packaging site (e.g., psi (Ψ)), RRE (e.g., to bind Rev and facilitate nuclear export). The 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, helper virus, or helper plasmid comprising one or more (e.g., all) of gag, pol, and env.

[0168] As used herein, "target cell" refers to the type of cell into which it is desired to deliver an exogenous agent by a fusosome (e.g., a lentiviral vector). In embodiments, the target cell is a specific tissue type or class of cells, e.g., CNS cells, e.g., neurons or glial cells. 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), leads to preferential delivery of an exogenous agent to the target cell as compared to non-target cells.

[0169] As used herein, "non-target cell" refers to a type of cell to which it is not desirable for the lentiviral vector to deliver an exogenous agent. In some embodiments, the non-target cell is a particular tissue type or class of cells. In some embodiments, the non-target cell is a non-diseased cell, e.g., a non-cancerous 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), leads to lower delivery of an exogenous agent to non-target cells as compared to target cells.

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

[0171] As used herein, "cytological material" refers to a part of a cell, including the lumen and cell membrane, or a cell having partial or complete nuclear inactivation. In some embodiments, the cytological material includes one or more of cytoskeletal components, organelles, and ribosomes. In embodiments, the cytological 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, for example, extracellular vesicles, microvesicles, nanovesicles, exosomes, apoptotic bodies (derived from apoptotic cells), microparticles (e.g., can be derived from platelets), ectosomes (e.g., can be induced from neutrophils and monocytes in serum), prostasomes (obtained from prostate cancer cells), cardiosomes (obtained from heart cells), or any combination thereof, or can include them. 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 have a diameter of about 10 - 10,000 nm, for example, a diameter of about 30 - 100 nm. In some embodiments, fusosomes contain one or more synthetic lipids.

[0173] In some embodiments, fusosomes are a virus, for example, a retrovirus, for example, a lentivirus, or include it. For example, in some embodiments, the bilayer of the fusosome of amphiphilic lipids is a viral envelope, or includes them. The viral envelope can contain fusogens, for example, fusogens endogenous to the virus or pseudotyped fusogens. In some embodiments, the lumen or cavity of the fusosome contains viral nucleic acids, for example, retroviral nucleic acids, for example, lentiviral nucleic acids. The viral nucleic acid can be a viral genome. In some embodiments, fusosomes further contain, for example, in their cavity or lumen, one or more viral non-structural proteins.

[0174] Fusosomes can have various properties that facilitate the delivery of payloads to target cells. For example, in some embodiments, the fusosome and the source cell together contain sufficient nucleic acid(s) to make particles that can fuse with the 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 fusogen activity.

[0175] Fusosomes can also contain various structures that facilitate the delivery of payloads to target cells. For example, in some embodiments, the fusosome and the source cell together contain sufficient nucleic acid(s) to make particles that can fuse with the 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 fusogen activity.

[0176] The fusosome can also include various structures that facilitate the delivery of the payload to the target cell. For example, in some embodiments, the fusosome (e.g., a virus, e.g., a retrovirus, e.g., a lentivirus) includes one or more (e.g., all) of the following proteins: gag polyprotein, polymerase (e.g., pol), integrase (e.g., a functional or non-functional variant), protease, and fusogen. In some embodiments, the fusosome further includes 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 by, for example, helper cells, helper viruses, or helper plasmids. In some embodiments, the fusosome nucleic acid (e.g., a retroviral nucleic acid) includes one or more (e.g., all) of the following nucleic acid sequences: 5' LTR (e.g., including U5 and lacking a functional U3 domain), Psi packaging element (Psi), central polypurine tract (cPPT) promoter operably linked to the payload gene, payload gene (optionally including an intron before the open reading frame), polyA tail sequence, WPRE, and 3' LTR (e.g., lacking U5 and a functional U3). In some embodiments, the fusosome nucleic acid (e.g., a retroviral nucleic acid) further includes one or more insulator sequences. In some embodiments, the fusosome nucleic acid (e.g., a retroviral nucleic acid) further includes one or more miRNA recognition sites. In some embodiments, one or more miRNA recognition sites are located downstream of the polyA tail sequence, e.g., between the polyA tail sequence and WPRE.

[0177] In some embodiments, the fusosomes provided herein are administered to a subject, such as a mammal, such as a human. In such embodiments, the subject can be identified as having a risk of, having symptoms of, or being diagnosable with a particular disease or condition (such as 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 fusosome comprises a nucleic acid sequence encoding an exogenous agent for treating a disease or condition, such as for treating a genetic defect.

[0178] A. Fusosomes generated from viruses. 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 variant), protease, and fusogen. In some embodiments, the fusosome further comprises rev. In some embodiments, one or more of the foregoing proteins are encoded by a retroviral genome, and in some embodiments, one or more of the foregoing proteins are provided in trans, e.g., by a helper cell, a helper virus, or a helper plasmid. In some embodiments, a fusosome nucleic acid (e.g., a retroviral nucleic acid) comprises one or more (e.g., all) of the following nucleic acid sequences: 5’ LTR (e.g., including U5 and lacking a functional U3 domain), Psi packaging element (Psi), central polypurine tract (cPPT) promoter operably linked to a payload gene, a payload gene (optionally including an intron upstream of the open reading frame), a polyA tail sequence, WPRE, and 3’ LTR (e.g., lacking U5 and a functional U3). In some embodiments, a fusosome nucleic acid (e.g., a retroviral nucleic acid) further comprises one or more insulator sequences. In some embodiments, a fusosome nucleic acid (e.g., a retroviral nucleic acid) further comprises one or more miRNA recognition sites. In some embodiments, one or more miRNA recognition sites are located downstream of the polyA tail sequence, e.g., between the polyA tail sequence and 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 the expression of the entire packaged RNA), a 5' LTR (e.g., U5 containing R (including the 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 the expression of the transgene, a transgene (or other exogenous effector element), a polypurine tract, and a 3' LTR (e.g., containing 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 covalently integrating 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, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV)), as well as lentiviruses.

[0181] In some embodiments, the retrovirus is a gammaretrovirus. In some embodiments, the retrovirus is an epsilonretrovirus. In some embodiments, the retrovirus is an alpharetrovirus. In some embodiments, the retrovirus is a betaretrovirus. In some embodiments, the retrovirus is a deltaretrovirus. In some embodiments, the retrovirus is a lentivirus. In some embodiments, the retrovirus is a spumaretrovirus. In some embodiments, the retrovirus is an endogenous retrovirus.

[0182] Exemplary lentiviruses include, but are not limited to, the following: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Visna - Maedi virus (VMV); Caprine arthritis - encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine 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, the vectors herein are nucleic acid molecules capable of importing or transporting another nucleic acid molecule. The imported nucleic acid is generally ligated, e.g., inserted, into the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication within a cell or may contain sequences sufficient to enable integration into the 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 can contain, for example, nucleic acid molecules (such as transfer plasmids) that typically include virus-derived nucleic acid elements that facilitate the transfer of nucleic acid molecules or the integration into viral particles that mediate genomic or nucleic acid transfer in cells. Viral particles typically contain various viral components in addition to the nucleic acid(s), and sometimes also host cell components. Viral vectors can contain, for example, viruses or viral particles that can transfer nucleic acids into cells or transfer the transferred nucleic acids (e.g., as naked DNA). Viral vectors and transfer plasmids can mainly contain structural and / or functional gene elements derived from viruses. Retroviral vectors can contain viral vectors or plasmids that mainly contain structural and functional genetic elements derived from retroviruses, or a part thereof. Lentiviral vectors can contain viral vectors or plasmids that mainly contain structural and functional genetic elements derived from lentiviruses, or a part thereof that includes LTRs.

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

[0186] In some vectors described herein, at least a part of one or more protein-coding regions that contribute to or are essential for replication may be absent compared to the corresponding wild-type virus. This results in a defect in the replication of the viral vector. In some embodiments, the vector can transduce a target non-dividing host cell and / or integrate its genome into the host genome.

[0187] The structure of the wild-type retroviral genome often includes 5' terminal repeats (LTRs) and 3' LTRs, between or within which are a packaging signal that enables packaging of the genome, a primer binding site, an integration site for enabling integration into the host cell genome, and the gag, pol, and env genes that encode packaging components that facilitate assembly of viral particles. More complex retroviruses have additional functions such as the rev and RRE sequences of HIV, which can efficiently export the RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of the infected target cell. In a provirus, regions called terminal repeats (LTRs) flank both ends of the viral genes. The LTRs are involved in the integration and transcription of the provirus. The LTR also functions as an enhancer-promoter sequence and can control the expression of viral genes. Capsid formation of retroviral RNA occurs by the psi sequence located at the 5' end of the viral genome.

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

[0189] In the case of the viral genome, the transcription start 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 any one or more of the following genes that encode proteins involved in the regulation of 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 processed by proteolysis into the mature proteins MA (matrix), CA (capsid), and NC (nucleocapsid). The pol gene encodes a reverse transcriptase (RT) that includes a DNA polymerase, an associated RNase H, and an integrase (IN) that mediates the replication of the genome. The env gene encodes the surface (SU) glycoprotein and the transmembrane (TM) protein of the virion that form a complex that specifically interacts with the cellular receptor protein. This interaction promotes infection, for example, by fusion of the viral membrane and the cell membrane.

[0191] In the replication-defective retroviral vector genome gag, pol and env 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 that encode proteins other than gag, pol, and env. Examples of additional genes include one or more of vif, vpr, vpx, vpu, tat, rev, and nef (in HIV). EIAV has, among others, an additional gene, S2. The proteins encoded by the additional genes perform various functions, some of which may overlap with functions provided by cellular proteins. For example, in EIAV, tat functions as a transcriptional activator of the viral LTR (Derse and Newbold 1993 Virology 194:530-6; Maury et al. 1994 Virology 200:632-42). Tat binds to a stable stem-loop RNA secondary structure called TAR. Rev regulates and coordinates the expression of viral genes 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 generally the same as those of similar mechanisms in primate viruses. In addition, the EIAV protein Ttm, encoded by the first exon of tat spliced into the env coding sequence at the start of the transmembrane protein, has been identified.

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

[0194] In an embodiment, a recombinant lentiviral vector (RLV) is a vector having retroviral genetic information sufficient to enable packaging of an RNA genome into viral particles capable of infecting target cells in the presence of packaging components. Infection of the target cells can include reverse transcription and integration into the target cell genome. RLVs typically carry non-viral coding sequences that are delivered to the target cells by the vector. In an embodiment, RLVs are unable to replicate independently to produce infectious retroviral particles within the target cells. Typically, RLVs lack functional gag-pol and / or env genes and / or other genes involved in replication. The vector can be configured as a split-intron vector, for example, as described in International Publication No. WO 99 / 15683, which is incorporated herein by reference in its entirety.

[0195] In some embodiments, the lentiviral vector comprises a minimal viral genome and is engineered, for example, as described in International Publication No. WO 98 / 17815, which is incorporated herein by reference in its entirety, to remove non-essential elements and retain essential elements in order to provide the functions necessary to infect, transduce, and deliver a nucleotide sequence of interest to a target host cell.

[0196] The smallest lentiviral genome can, for example, comprise (5’)R-U5-one or more first nucleotide sequences-U3-R(3’). However, plasmid vectors used to produce the lentiviral genome in a source cell can 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 can comprise native sequences associated with the transcribed retroviral sequences, such as the 5’ U3 region, or can comprise a heterologous promoter, such as a CMV promoter or the like, of another virus. Some lentiviral genomes comprise additional sequences to facilitate efficient virus production. For example, in the case of HIV, rev and RRE sequences may be included. Alternatively or in combination, codon optimization can be used, for example, genes encoding exogenous agents can be codon optimized as described in WO 01 / 79518, which is hereby incorporated by reference in its entirety. Alternative sequences that perform the same or similar function as the rev / RRE system can also be used. For example, a functional analog of the rev / RRE system is found in Mason Pfizer monkey virus. This is known as CTE and contains an RRE-type sequence within the genome that is thought to interact with factors in infected cells. The cellular factor can be considered a rev analog. Thus, CTE can be used as an alternative to 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 IRE-BP.

[0197] In some embodiments, a retroviral nucleic acid (e.g., a lentiviral nucleic acid, e.g., a primate or non - primate lentiviral nucleic acid) comprises: (1) a deleted gag gene in which the deletion 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 contains a leader sequence between the end of the 5′ LTR and the ATG of gag; (4) is a combination of (1), (2), and (3). In one embodiment, the lentiviral vector comprises all of features (1), (2), and (3). This strategy is described in more detail in International Publication No. WO 99 / 32646, which is incorporated herein by reference in its entirety.

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

[0199] Deletion of the additional genes may allow for the generation of vectors without genes associated with lentiviral (such as HIV) infectious diseases. In particular, tat is associated with disease. Second, deletion of the additional genes may allow the vector to package more heterologous DNA. Third, omitting genes of unknown function such as S2 can reduce the risk of unwanted effects. Examples of minimal lentiviral vectors are disclosed in International Publication No. WO 99 / ​​32646 and International Publication No. WO 98 / 17815.

[0200] In some embodiments, the retroviral nucleic acid lacks at least tat and S2 (when it is an EIAV vector system), and possibly also vif, vpr, vpx, vpu, and nef. In some embodiments, the retroviral nucleic acid also lacks rev, RRE, or both.

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

[0202] If the cells are different, the usage of specific codons is also different. This codon bias corresponds to the bias in the relative abundance of specific tRNAs in the cell type. It is possible to increase expression by changing the codons within the sequence so that they are adjusted to match the relative abundance of the corresponding tRNA. Similarly, it is possible to decrease expression by intentionally selecting codons for which the corresponding tRNA is known to be rare in a particular cell type. Thus, additional translational control is available. Additional explanations of codon optimization can be found, for example, in WO 99 / 41397, which is hereby incorporated by reference in its entirety.

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

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

[0205] In some embodiments, only the codons related to INS are codon-optimized. In other embodiments, the sequences are codon-optimized as a whole, except for the sequences containing the frameshift sites of gag-pol.

[0206] The gag-pol gene contains two overlapping reading frames that encode the gag-pol protein. Expression of both proteins depends on a frameshift during translation. This frameshift occurs as a result of "slippage" of the ribosome during translation. This slippage is thought to be caused, at least in part, by an RNA secondary structure that stalls the ribosome. Such secondary structures are present downstream of the frameshift site of the gag-pol gene. In the case of HIV, the overlapping region extends from nucleotide 1222 (nucleotide 1 is the A of the gag ATG) downstream of the start of gag to the end of gag (nt 1503). As a result, a 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 will allow for more efficient expression of the gag-pol protein. In the case of 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 the overlap of gag-pol 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 frequency in mammalian systems. The third and sometimes the second and third bases may be changed.

[0209] Those skilled in the art will appreciate that due to the degeneracy properties of the genetic code, a number of gag-pol sequences can be achieved. Also, many retroviral variants have been described that can be used as starting points for generating codon-optimized gag-pol sequences. The genomes of lentiviruses can vary considerably. For example, there are many pseudospecies still functioning in HIV-I. This also applies to EIAV. These variants can be used to enhance specific parts of the transduction process. Examples of HIV-I variants are in the HIV database managed by the Los Alamos National Laboratory. Details of EIAV clones are in the NCBI database managed by the National Institutes of Health.

[0210] The strategy of codon-optimized gag-pol sequences can be used in relation to retroviruses such as EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-I, HIV-2, etc. Further, this method can be used to increase the expression of genes from HTLV-I, HTLV-2, HFV, HSRV, and human endogenous retroviruses (HERV), 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. Further, in packaging, a short sequence of four stem-loops followed by a partial sequence from gag and env can be utilized as a packaging signal. Thus, (in addition to the complete gag sequence on the packaging construct) the gag sequence deleted from the retroviral vector genome can be included. In embodiments, the retroviral vector includes a packaging signal that includes a 255 - 360 nucleotide 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, the retroviral vector includes a gag sequence that includes one or more deletions, for example, the gag sequence includes approximately 360 nucleotides derived from the N-terminus.

