Fusosome compositions and uses thereof

Abstract

To provide fusosome compositions and uses thereof.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 re-targeted fusogen, a positive target cell-specific regulatory element, and a non-target cell-specific regulatory element. In some embodiments, the fusosome comprises one or more modifications that decrease an immune response against the fusosome.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent No. 62 / 671,838, filed May 15, 2018, and U.S. Patent No. 62 / 695,529, filed July 9, 2018, each of which is incorporated herein by reference in its entirety.

[0002] Including an array list by reference This application is filed with an electronic Sequence Listing, which is provided as a file titled V2050-7023WO Sequence Listing.TXT, created May 14, 2019, and is 651 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. [Background technology]

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

[0004] The present disclosure provides, at least in part, fusosome methods and compositions for in vivo delivery. In some embodiments, the fusosomes contain a combination of elements that promote specificity for target cells, such as one or more of a retargeting fusogen, a positive target cell-specific regulatory element, and a non-target cell-specific regulatory element. In some embodiments, the fusosomes contain one or more modifications that reduce an immune response to the fusosomes. [Brief explanation of the drawings]

[0005] [Figure 1A] This is a series of graphs showing results for cell lines, including targeted human hepatoma cell line (HepG2) and non-targeted (non-hepatic) cell lines, transduced with lentiviruses (LVs) encoding nucleic acid constructs containing positive TCSREs or NTCSREs. Figure 1A shows GFP expression in the human hepatoma cell line (HepG2), human embryonic kidney cell line (293LX), hematopoietic-derived human T cell line (Molt4.8), and mouse brain-derived endothelial cell line (bEND.3) transduced with LVs generated under the control of the PGK promoter with or without the miRT sequence (hPGK-eGFP+miRT). Figure 1B shows GFP expression in HepG2 and 293LX cells transduced with LVs generated under the control of the PGK promoter (hPGK-eGFP) or with LVs containing the miRT sequence and GFP under the control of the hepatocyte-specific promoter ApoE (hApoE-eGFP+miRT). Figure 1C shows quantification of phenylalanine (Phe) in the supernatants of HepG2 and 293LX cells transduced with LV containing the transgene phenylalanine ammonia-lyase (PAL) under the control of the SFFV promoter (SFFV-PAL) or LV containing the mirT sequence under the control of the hApoE promoter (hApoE-PAL+miRT). [Figure 1B] Same as above. [Figure 1C] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0006] Enumeration of Embodiments Provided herein are fusosomes containing retroviral vectors or particles, such as lentiviral vectors or particles, which generally result in increased expression of a desired exogenous agent (e.g., a therapeutic transgene) in target cells compared to non-target cells, e.g., in a subject, following introduction of the fusosomes into the cells. For example, in some instances, the increase in expression continues following in vivo administration of the provided fusosomes (e.g., retroviral vectors or particles) to a subject, e.g., a human subject. In particular, one of the major challenges to successful gene therapy is the ability to maintain stable, long-term expression of a therapeutic transgene (e.g., an exogenous agent) from genetically modified cells in vivo. Expression of a transgene in non-target cells, such as antigen-presenting cells (APCs), in some embodiments, results in activation of the adaptive immune response, leading to the generation of neutralizing antibodies against the transgene product by B cells and / or the elimination of transgene-producing cells by T cells. Thus, limiting transgene expression to target cells can, in some embodiments, substantially affect the durability of transgene expression by avoiding immune clearance. Furthermore, cell-type specific transgene expression can be highly relevant to disease biology, for example, restricting expression of pro-apoptotic genes to tumor cells or other target cells (eg, hepatocytes).

[0007] In particular, provided herein are fusosomes (e.g., retroviral vector particles) that, in some examples, contain expression of a nucleic acid sequence under the control of or regulated by a positive target cell-specific regulatory element (TCSRE, e.g., a tissue-specific promoter) and / or a negative target cell-specific regulatory element (negative TCSRE), e.g., a non-target cell-specific regulatory element (NTCSRE). In some embodiments, the negative TCSRE, such as an NCSRE, is due to miRNA-mediated gene silencing, such as by a nucleic acid sequence complementary to an miRNA sequence in a cell. In some embodiments, the provided fusosomes (e.g., retroviral vectors or particles) can specifically promote transgene (exogenous agent) expression in target cell lines (e.g., tumor or hepatocytes or other target cells) while restricting or limiting expression in non-target cells.

[0008] Among the embodiments provided are the following: 1. A fusosome, a) a lipid bilayer comprising a retargeted fusogen; b) a nucleic acid, (i) a positive target cell-specific regulatory element (e.g., a tissue-specific promoter) operably linked to a nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or an exogenous RNA), wherein the positive tissue-specific regulatory element increases expression of the exogenous agent in the target cell or tissue relative to a different fusosome lacking the positive tissue-specific regulatory element; or (ii) a fusosome comprising a nucleic acid that includes or encodes a non-target cell-specific regulatory element (e.g., a tissue-specific miRNA recognition sequence) operably linked to a nucleic acid encoding an exogenous agent, the non-target cell-specific regulatory element reducing expression of the exogenous agent in the non-target cell or tissue compared to an otherwise similar fusosome lacking the non-target cell-specific regulatory element.

[0009] 2. A fusosome, a) a lipid bilayer containing a retargeted fusogen; b) a nucleic acid, (i) a positive target cell-specific regulatory element (e.g., a tissue-specific promoter) operably linked to a nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or an exogenous RNA), wherein the positive tissue-specific regulatory element increases expression of the exogenous agent in the target cell or tissue relative to a different retroviral vector lacking the positive tissue-specific regulatory element; or (ii) a nucleic acid comprising or encoding a negative target cell-specific regulatory element (e.g., a tissue-specific miRNA recognition sequence) operably linked to a nucleic acid encoding an exogenous agent, wherein the negative tissue-specific regulatory element reduces expression of the exogenous agent in non-target cells or tissues compared to an otherwise similar retrovirus lacking the negative tissue-specific regulatory element.

[0010] 3. A fusosome, a) a lipid bilayer containing a fusogen (e.g., a retargeted fusogen); b) a nucleic acid, (i) a positive target cell-specific regulatory element (e.g., a tissue-specific promoter) operably linked to a nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or an exogenous RNA), wherein the positive tissue-specific regulatory element increases expression of the exogenous agent in the target cell or tissue relative to an otherwise similar fusosome lacking the positive tissue-specific regulatory element; and (ii) a fusosome comprising a nucleic acid that includes or encodes a non-target cell-specific regulatory element (e.g., a tissue-specific miRNA recognition sequence) operably linked to a nucleic acid encoding an exogenous agent, the non-target cell-specific regulatory element reducing expression of the exogenous agent in the non-target cell or tissue compared to an otherwise similar fusosome lacking the non-target cell-specific regulatory element.

[0011] 4. A fusosome, a) a lipid bilayer containing a fusogen (e.g., a retargeted fusogen); b) a nucleic acid, (i) a positive target cell-specific regulatory element (e.g., a tissue-specific promoter) operably linked to a nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or an exogenous RNA), wherein the positive tissue-specific regulatory element increases expression of the exogenous agent in the target cell or tissue relative to a different retroviral vector lacking the positive tissue-specific regulatory element; and (ii) a nucleic acid comprising or encoding a negative target cell-specific regulatory element (e.g., a tissue-specific miRNA recognition sequence) operably linked to a nucleic acid encoding an exogenous agent, wherein the negative tissue-specific regulatory element reduces expression of the exogenous agent in non-target cells or tissues compared to an otherwise similar retrovirus lacking the negative tissue-specific regulatory element.

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

[0013] 6. The fusosome of any of the above embodiments, wherein one or more (e.g., two or all three) of the following apply: the fusosome is a retroviral vector, the lipid bilayer is constituted by an envelope, e.g., a viral envelope, and the nucleic acid is a retroviral nucleic acid.

[0014] 7. The fusosome of any of the above embodiments, wherein the nucleic acid comprises one or more (e.g., all) of: a 5' LTR (e.g., comprising a U5 and lacking a functional U3 domain), a Psi packaging element (Psi), a central polypurine tract (cPPT) promoter operably linked to a payload gene, e.g., a nucleic acid encoding an exogenous agent, a payload gene, e.g., a nucleic acid encoding an exogenous agent (optionally comprising an intron before the open reading frame), a polyA tail sequence, a WPRE, and a 3' LTR (e.g., comprising a U5 and lacking a functional U3).

[0015] 8. The fusosome of any of the above 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 mutant), 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] 9. The fusosomes of any of the above embodiments, wherein when the fusosomes are administered to a subject: i) less than 10%, 5%, 4%, 3%, 2%, or 1% of the exogenous agent detectably present in the subject is in non-target cells; ii) at least 90%, 95%, 96%, 97%, 98%, or 99% of the cells of the subject that detectably contain the exogenous agent are target cells (e.g., cells of a single cell type, e.g., T cells); iii) fewer than 1,000,000, 500,000, 200,000, 100,000, 50,000, 20,000, or 10,000 cells of the subject detectably containing the exogenous agent are non-target cells; iv) the average level of the exogenous agent in all target cells of the subject is at least 100-fold, 200-fold, 500-fold, or 1,000-fold higher than the average level of the exogenous agent in all non-target cells of the subject; or v) the exogenous agent is not detectable in any non-target cells in the subject;

[0017] 10. The fusosome of any of the above embodiments, wherein the retargeted fusogen comprises sequences selected from Nipah virus F and G proteins, Measles virus F and H proteins, Tupaia paramyxovirus F and H proteins, Paramyxovirus F and G proteins or F and H proteins or F and HN proteins, Hendra virus F and G proteins, Henipavirus F and G proteins, Morbillivirus F and H proteins, Respirovirus F and HN proteins, Sendai virus F and HN proteins, Rubulavirus F and HN proteins, or Avulavirus F and HN proteins, or derivatives thereof, or any combination thereof.

[0018] 11. The fusosome of any of the above embodiments, wherein the fusogen comprises a domain of at least 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen, e.g., a sequence in Table 4 or Table 5, and optionally the wild-type paramyxovirus fusogen has a sequence set forth in any of SEQ ID NOs: 1-132.