[0212] A retroviral vector, helper cell, helper virus, or helper plasmid can contain 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 two or more retroviruses, such as 2, 3, 4, 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 a 55kD Gag precursor protein, also called p55. p55 is cleaved by the virus-encoded protease 4 (the product of the pol gene) during its maturation into four small proteins called MA (matrix [p17]), CA (capsid [p24]), NC (nucleocapsid [p9]), and p6. The pol precursor protein is cleaved from Gag by the virus-encoded protease and further digested to separate the activities of protease (p10), RT (p50), RNase H (p15), and integrase (p31).

[0214] The native Gag-Pol sequence can be used or modified in helper vectors (such as helper plasmids and helper viruses). These modifications include chimeric Gag-Pol, where the Gag and Pol sequences are obtained from different viruses (such as different species, subspecies, strains, clades, etc.) and / or the sequences are 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 includes a mutant INS1 inhibitory sequence that reduces the restriction of RNA nuclear export as compared to wild-type INS1, (ii) includes two nucleotide insertions that result in a frameshift and early termination, and / or (iii) does not include the INS2, INS3, and INS4 inhibitory sequences of gag.

[0216] In some embodiments, the vectors described herein are hybrid vectors that include both retroviral (e.g., lentiviral) sequences and non-lentiviral viral sequences. In some embodiments, the hybrid vector includes sequences of a retrovirus, such as a lentivirus, 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, such as HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used, or that combined and numerous substitutions and alterations in specific lentiviral sequences can be adapted without impairing the ability of the transfer vector to perform the functions described herein. Various lentiviral vectors are described in Naldini et al., (1996a, 1996b, and 1998), Zufferey et al., (1997), Dull et al., 1998, U.S. Patent No. 6,013,516, and U.S. Patent No. 5,994,136, many of which can be adapted to produce retroviral nucleic acids.

[0218] At both ends of the provirus, long terminal repeats (LTRs) are usually found. The LTR generally contains domains located at the ends of retroviral nucleic acids, which are direct repeats in the context of the natural sequence and include U3, R, and U5 regions. The LTR generally promotes the expression of retroviral genes (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. The LTR can contain a number of regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences for replication and integration of the viral genome. The viral LTR is usually divided into three regions called U3, R, and U5. The U3 region usually contains enhancer and promoter elements. The U5 region is usually the sequence between the primer binding site and the R region and can contain a polyadenylation sequence. The R (repeat) region can be adjacent to the U3 and U5 regions. The LTR is 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, there 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 the insertion of viral RNA into the viral capsid or particle. See, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109. Some retroviral vectors use a minimal packaging signal (psi [Ψ] sequence) for capsid formation 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 can comprise 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 a lentivirus or retroviral system by making the virus replication defective (e.g., a virus that cannot replicate completely and effectively such that infectious virions are not produced, such as the 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, wherein the right (3’) LTR enhancer-promoter region, known as the U3 region, is modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of virus replication. This is because during virus replication, the right (3’) LTR U3 region can be used as a template for the left (5’) LTR U3 region, and thus virus replication is inhibited in the absence of the U3 enhancer promoter. In embodiments, the 3’LTR is modified such that the U5 region is removed, altered, or substituted, e.g., with an exogenous poly(A) sequence. The 3’LTR, 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 production of viral particles. Examples of heterologous promoters that can be used include, for example, viral Simian Virus 40 (SV40) (e.g., early or late), Cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and Herpes simplex virus (HSV) (thymidine kinase) promoters. In some embodiments, the promoter can drive high levels of transcription in a Tat-independent manner. In certain embodiments, the heterologous promoter has additional advantages in controlling how the viral genome is transcribed. For example, the heterologous promoter can be inducible such that transcription of all or part of the viral genome occurs only in the presence of an inducer. Inducers include, but are not limited to, one or more compounds in which the host cell is cultured, or physiological conditions such as temperature or pH.

[0223] In some embodiments, the viral vector includes, for example, a TAR (trans-activation response) element located in the R region of a lentiviral (e.g., HIV) LTR. This element interacts with the lentiviral trans-activator (tat) gene element to enhance viral replication. However, this element is not required, for example, in embodiments where the U3 region of the 5’ LTR has been replaced by a heterologous promoter.

[0224] The R region, for example, the region within a retroviral LTR that begins at the start of the capping group (i.e., the start of transcription) and ends just prior to the start of the polyA tract, can be adjacent to the U3 and U5 regions. The R region plays a role during reverse transcription in the transfer of nascent DNA from one end of the genome to the other.

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

[0226] In some embodiments, a retroviral or lentiviral nucleic acid includes one or more export elements, for example, 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 hereby incorporated by reference in their entirety. Generally, an RNA export element is located within the 3’UTR of a gene and can be inserted as one or more copies.

[0227] In some embodiments, the expression of heterologous sequences in viral vectors is increased, for example, by incorporating into the vector one or more of post-transcriptional regulatory elements, polyadenylation sites, and transcription termination signals. Various post-transcriptional regulatory elements can increase the expression of heterologous nucleic acids in proteins, such as the post-transcriptional regulatory element of woodchuck hepatitis virus (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the post-transcriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864), etc. (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 post-transcriptional regulatory elements such as WPRE or HPRE.

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

[0229] Elements that direct the termination and polyadenylation of heterologous nucleic acid transcripts can be included, for example, to increase the expression of exogenous agents. Transcription termination signals may be found downstream of the polyadenylation signal. In some embodiments, the vector includes a polyadenylation sequence 3' of the polynucleotide encoding the exogenous agent. The polyA site can include a DNA sequence that directs both the termination and polyadenylation of nascent RNA transcripts by RNA polymerase II. The polyadenylation sequence promotes the stability of mRNA by adding a polyA tail to the 3' end of the coding sequence and contributes to the improvement of translation efficiency. 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, such as the insulator sequences 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 may include one or more modifications such as one or more nucleotide substitutions, additions, or deletions. The vector is one or more accessory elements (e.g., cPPT / FLAP) for enhancing transduction efficiency, viral packaging (e.g., Psi (Ψ) packaging signal, RRE), and / or other elements for increasing exogenous gene expression (e.g., poly(A) sequence), and optionally may include WPRE or HPRE.

[0232] In some embodiments, the lentiviral nucleic acid comprises one or more of a promoter (e.g., CMV), R sequence (e.g., including TAR), U5 sequence (e.g., for integration), PBS sequence (e.g., for reverse transcription), DIS sequence (e.g., for genomic dimerization), psi packaging signal, partial gag sequence, RRE sequence (e.g., for nuclear export), cPPT sequence (e.g., for nuclear import), a promoter driving expression of an exogenous agent, a gene encoding an exogenous agent, WPRE sequence (e.g., for efficient transgene expression), PPT sequence (e.g., for reverse transcription), R sequence (e.g., for polyadenylation and termination), and U5 signal (e.g., for integration), all in order, for example, from 5' to 3'.

[0233] ii) a vector engineered to remove splice sites Some lentiviral vectors have strong splicing and polyadenylation signals that can integrate within an active gene and lead to the formation of abnormal, sometimes truncated, transcripts.

[0234] The mechanism of proto-oncogene activation may involve the generation of chimeric transcripts resulting from the interaction between promoter elements or splice sites contained in the genome of the insertional mutagen and 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 mRNA can be generated by read-through transcription starting from the vector sequences and proceeding into adjacent cellular genes, or vice versa.

[0235] In some embodiments, the lentiviral nucleic acids described herein include a lentiviral backbone in which at least two splice sites have been removed, for example, to improve the safety profile of the lentiviral vector. The types and methods of identification of such splice sites are described in International Publication No. WO 2012 / 156839 A2, which are hereby incorporated by reference in their entirety.

[0236] iii) Method for producing retrovirus Large-scale production of virus particles is often useful to achieve the desired virus titer. Virus particles can be produced by transfecting a packaging cell line containing viral structural and / or accessory genes, such as the gag, pol, env, tat, rev, vif, vpr, vpu, vpx or nef genes, or other retroviral genes, with a transfer vector.

[0237] In an embodiment, the packaging vector is an expression vector or a viral vector that lacks a packaging signal and contains a polynucleotide encoding one, two, three, four, or more viral structures and / or accessory genes. Usually, the packaging vector is contained in a packaging cell and introduced into the cell via transfection, transduction, or infection. A transfer vector of a retrovirus, such as a lentivirus, can be introduced into a packaging cell line via transfection, transduction, or infection to generate a source cell or cell line. The packaging vector can be introduced into human cells or cell lines by standard methods including, for example, calcium phosphate transfection, lipofection, or electroporation. In some embodiments, the packaging vector is introduced into the cell together with a major selectable marker such as neomycin, hygromycin, puromycin, blasticidin, zeocin, thymidine kinase, DHFR, Gln synthetase, or ADA, and subsequently selected in the presence of the appropriate drug to isolate clones. The selectable marker gene can be physically linked to the gene encoded 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 replicases (e.g., gag, pol, and env) capable of packaging viral particles. Any suitable cell line can be used, such as mammalian cells, e.g., human cells. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In an embodiment, the packaging cell is a 293 cell, a 293T cell, or an A549 cell.

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

[0240] iv) Packaging of plasmids and cell lines In some embodiments, the source cell comprises one or more plasmids encoding viral structural proteins and replication enzymes (e.g., gag, pol, and env) that can package 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, the 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, the expression of the stably integrated viral structural genes is inducible.

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

[0243] In some embodiments, the expression of viral structural genes is regulated by a tetracycline (Tet)-dependent system, where the Tet-regulated transcriptional repressor (Tet-R) binds to the DNA sequence contained in the promoter and suppresses transcription by steric hindrance (Yao et al, 1998; Jones et al, 2005). When doxycycline (dox) is added, Tet-R is released and transcription becomes possible. A plurality of other suitable transcriptional regulatory promoters, transcription factors, and small molecule inducers are suitable for regulating the transcription of viral structural genes.

[0244] In some embodiments, an envelope under the control of third-generation lentiviral components, human immunodeficiency virus type 1 (HIV) Rev, Gag / Pol, and a Tet-regulated promoter and linked to an antibiotic resistance cassette is separately integrated into the source cell genome. In some embodiments, the source cell has only one copy each of Rev, Gag / Pol, and the envelope protein integrated into the 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 having Rev, Gag / Pol, and envelope proteins stably integrated into the genome. See, for example, Milani et al.EMBO Molecular Medicine, 2017, which is incorporated herein by reference in its entirety.

[0246] In some embodiments, the retroviral nucleic acids described herein are unable to undergo reverse transcription. Such nucleic acids can, in embodiments, transiently express an exogenous agent. The retrovirus or VLP may contain an inactivated reverse transcriptase protein or may not contain a reverse transcriptase protein. In embodiments, the retroviral nucleic acid contains an inactivated 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 inactivated or absent from the retroviral nucleic acid. In embodiments, one or more accessory genes selected from S2, rev, and tat are inactivated 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 that expresses the trans-acting retroviral gene sequences (e.g., gag, pol, and env) necessary for the production of viral particles. By completely separating the cis- and trans-acting vector sequences, the virus is unable to maintain replication over more than one cycle of infection. The generation of live virus can be avoided by several strategies, e.g., by minimizing overlap between cis- and trans-acting sequences to avoid recombination.

[0248] Viral vector particles lacking or containing sequences that lack viral RNA may be the result of removing or excluding viral RNA from the sequences. In one embodiment, this can be achieved by using the endogenous packaging signal binding site on gag. Alternatively, the endogenous packaging signal binding site is on pol. In this embodiment, the RNA to be delivered will contain the cognate packaging signal. In another embodiment, a heterologous binding domain located on the RNA to be delivered (heterologous to gag) and a cognate binding site located on gag or pol can be used to ensure the packaging of the RNA to be delivered. The heterologous sequence may be non-viral or viral, in which case it may be derived from a different virus. The vector particles can be used to deliver therapeutic RNA, in which case a functional integrase and / or reverse transcriptase is not required. These vector particles can also be used to deliver a therapeutic gene of interest, in which case pol is usually included.

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

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

[0251] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a viral gag protein or a retroviral gag and pol protein, and the gag protein or pol protein comprises a heterologous RNA-binding domain that can recognize a corresponding sequence in the RNA sequence and facilitate packaging of the RNA sequence into viral vector particles.

[0252] In some embodiments, the heterologous RNA-binding domain comprises an RNA-binding domain derived from a bacteriophage coat protein, Rev protein, protein of U1 small nuclear ribonucleoprotein particle, Nova protein, TFIII A protein, TIS11 protein, trp RNA-binding attenuation protein (TRAP), or pseudouridine synthase.

[0253] In some embodiments, the methods herein include detecting or confirming the absence of replication-competent retroviruses. The method can include assessing the RNA level of one or more target genes, such as structural genes or packaging genes, of a viral gene that is expressed in certain cells infected with a replication-competent retrovirus, such as a gammaretrovirus or lentivirus, but is absent from the viral vector used to transduce the cells with the heterologous nucleic acid, and is absent and / or not expressed in cells that do not contain a replication-competent retrovirus, or is not expected to be present and / or expressed. A replication-competent retrovirus can be determined to be present if the RNA level of one or more target genes is higher than a reference value that can be measured directly or indirectly, for example, from a positive control sample containing the target gene. For a detailed disclosure, see WO 2018 / 023094 A1.

[0254] vi) Suppression of genes encoding exogenous agents in source cells Proteins overexpressed in the source cells may indirectly or directly affect the assembly and / or infectivity of vector virions. The uptake of exogenous agents into vector virions may also affect downstream processing of vector particles.

[0255] In some embodiments, a tissue-specific promoter is used to restrict the expression of exogenous agents in the source cells. In some embodiments, a heterologous translation control system is used in eukaryotic cell culture to suppress the translation of exogenous agents in the source cells. More specifically, a retroviral nucleic acid may comprise a binding site operably linked to a gene encoding an exogenous agent, wherein the binding site is capable of interacting with an RNA-binding protein such that translation of the exogenous agent is suppressed 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, a tryptophan RNA-binding attenuation protein, TRAP) and the RNA target to which it binds suppresses or prevents the translation of a transgene within a source cell. This system is referred to as Transgene Repression In vector Production cell system or the TRIP system.

[0257] In an embodiment, by arranging a binding site for an RNA-binding protein (e.g., a TRAP-binding sequence, tbs) upstream of the NOI translation start codon, specific suppression of the translation of mRNA derived from an internal expression cassette can be achieved without adverse effects on the production or stability of vector RNA. The number of nucleotides between the tbs of a gene encoding an exogenous agent and the translation start codon can vary from 0 to 12 nucleotides. The tbs can be arranged downstream of an internal ribosome entry site (IRES) to suppress the translation of a gene encoding an exogenous agent in a polycistronic mRNA.

[0258] vii) Kill switch system and amplification In some embodiments, a polynucleotide or cell carrying a gene encoding an exogenous agent utilizes a suicide gene, e.g., an inducible suicide gene, to reduce the risk of direct toxicity and / or uncontrolled proliferation. In certain aspects, the suicide gene is not immunogenic to the host cell harboring the exogenous agent. Examples of suicide genes include caspase-9, caspase-8, or cytosine deaminase. Caspase-9 can be activated using a specific dimerization chemical inducer (CID).

[0259] In certain embodiments, the vector comprises a gene segment that renders a target cell, e.g., an immune effector cell, e.g., a T cell, sensitive to negative selection in vivo. For example, the transduced cells can be eliminated as a result of changes in the in vivo state of the individual. The negatively selectable phenotype can 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, inter alia: the herpes simplex virus type I thymidine kinase (HSV-I TK) gene that confers ganciclovir sensitivity (Wigler et al., Cell 11:223, 1977); the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and the 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 further comprising a positive marker that enables selection of cells with a negatively selectable phenotype in vitro. A positive selection marker can be a gene that expresses a dominant phenotype that enables positive selection of cells carrying the gene when introduced into a target cell. Examples of this type of gene include, inter alia: the hygromycin-B phosphotransferase gene (hph) that confers resistance to hygromycin B, the aminoglycoside phosphotransferase gene (neo or aph) derived from Tn5 that encodes resistance to the antibiotic G418, dihydrofolate reductase (DHFR), the adenosine deaminase gene (ADA), and the multidrug resistance (MDR) gene.

[0261] In some embodiments, the positive selectable marker and the negative selectable element are linked such that loss of the negative selectable element necessarily entails loss of the positive selectable marker. For example, the positive and negative selectable markers can be fused such that loss of one results in loss of the other. An example of a fusion polynucleotide that yields as an expression product a polypeptide that confers both of the desired positive and negative selection functions described above is the hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene produces a polypeptide that confers hygromycin B resistance for positive selection in vitro and ganciclovir sensitivity for negative selection in vivo. See Lupton S.D., et al, Mol. and Cell. Biology 1 1:3374-3378, 1991. Further, in embodiments, the polynucleotide encoding the chimeric receptor is present in a retroviral vector, such as the HyTK retroviral vector described in Lupton S.D., et al, (1991) supra, that includes a fusion gene, particularly a fusion gene that confers hygromycin B resistance for positive selection in vitro and ganciclovir sensitivity for negative selection in vivo. See also the publications of 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 can be derived from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT, and gpt. Other suitable markers are bifunctional selectable fusion genes in which the positive selectable marker is derived from hph or neo and the negative selectable marker is derived from the cytosine deaminase or TK gene, or the selectable marker.

[0263] viii) Strategies for modulating lentiviral integration Retroviral and lentiviral nucleic acids have been described in which important proteins / sequences are missing or disabled to prevent integration of the retroviral or lentiviral genome into the target cell genome, for example, the highly conserved DDE motif of retroviral integrase (Engelman and Craigie (1992) J. Virol. 66:6361-6369; Johnson et al. (1986) Proc. Natl. Acad. Sci. USA 83:7648-7652; Khan et al. al. (1991) Nucleic Acids Res. 19:851-860) allows for the production of integration-defective 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 the gene, or the DNAse1 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 introns.

[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 integration 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, 100.

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

[0270] ix) Maintenance of episomal virus In retroviruses with insufficient integration, the circular cDNA by-products of reverse transcription (e.g., 1-LTR and 2-LTR) may accumulate in the cell nucleus without integrating into the host genome (Yanez-Munoz R J et al., Nat. Med. 2006, 12:348-353). Next, like other exogenous DNAs, these intermediates can be integrated into cellular DNA at the same frequency (e.g., 10 3 ~10 5 / cell).

[0271] In some embodiments, the episomal retroviral nucleic acid does not replicate. The episomal viral DNA can be modified to be maintained in replicating cells by including an origin of replication for eukaryotes and a matrix attachment region (S / MAR) for association with the scaffold / nuclear matrix.

[0272] Thus, in some embodiments, the retroviral nucleic acids described herein include an origin of replication of a eukaryote or a variant thereof. Examples of origins of replication of eukaryotes of interest include the origin of replication of the β-globin gene described by Aladjem et al (Science, 1995, 270:815-819), a consensus sequence derived from an autonomously replicating sequence associated with an alpha-satellite sequence previously isolated from simian 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 is described by McWinney and Leffak (McWinney C. and Leffak M., Nucleic Acid Research 1990, 18(5):1233-42). In embodiments, the variant substantially maintains the ability to initiate replication in a eukaryote. The ability of a particular sequence to initiate replication can be determined by any suitable method, such as an autonomous replication assay based on bromodeoxyuridine incorporation and density shift (Araujo F.D. et al., supra; Frappier L.et al.)

[0273] In some embodiments, the retroviral nucleic acid includes a scaffold / matrix attachment region (S / MAR) or a variant thereof, such as a non-consensus-like AT-rich DNA element hundreds of base pairs in length, which organizes nuclear DNA of the eukaryotic genome into chromatin domains by periodic attachment of proteins to the scaffold or the matrix of the cell nucleus. They are typically found in non-coding regions such as adjacent regions, chromatin boundary regions, introns, etc. 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)'s 1.8 kbp S / MAR, the 0.7 Kbp minimal region of the S / MAR of the human IFN-γ gene described by Ramezani (Ramezani A. et al., Blood 2003, 101:4717-24) (hIFN-γ short) It is the 0.2 Kbp minimum region of the S / MAR of the human human dihydrofolate reductase gene (hDHFR) described by Mesner L.D. et al., Proc Natl Acad Sci USA, 2003, 100:3281-86). In embodiments, a functionally equivalent variant of the S / MAR is a sequence selected based on a set of six rules that have been shown to contribute to S / MAR function either together or alone (Kramer et al (1996) Genomics 33, 305; Singh et al (1997) Nucl. These rules were incorporated into the MAR-Wiz computer program, which is freely available at genomecluster.secs.oakland.edu / MAR-Wiz. In embodiments, the variant substantially maintains the same function of the S / MAR from which it is derived, in particular the ability to specifically bind to the nuclear matrix. One of ordinary skill in the art can determine whether a particular variant can specifically bind to the nuclear matrix, for example, by the in vitro or in vivo MAR assays described by Mesner et al. (Mesner L.D. et al, supra). In some embodiments, if a particular variant shows a tendency for DNA strand separation, the particular sequence is a variant of the S / MAR. This property can be determined using a particular program based on the methods of equilibrium statistical mechanics. The stress-induced duplex destabilization (SIDD) analysis technique, as defined by Bode et al (2005) J. Mol. Biol. 358, 597, "calculates the degree to which the imposed level of superhelical stress reduces the free energy required to open the duplex at each position along the DNA sequence. The results show sites of strong destabilization as deep minima and are presented as a SIDD profile [...]". The SIDD algorithm and mathematical basis (Bi and Benham (2004) Bioinformatics 20, 1477), as well as the analysis of SIDD profiles, can be performed using Internet resources freely available at WebSIDD (www.genomecenter.ucdavis.edu / benham).Thus, in some embodiments, a polynucleotide is considered a variant of an S / MAR sequence if it exhibits an SIDD profile similar to that of an S / MAR.

[0274] B. Cell-derived phososomes Phososome compositions can be generated from cells in culture, such as cultured mammalian cells, such as 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, such as human, cell lines described herein). In embodiments, the cultured cells are progenitor cells, such as bone marrow stromal cells, 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, cardiomyoblasts, neural progenitor cells, glial progenitor cells, neural progenitor cells, 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, such as induced pluripotent stem cells derived from a subject's cells), 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), progenitor cells (e.g., retinal progenitor cells, myeloblasts, bone marrow progenitor cells, thymocytes, meiocytes, megakaryoblasts, promegakaryoblasts, melanoblasts, lymphocytes, bone marrow progenitor cells, normoblasts, or angioblasts), progenitor cells (e.g., cardiac progenitor cells, satellite cells, radial glial cells, bone marrow stromal cells, pancreatic progenitor cells, endothelial progenitor cells, blast cells), or immortalized cells (e.g., HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR90, IMR91, PER.C6, HT-1080, or BJ cells).

[0276] The cultured cells can be cells derived from epithelial, connective, muscle, or nerve tissue or cells, and combinations thereof. The fusosomes can be obtained from cultured cells derived from any eukaryotic (e.g., mammalian) organ system, such as the cardiovascular system (heart, vascular system); digestive system (esophagus, stomach, liver, gallbladder, pancreas, intestine, colon, rectum, and anus); endocrine system (hypothalamus, pituitary gland, pineal gland or epiphysis, 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); genital system (ovaries, uterus, mammary glands, testes, vas deferens, seminal vesicles, prostate gland); respiratory system (pharynx, larynx, trachea, bronchi, lungs, diaphragm); skeletal system (bones, cartilage), and combinations thereof. In embodiments, the cells are derived from highly mitotic tissues (e.g., healthy tissues with high mitotic activity such as epithelium, embryonic tissue, bone marrow, intestinal crypts, etc.). In embodiments, the tissue sample is a tissue with high metabolism (e.g., skeletal tissue, nerve tissue, cardiomyocytes).

[0277] In some embodiments, the cells are derived from young donors, e.g., donors of 25 years old, 20 years old, 18 years old, 16 years old, 12 years old, 10 years old, 8 years old, 5 years old, 1 year old or younger. In some embodiments, the cells are derived from fetal tissue.

[0278] In some embodiments, the cells are derived from a subject and administered to the same subject or a subject having similar genetic characteristics (e.g., MHC compatibility).

[0279] In certain embodiments, the cells have telomeres with an average size exceeding 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 nucleotides (e.g., with a length of 4,000 - 10,000 nucleotides, with a length of 6,000 - 10,000 nucleotides).

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

[0281] The fusosome compositions described herein can be composed of fusosomes from one cell or tissue source, or a combination of sources. For example, the fusosome composition can include fusosomes from heterologous sources (e.g., animals, tissue cultures of cells of the aforementioned species), allogeneic, autologous, specific tissues (such as liver, skeleton, nerve, fat, etc.) that result in different protein concentrations and distributions, cells from different metabolic states (e.g., glycolytic, respiratory). The composition can also include fusosomes in different metabolic states, such as bound or unbound fusosomes, as described elsewhere herein.

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

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

[0284] In some embodiments, fusosomes are generated by inducing enucleation of the cell. Enucleation can be genetic, chemical (e.g., using actinomycin D, 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), assays such as mechanical methods (see, 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 combinations thereof can be used. Enucleation refers not only to the complete removal of the nucleus but also to the movement of the nucleus from its typical location such that the cell contains the nucleus but does not function.

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

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

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

[0288] i) Modification of cells prior to fusosome generation In some embodiments, the modification is performed on the cells, such as modification of a subject, tissue or cell, prior to fusosome generation. Such modifications can be effective, for example, in improving fusion, fusogen expression or activity, cargo structure or function, or target cell structure or function.

[0289] a) Physical modifications In some embodiments, the cells are physically modified prior to generating fusosomes. For example, as described elsewhere herein, fusogens can be linked to the cell surface.

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

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

[0292] In embodiments, fusosomes include an increase or decrease in the level of endogenous molecules. For example, fusosomes naturally exist in native source cells, but may contain endogenous molecules that are present at higher or lower levels than in fusosomes. In some embodiments, polypeptides are expressed from exogenous nucleic acids in source cells or fusosomes. In some embodiments, polypeptides are isolated from a source and loaded or bound to source cells or fusosomes.

[0293] In some embodiments, cells are treated with a chemical agent, such as a small molecule, before generating fusosomes to increase the expression or activity of an endogenous fusogen (e.g., endogenous to the source cell in some embodiments and endogenous to the target cell in some embodiments). In some embodiments, the small molecule can increase the expression or activity of a transcriptional activator of the endogenous fusogen. In some embodiments, the small molecule can 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 arrest compound, such as lysophosphatidylcholine. In some embodiments, fusosomes are generated from cells treated with a dissociation reagent that does not cleave fusogens, such as Accutase.

[0295] In some embodiments, source cells are physically modified, e.g., 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 an organelle, such as mitochondria, Golgi apparatus, endoplasmic reticulum, intracellular vesicles (lysosomes, autophagosomes, etc.).

[0297] b) Genetic recombination In some embodiments, the cell is genetically modified before generating the fusosome to increase the expression of an endogenous fusogen within the cell (e.g., endogenous to the source cell in some embodiments and endogenous to the target cell in some embodiments). In some embodiments, the genetic modification can increase the expression or activity of a transcriptional activator of the endogenous fusogen. In some embodiments, the genetic modification can decrease the expression or activity of a transcriptional repressor of the endogenous fusogen. In some aspects, the activator or repressor is 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 nuclease-inactive cas9 (dCas9) linked to an epigenetic modifier that targets the endogenous fusogen by a guide RNA.

[0298] In some embodiments, the cell is genetically modified before generating the fusosome to increase the expression of an exogenous fusogen within the cell, such as delivery of a transgene. In some aspects, a nucleic acid, such as DNA, mRNA, or siRNA, is introduced into the cell before generating the fusosome to increase or decrease the expression of a cell surface molecule (protein, glycan, lipid, or low molecular weight molecule) used, for example, for organ, tissue, or cell targeting. In some embodiments, the nucleic acid targets a repressor of the fusogen gene, such as an shRNA, siRNA construct. In some embodiments, the nucleic acid encodes an inhibitor of the fusogen repressor.

[0299] In some embodiments, the method includes introducing into the source cell a nucleic acid that is exogenous to the source cell encoding 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 and can include, for example, one or more backbone modifications, sugar modifications, non-canonical bases, or caps. Examples of backbone modifications include, for example, phosphorothioate, N3’ phosphoramidite, boranophosphate, phosphonoacetate, thio-PACE, morpholino phosphoramidite, or PNA. Examples of sugar modifications include, for example, 2’-O-Me, 2’F, 2’F-ANA, LNA, UNA, and 2’-O-MOE. Examples of non-canonical bases include, for example, 5-bromo-U, and 5-iodo-U, 2,6-diaminopurine, C-5 propynyl pyrimidine, difluorotoluene, difluorobenzene, dichlorobenzene, 2-thiouridine, pseudouridine, and dihydrouridine. Examples of caps include, for example, ARCA. Additional modifications are described, for example, in Deleavey et al., “Designing Chemically Modified Oligonucleotides for Targeted Gene Silencing” Chemistry&Biology Volume 19, Issue 8, 24 August 2012, Pages 937-954, which is incorporated herein by reference in its entirety.

[0300] In some embodiments, the cells are treated with a chemical agent, such as a small molecule, prior to generating fusosomes to increase the expression or activity of a fusogen that is exogenous compared to the source cells within the cell. In some embodiments, the small molecule can increase the expression or activity of a transcriptional activator of the exogenous fusogen. In some embodiments, the small molecule can 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 a modified fusogen. For example, a fusogen having a regulatable fusion activity, such as a specific cell type, tissue type, or local microenvironmental activity. Such regulatable fusion activities can include activation and / or initiation of fusogen 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 molecule, protein, or lipid is displayed on the target cell.

[0302] In some embodiments, the cells are genetically modified prior to generating fusosomes to alter (i.e., upregulate or downregulate) the expression of a signaling pathway (e.g., the Wnt / beta-catenin pathway). In some embodiments, the cells are genetically modified prior to generating fusosomes to alter (e.g., upregulate or downregulate) the expression of one or more genes of interest. In some embodiments, the cells are genetically modified prior to generating fusosomes to alter (e.g., upregulate or downregulate) the expression of a nucleic acid (e.g., miRNA or mRNA) or a nucleic acid of interest. In some embodiments, a nucleic acid, such as DNA, mRNA, or siRNA, is introduced into the cells prior to generating fusosomes to increase or decrease the expression of, for example, a signaling pathway, a gene, or a nucleic acid. In some embodiments, the nucleic acid targets a repressor of a signaling pathway, a gene, or a nucleic acid, or inhibits a signaling pathway, a gene, or a nucleic acid. In some embodiments, the nucleic acid encodes a transcription factor that upregulates or downregulates a signaling pathway, a gene, or a nucleic acid. In some embodiments, the activator or repressor is nuclease-inactive cas9 (dCas9) linked to a transcriptional activator or repressor that targets a signaling pathway, a gene, or a nucleic acid by a guide RNA. In some embodiments, the genetic modification epigenetically modifies an endogenous signaling pathway, a gene, or a nucleic acid with respect to its expression. In some embodiments, the epigenetic activator is nuclease-inactive cas9 (dCas9) linked to an epigenetic modifier that targets a signaling pathway, a gene, or a nucleic acid by a guide RNA. In some embodiments, the DNA of the cells is edited prior to generating fusosomes to change (e.g., upregulate or downregulate) the expression of a signaling pathway (e.g., the Wnt / beta-catenin pathway), a gene, or a nucleic acid. In some embodiments, the DNA is edited using a guide RNA and CRISPR-Cas9 / Cpf1 or other gene editing technology.