[0019] 12. The fusosome of embodiment 11, wherein the paramyxovirus is a Nipah virus, such as a Henipa virus.

[0020] 13. The fusosome of any of the above embodiments, wherein the positive target cell-specific regulatory element comprises a tissue-specific promoter, a tissue-specific enhancer, a tissue-specific splice site, a tissue-specific site that extends RNA or protein half-life, a tissue-specific mRNA export-promoting site, a tissue-specific translation-enhancing site, or a tissue-specific post-translational modification site.

[0021] 14. The fusosome of any of the above embodiments, wherein the non-target cell-specific regulatory element or negative TCSSE comprises a tissue-specific miRNA recognition sequence, a tissue-specific protease recognition site, a tissue-specific ubiquitin ligase site, a tissue-specific transcriptional repression site, or a tissue-specific epigenetic repression site.

[0022] 15. The fusosome of any of the above embodiments, wherein the non-target cell-specific regulatory element or negative TCSSE comprises a tissue-specific miRNA recognition sequence.

[0023] 16. The fusosome of embodiment 15, wherein the non-target cell-specific regulatory element or negative TCSSE is located or encoded within a transcribed region (e.g., a transcribed region encoding an exogenous agent), e.g., the RNA produced by the transcribed region comprises an miRNA recognition sequence within a UTR or coding region.

[0024] 17. The fusosome of any of the above embodiments, wherein the target cells are cancer cells and the non-target cells are non-cancerous cells.

[0025] 18. The fusosome of any of the above embodiments, wherein the nucleic acid, eg, retroviral nucleic acid, encodes a positive TCSRE and / or an NTCSRE or a negative TCSRE.

[0026] 19. The fusosome of any of the above embodiments, wherein the retroviral nucleic acid comprises a positive TCSRE and / or the complement of an NTCSRE or a negative TCSRE.

[0027] 20. The fusosome of any of the above embodiments, which does not deliver nucleic acid to a non-target cell, e.g., an antigen-presenting cell, an MHC class II+ cell, a professional antigen-presenting cell, a heterotypic antigen-presenting cell, a macrophage, a dendritic cell, a myeloid dendritic cell, a plasmacytoid dendritic cell, a CD11c+ cell, a CD11b+ cell, a splenocyte, a B cell, a hepatocyte, an endothelial cell, or a non-cancerous cell.

[0028] 21. The fusosomes of any of the above embodiments, wherein less than 10%, 5%, 2.5%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, or 0.000001% of the non-target cell type (e.g., antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, heterotypic antigen-presenting cells, macrophages, dendritic cells, myeloid dendritic cells, plasmacytoid dendritic cells, CD11c+ cells, CD11b+ cells, splenocytes, B cells, hepatocytes, endothelial cells, or non-cancerous cells) comprise nucleic acid, e.g., retroviral nucleic acid, using quantitative PCR, e.g., using the assay of Example 1.

[0029] 22. The fusosome of any of the above embodiments, wherein the target cell comprises 0.00001-10, 0.0001-10, 0.001-10, 0.01-10, 0.1-10, 0.5-5, 1-4, 1-3, or 1-2 copies of the nucleic acid, e.g., a retroviral nucleic acid or portion thereof, per host cell genome, e.g., the copy number of the nucleic acid is assessed following administration in vivo.

[0030] 23. The fusosome of any of the above embodiments, less than 10%, 5%, 2.5%, 1%, 0.5%, 0.1%, 0.01% of the non-target cells (e.g., antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, atypical antigen-presenting cells, macrophages, dendritic cells, myeloid dendritic cells, plasmacytoid dendritic cells, CD11c+ cells, CD11b+ cells, splenocytes, B cells, hepatocytes, endothelial cells, or non-cancerous cells) contain the exogenous agent; or Fusosomes in which an exogenous agent (e.g., a protein) is not detectably present in non-target cells, such as antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, allotypic 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.

[0031] 24. The fusosome of any of the above embodiments, which delivers a nucleic acid, e.g., a retroviral nucleic acid, to a target cell, e.g., a T cell, a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a hepatocyte, a hematopoietic stem cell, a CD34+ hematopoietic stem cell, a CD105+ hematopoietic stem cell, a CD117+ hematopoietic stem cell, a CD105+ endothelial cell, a B cell, a CD20+ B cell, a CD19+ B cell, a cancer cell, a CD133+ cancer cell, an EpCAM+ cancer cell, a CD19+ cancellous cell, a Her2 / Neu+ cancer cell, a GluA2+ neuron, a GluA4+ neuron, an NKG2D+ natural killer cell, an SLC1A3+ astrocyte, an SLC7A10+ adipocyte, or a CD30+ lung epithelial cell.

[0032] 25. The fusosome of any of the above embodiments, wherein the fusosome is expressed on a target cell (e.g., a T cell, a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a hepatocyte, a hematopoietic stem cell, a CD34+ hematopoietic stem cell, a CD105+ hematopoietic stem cell, a CD117+ hematopoietic stem cell, a CD105+ endothelial cell, a B cell, a CD20+ B cell, a CD19+ B cell, a cancer cell, a CD133+ cancer cell, an EpCAM+ cancer cell, a CD19+ cancer cell, a Her2 / Neu+ cancer cell, a GluA2+ neuron, a GluA4+ neuron, a GluA5+ neuron, a GluA6+ neuron, a GluA7+ neuron, a GluA8+ neuron, a GluA9+ neuron, a GluA10+ neuron, a GluA10+ neuron, a GluA2 ... at least 0.00001%, 0.0001%, 0.001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fusosomes (one or more of: NKG2D+ natural killer cells, SLC1A3+ astrocytes, SLC7A10+ adipocytes, or CD30+ lung epithelial cells) contain nucleic acids, e.g., using quantitative PCR, e.g., using the assay of Example 3.

[0033] 26. The fusosome of any of the above embodiments, wherein the fusosome is attached to a target cell (e.g., a T cell, a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a hepatocyte, a hematopoietic stem cell, a CD34+ hematopoietic stem cell, a CD105+ hematopoietic stem cell, a CD117+ hematopoietic stem cell, a CD105+ endothelial cell, a B cell, a CD20+ B cell, a CD19+ B cell, a cancer cell, a CD133+ cancer cell, an EpCAM+ cancer cell, a CD19+ cancer cell, a Her2 / Neu+ cancer cell, GluA2+ neurons, GluA4+ neurons, NKG2D+ natural killer cells, SLC1A3+ astrocytes, SLC7A10+ adipocytes, or CD30+ lung epithelial cells), in which at least 0.00001%, 0.0001%, 0.001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fusosomes contain the exogenous agent.

[0034] 27. The fusosome of any of the above embodiments, wherein upon administration, the ratio of target cells containing said nucleic acid to non-target cells containing said nucleic acid is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by a quantitative PCR assay, e.g., using the assays of Examples 1 and 3.

[0035] 28. The fusosome of any of the above embodiments, wherein the ratio of the average copy number of said nucleic acid or portion thereof in target cells to the average copy number of said nucleic acid or portion thereof in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by a quantitative PCR assay, e.g., using the assays of Examples 1 and 3.

[0036] 29. The fusosome of any of the above embodiments, wherein the ratio of the median copy number of said nucleic acid or portion thereof in target cells to the median copy number of said nucleic acid or portion thereof in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by a quantitative PCR assay, e.g., using the assays of Examples 1 and 3.

[0037] 30. The fusosome of any of the above embodiments, wherein the ratio of target cells containing the exogenous RNA agent to non-target cells containing the exogenous RNA agent is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by reverse transcription quantitative PCR assay.

[0038] 31. The fusosome of any of the above embodiments, wherein the ratio of the average level of the exogenous RNA agent in target cells to the average level of the exogenous RNA agent in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by reverse transcription quantitative PCR assay.

[0039] 32. The fusosome of any of the above embodiments, wherein the ratio of median exogenous RNA agent level in target cells to median 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, e.g., by reverse transcription quantitative PCR assay.

[0040] 33. The fusosome of any of the above embodiments, wherein the ratio of target cells comprising the exogenous protein agent to non-target cells comprising the exogenous protein agent is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by FACS assay, e.g., using the assay of Example 2 and / or Example 4.

[0041] 34. The fusosome of any of the above embodiments, wherein the ratio of the average level of the exogenous protein agent in target cells to the average level of the exogenous protein agent in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by FACS assay, e.g., using the assay of Example 2 and / or Example 4.

[0042] 35. The fusosome of any of the above embodiments, wherein the ratio of median exogenous protein agent level in target cells to median exogenous protein agent level in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by FACS assay, e.g., using the assay of Example 2 and / or Example 4.

[0043] 36. The fusosome of any of the above embodiments, i) exogenous or overexpressed immunosuppressive proteins in the lipid bilayer, e.g., envelope, and ii) fusosomes containing one or both of the following immunostimulatory proteins that are absent or present at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) compared to fusosomes produced from an otherwise similar source cell:

[0044] 37. The fusosome of any of the above embodiments, i) a first exogenous or overexpressed immunosuppressive protein in a lipid bilayer, e.g., an envelope, and a second exogenous or overexpressed immunosuppressive protein in a lipid bilayer, e.g., an envelope; ii) a first exogenous or overexpressed immunosuppressive protein present in the lipid bilayer, e.g., envelope, and a second immunostimulatory protein present at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) relative to the absence or relative to fusosomes produced from an otherwise unmodified similar source cell; or iii) fusosomes comprising one or more of: 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 an otherwise similar source cell, 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 an otherwise similar source cell.

[0045] 38. The fusosome of any of the above embodiments, wherein the nucleic acid comprises one or more insulator elements.