[0303] Cells can be genetically modified using recombinant methods. A nucleic acid sequence encoding a desired gene can be obtained using recombinant methods, for example, by screening a library from cells that express the gene, by deriving the gene from a vector known to contain it, or by directly isolating it from cells and tissues that contain it using standard techniques. Alternatively, the gene of interest can be made 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. A typical cloning vector contains a transcriptional terminator and a translational terminator, an initiation sequence, and a promoter useful for expression of the desired nucleic acid sequence.

[0305] In some embodiments, the cells can be genetically modified with one or more expression regions, such as genes. In some embodiments, the cells can be genetically modified with an exogenous gene (e.g., capable of expressing an exogenous gene product such as an RNA or polypeptide product) and / or an exogenous regulatory nucleic acid. In some embodiments, the cells can be genetically modified with an exogenous sequence encoding a gene product that is endogenous to the target cell and / or an exogenous regulatory nucleic acid capable of regulating the expression of an endogenous gene. In some embodiments, the cells can be genetically modified with an exogenous gene and / or a regulatory nucleic acid that regulates the expression of the exogenous gene. In some embodiments, the cells can be genetically modified with an exogenous gene and / or a regulatory nucleic acid that regulates the expression of an endogenous gene. It will be understood by those skilled in the art that the cells described herein can be genetically modified to express various exogenous genes encoding proteins or regulatory factors that can act on endogenous or exogenous genomic gene products of the target cell. In some embodiments, such genes confer characteristics to the fusosome and, for example, regulate fusion with the target cell. In some embodiments, the cells can be genetically modified to express an endogenous gene and / or a regulatory nucleic acid. In some embodiments, the endogenous gene or regulatory nucleic acid regulates the expression of other endogenous genes. In some embodiments, the cells can be genetically modified to express an endogenous gene and / or a regulatory nucleic acid that is expressed differently (e.g., inductively, tissue-specifically, constitutively, or at a higher or lower level) from a version of the endogenous gene and / or regulatory nucleic acid on another chromosome.

[0306] Promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these 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, such that promoter function is conserved when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased up to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to be able to function either cooperatively or independently to activate transcription.

[0307] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. 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 leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and 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 the expression of a polynucleotide sequence operably linked thereto when such expression is desired, or turn off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to, tissue-specific promoters, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. In some embodiments, the expression of the fusogen is upregulated before the fusosome is generated, for example, 3, 6, 9, 12, 24, 26, 48, 60, or 72 hours before the fusosome is generated.

[0309] The expression vector introduced into the source can also contain either or both of a selectable marker gene and a reporter gene to facilitate the identification and selection of expressing cells from a population of cells that are required to be transfected or infected via a viral vector. In other embodiments, the selectable marker can be carried on a separate DNA fragment and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be adjacent to appropriate regulatory sequences to allow 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 to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that does not exist in the recipient source or is not expressed by the recipient source and encodes a polypeptide whose expression is revealed by some readily detectable property, such as enzyme activity. The expression of the reporter gene is assayed at an appropriate time after the DNA has been introduced into the recipient cells. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, a construct having a minimal 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be ligated to the reporter gene and used to evaluate agents for their ability to regulate 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 modified for a particular time, e.g., due to the developmental or differentiation state of the source. In some embodiments, fusosomes are generated from a source of cells that have been genetically modified to alter the expression of one or more proteins that affect fusogenic activity, structure, or function, such as fusogenic or non-fusogenic proteins. The expression of one or more proteins may be restricted to a particular location(s) or may be widespread throughout the source.

[0312] In some embodiments, the expression of fusogenic proteins is modified. In some embodiments, the fusosomes are generated from cells in which the expression of fusogenic proteins is modified, e.g., having an increase or decrease in the expression of fusogens of at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more.

[0313] In some embodiments, the cells can be engineered to express a cytoplasmic enzyme (e.g., protease, phosphatase, kinase, demethylase, methyltransferase, acetylase) that targets the fusogenic protein. In some aspects, the cytoplasmic enzyme affects one or more fusogens by altering post-translational modifications. Post-translational protein modifications can affect the responsiveness to nutrient availability and redox conditions, as well as protein-protein interactions. In some embodiments, the fusosomes contain fusogens with altered post-translational modifications, e.g., having 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 the modifications into cells include physical, biological, and chemical methods. For example, Geng.&Lu, Microfluidic electroporation for cellular analysis and delivery. Lab on a Chip. 13(19):3803 - 21. 2013; Sharei, A. et al. A vector - free microfluidic platform for intracellular delivery. PNAS vol.110 no.6. 2013; Yin, H. et al., Non - viral vectors See Nature Reviews Genetics. 15:541-555. 2014. Suitable methods for modifying cells for use in generating the fusosomes described herein include, for example, diffusion, osmotic pressure, osmotic pulses, 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 genetic modification includes various assays. Such assays include, for example, molecular biology assays such as Southern blotting and Northern blotting, RT-PCR and PCR; biochemical assays such as detecting the presence or absence of a specific peptide 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) an inner lumen surrounded by the lipid bilayer (e.g., containing cytosolic content), (c) an exogenous or overexpressed fusogen (e.g., disposed in the fusogen lipid bilayer and the fusosome is derived from a source cell), wherein the fusosome has a partial or complete nuclear inactivation (e.g., nuclear removal).

[0317] In some aspects, the present disclosure provides a fusosome composition comprising a plurality of fusosomes derived from source cells, wherein the plurality of fusosomes comprise: (a) a lipid bilayer; (b) an inner lumen containing a cytoplasmic sol, the inner lumen being surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen disposed within the lipid bilayer; (d) a cargo, such as a nucleic acid containing a payload gene; the fusosome does not contain 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 are contacted with a cell population comprising target cells and non-target cells, the cargo is present in the target cells at least 10-fold more than in the non-target cells or reference cells, or (ii) the plurality of fusosomes fuse with at least at least 50% of the non-target cells or reference cells at a faster rate than with the target cells, and 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, providing a fusosome.

[0318] In some aspects, the present disclosure provides a fusosome composition comprising a plurality of fusosomes derived from source cells, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) an inner lumen containing a cytoplasmic sol, the inner lumen being surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen disposed within the lipid bilayer; and (d) a nucleic acid containing a payload gene encoding an exogenous agent of Table 5 or Table 6, the fusosome does not contain a nucleus, and the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein, providing a fusosome composition.

[0319] In some aspects, the present disclosure provides a fusosome composition comprising a plurality of fusosomes derived from source cells, wherein the plurality of fusosomes comprise: (a) a lipid bilayer; (b) an inner lumen containing a cytoplasmic sol, the inner lumen being 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 a miRNA present in non-target cells at a higher level than in target cells, e.g., a non-target cell-specific miRNA recognition sequence bound by a miRNA of Table 4, wherein the target cells are a first type of CNS cells, and optionally, the non-target cells are a second different type of CNS cells or non-CNS cells, the fusosome does not contain 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 non-target cells (e.g., the non-target cells described herein) at a level that is at least 10, 100, 1,000, or 10,000 times higher than the level of miRNA present in target cells (e.g., CNS cells). In some embodiments, the miRNA is not detectably present in target cells (e.g., CNS cells, e.g., the CNS cells described herein). In some aspects, the miRNA is not present in target cells (e.g., CNS cells, e.g., the CNS cells described herein).

[0321] In some aspects, the present disclosure provides a fusosome composition comprising a plurality of fusosomes derived from source cells, wherein the plurality of fusosomes comprise: (a) a lipid bilayer; (b) an inner lumen containing a cytoplasmic sol, the inner lumen being 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, for example, a promoter specific for 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, the fusosome does not contain 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 source cells, wherein the plurality of fusosomes comprise: (a) a lipid bilayer; (b) an inner lumen containing a cytoplasmic sol, the inner lumen being 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 the promoter in Table 3, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, the fusosome does not contain a nucleus, and the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein.

[0323] In some aspects, the present disclosure provides a fusosome composition comprising a plurality of fusosomes derived from source cells, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) an inner lumen containing a cytoplasmic sol, the inner 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, for example, a non-target cell-specific miRNA recognition sequence, such as a non-target cell-specific miRNA recognition sequence bound by the miRNA of Table 4. (iii) Optionally, a nucleic acid comprising a positive target cell-specific regulatory element, for example, a positive target cell-specific regulatory element (such as a target cell-specific promoter) operably linked to the payload gene, wherein the positive target cell-specific regulatory element increases the expression of the payload gene in the target cell as compared to otherwise similar fusosomes lacking the positive target cell-specific regulatory element, the target cell being a first type of CNS cell, optionally, the non-target cell being a second different type of CNS cell or a non-CNS cell, optionally, the target cell being a neuron and the non-target cell being a glial cell (such as an oligodendrocyte, an astrocyte or a microglial cell), or the target cell being a glial cell (such as an oligodendrocyte, an astrocyte or a microglial cell) and the non-target cell being a neuron, the fusosome not containing a nucleus, and the amount of viral capsid protein in the fusosome composition being less than 1% of the total protein, to provide a fusosome composition.

[0324] In some embodiments, one or more of the following exist: i) the fusosome contains or is contained by a cell biological agent; ii) the fusosome contains enucleated cells; iii) the fusosome contains inactivated nuclei; iv) the fusosome fuses with target cells at a higher rate, e.g., at least at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher than non-target cells, e.g., in the assay of Example 42; v) the fusosome fuses with target cells at a higher rate, 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 higher than other fusosomes, e.g., in the assay of Example 42; vi) the agent within the fusosome fuses with target cells at a rate such that the agent within the fusosome 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, e.g., in the assay of Example 42; vii) the fusogen is present at a copy number of at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,00,000, or 1,000,000,000 copies, or less, as measured by the assay of Example 26; viii) the fusosome contains a therapeutic agent at a copy number of at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies, or less, as measured by the assay of Example 88;(ix) The ratio of the copy number of fusogen to the copy number of 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, 1:10,000 to 1:100,000, or 1:100,000 to 1:1,000,000; (x) The fusosome contains a lipid composition that is substantially similar to that of the source cell, 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 is within 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the corresponding lipid level of the source cell; (xi) The fusosome contains a proteomic composition similar to that of the source cell, for example, using the assay of Example 87; (xii) The fusosome contains a ratio of lipid to protein within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cell when measured using the assay of Example 40; (xiii) The fusosome contains a ratio of protein to nucleic acid (e.g., DNA) within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cell when measured using the assay of Example 41; (xiv) The fusosome contains a ratio of lipid to nucleic acid (e.g., DNA) within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cell when measured using the assay of Example 91; (xv) The fusosome has a half-life within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of that of a reference cell, e.g., the source cell, in a subject, e.g., a mouse, as determined by the assay of Example 60;(xvi) When measured using, for example, the assay of Example 50, the fusosome transports glucose (e.g., labeled glucose, e.g., 2-NBDG) across the membrane in the absence of glucose, 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., other similar fusosomes; (xvii) The fusosome contains an esterase activity within the 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 a reference cell, e.g., a source cell or mouse embryonic fibroblasts, when measured using, for example, the assay of Example 51; (xviii) The fusosome contains 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, as described in, for example, Example 53; (xix) The fusosome contains a respiration level (e.g., oxygen consumption rate) that is 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, as described in, for example, Example 54; (xx) The fusosome contains an annexin V staining level of up to 18,000, 17,000, 16,000, 15,000, 14,000, 13,000, 12,000, 11,000, or 10,000 when measured using, for example, the assay of Example 55, or the fusosome contains an annexin-V staining level that is at least 5%, 10%, 20%, 30%, 40%, or 50% lower than the annexin-V staining level of other similar fusosomes treated with menadione in the assay of Example 55, or the fusosome contains an annexin-V staining level that is at least 5%, 10%, 20%, 30%, 40%, or 50% lower than the annexin-V staining level of macrophages treated with menadione in the assay of Example 55;(xxi) The fusosome has a miRNA content level of at least at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher than that of the source cells, for example, as determined by the assay of Example 33; (xxii) The fusosome has a soluble:insoluble protein ratio within or greater than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the source cells, for example, 1% - 2%, 2 - 3%, 3% - 4%, 4% - 5%, 5% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, or 80% - 90% of the source cells, for example, as determined by the assay of Example 38; (xxiii) The fusosome has an LPS level less than 5%, 1%, 0.5%, 0.01%, 0.005%, 0.0001%, 0.00001% or lower than that of the source cells, for example, as measured by mass spectrometry in Example 39; (xxiv) The fusosome can, for example, using the assay of Example 49, exhibit at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% more signal transduction, for example, extracellular signals in response to insulin such as AKT phosphorylation, or uptake of glucose (e.g., labeled glucose such as 2-NBDG) in response to insulin, in the absence of insulin, compared to negative controls such as other similar fusosomes; (xxv) When the fusosome is administered to a subject, for example, a mouse, targeting tissues such as the liver, lung, heart, spleen, pancreas, digestive tract, kidney, testis, ovary, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye, 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 are present in the target tissue 24, 48, or 72 hours later, for example, as determined by the assay of Example 64;(xxvi) Fusosomes have juxtacrine signaling levels that are at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than those of reference cells, such as source cells or bone marrow mesenchymal cells (BMSCs), as determined by the assay of Example 56; (xxvii) Fusosomes have paracrine signaling levels that are at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than those of reference cells, such as source cells or C2C12 cells, as determined by the assay of Example 57; (xxviii) Fusosomes polymerize actin at levels within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the level of polymerized actin in reference cells, such as source cells or C2C12 cells, as determined by the assay of Example 58; (xxix) Fusosomes have a membrane potential within about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the membrane potential of reference cells, such as source cells or C2C12 cells, as determined by the assay of Example 59, or fusosomes have a membrane potential of about -20 to 150 mV, -20 to -50 mV, -50 to -100 mV, or -100 to -150 mV; (xxx) Fusosomes can extravasate from blood vessels at an extravasation rate of at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of source cells or cells of the same type as source cells, using, for example, the assay of Example 44 (where the source cells are neutrophils, lymphocytes, B cells, macrophages, or NK cells); (xxxi) Fusosomes can pass through cell membranes, such as endothelial cell membranes or the blood-brain barrier; (xxxii) Fusosomes can secrete proteins 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 that of reference cells, using, for example, the assay of Example 48; (xxxiii) Fusosomes meet the standards of pharmaceuticals or Good Manufacturing Practice (GMP).(xxxiv) The fusosome was manufactured in accordance with Good Manufacturing Practice (GMP); (xxxv) The fusosome has a pathogen level below a predetermined reference value, for example, is substantially free of pathogens; (xxxiv) The fusosome has a contaminant level below a predetermined reference value, for example, is substantially free of contaminants; (xxxvii) The fusosome has low immunogenicity, for example, 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, for example, retinal neurons; or (xxxix) The source cells are other than 293 cells, HEK cells, human endothelial cells or human epithelial cells, monocytes, macrophages, dendritic cells, or stem cells.;

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

[0326] In some embodiments, one or more of the following exist: i) the source cell is selected from endothelial cells, macrophages, neutrophils, granulocytes, leukocytes, stem cells (e.g., mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells), myeloblasts, myoblasts, hepatocytes, or neurons, such as retinal neurons; ii) the organelle is selected from the Golgi apparatus, lysosomes, endoplasmic reticulum, mitochondria, vacuoles, endosomes, precursors, autophagosomes, centrosomes, glycosomes, glyoxysomes, hydrogenosomes, melanosomes, mitosomes, cnidocysts, peroxisomes, proteasomes, vesicles, and stress granules; iii) the fusosome has a size greater than 5um, 10um, 20um, 50um, or 100um; iv) the fusosome, or a composition or preparation containing a plurality of fusosomes, has a density other than 1.08 g / ml to 1.12 g / ml, for example, the fusosome has a density of 1.25 g / ml ± 0.05 as measured by the assay of Example 30; v) the fusosome is not captured by the circulating scavenger system or Kupffer cells of the liver sinusoid; vi) the source cell is other than 293 cells; vii) the source cell is not transformed or immortalized; viii) the source cell is transformed or immortalized using a method other than adenovirus-mediated immortalization, for example, immortalized by natural mutation or telomerase expression; ix) the fusogen is other than VSVG, SNARE protein, or secretory granule protein; x) the fusosome does not contain Cre or GFP, such as EGFP; xi) the fusosome further contains an exogenous protein other than Cre or GFP, such as EGFP; xii) the fusosome further contains an exogenous nucleic acid (e.g., RNA, e.g., mRNA, miRNA, or siRNA) or an exogenous protein (e.g., an antibody, e.g., an antibody) in, for example, the lumen; or xiii) the fusosome does not contain mitochondria.