[0046] 39. A fusosome, a) a lipid bilayer containing a fusogen (e.g., a retargeted fusogen); b) an exogenous agent (e.g., an exogenous polypeptide or exogenous RNA) or a nucleic acid encoding an exogenous agent (e.g., a retroviral nucleic acid); c) i) a first exogenous or overexpressed immunosuppressive protein in a lipid bilayer, e.g., an envelope, and a second exogenous or overexpressed immunosuppressive protein in a lipid bilayer, e.g., an envelope; ii) a first exogenous or overexpressed immunosuppressive protein present in the lipid bilayer, e.g., envelope, and a second immunostimulatory protein present at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) relative to the absence or relative to fusosomes produced from an otherwise unmodified similar source cell; or iii) one or more of: a first immunostimulatory protein that is absent or present at reduced levels relative to fusosomes produced from an otherwise unmodified similar source cell (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced); and a second immunostimulatory protein that is absent or present at reduced levels relative to fusosomes produced from an otherwise unmodified similar source cell (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced); When administered to a subject (e.g., a human subject or a mouse): i) the fusosomes do not produce a detectable antibody response (e.g., after a single dose or multiple doses), e.g., by a FACS antibody detection assay, e.g., the assay of Example 13 or Example 14, or antibodies to the fusosomes are present at levels less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels; ii) the fusosomes do not elicit a detectable cellular immune response (e.g., a T cell response, an NK cell response, or a macrophage response), or there is a cellular immune response against the fusosomes at a level less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels, e.g., by a PBMC lysis assay (e.g., the assay of Example 5), by a NK cell lysis assay (e.g., the assay of Example 6), by a CD8 killer T cell lysis assay (e.g., the assay of Example 7), or by a macrophage phagocytosis assay (e.g., the assay of Example 8); iii) the fusosomes do not elicit a detectable innate immune response, e.g., complement activation (e.g., after a single dose or multiple doses), e.g., by a complement activity assay (e.g., the assay of Example 9), or the innate immune response to the fusosomes is present at a level less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels; iv) less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.002%, or 0.001% of the fusosomes are inactivated by serum, e.g., by a serum inactivation assay, e.g., the assay of Example 11 or Example 12; v) target cells that receive the exogenous agent from the fusosomes do not develop a detectable antibody response (e.g., after a single dose or multiple doses), e.g., by a FACS antibody detection assay, e.g., the assay of Example 15, or antibodies to the target cells are present at levels less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels; or vi) The target cells that received the exogenous agent from the fusosomes do not produce a detectable cellular immune response (e.g., a T cell response, an NK cell response, or a macrophage response), or there is a cellular response against the target cells at a level 10%, 5%, 4%, 3%, 2%, or 1% above background levels, as determined by, 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).

[0047] 40. The fusosome of any of the above embodiments, wherein one or more (e.g., two or all three) of the following apply: the fusosome is a retroviral vector, the lipid bilayer is constituted by an envelope, e.g., a viral envelope, and the nucleic acid is a retroviral nucleic acid.

[0048] 41. The fusosome of embodiment 39 or 40, wherein the background level is the corresponding level in the same subject before administration of the particle or vector.

[0049] 42. The fusosome of any of embodiments 39 to 41, wherein the immunosuppressive protein is a complement regulatory protein or CD47.

[0050] 43. The fusosome of any of embodiments 39-42, wherein the immunostimulatory protein is an MHC (e.g., HLA) protein.

[0051] 44. The fusosome of any of embodiments 39 to 43, wherein one or both of the first exogenous or overexpressed immunosuppressive proteins is other than CD47 and the second immunostimulatory protein is other than MHC.

[0052] 45. A fusosome, a) a lipid bilayer containing a fusogen; b) a nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or an exogenous RNA); c) Fusosomes comprising exogenous or overexpressed MHC, such as HLA (e.g., HLA-G or HLA-E), or a combination thereof, in a lipid bilayer.

[0053] 46. ​​A fusosome, a) a lipid bilayer comprising a fusogen, wherein the fusogen comprises a domain of at least 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen, e.g., a sequence in Table 4 or Table 5, and optionally the wild-type paramyxovirus fusogen is set forth in any one of SEQ ID NOs: 1-132; b) a nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or an exogenous RNA); c) Fusosomes containing exogenous or overexpressed CD47 or complement regulatory proteins, or a combination thereof, in their envelopes.

[0054] 47. A fusosome, a) a lipid bilayer comprising a fusogen, wherein the fusogen comprises a domain of at least 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen, e.g., a sequence in Table 4 or Table 5, and optionally, the wild-type paramyxovirus fusogen has a sequence of amino acids set forth in any one of SEQ ID NOs: 1-132; b) a nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or an exogenous RNA); c) Fusosomes comprising MHC1 (e.g., HLA-A, HLA-B, or HLA-C) or MHCII (e.g., HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR) that are absent or present at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) compared to fusosomes produced from an otherwise unmodified similar source cell.

[0055] 48. A fusosome, a) a lipid bilayer comprising a fusogen, wherein the fusogen comprises a domain of at least 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen, e.g., a sequence in Table 4 or Table 5, and optionally, the wild-type paramyxovirus fusogen has a sequence set forth in any one of SEQ ID NOs: 1-132; b) a nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or an exogenous RNA); c) fusosomes comprising one or both of an exogenous or overexpressed immunosuppressive or 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 an otherwise similar source cell that is not modified.

[0056] 49. The fusosome of any of embodiments 45 to 48, wherein one or more (e.g., two or all three) of the following applies: the fusosome is a retroviral vector, the lipid bilayer is constituted by an envelope, e.g., a viral envelope, and the nucleic acid is a retroviral nucleic acid.

[0057] 50. The fusosome of any of the above embodiments, wherein the fusosome circulates for at least 0.5, 1, 2, 3, 4, 6, 12, 18, 24, 36, or 48 hours after administration to a subject.

[0058] 51. The fusosomes of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 30 minutes after administration.

[0059] 52. The fusosomes of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 1 hour after administration.

[0060] 53. The fusosomes of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 2 hours after administration.

[0061] 54. The fusosomes of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 4 hours after administration.

[0062] 55. The fusosomes of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 8 hours after administration.

[0063] 56. The fusosomes of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 12 hours after administration.

[0064] 57. The fusosomes of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 18 hours after administration.

[0065] 58. The fusosomes of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 24 hours after administration.

[0066] 59. The fusosomes of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 36 hours after administration.

[0067] 60. The fusosomes of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 48 hours after administration.

[0068] 61. The fusosomes of any of the above embodiments, which have reduced immunogenicity as measured by a reduction in humoral response compared to a control retrovirus, e.g., an unmodified fusosome but similar to the fusosome, after one or more administrations of the fusosomes to a suitable animal model, e.g., an animal model described herein.

[0069] 62. The fusosome of any of the above embodiments, wherein the reduction in humoral response is measured in a serum sample by anti-cellular antibody titers, e.g., anti-retroviral antibody titers, e.g., by ELISA.

[0070] 63. The fusosome of any of the above embodiments, wherein a serum sample from an animal administered the retroviral composition has a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in anti-fusosome antibody titer compared to a serum sample from a subject administered unmodified cells.

[0071] 64. The fusosomes of any of the above embodiments, wherein a serum sample from a subject to which the fusosomes have been administered has an increased anti-cellular antibody titer, e.g., a 1%, 2%, 5%, 10%, 20%, 30%, or 40% increase, from baseline, e.g., a baseline referenced to a serum sample from the same subject prior to administration of the fusosomes.

[0072] 65. The fusosome of any of the above embodiments, the subject to whom the fusosomes are to be administered has, is known to have, or is tested to have pre-existing antibodies (e.g., IgG or IgM) reactive with fusosomes; the subject to whom the fusosomes are to be administered does not have detectable levels of pre-existing antibodies reactive with fusosomes; the subject receiving the fusosomes has, is known to have, or is tested to have, antibodies (e.g., IgG or IgM) reactive with the fusosomes; subjects receiving fusosomes (e.g., at least once, twice, three times, four times, five times or more) do not have detectable levels of antibodies reactive with fusosomes; or Fusosomes in which antibody levels do not increase by more than 1%, 2%, 5%, 10%, 20%, or 50% between two time points: a first time point before the first administration of fusosomes and a second time point after one or more administrations of fusosomes.

[0073] 66. The fusosome of any of the above embodiments, wherein the fusosome is a retroviral vector produced by the method of Example 5, 6, or 7, e.g., from cells transfected with HLA-G or HLA-E cDNA.

[0074] 67. The fusosome of any of the above embodiments, wherein the fusosome is a retroviral vector produced from NMC-HLA-G cells and wherein the rate of lysis, e.g., PBMC-mediated lysis, NK cell-mediated lysis, and / or CD8+ T cell-mediated lysis, is reduced at a particular time point compared to retroviral vector produced from NMC or an NMC empty vector.

[0075] 68. The fusosome of any of the above embodiments, wherein the modified fusosome avoids phagocytosis by macrophages.

[0076] 69. The fusosome of any of the above embodiments, wherein the fusosome is produced by the method of Example 8, e.g., from cells transfected with CD47 cDNA.

[0077] 70. The fusosome of any of the above embodiments, wherein the fusosome is a retroviral vector, and when macrophages are incubated with the retroviral vector derived from NMC-CD47, the phagocytic index is reduced compared to that derived from NMC or an NMC empty vector.

[0078] 71. The fusosome of any of the above embodiments, having a reduction in macrophage phagocytosis, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in macrophage phagocytosis, compared to control fusosomes, e.g., unmodified fusosomes but similar to the fusosomes, wherein the reduction in macrophage phagocytosis is determined by an in vitro phagocytic index, e.g., assayed as described in Example 8.

[0079] 72. The fusosome of any of the above embodiments, wherein a composition comprising a plurality of fusosomes, when incubated with macrophages in an in vitro assay of macrophage phagocytosis, has a phagocytic index of 0, 1, 10, 100 or greater, as measured, for example, by the assay of Example 8.

[0080] 73. The fusosome of any of the above embodiments, wherein the fusosome has a modified complement activity compared to an unmodified retroviral vector.

[0081] 74. The fusosome of any of the above embodiments, produced by the method of Example 9, e.g., from cells transfected with a cDNA encoding a complement regulatory protein, e.g., DAF.

[0082] 75. The fusosome of any of the above embodiments, wherein the fusosome is a retroviral vector, and the dose of the retroviral vector is greater when the modified retroviral vector (e.g., HEK293-DAF) is incubated with the corresponding mouse serum (e.g., HEK-293DAF mouse serum) than when the control retroviral vector (e.g., HEK293 retroviral vector) is incubated with the corresponding mouse serum (e.g., HEK293 mouse serum) in the presence of 200 pg / ml of C3a.

[0083] 76. The fusosome of any of the above embodiments, wherein the fusosome is a retroviral vector, and the dose of retroviral vector is greater for the modified retroviral vector (e.g., HEK293-DAF) incubated with naive mouse serum than for a control retroviral vector (e.g., HEK293 retroviral vector) incubated with naive mouse serum in the presence of 200 pg / ml of C3a.

[0084] 77. The fusosomes of any of the above embodiments, which are resistant to complement-mediated inactivation in patient serum 30 minutes after administration by the assay of Example 9.

[0085] 78. The fusosomes of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are resistant to complement-mediated inactivation.

[0086] 79. The fusosome of any of the above embodiments, wherein the complement regulatory protein comprises one or more of a protein that binds decay-accelerating factor (DAF, CD55), e.g., factor H (FH)-like protein-1 (FHL-1), e.g., C4b-binding protein (C4BP), e.g., complement receptor 1 (CD35), e.g., a membrane cofactor protein (MCP, CD46), e.g., protectin (CD59), e.g., a protein that inhibits classical and alternative complement pathway CD / C5 convertases, e.g., a protein that regulates MAC assembly.

[0087] 80. The fusosome of any of the above embodiments, produced by the method of Example 10, e.g., from cells transfected with DNA encoding an shRNA targeting MHC class I, e.g., a retroviral vector derived from NMC-shMHC class I, having reduced expression of MHC class I relative to NMC and NMC vector control.

[0088] 81. The fusosome of any of the above embodiments, wherein the measure of immunogenicity in the fusosome (e.g., retroviral vector) is serum inactivation, e.g., serum inactivation measured as described herein, e.g., as described in Example 11.

[0089] 82. The fusosome of any of the above embodiments, wherein the percentage of cells that receive the exogenous agent does not differ between fusosome samples that have been incubated with serum from fusosome-naive mice and heat-inactivated serum.

[0090] 83. The fusosome of any of the above embodiments, wherein the percentage of cells that receive the exogenous agent does not differ between fusosome samples that have been incubated with serum from fusosome-naive mice and serum-free control incubations.

[0091] 84. The fusosomes of any of the above embodiments, wherein the percentage of cells that receive the exogenous agent is lower in fusosome samples incubated with positive control serum than in fusosome samples incubated with serum from fusosome-naive mice.

[0092] 85. The fusosome of any of the above embodiments, e.g., wherein the modified retroviral vector, modified by a method described herein, has reduced serum inactivation (reduced compared to administration of an unmodified retroviral vector) after multiple (e.g., more than one, e.g., two or more) administrations of the modified retroviral vector.

[0093] 86. The fusosome of any of the above embodiments, wherein the fusosomes described herein are not inactivated by serum after multiple administrations.

[0094] 87. The fusosome of any of the above embodiments, wherein the measure of immunogenicity in the fusosome is serum inactivation, e.g., serum inactivation after multiple doses, e.g., measured as described herein, e.g., as described in Example 12.

[0095] 88. The fusosome of any of the above embodiments, wherein the percentage of cells that receive the exogenous agent does not differ between fusosome samples that have been incubated with serum from mice treated with modified (e.g., HEK293-HLA-G) fusosomes and heat-inactivated serum.

[0096] 89. The fusosomes of any of the above embodiments, wherein the percentage of cells that receive the exogenous agent does not differ between fusosome samples that have been incubated with serum from mice treated 1, 2, 3, 5, or 10 times with the modified (e.g., HEK293-HLA-G) retroviral vector.

[0097] 90. The fusosome of any of the above embodiments, wherein the percentage of cells that receive the exogenous agent does not differ between fusosome samples incubated with serum from vehicle-treated mice and mice treated with modified (e.g., HEK293-HLA-G) fusosomes.

[0098] 91. The fusosome of any of the above embodiments, wherein the percentage of cells that receive the exogenous agent is lower in fusosomes derived from control cells (e.g., HEK293) than in modified (e.g., HEK293-HLA-G) fusosomes.

[0099] 92. The fusosome of any of the above embodiments, wherein the measure of immunogenicity in the fusosome is an antibody response.

[0100] 93. The fusosome of any of the above embodiments, wherein the subject receiving the fusosome described herein has pre-existing antibodies that bind to and recognize the fusosome, e.g., as described herein, e.g., as measured as described in Example 13.

[0101] 94. The fusosomes of any of the above embodiments, wherein serum from fusosome-naive mice shows more signal (e.g., fluorescence) than a negative control, e.g., serum from mice depleted of IgM and IgG, indicating that immunogenicity is occurring.

[0102] 95. The fusosomes of any of the above embodiments, wherein serum from fusosome-naive mice exhibits a similar signal (e.g., fluorescence) compared to a negative control, indicating, for example, that no detectable immunogenicity occurred.

[0103] 96. The fusosome of any of the above embodiments, comprising a retroviral vector that has been modified, e.g., modified by a method described herein, and which has a reduced humoral response (e.g., reduced compared to administration of an unmodified retrovirus) after multiple (e.g., more than one, e.g., two or more) administrations of the modified retrovirus, e.g., measured as described herein, e.g., as described in Example 14.

[0104] 97. The fusosome of any of the above embodiments, wherein the fusosome, e.g., a retroviral vector, is produced from a cell transfected with, e.g., HLA-G or HLA-E cDNA, by the method of Example 5, 6, 7, or 14.

[0105] 98. The fusosome of any of the above embodiments, wherein the humoral response is assessed by determining the value of the level of anti-fusosome antibodies (e.g., IgM, IgG1, and / or IgG2 antibodies).

[0106] 99. The fusosome of any of the above embodiments, wherein the modified (e.g., NMC-HLA-G) fusosome, e.g., retroviral vector, reduces anti-viral IgM or IgG1 / 2 antibody titers (e.g., as measured by FACS fluorescence intensity) after infusion compared to a control, e.g., an NMC retroviral vector or an NMC empty retroviral vector.

[0107] 100. The fusosome of any of the above embodiments, wherein the recipient cell is not targeted by an antibody response or the antibody response is below a control level, e.g., measured as described herein, e.g., as described in Example 15.

[0108] 101. The fusosome of any of the above embodiments, wherein the signal (e.g., mean fluorescence intensity) is similar for recipient cells derived from retroviral vector-treated mice and PBS-treated mice.

[0109] 102. The fusosome of any of the above embodiments, wherein the measure of immunogenicity of the recipient cell is a macrophage response.

[0110] 103. The fusosome of any of the above embodiments, wherein the recipient cells are not targeted by macrophages or are targeted below control levels.

[0111] 104. The fusosome of any of the above embodiments, wherein the phagocytic index, e.g., measured as described herein, e.g., as described in Example 16, is similar for recipient cells derived from fusosome-treated mice and PBS-treated mice.

[0112] 105. The fusosome of any of the above embodiments, wherein the measure of immunogenicity of the recipient cells is a PBMC response.

[0113] 106. The fusosome of any of the above embodiments, wherein the recipient cell does not induce a PBMC response.

[0114] 107. The fusosome of any of the above embodiments, wherein the percentage of CD3+ / CMG+ cells is similar in recipient cells derived from fusosome-treated mice and PBS-treated mice, e.g., when measured as described herein, e.g., as described in Example 17.

[0115] 108. The fusosome of any of the above embodiments, wherein the measure of recipient cell immunogenicity is a natural killer cell response.

[0116] 109. The fusosome of any of the above embodiments, wherein the recipient cells do not induce a natural killer cell response, or induce a lower natural killer cell response, e.g., below a control value.

[0117] 110. The fusosome of any of the above embodiments, wherein the percentage of CD3+ / CMG+ cells is similar in recipient cells derived from fusosome-treated mice and PBS-treated mice, e.g., when measured as described herein, e.g., as described in Example 18.

[0118] 111. The fusosome of any of the above embodiments, wherein the measure of immunogenicity of the recipient cells is a CD8+ T cell response.

[0119] 112. The fusosome of any of the above embodiments, wherein the recipient cells do not induce a CD8+ T cell response, or induce a lower CD8+ T cell response, e.g., below control values.

[0120] 113. The fusosome of any of the above embodiments, wherein the percentage of CD3+ / CMG+ cells is similar in recipient cells derived from fusosome-treated mice and PBS-treated mice, e.g., when measured as described herein, e.g., as described in Example 19.

[0121] 114. The fusosome of any of the above embodiments, wherein the fusogen is a retargeted fusogen.

[0122] 115. The fusosome of any of the above embodiments, comprising a retroviral nucleic acid encoding one or both of: (i) a positive target cell-specific regulatory element operably linked to a nucleic acid encoding an exogenous agent, or (ii) a non-target cell-specific regulatory element or a negative TCSSE operably linked to a nucleic acid encoding an exogenous agent.

[0123] 116. A fusosome, a) a lipid bilayer comprising a fusogen, wherein the fusogen comprises a domain of at least 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen, e.g., to a sequence in Table 4 or Table 5, and optionally, the wild-type paramyxovirus has a sequence of amino acids set forth in any one of SEQ ID NOs: 1-132; b) a nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or an exogenous RNA), wherein the retroviral nucleic acid comprises one or more insulator elements.

[0124] 117. The fusosome of embodiment 116, wherein one or more (e.g., two or all three) of the following applies: the fusosome is a retroviral vector, the lipid bilayer is constituted by an envelope, e.g., a viral envelope, e.g., a pseudotyped envelope, and the nucleic acid is a retroviral nucleic acid.

[0125] 118. The fusosome of embodiment 116 or 117, wherein the nucleic acid comprises two insulator elements, e.g., a first insulator element upstream of the region encoding the exogenous agent, and a second insulator element downstream of the region encoding the exogenous agent, e.g., the first insulator element and the second insulator element comprise the same or different sequences.