[0327] The present disclosure also provides, in some embodiments, a fusosome comprising: (a) a lipid bilayer; (b) an inner lumen surrounded by the lipid bilayer (e.g., containing a cytosolic solution); (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 fertrin beta; iv) the fusosome has, e.g., low immunogenicity as described herein; v) the fusosome meets pharmaceutical or good manufacturing practice (GMP) standards; vi) the fusosome is manufactured according to good manufacturing practice (GMP) standards; vii) the fusosome has a pathogen level below a predetermined reference value, e.g., is substantially free of pathogens; or viii) the fusosome has a contaminant level below a predetermined reference value, e.g., is substantially free of contaminants.

[0329] The present disclosure also provides, in some embodiments, 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 containing a cytosolic solution, the inner lumen being 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 contain a nucleus, the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein, and the plurality of fusosomes deliver the cargo to at least 30% of the number of cells in the target cell population when contacting the target cell population in the presence of an inhibitor of endocytosis and when contacting a reference target cell population not treated with the inhibitor of endocytosis, as compared to the reference target cell population.

[0330] The present disclosure also, in some aspects, provides a fusosome composition comprising a plurality of fusosomes derived from source cells, wherein the plurality of fusosomes comprise: (a) a lipid bilayer; (b) an inner lumen containing cytosol, the inner lumen being surrounded by the lipid bilayer; (c) an exogenous or overexpressed retargeted fusogen disposed in the lipid bilayer; and (d) a cargo, wherein the fusosomes do not contain a nucleus, and 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 the target cells at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold more than in the non-target cells, or (ii) the plurality of fusosomes fuse with the target cells at a rate at least 50% higher than with the non-target cells.

[0331] The present disclosure also provides, in some embodiments, a fusosome composition comprising a plurality of fusosomes derived from source cells, wherein the plurality of fusosomes comprise: (a) a lipid bilayer, (b) an inner lumen surrounded by the lipid bilayer, (c) an exogenous or overexpressed fusogen disposed within the lipid bilayer, and (d) a cargo, and the fusosomes do not contain a nucleus, and comprise one or more of the following (e.g., at least 2, 3, 4, or 5): i) the fusogen is present at a copy number of at least 1,000 copies; ii) the fusosomes contain a therapeutic agent at 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 of the source cells; iv) the fusosomes contain a proteomic composition similar to that of the source cells; v) the fusosomes can transmit 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, which is, for example, at least 10% more than that of a negative control, e.g., other similar fusosomes, in the absence of insulin; vi) the fusosomes target a 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, 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 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 neurons, to provide the fusosome composition.

[0332] In embodiments, one or more of the following: i) the source cell is other than a 293 cell; ii) the source cell is not transformed or immortalized; iii) a method other than adenovirus-mediated immortalization, for example, a method of immortalizing by natural mutation or telomerase expression, is used to transform or immortalize the source cell; 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 intravascular nucleic acid (e.g., RNA, e.g., mRNA, miRNA, or siRNA) or an exogenous protein (e.g., an antibody, e.g., an antibody); or vii) the fusosome does not contain mitochondria.

[0333] In embodiments, one or more of the following: i) the source cell is other than a 293 cell or a HEK cell; ii) the source cell is not transformed or immortalized; iii) a method other than adenovirus-mediated immortalization, for example, a method of immortalizing by natural mutation or telomerase expression, is used to transform or immortalize the source cell; iv) the fusogen is not a viral fusogen; or v) the fusosome has a size other than 40 - 150 nm, for example, a size exceeding 150 nm, 200 nm, 300 nm, 400 nm, or 500 nm.

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

[0335] In an embodiment, the fusosome is: i) free of virus, non-infectious, or non-proliferative in a host cell; ii) not a viral vector; iii) not a VLP (virus-like particle); iv) free of viral structural proteins, such as proteins derived from gag, such as viral capsid proteins, such as viral capsule proteins, such as viral nucleocapsid proteins, or the amount of viral capsid protein is less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% of the total protein, for example, by mass spectrometry, for example, using the assay of Example 93; v) free of viral matrix proteins; vi) free of viral non-structural proteins, such as pol or its fragments or variants, viral reverse transcriptase proteins, viral integrase proteins, or viral protease proteins; vii) free of viral nucleic acids, such as viral RNA or viral DNA; viii) contains 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 fusosome comprises (or is 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% viral capsid protein (e.g., about 0.05% viral capsid protein). In embodiments, the viral capsid protein is a complex of rabbit endogenous lentivirus (RELIK) capsid and cyclophilin A. In embodiments, the viral capsid protein:total protein ratio is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 (or is identified as such).

[0337] In some embodiments, the fusosome does not contain (or is identified as not containing) the gag protein, or a fragment or variant thereof, or the amount of the 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, for example, by the assay of Example 93.

[0338] In embodiments, the ratio of the copy number of fusogen to the copy number of 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 is between 100:1 and 50:1, 50:1 and 20:1, 20:1 and 10:1, 10:1 and 5:1, or is 1:1. In embodiments, the ratio of the copy number of fusogen to the copy number of 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: i) the fusosome does not contain droplets immiscible with water; 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 mitochondria, Golgi apparatus, lysosomes, endoplasmic reticulum, vacuoles, endosomes, precursors, autophagosomes, centrosomes, glycosomes, glyoxysomes, hydromenosomes, melanosomes, mitosomes, cnidocysts, peroxisomes, proteasomes, vesicles, and granules.

[0340] In embodiments, one or more of the following: i) the fusogen is a mammalian fusogen or a viral fusogen; ii) the fusosome is not made by loading a therapeutic or diagnostic substance onto the fusosome; iii) the source cell is not loaded with a therapeutic or diagnostic substance; iv) the fusosome does not contain doxorubicin, dexamethasone, cyclodextrin, polyethylene glycol, microRNA, such as miR125, VEGF receptor, ICAM-1, E-selectin, iron oxide, fluorescent protein, such as GFP or RFP, nanoparticles, or RNase, or does not contain any of the foregoing in exogenous form; or v) the fusosome further comprises an exogenous therapeutic agent having one or more post-translational modifications, such as glycosylation.

[0341] In embodiments, the fusosome is monolayer or multilayer.

[0342] In an embodiment, one or more of the following: i) the fusosome is not an exosome; ii) the fusosome is a microvesicle; iii) the fusosome contains a non-mammalian fusogen; iv) the fusosome is designed to incorporate a fusogen; v) the fusosome contains an exogenous fusogen; vi) the fusosome has a size of at least 80 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm, or a 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; vii) the fusosome contains one or more organelles, such as mitochondria, Golgi apparatus, lysosomes, endoplasmic reticulum, vacuoles, endosomes, acrosomes, autophagosomes, centrosomes, glycosomes, glyoxysomes, hydrogenosomes, melanosomes, mitosomes, cnidosomes, peroxisomes, proteasomes, vesicles, and stress granules; viii) the fusosome contains a cytoskeleton or its components, such as actin, Arp2 / 3, formin, coronin, dystrophin, keratin, myosin, or tubulin; ix) the fusosome, or a composition or preparation containing a plurality of fusosomes, does not have a buoyant density of 1.08 - 1.22 g / ml, or has a buoyant density of at least 1.18 - 1.25 g / ml, or 1.05 - 1. in a sucrose gradient centrifugation assay, as described, for example, in Thery et al., "Isolation and characterization of exosomes from cell culture supernatants and biological fluids." Curr Protoc Cell Biol. 2006 Apr; Chapter 3: Unit 3.22Having a density of 12 g / ml; x) the lipid bilayer is enriched in ceramide or sphingomyelin or a combination thereof compared to the source cells, or the lipid bilayer is not enriched (e.g., depleted) in glycolipids, free fatty acids, or phosphatidylserine, or a combination thereof compared to the source cells; xi) the fusosome contains, for example, phosphatidylserine (PS) or CD40 ligand, or both PS and CD40 ligand as measured in the assay of Example 92; xii) the fusosome is enriched in PS compared to the source cells, for example, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% are positive for PS in a population of fusosomes, as determined 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; xiii) the fusosome is substantially free of acetylcholinesterase (AChE) or contains 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, as determined by the assay of Example 52; xiv) the fusosome is substantially free of tetraspanin family proteins (e.g., CD63, CD9, or CD81), ESCRT-related proteins (e.g., TSG101, CHMP4A-B, or VPS4B), Alix, TSG101, MHC I, MHC II, 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 less than 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 5% or 10% of the individual exosome marker proteins and / or 0.05%, 0.1%, 0.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 -4less than, or the fusosome contains an amount of calnexin per total protein that is about 70%, 75%, 80%, 85%, 88%, 90%, 95%, 99%, or more less than that of the parent cell; xvii) the fusosome contains an exogenous agent (e.g., an exogenous protein, mRNA, or siRNA), measured, for example, using the assay of Example 34; or xviii) the fusosome can be immobilized on a mica surface for at least 30 minutes by atomic force microscopy, 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.

[0343] In embodiments, one or more of the following: i) the fusosome is an exosome; ii) the fusosome is not a microvesicle; iii) the size of the fusosome is less than 80 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm, or the average size of 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 fusosome does not contain organelles; v) the fusosome does not contain the cytoskeleton or its components, such as actin, Arp2 / 3, formin, coronin, dystrophin, keratin, myosin, or tubulin; vi) the fusosome, or a composition or preparation containing a plurality of fusosomes, has a buoyant density of 1.08 - 1.22 g / ml in a sucrose gradient centrifugation assay, as described, for example, in Thery et al., "Isolation and characterization of exosomes from cell culture supernatants and biological fluids." Curr Protoc Cell Biol. 2006 Apr;Chapter 3:Unit 3.22; vii) the lipid bilayer is not enriched (e.g., depleted) for ceramide or sphingomyelin or a combination thereof compared to the source cell, or the lipid bilayer is enriched for glycolipid, free fatty acid, or phosphatidylserine, or a combination thereof compared to the source cell; viii) the fusosome does not contain phosphatidylserine (PS) or CD40 ligand, or both PS and CD40 ligand, or is depleted for the source cell, as measured, for example, in the assay of Example 92; ix) the fusosome is not enriched (e.g., depleted) for PS compared to the source cell, for example, as in Kanada M, et al. (2015) Differential fates of biomolecules delivered to target cells via Positive for PS in a population of phososomes, for example, less than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as assayed by Proc Natl Acad Sci USA 112:E1433-E1442. x) Phososomes substantially contain acetylcholinesterase (AChE), for example, contain at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, or less than 1000 AChE activity units / μg protein as assayed by, for example, the assay of Example 52; xi) Phososomes contain tetraspanin family proteins (e.g., CD63, CD9, or CD81), ESCRT-related proteins (e.g., TSG101, CHMP4A-B, or VPS4B), Alix, TSG101, MHCI, MHCII, GP96, actinin-4, mitofilin, syntenin-1, TSG101, ADAM10, EHD4, syntenin-1, TSG1O1, EHD1, flotillin-1, heat shock 70-kDa protein (HSC70 / HSP73, HSP70 / HSP72), or any combination thereof, for example, contain less than 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 5% or 10% of individual exosome marker proteins and / or 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20% or 25% of the total exosome marker proteins of any of the above proteins, or, for example, any one or more of these proteins are enriched as compared to the source cells as assayed by the assay of Example 89; xii) Phososomes have a level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) less than 500, 250, 100, 50, 20, 10, 5, or 1 ng GAPDH / μg total protein, or, for example, using the assay of Example 36, the level of GAPDH per μg of total protein in ng / ug units is at least 1%, 2.Levels of GAPDH less than the levels of GAPDH in the source cells, exceeding 5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%; xiii) The fusosome is 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., the amount of calnexin is less than 500, 250, 100, 50, 20, 10, 5, or 1 ng calnexin / μg total protein, or the fusosome contains, for example, 1%, 2.5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less calnexin per μg of total protein compared to the source cells, as determined using the assay of Example 90, or the average content of calnexin within the fusosome is about 1×10. -4 , 1.5×10 -4 , 2×10 -4 , 2.1×10 -4 , 2.2×10 -4 , 2.3×10 -4 , 2.4×10 -4 , 2.43×10 -4 , 2.5×10 -4 , 2.6×10 -4 , 2.7×10 -4 , 2.8×10 -4 , 2.9×10 -4 , 3×10 -4 , 3.5×10 -4 , or 4×10 -4 less, or the fusosome contains about 70%, 75%, 80%, 85%, 88%, 90%, 95%, 99%, or more less calnexin per μg of total protein than the parental cells, or xiv) By atomic force microscopy, e.g., Kanada M, et al. (2015) Differential fates of biomolecules delivered to target Cells via extracellular vesicles. By the assay of Proc Natl Acad Sci USA 112:E1433-E1442, fusosomes cannot be immobilized on the mica surface for at least 30 minutes.

[0344] In embodiments, one or more of the following: i) the fusosome does not contain VLPs; ii) the fusosome does not contain viruses; iii) the fusosome does not contain replicable viruses; iv) the fusosome does not contain viral proteins, such as viral structural proteins, such as capsid proteins or viral substrate proteins; v) the fusosome does not contain capsid proteins derived from enveloped viruses; vi) the fusosome does not contain nucleocapsid proteins; or vii) the fusogen is not a viral fusogen.

[0345] In embodiments, the fusosome contains cytosolic content.

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

[0347] In an embodiment, after administration of the fusosome or fusosome composition to a subject, such as a human subject, it retains one, two, three, four, five, six, or more of any of the above characteristics for 5 days or less, such as 4 days or less, 3 days or less, 2 days or less, 1 day or less, for example, over about 12 to 72 hours.