[0126] 119. The fusosome of any of embodiments 116-118, wherein the variation in median exogenous agent level in samples of cells isolated after administering the fusosomes to a subject at a first time point is at least about 10,000%, 5,000%, 2,000%, 1,000%, 500%, 200%, 100%, 50%, 20%, 10%, or 5%, or less, of the median exogenous agent level in samples of cells isolated after administering the fusosomes to a subject at a second, later time point.

[0127] 120. The fusosome of embodiment 119, wherein the median expression level per cell is assessed only in cells having a retroviral genome copy number of at least 1.0.

[0128] 121. The fusosome of any of embodiments 116-120, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of target cells in a subject detectably contain the exogenous agent.

[0129] 122. The fusosome of any of embodiments 119 to 121, wherein the median payload gene expression level is assessed across cells isolated from the subject at 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, and 1095 days after administration of the fusosome to the subject.

[0130] 123. The fusosome of any of embodiments 116-122, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of target cells in a subject that detectably contain the exogenous agent at a first time point still detectably contain the exogenous agent at a later, second time point, wherein the first time point is 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days after administration of the fusosome to the subject.

[0131] 124. The fusosome of embodiment 123, wherein the second time point is 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days after the first time point.

[0132] 125. The fusosomes of any of embodiments 116 to 124, which are not genotoxic or do not increase the rate of tumor formation in target cells compared to target cells not treated with the fusosomes.

[0133] 126. The fusosome of any of embodiments 116 to 125, wherein the median exogenous drug level is assessed in a population of cells from a subject that received the fusosome.

[0134] 127. The fusosome of any of embodiments 116-126, wherein the median exogenous agent level assessed in a population of cells collected (e.g., isolated) from the subject on different days after administration differs from the median exogenous agent level in a population of cells assessed at 7 days, 14 days, 28 days, or 56 days by less than about 10,000%, 1000%, 100%, or 10%, e.g., 10,000% to 1000%, 1000% to 100%, or 100% to 10%, and wherein the cells in the population have a vector copy number of at least 1.0.

[0135] 128. The fusosome of any of embodiments 116 to 127, wherein exogenous drug levels are assessed across cells from the subject receiving the fusosome.

[0136] 129. The fusosome of any of embodiments 116-128, wherein the percentage of cells containing the exogenous agent is assessed in a plurality of cells collected (e.g., isolated) from the subject at 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, and 1095 days after administration of the fusosome.

[0137] 130. The fusosome of any of embodiments 116 to 129, wherein the difference in the percentage of cells containing the exogenous agent assessed in cells isolated on two different days after administration is less than 1%, 5%, 10%, 20%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 1500%, or 2000%.

[0138] 131. The fusosome of any of embodiments 116 to 130, wherein the percentage of target cells positive for the exogenous agent is similar across cells collected at 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days.

[0139] 132. The fusosome of any of embodiments 116-131, At least the same number of target cells as on day 7 were positive for the exogenous agent on days 14, 28, 56, 112, 365, 730, or 1095; At least the same number of target cells were positive for the exogenous agent at 28, 56, 112, 365, 730, or 1095 days as at 14 days; At least the same number of target cells as on day 28 were positive for the exogenous agent on days 56, 112, 365, 730, or 1095; At least as many target cells as on day 56 were positive for the exogenous agent on days 112, 365, 730, or 1095; At least as many target cells as on day 112 were positive for the exogenous agent on days 365, 730, or 1095; At least the same number of target cells were positive for the exogenous agent at 730 or 1095 days as at 365 days; At least as many target cells were positive for exogenous agent at 1095 days as at 730 days, fusosomes.

[0140] 133. The fusosome of any of embodiments 116 to 132, the median exogenous agent levels in target cells containing the exogenous agent are similar in cells collected at 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days; the median exogenous agent level in target cells containing the exogenous agent at 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days is at least as high as at 7 days; the median exogenous agent level in target cells containing the exogenous agent at 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days is at least as high as at 14 days; the median exogenous agent levels in target cells containing the exogenous agent at 56 days, 112 days, 365 days, 730 days, or 1095 days are at least as high as at 28 days; the median exogenous agent levels in target cells containing the exogenous agent at 112 days, 365 days, 730 days, or 1095 days are at least as high as at 56 days; the median exogenous agent levels in target cells containing the exogenous agent at 365 days, 730 days, or 1095 days are at least as high as at 112 days; the median exogenous drug levels in target cells containing the exogenous drug at 730 days or 1095 days are at least as high as at 365 days; or fusosomes, where the median exogenous drug levels in target cells containing exogenous drug at 1095 days were at least as high as at 730 days.

[0141] 134. A method of delivering an exogenous agent to a subject (e.g., a human subject), comprising administering to the subject a fusosome of any of the above embodiments, thereby delivering the exogenous agent to the subject.

[0142] 135. A method of modulating a function in a subject (e.g., a human subject), target tissue, or target cell, comprising contacting, e.g., administering to the subject, target tissue, or target cell, a fusosome of any of the above embodiments.

[0143] 136. The method of embodiment 135, wherein the target tissue or target cell is present in a subject.

[0144] 137. A method of treating or preventing a disorder, e.g., cancer, in a subject (e.g., a human subject), comprising administering to the subject a fusosome of any of the above embodiments.

[0145] 138. A method of producing fusosomes according to any of the above embodiments, comprising: a) providing a source cell containing nucleic acid and a fusogen (e.g., a retargeted fusogen); b) culturing the source cells under conditions that allow the production of fusosomes; c) isolating, enriching or purifying fusosomes from source cells, thereby producing fusosomes.

[0146] 139. A source cell for producing fusosomes, a) a nucleic acid; b) structural proteins capable of packaging nucleic acids, wherein at least one structural protein comprises a fusogen that binds to a fusogen receptor; c) source cells comprising fusogens that are absent or present at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) compared to otherwise similar source cells.

[0147] 140. The method or source cell of embodiment 138 or the source cell of embodiment 139, wherein one or more (e.g., two or all three) of the following are applied: the fusosome is a retroviral vector, the nucleic acid is a retroviral nucleic acid, and the structural protein is a viral structural protein.

[0148] 141. A source cell of embodiment 139 or 140, wherein binding of a fusogen to a fusogen receptor results in fusion of a fusosome with a target cell.

[0149] 142. The source cell of any of embodiments 139-141, which binds to a second similar source cell, e.g., a fusogen from the source cell binds to a fusogen receptor on the second source cell.

[0150] 143. A population of source cells of any of embodiments 139-142.

[0151] 144. The population of source cells of embodiment 143, wherein less than 10%, 5%, 4%, 3%, 2%, or 1% of the cells in the population are multinucleated.

[0152] 145. A source cell or population of source cells of any of embodiments 139-144, which has been modified to reduce (e.g., not fuse with) other source cells during the production of fusosomes as described herein.

[0153] 146. A source cell or population of source cells of any of embodiments 139 to 145, wherein the fusogen (e.g., the retargeted fusogen) does not bind to a protein contained by the source cell, e.g., to a protein on the surface of the source cell.

[0154] 147. A source cell or population of source cells of any of embodiments 139-146, wherein a fusogen (e.g., a retargeted fusogen) binds to a protein contained by the source cell but does not fuse with the cell.

[0155] 148. A source cell or population of source cells of any of embodiments 139-147, wherein the fusogen does not induce fusion with the source cells.

[0156] 149. A source cell or population of source cells of any of embodiments 139-148, wherein the source cell does not express a protein (e.g., an antigen) that binds to a fusogen.

[0157] 150. The source cell or population of source cells of any of embodiments 139-149, wherein a plurality of source cells do not form syncytia when expressing a fusogen, or wherein less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the cells in the population are multinucleated (e.g., containing two or more nuclei).

[0158] 151. The source cell or population of source cells of any of embodiments 139-150, wherein a plurality of source cells do not form syncytia when producing fusosomes, or less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the cells in the population are multinucleated.

[0159] 152. The source cell or population of source cells of any of embodiments 139-151, wherein less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the nuclei in the population are in syncytia.

[0160] 153. The source cell or population of source cells of any of embodiments 139-152, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of the nuclei in the population are within mononuclear cells.

[0161] 154. A source cell or population of source cells of any of embodiments 139-153, wherein the percentage of multinucleated cells is lower, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower, compared to a population of otherwise similar, unmodified source cells.

[0162] 155. A source cell or population of source cells of any of embodiments 139-154, wherein the percentage of nuclei present in syncytia is lower, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower, compared to an otherwise similar source cell that is unmodified.

[0163] 156. A source cell or population of source cells of any of embodiments 139 to 155, wherein multinucleated cells (e.g., cells with two or more nuclei) are detected by a microscopic assay, e.g., the assay of Example 20, using a DNA stain.

[0164] 157. A source cell or population of source cells of any of embodiments 139-156, wherein the number of functional fusosomes (e.g., viral particles) obtained from the modified source cells is at least 10%, 20%, 40%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 5-fold, or 10-fold greater than the number of fusosomes obtained from otherwise similar, unmodified source cells, e.g., using the assay of Example 20.

[0165] 158. A fusosome comprising a fusogen receptor that lacks the fusogen receptor or is present at a reduced level (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) compared to unmodified fusosomes derived from an otherwise similar source cell.

[0166] 159. A method for producing fusosomes, comprising: a) providing source cells containing a fusogen (e.g., a retargeted fusogen), wherein the source cells lack fusogen receptors or contain fusogen receptors present at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) compared to otherwise similar source cells that are unmodified; b) culturing the source cells under conditions that allow the production of fusosomes; c) isolating, enriching or purifying fusosomes from source cells, thereby producing fusosomes.

[0167] 160. The method of embodiment 159, wherein providing the source cells comprises knocking down or knocking out a fusogen receptor in the source cells or their precursors.

[0168] 161. The fusosome of embodiment 158, or the method of embodiment 159 or 160, wherein the fusosome is a retroviral vector or a retrovirus-like particle.