[0348] In an embodiment, the fusosome has one or more of the following characteristics: a) contains one or more endogenous proteins from the source cell, such as a membrane protein or a cytosolic protein; b) contains at least 10, 20, 50, 100, 200, 500, 1000, 2000, or 5000 different proteins; c) contains at least 1, 2, 5, 10, 20, 50, or 100 different glycoproteins; d) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the proteins within the fusosome are naturally occurring proteins; e) contains at least 10, 20, 50, 100, 200, 500, 1000, 2000, or 5000 different RNAs; or f) contains 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 an embodiment, the fusosome is engineered to have one, two, three, four, five or more of the following characteristics, or the fusosome is not a naturally occurring cell, or the nucleus does not naturally have one, two, three, four, five or more of the following characteristics: a) partial nuclear inactivation results in a reduction of at least 50%, 60%, 70%, 80%, 90% or more of nuclear function, e.g., transcription as measured by the assay of Example 24 and DNA replication as measured by the assay of Example 25, e.g., a reduction of transcription or DNA replication, or both; b) the fusosome is unable to transcribe or the transcriptional activity is less than 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the transcriptional activity of a reference cell, e.g., a source cell, using the assay of Example 24; c) the fusosome is unable to replicate nuclear DNA or the nuclear DNA replication is less than 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the nuclear DNA replication of a reference cell, e.g., a source cell, using the assay of Example 25; d) the fusosome lacks chromatin or the chromatin content is less than 1%, 2.less than 5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%; e) the fusosome lacks a nuclear membrane, e.g., by the assay of Example 31, less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the amount of the nuclear membrane of a reference cell, e.g., a source cell or a Jurkat cell; f) the fusosome lacks a functional nuclear pore complex, or by the assay of Example 31, the activity of nuclear import or nuclear export is reduced by at least 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%, or it lacks one or more nuclear pore proteins, e.g., NUP98 or importin 7; g) the fusosome does not contain histones, or the histone level is less than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the histone level of the source cell (e.g., of H1, H2a, H2b, H3, or H4), e.g., by the assay of Example 32; h) the fusosome contains less than 20, 10, 5, 4, 3, 2, or 1 chromosome; i) nuclear function is excluded; j) the fusosome is an enucleated mammalian cell; k) the nucleus has been removed or inactivated, e.g., by mechanical force, by radiation, or by chemical ablation; or l) the fusosome is derived from a mammalian cell having DNA that has been completely or partially removed during interphase or mitosis.

[0350] In embodiments, the fusosome contains mtDNA or vector DNA. In embodiments, the fusosome does not contain DNA.

[0351] In an embodiment, the source cell is a primary cell, an immortalized cell, or a cell line (e.g., a myeloblast cell line, e.g., C2C12). In an embodiment, the fusosome is derived from a source cell having a modified genome with reduced immunogenicity (e.g., by genome editing, for example, to remove MHC proteins or MHC complexes). In an embodiment, the source cell is derived from a cell culture treated with an anti-inflammatory signal. In an embodiment, the source cell is derived from a cell culture treated with an immunosuppressive substance. In an embodiment, the source cell is substantially non-immunogenic, for example, using the assays described herein. In an embodiment, the source cell contains an exogenous agent, for example, a therapeutic agent. In an embodiment, the source cell is a recombinant cell.

[0352] In embodiments, the fusosome further comprises an exogenous agent, such as a therapeutic agent, such as a protein or nucleic acid (e.g., DNA, chromosome (e.g., human artificial chromosome), RNA, such as mRNA or miRNA). In embodiments, the exogenous agent is present at least at 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies, for example, contained by the fusosome, or 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 fusosome has one or more endogenous molecules, such as a protein or nucleic acid, whose levels are altered, such as increased or decreased, for example, by treating mammalian cells with siRNA or a gene editing enzyme. In embodiments, the endogenous agent is present at an average level of at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies (e.g., contained by the fusosome), or 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 at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 500, 10 3 5.0×10 3 10 4, 5.0×10 4 , 10 5 , 5.0×10 5 , 10 6 , 5.0×10 6 , 1.0×10 7 , 5.0×10 7 , or 1.0×10 8 are present at 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, 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, such as HA, HIV-1 ENV, HHV-4, gp120, or VSV-G. In embodiments, the fusogen is a mammalian fusogen, such as SNARE, Syncytin, myomaker, myomixer, myomerger, or FGFRL1. In embodiments, the fusogen is active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. In embodiments, the fusogen is not active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. In embodiments, the fusosome fuses with the target cell at the surface of the target cell. In embodiments, the fusogen promotes fusion in a lysosome-independent manner. In embodiments, the fusogen is a protein fusogen. In embodiments, the fusogen is a lipid fusogen, such as oleic acid, glycerol monooleate, glyceride, diacylglycerol, or a modified unsaturated fatty acid. In embodiments, the fusogen is a chemical fusogen, such as PEG. In embodiments, the fusogen is a small molecule fusogen, such as NSAIDs including halothane, meloxicam, piroxicam, tenoxicam, and chlorpromazine. In embodiments, the fusogen is a recombinant. In embodiments, the fusogen is biochemically incorporated, for example, 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, for example, expressed in a source cell under conditions that allow the fusogen to bind to the lipid bilayer.

[0355] In embodiments, the fusosome binds to the 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 fusosome compositions, the plurality of fusosomes are the same. In some embodiments, the plurality of fusosomes are different. In some embodiments, the plurality of fusosomes are derived from one or more source cells. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the plurality of fusosomes 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 plurality of 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 within 10%, 50%, or 90% of the variation in the source cell population, and has a variation in size distribution of about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, less than 5%, 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 fusogen copy number within 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the average fusogen copy number in the fusosomes in the fusosome composition. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the plurality of fusosomes have a therapeutic agent copy number within 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the average therapeutic agent copy number in the fusosomes in the fusosome composition. In some embodiments, the fusosome composition is at least 10 5 、10 6 、10 7 、10 8 、10 9 、10 10 、10 11 、10 12 、10 13 、10 14 、or 10 15or contains one or more fusosomes. In some embodiments, the fusosome composition is in a volume of at least 1 μl, 2 μl, 5 μl, 10 μl, 20 μl, 50 μl, 100 μl, 200 μl, 500 μl, 1 ml, 2 ml, 5 ml, or 10 ml.

[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 number of cells in the target cell population as 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% of the cargo to the target cell population as 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 the target cell population as 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 an inhibitor of lysosomal acidification, such as bafilomycin A1. In some embodiments, the inhibitor of endocytosis is a dynamin inhibitor, such as dynasore.

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

[0362] In some embodiments, the cargo to be 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 lysosome acidification-independent pathway, a macropinocytosis-independent pathway (e.g., an inhibitor of endocytosis is, for example, a macropinocytosis inhibitor at a concentration of, for example, 25 μM, such as 5-(N-ethyl-N-isopropyl)amiloride (EIPA)), or an actin-independent pathway (e.g., an inhibitor of endocytosis is, for example, an inhibitor of actin polymerization at a concentration of, for example, 6 μM, such as latrunculin B).

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

[0365] In some embodiments, when the plurality of fusosomes contacts a cell population comprising target cells and non-target cells, the cargo is present in the target cells at least 10-fold more than in the non-target cells.

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

[0367] In some embodiments, the plurality of fusosomes fuses with the target cells at a rate 50% higher than with the non-target cells.

[0368] In some embodiments, upon contact with the target cell population, the fusosome delivers the cargo to a target cell location other than an endosome or lysosome, such as the cytosol. In an embodiment, less than 50%, 40%, 30%, 20%, or 10% of the cargo is delivered to an endosome or lysosome.

[0369] In some embodiments, the plurality of fusosomes include exosomes, microvesicles, or combinations thereof.

[0370] In some embodiments, the plurality of fusosomes have an average size of at least 50 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm. In other embodiments, the plurality of fusosomes have 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) includes a mammalian fusogen. In some embodiments, the fusogen (e.g., the retargeted fusogen) includes 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, Sendai virus F protein, rubella virus F protein, or alphavirus F protein, or a sequence selected from derivatives thereof.

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

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

[0374] In some embodiments, the fusogen (e.g., the 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, Sendai virus HN protein, rubulavirus HN protein, abulavirus HN protein, or derivatives thereof. In some embodiments, the fusogen (e.g., the retargeted fusogen) is 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 abulavirus 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 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 from 1000:1 to 1:1, or from 500:1 to 1:1, or from 250:1 to 1:1, or from 150:1 to 1:1, or from 100:1 to 1:1, or from 75:1 to 1:1, 50:1 to 1:1, 25:1 to 1:1, 20:1 to 1:1, or from 15:1 to 1:1, or from 10:1 to 1:1, or from 5:1 to 1:1, or from 2:1 to 1:1, or from 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, the fusosome can deliver nucleic acids to target cells, for example, for gene therapy, for example, to stably modify the genome of the target cells.

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

[0379] In an embodiment, 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 fusosomes. In an embodiment, 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 an embodiment, the fusosome is derived from mammalian cells having a genome modified (e.g., by genome editing) to reduce immunogenicity (e.g., to remove MHC proteins or MHC complexes). In an embodiment, the source cells are derived from a cell culture treated with an anti-inflammatory signal. In an embodiment, the method further comprises contacting the source cells of step a) with an immunosuppressive substance or an anti-inflammatory signal, for example, to inactivate the nucleus, for example, before or after enucleating the cells.

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

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

[0383] C. Fusogen and Pseudotyping In some embodiments, the fusosomes described herein (e.g., vesicles or a part of a cell) include one or more fusogens, for example, to facilitate the fusion of the fusosome to a membrane, for example, a cell membrane. These compositions may also include surface modifications that are performed to include one or more fusogens during or after synthesis. The surface modification may include modification to the membrane, for example, insertion of a lipid or a protein into the membrane.

[0384] In some embodiments, fusosomes contain one or more fusogens (e.g., incorporated into the cell membrane) on their outer surfaces to target specific cell or tissue types (e.g., CNS cells). In some aspects, the specific cell types targeted by 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. The fusosome may contain a targeting domain. Fusogens include, but are not limited to, protein-based, lipid-based, and chemical-based fusogens. A fusogen may bind to a partner, such as a feature on the surface of a target cell. In some embodiments, the partner on the surface of the target cell is a target cell moiety. In certain embodiments, the fusogen is a fusogen or retargeting fusogen that binds to a target cell from 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, the fusosome containing the fusogen will integrate the membrane into the lipid bilayer of the target cell. In some embodiments, one or more fusogens described herein may be included in the fusosome.

[0385] The fusosomes described herein (e.g., retroviral vectors) can contain fusogens, such as endogenous fusogens or pseudotyped fusogens.

[0386] i) Protein fusogens In some embodiments, the fusogen comprises a protein (e.g., glycoprotein), lipid, or small molecule. The fusogen can be, for example, a mammalian fusogen or a viral fusogen. In some embodiments, the fusogen comprises a protein fusogen, such as 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 natural protein or a derivative of a natural protein, a synthetic protein, a fragment thereof, a variant thereof, a protein fusion of one or more fusogens or fragments, and any combination thereof. In some embodiments, the viral fusogen is a class I viral membrane fusion protein, a class II viral membrane protein, a class III viral membrane fusion protein, a viral membrane glycoprotein, or another 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, such as HA, HIV-1 ENV, HHV-4, gp120, or VSV-G. In embodiments, the fusogen is a mammalian fusogen, such as SNARE, Syncytin, myomaker, myomixer, myomerger, or FGFRL1. In embodiments, the fusogen is active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. In embodiments, the fusogen is not active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. In embodiments, the fusosome fuses with the target cell at the surface of the target cell. In embodiments, the fusogen promotes fusion in a lysosome-independent manner. In embodiments, the fusogen is a protein fusogen. In embodiments, the fusogen is a lipid fusogen, such as oleic acid, glycerol monooleate, glyceride, diacylglycerol, or a modified unsaturated fatty acid. In embodiments, the fusogen is a chemical fusogen, such as PEG. In embodiments, the fusogen is a small molecule fusogen, such as NSAIDs including halothane, meloxicam, piroxicam, tenoxicam, and chlorpromazine. In embodiments, the fusogen is a recombinant. In embodiments, the fusogen is biochemically incorporated, for example, 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, for example, expressed in a source cell under conditions that allow the fusogen to bind to the lipid bilayer.

[0388] In some embodiments, the fusogen (e.g., a retargeted fusogen) comprises a mammalian fusogen. In some embodiments, the fusogen (e.g., a retargeted fusogen) comprises a viral fusogen. In some embodiments, the fusogen (e.g., a retargeted fusogen) is a protein fusogen. In some embodiments, the fusogen (e.g., a retargeted fusogen) is an 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, or comprises a sequence selected from derivatives thereof.

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

[0390] In some embodiments, the fusogen is present at 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, Sendai virus HN protein, rubulavirus HN protein, abulavirus HN protein, or derivatives thereof. In some embodiments, the fusogen (e.g., a retargeted fusogen) is selected from the group consisting of 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 abulavirus F and HN proteins, or derivatives thereof, or any combination thereof.

[0392] Non-mammalian fusogens include viral fusogens, their homologs, their fragments, and fusion proteins containing one or more proteins or their fragments. Viral fusogens include class I fusogens, class II fusogens, class III fusogens, and class IV fusogens. In embodiments, class I fusogens such as human immunodeficiency virus (HIV) gp41 have a characteristic post-fusion conformation with a characteristic trimer of α-helical hairpins having a central coiled-coil structure. Class I viral fusion proteins include proteins having a central post-fusion six-helix bundle. Examples of class I viral fusion proteins include influenza HA, parainfluenza F, HIV Env, Ebola GP, orthomyxovirus-derived hemagglutinin, paramyxovirus-derived F protein (e.g., measles (Katoh et al. BMC Biotechnology 2010, 10:37)), retrovirus-derived ENV protein, and filovirus and coronavirus fusogens. In embodiments, class II viral fusogens such as dengue E glycoprotein have structural features of β-sheets that refold to form a trimer of hairpins with an elongated external domain. 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 derived from Semliki Forest virus, Sindbis, rubella virus, and dengue virus. In embodiments, class III viral fusogens such as vesicular stomatitis virus G glycoprotein have both structural features found in class I and II. In embodiments, class III viral fusogens include an α-helix (e.g., forming a six-helix bundle and folding the protein similar to class I viral fusogens), and a β-sheet having 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 virus fusogen is the fusion-associated small transmembrane (FAST) protein encoded by an unenveloped 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 virus fusogen is small enough not to form a hairpin (doi:10.1146 / annurev-cellbio-101512-122422, doi:10.1016 / j.devcel.2007.12.008).

[0393] Fusogens that include the viral envelope protein (env) generally determine the range of host cells that can be infected and transformed by fusosomes. In the case of lentiviruses such as HIV-1, HIV-2, SIV, FIV, EIV, etc., the native env protein includes gp41 and gp120. In some embodiments, the viral env protein expressed by the source cells described herein is encoded in a vector separate from the viral gag and pol genes, as previously described.

[0394] Examples of retrovirus-derived env genes that can be used include, but are not limited to, MLV envelope, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV (poultry disease virus), and influenza virus envelope. Similarly, genes encoding envelopes derived from DNA viruses (viral families of Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae) can be utilized alongside RNA viruses (e.g., RNA virus families of Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae). Representative examples include FeLV, VEE, HFVW, WDSV, SFV, rabies, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10, EIAV, etc.

[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, D virus, Hepatitis E virus, Rotavirus, any virus of the Norwalk virus group, Enteric adenovirus, Parvovirus, Dengue virus, Monkeypox, Mononegavirus, Lyssavirus such as Rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 and 2, Australian bat virus, Ebolavirus, Vesiculovirus, Herpesvirus such as Herpes simplex virus type 1 and 2, Varicella zoster, Cytomegalovirus, Epstein-Barr virus (EBV), Human herpesvirus (HHV), Human herpesvirus 6 and 8, Human immunodeficiency virus (HIV), Papillomavirus, Murine gammaherpesvirus, Arenavirus such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-related hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, Lymphocytic choriomeningitis virus (LCMV), Bunyaviridae such as Crimean-Congo hemorrhagic fever virus, Hantavirus, virus causing hemorrhagic nephrotic syndrome, Rift Valley fever virus, Filoviridae (filovirus) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae including Kyasanur Forest disease virus, Omsk hemorrhagic fever virus, virus causing tick-borne encephalitis, and Paramyxoviridae such as Hendra virus and Nipah virus, variola major and variola minor (smallpox), Alphavirus such as Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, SARS-related coronavirus (SARS-CoV), West Nile virus, any virus causing encephalitis.

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

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

[0398] Furthermore, the fusogen or viral envelope protein can be modified or engineered to include a polypeptide sequence that enables the transduction vector to target and infect host cells outside its normal range, or more specifically, enables restriction of transduction into a cell or tissue type. For example, the fusogen or envelope protein can be in-frame with a target sequence such as a receptor ligand, an antibody (using recombinant antibody-type molecules such as the antigen-binding portion of an antibody or a single-chain antibody), and a polypeptide moiety or a modification thereof (e.g., if a glycosylation site is present in the target sequence) that facilitates direct delivery of virion particles to the target cell of interest when displayed on the transduction vector coat. Further, the envelope protein can further include a sequence that modulates cell function. Modulating cell function with a transduction vector can increase or decrease the transduction efficiency of a particular cell type in a mixed cell population. For example, stem cells can be more specifically transduced with an envelope sequence that includes a ligand or binding partner that specifically binds to stem cells, rather than other cell types found in blood or bone marrow. Non-limiting examples are stem cell factor (SCF) and Flt-3 ligand. Other examples include, for example, antibodies (e.g., single-chain antibodies specific for a cell type), and essentially any antigen (including receptors) that binds to tissues such as lung cancer, liver, pancreas, heart, endothelial, smooth, breast, prostate, epithelial, vascular cancers, etc.

[0399] A protein fusogen or viral envelope protein can be retargeted by mutating amino acid residues of the fusogen protein or targeting protein (e.g., hemagglutinin protein). In some embodiments, the fusogen is randomly mutated. In some embodiments, the fusogen is rationally mutated. In some embodiments, the fusogen is subjected to directed evolution. In some embodiments, the fusogen is truncated and only a subset of the peptides are used in the retroviral vector or VLP. For example, the amino acid residues of the measles hemagglutinin protein can be mutated to change the binding properties of the protein 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 contain 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 (e.g., incorporated into the cell membrane) on their outer surfaces to target specific cell or tissue types. Fusogens include, but are not limited to, protein-based, lipid-based, and chemical-based fusogens. The fusogen may be capable of binding to a partner on the surface of the target cell. In some embodiments, fusosomes containing fusogens will integrate the membrane into the lipid bilayer of the target cell.

[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 poxvirus family fusogen.

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

[0405] In some embodiments, the fusogen described herein comprises 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 of such sequence, e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to 100, 200, 300, 400, 500, or 600 amino acids in length. For example, in some embodiments, the fusogen described herein comprises an amino acid sequence having at least 80% identity to any amino acid sequence of Table 1. In some embodiments, the nucleic acid sequence described herein encodes 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 of such sequence, e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length.

[0406] In some embodiments, the fusogens described herein include an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 57, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or 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 to a sequence that is 100, 200, 300, 400, 500, or 600 amino acids in length. For example, in some embodiments, the fusogens described herein include an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 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 to 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 such a sequence, e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence that is 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

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

Table 1-26

Table 1-27

Table 1-28

Table 1-29

[0407] In some embodiments, the fusogens described herein include the amino acid sequences of Table 2, or amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or portions of such sequences, e.g., amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to 100, 200, 300, 400, 500, or 600 amino acids in length. For example, in some embodiments, the fusogens described herein include amino acid sequences having at least 80% identity to any of the amino acid sequences of Table 2. In some embodiments, the nucleic acid sequences described herein encode the amino acid sequences of Table 2, or amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or portions of such sequences, e.g., amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length.

[0408] In some embodiments, the fusogens described herein include an amino acid sequence set forth in any one of SEQ ID NOs: 58 to 133, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or 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 to a sequence that is 100, 200, 300, 400, 500, or 600 amino acids in length. For example, in some embodiments, the fusogens described herein include an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 58 to 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 to 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 such a sequence, e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence that is 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

Table 2-29

[0409] ii) Lipid fusogen In some embodiments, the liposomes can be treated with fusogenic lipids such as saturated fatty acids. In some embodiments, the saturated fatty acids have 10 to 14 carbons. In some embodiments, the saturated fatty acids have longer chain carboxylic acids. In some embodiments, the saturated fatty acids are monoesters.

[0410] In some embodiments, the liposomes can be treated with unsaturated fatty acids. In some embodiments, the unsaturated fatty acids have C16-C18 unsaturated fatty acids. In some embodiments, the unsaturated fatty acids include oleic acid, glycerol monooleate, glycerides, diacylglycerol, 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, the liposomes contain, in the membrane, one or more negatively curved lipids, such as negatively curved lipids that are exogenous to the source cell. In embodiments, the negatively curved lipid or its precursor is added to the source cell or the medium containing the liposomes. In embodiments, the source cell is engineered to express or overexpress one or more lipid synthesis genes. The negatively curved lipid can be, for example, diacylglycerol (DAG), cholesterol, phosphatidic acid (PA), phosphatidylethanolamine (PE), or fatty acid (FA).

[0412] While not wishing to be bound by theory, in some embodiments, positive-curvature lipids inhibit membrane fusion. In some embodiments, fusosomes comprise a decrease in the level of one or more positive-curvature lipids, such as exogenous positive-curvature lipids, in the membrane. In embodiments, the level decreases, for example, by inhibiting lipid synthesis, such as by knockout or knockdown of lipid synthesis genes in the source cells. Positive-curvature lipids can be, for example, lysophosphatidylcholine (LPC), phosphatidylinositol (PtdIns), lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), or monoacylglycerol (MAG).

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

[0414] In some embodiments, chemical fusogens induce local dehydration between two membranes, which leads to unfavorable molecular packing of the bilayer. In some embodiments, chemical fusogens induce dehydration of regions near the lipid bilayer, causing displacement of aqueous molecules between cells and enabling interaction between the two membranes.

[0415] In some embodiments, chemical fusogens are cations. Some non-limiting examples of cations include Ca2+, Mg2+, Mn2+, Zn2+, La3+, Sr3+, and H+.

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

[0417] In some embodiments, chemical fusogens are soluble and lipid-soluble. Some non-limiting examples include oleoylglycerol, dioleoylglycerol, trioleoylglycerol, and their variants and derivatives.

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

[0419] In some embodiments, the chemical fusogen is a small organic molecule. Non-limiting examples include n-hexyl bromide.

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

[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 their variants and derivatives.

[0422] In some embodiments, the fusosome contains actin and an agent that stabilizes polymerized actin. Without wishing to be bound by theory, the stabilized actin within the fusosome can promote fusion with target cells. In embodiments, the agent that stabilizes polymerized actin is selected from actin, myosin, biotin-streptavidin, ATP, neuronal Wiskott-Aldrich syndrome protein (N-WASP), or formin. See, for example, Langmuir. 2011 Aug 16;27(16):10061-71 and Wen et al., Nat Commun. 2016 Aug 31;7. In embodiments, the fusosome contains actin that is exogenous or overexpressed relative to the source cell, such as wild-type actin or actin containing a mutation that promotes polymerization. In embodiments, the fusosome contains ATP or phosphocreatine, such as exogenous ATP or phosphocreatine. iv) Small molecule fusogen

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

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

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

[0426] In some embodiments, fusogens are linked to affinity tags. 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 is distinct from the fusogen.

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

[0428] In some embodiments, the fusogen can be modified with a protease domain that recognizes a specific protein, e.g., overexpression of a protease, e.g., an engineered fusion protein having protease activity. For example, a protease or a protease domain derived from a protease, e.g., MMP, mitochondrial processing peptidase, mitochondrial intermediate peptidase, inner membrane peptidase.

[0429] Alfonzo, J.D. & Soll, D. Mitochondrial tRNA import - the challenge to understand has just begun. Biological Chemistry 390:717 - 722, 2009; Langer, T. et al. Characterization of Peptides Released from Mitochondria. THE JOURNAL OF BIOLOGICAL CHEMISTRY. Vol. 280, No. 4, 2691 - 2699, 2005; Vliegh, P. et al. Synthetic therapeutic peptides: science and market. Drug Discovery Today. 15(1 / 2), 2010; Quiros P.M.m et al., New roles for mitochondrial proteases in health, ageing and disease. Nature Reviews Molecular Cell Biology. V16, 2015; Weber - Lotfi, F. et al. DNA import competence and mitochondrial genetics. Biopolymers and Cell. Vol. 30, N 1, 71 - 73, 2014. See also

[0430] III. Positive target cell - specific regulatory elements In some embodiments, the fusosomes described herein, such as viruses, such as retroviruses, comprise nucleic acids (e.g., genes encoding exogenous agents), such as retroviral nucleic acids, that include positive target cell - specific regulatory elements such as tissue - specific promoters, tissue - specific enhancers, tissue - specific splice sites, tissue - specific sites that extend the half - life of RNA or proteins, tissue - specific mRNA nuclear export promoting sites, tissue - specific translation enhancing sites, or tissue - specific post - translational modification sites.

[0431] In some embodiments, the fusosomes described herein, such as viruses, such as retroviruses, include nucleic acids, such as retroviral nucleic acids, that can include regions such as origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine-Dalgarno sequences or Kozak sequences), introns, polyadenylation sequences, untranslated regions such as 5' and 3' untranslated regions, which interact with host cell proteins to effect transcription and translation and can direct, increase, modulate, or control the transcription or expression of operably linked polynucleotides. 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 can be used, including ubiquitous promoters and inducible promoters.

[0432] In certain embodiments, the control elements can direct, increase, modulate, or control the transcription or expression of operably linked polynucleotides in a cell-specific manner. In certain embodiments, the nucleic acid, such as a retroviral nucleic acid, includes one or more expression control sequences specific to a particular cell, cell type, or cell lineage, such as a target cell, i.e., the expression of a polynucleotide operably linked to an expression control sequence specific to a particular cell, cell type, or cell lineage is expressed in the target cell but not (or is expressed at a lower level) in non-target cells.

[0433] In certain embodiments, the nucleic acid, such as a retroviral nucleic acid, can include exogenous, endogenous, or heterologous control sequences such as promoters and / or enhancers.

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

[0435] In embodiments, an enhancer can include a segment of DNA that can provide enhanced transcription and, in some cases, can function independently of its orientation relative to another control sequence. The enhancer can function cooperatively or additively with a promoter and / or other enhancer elements. In some embodiments, a promoter / enhancer segment of DNA can include a sequence that can provide both promoter and enhancer functions.

[0436] Exemplary ubiquitous expression control sequences include the cytomegalovirus (CMV) immediate early promoter, the viral simian virus 40 (SV40) (e.g., early or late), the Moloney murine leukemia virus (MoMLV) LTR promoter, the Rous sarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5, and P11 promoters from vaccinia virus, the elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), beta-kinase (β-KIN), the human ROSA 26 locus (Orions et al., Nature Biotechnology 25, 1477-1482 (2007)), the ubiquitin C promoter (UBC), the phosphoglycerate kinase-1 (PGK) promoter, the cytomegalovirus enhancer / chicken beta-actin (CAG) promoter, the beta-actin promoter and the myeloproliferative sarcoma virus enhancer, the negative control region deletion, the d1587rev primer binding site replacement (MND) promoter (Challita et al., J Virol. 69(2):748-55 (1995)).

[0437] In some embodiments, a promoter can be paired with a heterologous gene to confer its regulatory function on the heterologous gene. In some embodiments, cis-regulatory elements from the promoter of a first gene can be linked to a segment of the promoter of a different gene to create a chimeric promoter having the characteristics of both promoters.

[0438] In some aspects, the promoter is a tissue-specific promoter, such as 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 fusosome (e.g., viral vector) described herein includes, in its nucleic acid, a promoter having the sequence of the 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 fusosome (e.g., viral vector) described herein includes, in its nucleic acid, a promoter having a transcription factor binding site derived from a region within 3 kb of the transcription start site of the gene listed in Table 3. In some embodiments, the fusosome (e.g., viral vector) described herein includes, in its 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 the 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), for example, the CNS cell-specific promoter is the SYN, NSE, CaMKII, tubulin or PDGF promoter. In some aspects, the CNS cell-specific promoter is the 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), for example, the CNS cell-specific promoter is the fSST or fNPY promoter. In some aspects, the CNS cell-specific promoter is the promoter described in Delzor et al., Hum Gene Ther Methods. 2012 Aug;23(4):242-54 (incorporated herein by reference in its entirety), for example, the CNS cell-specific promoter is the GAD67 or DLX5 / 6 promoter. In some aspects, the CNS cell-specific promoter is the promoter described in Egashira et al., Sci Rep. 2018 Oct 11;8(1):15156 (incorporated herein by reference in its entirety), for example, the CNS cell-specific promoter is the VGLUT1 or Dock10 promoter. In some embodiments, the CNS cell-specific promoter is the promoter described in Naciff et al., J. Neurochem., 1999 Jan; seventy-two (1):17-28 (incorporated herein by reference in its entirety), for example, the CNS cell-specific promoter is the ChAT promoter. In some aspects, the CNS cell-specific promoter is the VAChT promoter. In some aspects, the CNS cell-specific promoter is Delzor et al., Hum The promoter described in Gene Ther Methods. 2012 Aug; 23(4):242-254 (which is incorporated herein by reference in its entirety), for example, the CNS cell-specific promoter is the Drd1a promoter. In some embodiments, the CNS cell-specific promoter is the promoter described in Benzekhroufa et al., Gene Ther. 2009 May; 16(5):681-8 (which is incorporated herein by reference in its entirety), for example, the CNS cell-specific promoter is the TPH-2 promoter. In some embodiments, the CNS cell-specific promoter is the promoter described in Merienne et al., Gene Ther. 2015 Oct; 22(10):830-9 (which is incorporated herein by reference in its entirety), for example, the CNS cell-specific promoter is the GFAP, EAAT1, or GS promoter. In some aspects, the CNS cell-specific promoter is the promoter described in the Immgen consortium (which is incorporated herein by reference in its entirety), for example, 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 the promoter described in McIver et al., J Neurosci Res. 2005 Nov 1; 82(3):397-403 (which is incorporated herein by reference in its entirety), for example, the CNS cell-specific promoter is the MBP promoter. In some embodiments, the CNS cell-specific promoter is the promoter described in Kagiava et al., J Gene Med. 2014 Nov-Dec; 16(11-12):364-73 (which is incorporated herein by reference in its entirety), for example, the CNS cell-specific promoter is the MBP or CNP promoter. In some aspects, the CNS cell-specific promoter is Regev et a promoter as described in al., Proc Natl Acad Sci U S A. 2010 Mar 2;107(9):4424-9 (which is incorporated herein by reference in its entirety), for example, the CNS cell-specific promoter is the CRFR2β promoter. In some embodiments, the CNS cell-specific promoter is any of the aforementioned transcriptionally active fragments. 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 with any of the aforementioned.

[0440] Internal ribosome entry sites (IRES) typically facilitate direct internal ribosome entry into the start codon (such as ATG) of a cistron (protein-coding region), thereby resulting in cap-independent translation of the gene. See, for example, Jackson et al, (1990) Trends Biochem Sci 15(12):477-83), and Jackson and Kaminski. (1995) RNA 1(10):985-1000. In certain embodiments, the vector comprises one or more exogenous genes encoding one or more exogenous agents. In certain embodiments, to achieve efficient translation of each of a plurality of exogenous protein agents, the polynucleotide sequence can be separated by one or more IRES sequences or a polynucleotide sequence encoding a self-cleaving polypeptide.

[0441] As used herein, a nucleic acid, such as a retroviral nucleic acid, can also include one or more Kozak sequences, for example, short nucleotide sequences that facilitate the initial binding of mRNA to the small subunit of the ribosome and increase translation. The consensus Kozak sequence is (GCC)RCCATGG, where R is 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, the retroviral nucleic acid, includes elements that enable any type of conditional expression, including but not limited to conditional expression of an exogenous agent, such as inducible expression, repressible expression, cell-type specific expression, or tissue-specific expression. In some embodiments, to achieve conditional expression of an exogenous agent, expression is controlled by subjecting a cell, tissue, or organism to a treatment or condition that causes the exogenous agent to be expressed, or causes an increase or decrease in the expression of the exogenous agent.