[0169] 162. Retroviral vectors (e.g., suitable for in vivo use in human subjects), a) an envelope containing a retargeted fusogen; b) (i) a positive target cell-specific regulatory element (e.g., a tissue-specific promoter) operably linked to a nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or an exogenous RNA), wherein the positive tissue-specific regulatory element increases expression of the exogenous agent in the target cell or tissue relative to a different retroviral vector lacking the positive tissue-specific regulatory element; or (ii) a retroviral vector comprising a retroviral nucleic acid that comprises or encodes a negative target cell-specific regulatory element (e.g., a tissue-specific miRNA recognition sequence) operably linked to a nucleic acid encoding an exogenous agent, wherein the negative tissue-specific regulatory element reduces expression of the exogenous agent in non-target cells or tissues compared to an otherwise similar retrovirus lacking the negative tissue-specific regulatory element.

[0170] 163. Retroviral vectors (e.g., suitable for in vivo use in human subjects), a) an envelope comprising a fusogen (e.g., a retargeted fusogen); b) (i) a positive target cell-specific regulatory element (e.g., a tissue-specific promoter) operably linked to a nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or an exogenous RNA), wherein the positive tissue-specific regulatory element increases expression of the exogenous agent in the target cell or tissue relative to a different retroviral vector lacking the positive tissue-specific regulatory element; and (ii) a retroviral vector comprising a retroviral nucleic acid that includes or encodes a negative target cell-specific regulatory element (e.g., a tissue-specific miRNA recognition sequence) operably linked to a nucleic acid encoding an exogenous agent, wherein the negative tissue-specific regulatory element reduces expression of the exogenous agent in non-target cells or tissues compared to an otherwise similar retrovirus lacking the negative tissue-specific regulatory element.

[0171] 164. The retroviral vector of any of the above embodiments, which, when administered to a subject, i) less than 10%, 5%, 4%, 3%, 2%, or 1% of the exogenous agent detectably present in the subject is in non-target cells; ii) at least 90%, 95%, 96%, 97%, 98%, or 99% of the cells of the subject that detectably contain the exogenous agent are target cells (e.g., cells of a single cell type, e.g., T cells); iii) fewer than 1,000,000, 500,000, 200,000, 100,000, 50,000, 20,000, or 10,000 cells of the subject detectably containing the exogenous agent are non-target cells; 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) A retroviral vector, wherein the exogenous agent is not detectable in any non-target cells in the subject.

[0172] 165. The retroviral vector of any of the above embodiments, wherein the retargeting fusogen comprises sequences selected from Nipah virus F and G proteins, measles virus F and H proteins, Tupaia paramyxovirus F and H proteins, paramyxovirus F and G proteins or F and H proteins or F and HN proteins, Hendra virus F and G proteins, Henipavirus F and G proteins, Morbillivirus F and H proteins, Respirovirus F and HN proteins, Sendai virus F and HN proteins, Rubulavirus F and HN proteins, or Avulavirus F and HN proteins, or derivatives thereof, or any combination thereof.

[0173] 166. The retroviral vector of any of the above embodiments, wherein the fusogen comprises a domain of at least 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen, e.g., a sequence in Table 4 or Table 5, and optionally, the wild-type paramyxovirus fusogen has a sequence of amino acids set forth in any of SEQ ID NOs: 1-132.

[0174] 167. The retroviral vector of embodiment 166, wherein the paramyxovirus is a Nipah virus, such as a Henipa virus.

[0175] 168. The retroviral vector of any of the above embodiments, wherein the positive target cell-specific regulatory element comprises a tissue-specific promoter, a tissue-specific enhancer, a tissue-specific splice site, a tissue-specific site that extends the half-life of RNA or protein, a tissue-specific mRNA export-promoting site, a tissue-specific translation-enhancing site, or a tissue-specific post-translational modification site.

[0176] 169. The retroviral vector of any of the above embodiments, wherein the negative target cell-specific regulatory element comprises a tissue-specific miRNA recognition sequence, a tissue-specific protease recognition site, a tissue-specific ubiquitin ligase site, a tissue-specific transcriptional repression site, or a tissue-specific epigenetic repression site.

[0177] 170. The retroviral vector of any of the above embodiments, wherein the negative target cell-specific regulatory element comprises a tissue-specific miRNA recognition sequence.

[0178] 171. The retroviral vector of embodiment 170, wherein the negative target cell-specific regulatory element is positioned or encoded within the transcribed region (e.g., the transcribed region encoding the exogenous agent), e.g., whereby the RNA produced by the transcribed region contains an miRNA recognition sequence within the UTR or coding region.

[0179] 172. The retroviral vector of any of the above embodiments, wherein the target cells are cancer cells and the non-target cells are non-cancerous cells.

[0180] 173. The retroviral vector of any of the above embodiments, wherein the retroviral nucleic acid encodes a positive TCSSE and / or a negative TCSSE.

[0181] 174. The retroviral vector of any of the embodiments, wherein the retroviral nucleic acid comprises the complement of the positive TCSSRE and / or the negative TCSSRE.

[0182] 175. The retroviral vector of any of the above embodiments, wherein the retroviral vector does not deliver nucleic acid to a non-target cell, such as an antigen-presenting cell, an MHC class II+ cell, a professional antigen-presenting cell, a heterotypic antigen-presenting cell, a macrophage, a dendritic cell, a myeloid dendritic cell, a plasmacytoid dendritic cell, a CD11e+ cell, a CD11b+ cell, a splenocyte, a B cell, a hepatocyte, an endothelial cell, or a non-cancerous cell.

[0183] 176. The retroviral vector of any of the above embodiments, wherein less than 10%, 5%, 2.5%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, or 0.000001% of a non-target cell type (e.g., antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, heterotypic antigen-presenting cells, macrophages, dendritic cells, myeloid dendritic cells, plasmacytoid dendritic cells, CD11le+ cells, CD11b+ cells, splenocytes, B cells, hepatocytes, endothelial cells, or non-cancerous cells) comprise retroviral nucleic acid using quantitative PCR, e.g., using the assay of Example 1.

[0184] 177. The retroviral vector of any of the above embodiments, wherein the target cell comprises 0.00001-10, 0.0001-10, 0.001-10, 0.01-10, 0.1-10, 0.5-5, 1-4, 1-3, or 1-2 copies of retroviral nucleic acid or a portion thereof per host cell genome, e.g., the copy number of the retroviral nucleic acid is assessed in vivo after administration.

[0185] 178. The retroviral vector of any of the above embodiments, wherein less than 10%, 5%, 2.5%, 1%, 0.5%, 0.1%, 0.01% of the non-target cells (e.g., antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, heterotypic antigen-presenting cells, macrophages, dendritic cells, myeloid dendritic cells, plasmacytoid dendritic cells, CD11c+ cells, CD11b+ cells, splenocytes, B cells, hepatocytes, endothelial cells, or non-cancerous cells) comprise the exogenous agent, or the exogenous agent (e.g., protein) is not detectably present in the non-target cells, e.g., antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, heterotypic 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.

[0186] 179. The retroviral vector of any of the above embodiments, wherein the retroviral vector delivers the retroviral nucleic acid to a target cell, e.g., a T cell, a CD3+ T cell, a CD4+ T cell, a CD5+ T cell, a hepatocyte, a hematopoietic stem cell, a CD34+ hematopoietic stem cell, a CD105+ hematopoietic stem cell, a CD117+ hematopoietic stem cell, a CD105+ endothelial cell, a B cell, a CD20+ B cell, a CD19+ B cell, a cancer cell, a CD133+ cancer cell, an EpCAM+ cancer cell, a CD19+ cancellous cell, a Her2 / Neu+ cancer cell, a GluA2+ neuron, a GluA4+ neuron, an NKG2D+ natural killer cell, an SLCIA3+ astrocyte, an SLC7A10+ adipocyte, or a CD30+ lung epithelial cell.

[0187] 180. The retrovirus of any of the above embodiments, wherein the retrovirus is capable of targeting at least 0.00001%, 20%, or more of target cells (e.g., one or more of T cells, CD3+ T cells, CD4+ T cells, CD5+ T cells, hepatocytes, hematopoietic stem cells, CD34+ hematopoietic stem cells, CD105+ hematopoietic stem cells, CD117+ hematopoietic stem cells, CD105+ endothelial cells, B cells, CD20+ B cells, CD19+ B cells, cancer cells, CD133+ cancer cells, EpCAM+ cancer cells, CD19+ cancer cells, Her2 / Neu+ cancer cells, GluA2+ neurons, GluA4+ neurons, NKG2D+ natural killer cells, SLCIA3+ astrocytes, SLC7A10+ adipocytes, or CD30+ lung epithelial cells). 0.0001%, 0.001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the retroviral vectors contain retroviral nucleic acid, e.g., using quantitative PCR, e.g., using the assay of Example 3.

[0188] 181. The retrovirus of any of the above embodiments, wherein the retrovirus is capable of targeting a target cell (e.g., a T cell, a CD3+ T cell, a CD4+ T cell, a CD5+ T cell, a hepatocyte, a hematopoietic stem cell, a CD34+ hematopoietic stem cell, a CD105+ hematopoietic stem cell, a CD117+ hematopoietic stem cell, a CD105+ endothelial cell, a B cell, a CD20+ B cell, a CD19+ B cell, a cancer cell, a CD133+ cancer cell, an EpCAM+ cancer cell, a CD19+ cancer cell, a Her2 / Neu+ cancer cell, a G1 At least 0.00001%, 0.0001%, 0.001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the following cells (uA2+ neurons, GluA4+ neurons, NKG2D+ natural killer cells, SLCIA3+ astrocytes, SLC7AlO+ adipocytes, or CD30+ lung epithelial cells) contain the exogenous agent, a retroviral vector.

[0189] 182. The retrovirus of any of the above embodiments, wherein upon administration, the ratio of target cells containing retroviral nucleic acid to non-target cells containing retroviral nucleic acid is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by a quantitative PCR assay, e.g., using the assays of Examples 1 and 3.

[0190] 183. The retroviral vector of any of the above embodiments, wherein the ratio of the average copy number of the retroviral nucleic acid, or a portion thereof, in target cells to the average copy number of the retroviral nucleic acid, or a portion thereof, in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by a quantitative PCR assay, e.g., using the assays of Examples 1 and 3.

[0191] 184. The retroviral vector of any of the above embodiments, wherein the ratio of the median copy number of the retroviral nucleic acid, or a portion thereof, in target cells to the median copy number of the retroviral nucleic acid, or a portion thereof, in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by a quantitative PCR assay, e.g., using the assays of Examples 1 and 3.

[0192] 185. The retroviral vector of any of the above embodiments, wherein the ratio of target cells containing the exogenous RNA agent to non-target cells containing the exogenous RNA agent is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by reverse transcription quantitative PCR assay.

[0193] 186. The retroviral vector of any of the above embodiments, wherein the ratio of the average level of the exogenous RNA agent in target cells to the average level of the exogenous RNA agent in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by reverse transcription quantitative PCR assay.

[0194] 187. The retroviral vector of any of the above embodiments, wherein the ratio of median exogenous RNA agent level in target cells to median 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, by reverse transcription quantitative PCR assay.

[0195] 188. The retroviral vector of any of the above embodiments, wherein the ratio of target cells containing the exogenous protein agent to non-target cells containing the exogenous protein agent is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by FACS assay, e.g., using the assay of Example 2 and / or Example 4.

[0196] 189. The retroviral vector of any of the above embodiments, wherein the ratio of the average level of the exogenous protein agent in target cells to the average level of the exogenous protein agent in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by FACS assay, e.g., using the assay of Example 2 and / or Example 4.

[0197] 190. The retroviral vector of any of the above embodiments, wherein the ratio of median exogenous protein agent level in target cells to median exogenous protein agent level in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by FACS assay, e.g., using the assay of Example 2 and / or Example 4.

[0198] 191. The retroviral vector of any of the above embodiments, i) exogenous or overexpressed immunosuppressive proteins in the envelope, and ii) a retroviral vector comprising one or both of the following immunostimulatory proteins that are absent or present at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) compared to a retroviral vector produced from an otherwise similar source cell that is unmodified.

[0199] 192. The retroviral vector of any of the above embodiments, i) a first exogenous or overexpressed immunosuppressive protein in the envelope, and a second exogenous or overexpressed immunosuppressive protein in the envelope; ii) a first exogenous or overexpressed immunosuppressive protein in the envelope and a second immunostimulatory protein present at a reduced level (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) relative to the absence or relative to a retrovirus-like particle or retroviral vector produced from an otherwise similar source cell, or iii) a retroviral vector comprising one or more of: 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 a retrovirus-like particle or retroviral vector produced from an otherwise unmodified similar source cell; 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 a retrovirus-like particle or retroviral vector produced from an otherwise unmodified similar source cell.

[0200] 193. The retroviral vector of any of the above embodiments, wherein the retroviral nucleic acid comprises one or more insulator elements.

[0201] 194. A retrovirus-like particle or retroviral vector (e.g., a particle or vector suitable for in vivo use in a human subject), comprising: a) an envelope comprising a fusogen (e.g., a retargeted fusogen); b) an exogenous agent (e.g., an exogenous polypeptide or exogenous RNA) or a nucleic acid encoding an exogenous agent (e.g., a retroviral nucleic acid); c) i) a first exogenous or overexpressed immunosuppressive protein in the envelope, and a second exogenous or overexpressed immunosuppressive protein in the envelope; ii) a first exogenous or overexpressed immunosuppressive protein in the envelope and a second immunostimulatory protein present at a reduced level (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) relative to the absence or relative to a retrovirus-like particle or retroviral vector produced from an otherwise similar source cell, or iii) one or more of: 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) relative to a retrovirus-like particle or retroviral vector produced from an otherwise similar source cell, and a second immunostimulatory protein that is absent or present at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) relative to a retrovirus-like particle or retroviral vector produced from an otherwise similar source cell, and when administered to a subject (e.g., a human subject or a mouse), i) the particle or vector does not generate a detectable antibody response (e.g., after a single dose or multiple doses), e.g., by a FACS antibody detection assay, e.g., the assay of Example 13 or Example 14, or antibodies to the particle or vector are present at levels less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels; ii) the particle or vector does not produce a detectable cellular immune response (e.g., a T cell response, an NK cell response, or a macrophage response), or there is a cellular immune response to the particle or vector at a level less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels, e.g., by a PBMC lysis assay (e.g., the assay of Example 5), by a NK cell lysis assay (e.g., the assay of Example 6), by a CDS killer T cell lysis assay (e.g., the assay of Example 7), or by a macrophage phagocytosis assay (e.g., the assay of Example 8); iii) the particle or vector does not elicit a detectable innate immune response, e.g., complement activation (e.g., after a single dose or multiple doses), e.g., by a complement activity assay (e.g., the assay of Example 9), or the innate immune response to the particle or vector is present at a level less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels; iv) less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.002%, or 0.001% of the virus is inactivated by serum, e.g., by a serum inactivation assay, e.g., by the assay of Example 11 or Example 12; v) target cells that receive an exogenous agent from a particle or vector do not produce a detectable antibody response (e.g., after a single dose or multiple doses), e.g., by a FACS antibody detection assay, e.g., the assay of Example 15, or antibodies to the target cells are present at levels less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels; or vi) A retrovirus-like particle or retroviral vector in which target cells that receive an exogenous agent from the particle or vector do not produce a detectable cellular immune response (e.g., a T cell response, an NK cell response, or a macrophage response), or there is a cellular response against the target cells at a level that is less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels, e.g., by 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 CDS killer T cell lysis assay (e.g., the assay of Example 19).

[0202] 195. The retrovirus-like particle or retroviral vector of embodiment 194, wherein the background level is the corresponding level in the same subject before administration of the particle or vector.

[0203] 196. The retrovirus-like particle or retroviral vector of embodiment 194 or 195, wherein the immunosuppressive protein is a complement regulatory protein or CD47.

[0204] 197. The retrovirus-like particle or retroviral vector of any of embodiments 194-196, wherein the immunostimulatory protein is an MHC (e.g., HLA) protein.

[0205] 198. The retrovirus-like particle or retroviral vector of any of embodiments 194 to 197, wherein one or both of the first exogenous or overexpressed immunosuppressive proteins is other than CD47 and the second immunostimulatory protein is other than MHC.

[0206] 199. A retrovirus-like particle or retroviral vector (e.g., a particle or vector suitable for in vivo use in a human subject), comprising: a) an envelope containing a fusogen; b) a retroviral nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or exogenous RNA); and c) A retrovirus-like particle or retroviral vector comprising an exogenous or overexpressed MHC, such as HLA (e.g., HLA-G or HLA-E), or a combination thereof, in the envelope.

[0207] 200. A pseudotyped retrovirus-like particle or retroviral vector (e.g., a particle or vector suitable for in vivo use in a human subject), comprising: a) a pseudotyped envelope comprising a fusogen, wherein the fusogen comprises a domain of at least 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen, e.g., a sequence in Table 4 or Table 5, and optionally, the wild-type paramyxovirus fusogen has a sequence of amino acids set forth in any one of SEQ ID NOs: 1-132; b) a retroviral nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or exogenous RNA); and c) A pseudotyped retrovirus-like particle or retroviral vector comprising exogenous or overexpressed CD47 or complement regulatory protein, or a combination thereof, in the envelope.

[0208] 201. A pseudotyped retrovirus-like particle or retroviral vector (e.g., a particle or vector suitable for in vivo use in a human subject), comprising: a) a pseudotyped envelope comprising a fusogen, wherein the fusogen comprises a domain of at least 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen, e.g., a sequence in Table 4 or Table 5, and optionally, the wild-type paramyxovirus fusogen has a sequence of amino acids set forth in any one of SEQ ID NOs: 1-132; b) a retroviral nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or exogenous RNA); and c) A pseudotyped retrovirus-like particle or retrovirus comprising an MHC I (e.g., HLA-A, HLA-B, or HLA-C) or MHC II (e.g., HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR) 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 a retrovirus-like particle or retrovirus produced from an otherwise unmodified identical source cell.

[0209] 202. A pseudotyped retrovirus-like particle or retroviral vector (e.g., a particle or vector suitable for in vivo use in a human subject), comprising: a) a pseudotyped envelope comprising a fusogen, wherein the fusogen comprises a domain of at least 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen, e.g., a sequence in Table 4 or Table 5, and optionally, the wild-type paramyxovirus fusogen has a sequence of amino acids set forth in any one of SEQ ID NOs: 1-132; b) a retroviral nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or exogenous RNA); and c) a pseudo-retrovirus-like particle or retroviral vector comprising one or both of an exogenous or overexpressed immunosuppressive or immunostimulatory protein that is absent or at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) compared to a retrovirus-like particle or retroviral vector produced from an otherwise unmodified similar source cell.

[0210] 203. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the retrovirus-like particle or retroviral vector circulates for at least 0.5, 1, 2, 3, 4, 6, 12, 18, 24, 36, or 48 hours after administration to a patient.

[0211] 204. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the retrovirus is in circulation 30 minutes after administration.

[0212] 205. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the retrovirus is in circulation 1 hour after administration.

[0213] 206. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the retrovirus is in circulation 2 hours after administration.

[0214] 207. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the retrovirus is in circulation 4 hours after administration.

[0215] 208. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the retrovirus is in circulation 8 hours after administration.

[0216] 209. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the retrovirus is in circulation 12 hours after administration.

[0217] 210. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the retrovirus is in circulation 18 hours after administration.

[0218] 211. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the retrovirus is in circulation 24 hours after administration.

[0219] 212. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the retrovirus is in circulation 36 hours after administration.

[0220] 213. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the retrovirus is in circulation 48 hours after administration.

[0221] 214. The retrovirus-like particle or retroviral vector of any of the above embodiments, which has reduced immunogenicity compared to a control retrovirus, e.g., a retrovirus that is similar to the retrovirus but unmodified, as measured by a reduction in the humoral response following one or more administrations of the retrovirus to a suitable animal model, e.g., an animal model described herein.