[0443] Examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as the promoter of the gene encoding glucocorticoid or estrogen receptor (inducible by treatment with the corresponding hormone), metallothionein promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), "GeneSwitch" mifepristone regulatable system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switch (International Publication No. WO 2002 / 088346), tetracycline-dependent regulatory system, and the like.

[0444] Expression of the 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 some 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) regulatory system. The Tet system is based on the co-expression of two elements in each target cell: (i) a tetracycline response element containing repeats of the Tet operator sequence (TetO) fused to a minimal promoter and linked to the gene of interest (e.g., a gene encoding an exogenous agent) and (ii) a transcriptional transactivator (tTA), a fusion protein of the Tet repressor (TetR) and the transactivation domain of the herpes simplex virus-derived VP16 protein. In the first described version, expression of the transgene was active in the absence of tetracycline or its potent analog doxycycline (Do) (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 (Tet-ON system). In some embodiments, in the transactivator, the VP16 domain is replaced by a minimal activation domain, potential splice donor and splice acceptor sites are removed, the protein is codon-optimized, is highly sensitive to Dox, and has reduced baseline activity, resulting in an improved transactivator variant rtTA2S-M2. Furthermore, various Tet-responsive promoter elements have been generated, including modifying TetO at 36 nucleotide intervals from adjacent operators to enhance regulation. Additional modifications may serve to further reduce basal activity and increase the expression dynamic range. As an example, the pTet-T11 (abbreviation: TII) variant exhibits a high dynamic range and low background activity.

[0446] Conditional expression can also be achieved by using a site-specific DNA recombinase. According to certain embodiments, a nucleic acid, such as a retroviral nucleic acid, contains at least one (typically two) sites for recombination mediated by an excision or integration protein, enzyme, cofactor, or related protein involved in the recombination reaction, which contains a site-specific recombinase, such as one or more recombination sites (e.g., 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 50, etc.) that may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing a recombinant protein sequence or a fragment 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 comprising one or more riboswitches. Riboswitches are a common bacterial function for regulating gene expression and are a means of achieving RNA control of biological functions. Riboswitches can be present in the 5' untranslated region of mRNA and can enable the regulatory control of gene expression through the binding of small molecule ligands that induce or suppress riboswitch activity. In some embodiments, the riboswitch controls gene products involved in the production of small molecule ligands. Riboswitches typically operate in cis, although trans-acting riboswitches have been identified. Natural riboswitches are composed of two domains: an aptamer domain that binds ligands via a three-dimensional folded RNA structure, and a functional switching domain that induces or suppresses riboswitch activity based on the presence or absence of the ligand. Thus, there are two ligand-sensitive conformations achieved by riboswitches, representing an on-state and an off-state (Garst et al., 2011). The functional switching domain can affect polynucleotide expression by regulating internal ribosome entry sites, the accessibility of pre-mRNA splice donors in retroviral gene constructs, translation, transcription termination, transcript degradation, miRNA expression, or shRNA expression (Dambach and Winkler 2009). The aptamer and functional switching domains can be used as modular components to enable 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 found (Mandal et al 2003; US Patent Application Publication No. 20080269258; and International Publication No. 2006055351). Purine riboswitches share a similar structure consisting of three conserved helical / stem structures (P1, P2, P3) and intervening loop / junction elements (J1-2, L2, J2-3, L3, J3-1). The aptamer domain of the purine family of riboswitches naturally varies in affinity / regulation by various purine compounds such as adenine, guanine, adenosine, guanosine, deoxyadenosine, deoxyguanosine etc. due to sequence variations (Kim et al. 2007).

[0449] In some embodiments, a nucleic acid, such as a retroviral nucleic acid described herein, comprises a polynucleotide encoding an exogenous agent operably linked to a promoter and a riboswitch. The riboswitch comprises one or more, for example, all of the following: a.) an aptamer domain, for example, an aptamer domain that can bind to a nucleoside analog antiviral agent and has a reduced binding to guanine or 2'-deoxyguanosine compared to the nucleoside analog antiviral agent; b.) a functional switching domain, for example, a functional switching domain that can regulate the expression of an exogenous agent, wherein the binding of a nucleoside analog by the aptamer domain induces or suppresses the expression regulatory activity of the functional switching domain, thereby regulating the 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 the non-limiting examples provided herein, the exogenous gene encodes one or more engineered signaling polypeptides. For example, the riboswitch and a target polynucleotide encoding 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 an NK cell.

[0450] The aptamer domain can be used, for example, as a modular component and can be combined with any of the functional switching domains to affect the RNA transcript. In any of the embodiments disclosed herein, the riboswitch can affect the RNA transcript by regulating any of the following activities: internal ribosome entry site (IRES), accessibility of the pre-mRNA splice donor, translation, transcription termination, transcript degradation, miRNA expression, or shRNA expression. In some embodiments, the functional 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 results in the riboswitch having the potential to affect the RNA transcript. In some embodiments, the riboswitch can include a ribozyme. A riboswitch having a ribozyme can inhibit or enhance the degradation of the transcript of a target polynucleotide in the presence of a small molecule ligand. In some embodiments, the ribozyme can be of the ribozyme pistol class, ribozyme hammerhead class, ribozyme twist class, ribozyme hatchet class, 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 TCSRE includes a tissue-specific miRNA recognition sequence, a tissue-specific protease recognition site, a tissue-specific ubiquitin ligase site, a tissue-specific transcription repression site, or a tissue-specific epigenetic repression site.

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

[0453] In some embodiments, miRNA is a small non-coding RNA of 20-22 nucleotides, typically excised from an approximately 70-nucleotide fold-back RNA precursor structure known as pre-miRNA. Generally, miRNA negatively regulates targets in one of two ways, depending on the degree of complementarity between the miRNA and the target. First, miRNAs that bind to the protein-coding mRNA sequence with complete or near-complete complementarity usually induce the RNA interference (RNAi) pathway. miRNAs that exert a regulatory effect by binding to imperfect complementary sites within the 3'untranslated region (UTR) of the mRNA target usually suppress the expression of the target gene at the post-transcriptional and apparently translational levels through a RISC complex similar to or perhaps the same as those used in the RNAi pathway. Consistent with translational control, miRNAs that use this mechanism reduce the protein level of the target gene, but the mRNA levels of these genes are minimally affected. miRNAs (e.g., naturally occurring miRNAs or artificially designed miRNAs) can specifically target any mRNA sequence. For example, in one embodiment, one skilled in the art can design a short hairpin RNA construct expressed as a human miRNA (e.g., miR-30 or miR-21) primary transcript. This design has been shown to significantly improve knockdown efficiency by adding a Drosha processing site to the hairpin structure (Pusch et al., 2004). The hairpin stem consists of a 22-nt dsRNA (e.g., the antisense has complete complementarity to the target of interest) and a 15-19-nt loop derived from a human miR. Adding miR loops and miR30 flanking sequences to one or both sides of the hairpin results in a greater than 10-fold increase in Drosha and Dicer processing of the expressed hairpin compared to conventional shRNA designs that do not use microRNAs. Increased Drosha and Dicer processing leads to increased siRNA / miRNA production and increased potency of the expressed hairpin.

[0454] Hundreds of different miRNA genes are differentially expressed during development and between tissue types. Several studies have suggested an important regulatory role for miRNAs in a wide range of biological processes, such as the timing of development, 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 analysis has shown that miRNAs have different expression profiles in different tissues. Using computational methods, the expression of approximately 7,000 predicted human miRNA targets was analyzed. The data suggest that miRNA expression broadly contributes to the tissue specificity of mRNA expression in many human tissues. (See Sood et al. 2006 PNAS USA 103(8):2746-51)

[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 the expression of exogenous agents in non-target cell types. MicroRNAs induce sequence-specific post-transcriptional gene silencing in many organisms by inhibiting the translation of messenger RNA (mRNA) or by causing the degradation of mRNA. See, for example, Brown et al. 2006 Nature Med. 12(5):585-91., and International Publication No. WO 2007 / 000668, each of which is incorporated herein by reference in its entirety. In some embodiments, a nucleic acid, such as 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 about 20-25, 21-24, or 23 nucleotides in length. In an embodiment, the tissue-specific miRNA recognition sequence has perfect complementarity to the 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, the methods herein include tissue-specific expression of an exogenous agent in a target cell, comprising contacting a plurality of fusosomes, such as a virus, such as a retroviral vector, comprising nucleotides encoding the exogenous agent and at least one tissue-specific microRNA (miRNA) with a plurality of cells comprising the target cell and non-target cells, wherein the exogenous agent is preferentially, e.g., restrictedly, expressed in the target cell.

[0457] For example, a nucleic acid, such as a retroviral nucleic acid, may comprise at least one miRNA recognition sequence operably linked to a nucleotide sequence having a corresponding miRNA in a non-target cell, such as a hematopoietic progenitor cell (HSPC), a hematopoietic stem cell (HSC), which prevents or reduces the expression of the nucleotide sequence in the non-target cell but does not prevent or reduce the expression in a target cell, such as a differentiated cell. In some embodiments, the nucleic acid, such as a retroviral nucleic acid, comprises at least one miRNA sequence target for a miRNA present in an effective amount in the non-target cell (e.g., the concentration of the endogenous miRNA is sufficient to reduce or prevent the expression of the transgene), and also comprises a transgene. In an embodiment, the miRNA used in this system is strongly expressed in non-target cells such as HSPC and HSC, but is not expressed in, for example, differentiated progeny of the myeloid and lymphoid lineages, and prevents or reduces the expression of the transgene in the sensitive stem cell population while maintaining the 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., fusosomal nucleic acid or retroviral nucleic acid) comprises a sequence that is complementary to the miRNA of Table 4 or has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementarity thereto. In some embodiments, the nucleic acid (e.g., fusosomal nucleic acid or retroviral nucleic acid) comprises a sequence that is completely complementary to an endogenous miRNA, such as the seed sequence within the miRNA of Table 4. In some embodiments, the miRNA comprises a sequence set forth in any one of SEQ ID NOs: 156-162. In an embodiment, the seed sequence is at least 6, 7, 8, 9, or 10 nucleotides in length.

Table 4

[0459] In some embodiments, negative TSCRE or NTSCRE contains miRNA recognition sites for miRNAs 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), for example, miR-124.

[0460] In some embodiments, the fusosomes described herein contain a nucleic acid comprising a payload gene and a positive target cell-specific regulatory element. For example, the target cells are neurons, such as pan-neuronal cells, GABAergic neurons, glutamatergic neurons, cholinergic neurons, dopaminergic neurons, or serotonergic neurons. In some aspects, the nucleic acid further comprises a non-target cell-specific regulatory element (NTCSRE). For example, NTSCRE contains miRNA recognition sites for miRNAs expressed in glial cells (e.g., astrocytes, microglial cells, or oligodendrocytes).

[0461] In some embodiments, the fusosomes described herein contain a nucleic acid comprising a payload gene and a positive target cell-specific regulatory element. For example, the target cells are glial cells, such as astrocytes, microglial cells, or oligodendrocytes. In some aspects, the nucleic acid further comprises a non-target cell-specific regulatory element (NTCSRE). For example, NTSCRE contains miRNA recognition sites for miRNAs expressed in neurons, such as pan-neuronal cells, GABAergic neurons, glutamatergic neurons, cholinergic neurons, dopaminergic neurons, or serotonergic neurons.

[0462] In some embodiments, the negative TSCRE or NTSCRE comprises miRNA recognition sites for the miRNAs described herein. Exemplary miRNAs include those described in 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 U S A. 2004 Mar 2; 101(9):2999-3004; Sempere et al. Genome Biol. 2004; 5(3):R13; Metzler et al., Genes Chromosomes Cancer. 2004 Feb; 39(2):167-9; Calin et al., Proc Natl Acad Sci U S 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 those found at www.miRNA.org.

[0463] In some embodiments, the negative TSCRE or NTSCRE comprises miRNA recognition sites for miRNAs selected from miR-1b, miR-189b, miR-93, miR-125b, miR-130, miR-32, miR-128, miR-22, miR124a, miR-296, miR-143, miR-15, miR-141, miR-143, miR-16, miR-127, miR99a, miR-183, miR-19b, miR-92, miR-9, miR-130b, miR-21, miR-30b, miR-16, miR-99a, miR-212, miR-30c, miR-213, miR-20, miR-155, miR-152, miR-139, miR-30b, miR-7, miR-30c, miR-18, miR-137, miR-219, miR-1d, miR-178, miR-24, miR-122a, miR-215, miR-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, 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., 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 the miRNAs of Table 4. In some embodiments, one or more miRNA recognition sites on the fusosomal nucleic acid (e.g., retroviral nucleic acid) are transcribed in cis with an exogenous agent. In some embodiments, one or more miRNA recognition sites on the fusosomal nucleic acid (e.g., retroviral nucleic acid) are located downstream of the polyA tail sequence, e.g., between the polyA tail sequence and WPRE. In some embodiments, one or more miRNA recognition sites on the fusosomal nucleic acid (e.g., retroviral nucleic acid) are located downstream of WPRE.

[0465] V. Immunomodulation In some embodiments, the fusosomes described herein, such as retroviral vectors or VLPs, include upregulation of CD47. See, e.g., U.S. Patent No. 9,050,269, which is incorporated herein by reference in its entirety. In some embodiments, the fusosomes described herein, such as retroviral vectors or VLPs, include upregulation of complement regulatory proteins. See, e.g., Spanish Patent No. 2627445 T3 and U.S. Patent No. 6790641, each of which is incorporated herein by reference in its entirety. In some embodiments, the fusosomes described herein, such as retroviral vectors or VLPs, lack or include a decrease in level of MHC proteins, such as MHC class I or class II. See, e.g., U.S. Patent Application Publication No. 20170165348, which is incorporated herein by reference in its entirety.

[0466] Fusosomes, such as retroviral vectors or VLPs, may be recognized by the immune system of a subject. In the case of enveloped viral vector particles (e.g., retroviral vector particles), the membrane-bound proteins presented on the surface of the viral envelope may be recognized and the viral particles themselves may be neutralized. Further, upon infection of a target cell, the viral envelope may integrate with the cell membrane, such that the viral envelope proteins may be presented on the surface of the cell or remain in close association with the cell surface. Thus, the immune system may also target cells infected with the viral vector particles. Both effects may lead to a decrease in the effectiveness of delivery of exogenous agents by the viral vector.

[0467] The viral particle envelope is typically derived from the membrane of the source cell. Thus, membrane proteins expressed on the cell membrane from which the viral particles bud may be incorporated into the viral envelope. The immunomodulatory protein CD47

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

[0469] Based on studies using knockout mice lacking the membrane receptor CD47, professional phagocytes have been shown to distinguish between non-self and 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 SIRPα (signal regulatory protein), which is 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). The CD47-SIRPα interaction appears to inactivate autologous macrophages in mice, but a severe reduction (presumably 90%) of CD47 is seen in human blood cells from some Rh genotypes that show little or no evidence of anemia (Mouro-Chanteloup et al., 2003, Blood 101(1):338-344), and there is little or no evidence of enhanced cell interaction with phagocytic monocytes (Arndt et al., 2004, Br. J. Haematol. 125(3):412-4).

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

[0471] The methods described herein can include avoiding phagocytosis of particles by phagocytes. The method includes exposing a fusosome containing CD47, such as a retroviral vector or VLP, to phagocytes, and the fusosome, such as a viral particle, avoids phagocytosis by phagocytes or, in other respects, shows a decrease in phagocytosis compared to a similar unmodified fusosome, such as a retroviral vector or VLP. It may include expressing at least one peptide containing at least a biologically active portion of CD47 in the fusosome, such as a retroviral vector or VLP. In some embodiments, the half-life of the fusosome, such as a retroviral vector or VLP, in a subject is extended compared to a fusosome that is otherwise the same but unmodified, such as a retroviral vector or VLP.

[0472] Delet...

Claims

【Claim 1】 The invention described in the specification.

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