[0222] 215. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the reduction in humoral response is measured in a serum sample by anti-cellular antibody titer, e.g., anti-retroviral antibody titer, e.g., ELISA.

[0223] 216. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein a serum sample from an animal administered the retroviral composition has a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in anti-retroviral antibody titers compared to a serum sample from a subject administered the unmodified cells.

[0224] 217. The retroviral-like particle or retroviral vector of any of the above embodiments, wherein a serum sample from a subject to which the retroviral composition has been administered has an increased anti-cellular antibody titer, e.g., a 1%, 2%, 5%, 10%, 20%, 30%, or 40% increase, from baseline, e.g., a baseline referenced to a serum sample from the same subject prior to administration of the retroviral composition.

[0225] 218. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the subject to be administered the retrovirus or pharmaceutical composition has, or is known to have, or is tested to have, pre-existing antibodies (e.g., IgG or IgM) reactive with the retrovirus, the subject to be administered the retroviral composition does not have detectable levels of pre-existing antibodies reactive with the retrovirus, the subject receiving the retrovirus or pharmaceutical composition has, or is known to have, or is tested to have, antibodies (e.g., IgG or IgM) reactive with the retrovirus, the subject receiving the retrovirus or pharmaceutical composition (e.g., at least 1, 2, 3, 4, 5 or more times) does not have detectable levels of antibodies reactive with the retrovirus, or the level of antibodies does not increase by more than 1%, 2%, 5%, 10%, 20%, or 50% between two time points, a first time point that is before the first administration of the retrovirus, and a second time point that is after one or more administrations of the retrovirus.

[0226] 219. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the retroviral vector is produced by the method of Example 5, 6, or 7, e.g., from cells transfected with HLA-G or HLA-E cDNA.

[0227] 220. The retroviral-like particle or retroviral vector of any of the above embodiments, wherein the retroviral vector produced from NMC-HLA-G cells exhibits a reduced percent of lysis, e.g., PBMC-mediated lysis, NK cell-mediated lysis, and / or CDS+ T cell-mediated lysis, at a particular time point compared to retroviral vector produced from NMC or NMC empty vector.

[0228] 221. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the modified retroviral vector avoids phagocytosis by macrophages.

[0229] 222. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the retroviral vector is produced by the method of Example 8, e.g., from cells transfected with CD47 cDNA.

[0230] 223. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein when macrophages are incubated with a retroviral vector derived from NMC-CD47, the phagocytic index is reduced compared to that derived from NMC or an NMC empty vector.

[0231] 224. The retrovirus-like particle or retroviral vector of any of the above embodiments, which has a reduction in macrophage phagocytosis, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in macrophage phagocytosis, compared to a control retrovirus, e.g., a retrovirus that is unmodified but similar to the retrovirus, wherein the reduction in macrophage phagocytosis is determined by assaying the phagocytic index in vitro, e.g., as described in Example 8.

[0232] 225. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the retroviral composition, when incubated with macrophages in an in vitro assay of macrophage phagocytosis, has a phagocytic index of 0, 1, 10, 100 or greater when measured, e.g., by the assay of Example 8.

[0233] 226. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the retrovirus-like particle or retroviral vector has been modified to have reduced complement activity compared to the unmodified retroviral vector.

[0234] 227. The retrovirus-like particle or retroviral vector of any of the above embodiments, produced by the method of Example 9, e.g., from a cell transfected with a cDNA encoding a complement regulatory protein, e.g., DAF.

[0235] 228. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the dose of retroviral vector is greater when the modified retroviral vector (e.g., HEK293-DAF) is incubated with corresponding mouse serum (e.g., HEK-293DAF mouse serum) than when the control retroviral vector (e.g., HEK293 retroviral vector) is incubated with corresponding mouse serum (e.g., HEK293 mouse serum) in the presence of 200 pg / ml C3a.

[0236] 229. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the dose of retroviral vector is greater when the modified retroviral vector (e.g., HEK293-DAF) is incubated with naive mouse serum than when a control retroviral vector (e.g., HEK293 retroviral vector) is incubated with naive mouse serum in the presence of 200 pg / ml C3a.

[0237] 230. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the retroviral composition is resistant to complement-mediated inactivation in patient serum 30 minutes after administration by the assay of Example 9.

[0238] 231. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the retrovirus is resistant to complement-mediated inactivation.

[0239] 232. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the complement regulatory protein comprises one or more of a protein that binds decay accelerating factor (DAF, CD55), e.g. factor H (FH)-like protein-I (FHL-I), e.g. C4b-binding protein (C4BP), e.g. complement receptor I (CD35), e.g. membrane cofactor protein (MCP, CD46), e.g. protectin (CD59), e.g. a protein that inhibits classical and alternative complement pathway CD / C5 convertases, e.g. a protein that regulates MAC assembly.

[0240] 233. The retrovirus-like particle or retroviral vector of any of the above embodiments, produced by the method of Example 10, e.g., from cells transfected with DNA encoding an shRNA that targets MHC class I, e.g., wherein the NMC-shMHC class I-derived retroviral vector has reduced expression of MHC class I compared to NMC and an NMC vector control.

[0241] 234. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the measure of immunogenicity in the retroviral vector is serum inactivation, e.g., serum inactivation measured as described herein, e.g., as described in Example 11.

[0242] 235. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the percentage of cells that receive the exogenous agent does not differ between retrovirus samples that have been incubated with serum from retroviral vector-naive mice and heat-inactivated serum.

[0243] 236. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the percentage of cells that receive the exogenous agent does not differ between retroviral vector samples that have been incubated with serum from retroviral vector-naive mice and serum-free control incubations.

[0244] 237. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the percentage of cells that receive the exogenous agent is lower in the retroviral vector sample incubated with positive control serum than in the retroviral vector sample incubated with serum from retroviral vector-naive mice.

[0245] 238. The retrovirus-like particle or retroviral vector of any of the above embodiments, e.g., a modified retroviral vector modified by a method described herein, has reduced serum inactivation (reduced compared to administration of an unmodified retroviral vector) after multiple (e.g., more than one, e.g., two or more) administrations of the modified retroviral vector.

[0246] 239. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the retroviral vector described herein is not inactivated by serum after multiple administrations.

[0247] 240. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the measure of immunogenicity in the retroviral vector is serum inactivation, e.g., serum inactivation after multiple doses, e.g., measured as described herein, e.g., as described in Example 12, after multiple doses.

[0248] 241. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the percentage of cells that receive the exogenous agent does not differ between retroviral vector samples that have been incubated with serum from mice treated with the modified (e.g., HEK293-HLA-G) retroviral vector and heat-inactivated serum.

[0249] 242. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the percentage of cells that receive the exogenous agent does not differ between retroviral vector samples that have been incubated from mice treated 1, 2, 3, 5, or 10 times with the modified (e.g., HEK293-HLA-G) retroviral vector.

[0250] 243. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the percentage of cells that receive the exogenous agent does not differ between retroviral vector samples that have been incubated with serum from vehicle-treated mice and mice treated with a modified (e.g., HEK293-HLA-G) retroviral vector.

[0251] 244. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the percentage of cells that receive the exogenous agent is lower in the retroviral vector derived from control cells (e.g., HEK293) than in the modified (e.g., HEK293-HLA-G) retroviral vector.

[0252] 245. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the measure of immunogenicity in the retroviral vector is an antibody response.

[0253] 246. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein a subject receiving the retroviral vector described herein has pre-existing antibodies that bind to and recognize the retroviral vector, e.g., as described herein, e.g., as measured as described in Example 13.

[0254] 247. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein serum from retroviral vector-naive mice exhibits a greater signal (e.g., fluorescence) than a negative control, e.g., serum from mice depleted of IgM and IgG, indicating that immunogenicity has occurred.

[0255] 248. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein serum from retroviral vector-naive mice exhibits a similar signal (e.g., fluorescence) compared to a negative control, indicating, for example, that no detectable immunogenicity occurred.

[0256] 249. The retrovirus-like particle or retroviral vector of any of the above embodiments, which has been modified, e.g., modified by a method described herein, and which has a reduced humoral response (e.g., reduced compared to administration of an unmodified retrovirus) after multiple (e.g., more than one, e.g., two or more) administrations of the modified retroviral vector, e.g., measured as described herein, e.g., as described in Example 14.

[0257] 250. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the retroviral vector is produced by the method of Example 5, 6, 7, or 14, e.g., from cells transfected with HLA-G or HLA-E cDNA.

[0258] 251. The retroviral-like particle or retroviral vector of any of the above embodiments, wherein the humoral response is assessed by determining a value for the level of anti-retroviral vector antibodies (e.g., IgM, IgG1, and / or IgG2 antibodies).

[0259] 252. The retroviral-like particle or retroviral vector of any of the above embodiments, wherein the modified (e.g., NMC-HLA-G) retroviral vector reduces anti-viral IgM or IgG1 / 2 antibody titers (e.g., as measured by FACS fluorescence intensity) after infusion compared to a control, e.g., an NMC retroviral vector or an NMC empty retroviral vector.

[0260] 253. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the recipient cells are not targeted by an antibody response, or the antibody response is below a control level, e.g., measured as described herein, e.g., as described in Example 15.

[0261] 254. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the signal (e.g., mean fluorescence intensity) is similar for recipient cells derived from mice treated with the retroviral vector and mice treated with PBS.

[0262] 255. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the measure of recipient cell immunogenicity is a macrophage response.

[0263] 256. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the recipient cells are not targeted by macrophages, or are targeted below control levels.

[0264] 257. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the phagocytic index is similar for recipient cells derived from retroviral vector-treated mice and PBS-treated mice, e.g., when measured as described herein, e.g., as described in Example 16.

[0265] 258. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the measure of recipient cell immunogenicity is a PBMC response.

[0266] 259. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the recipient cells do not elicit a PBMC response.

[0267] 260. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the percentage of CD3+ / CMG+ cells is similar in recipient cells derived from retroviral vector-treated mice and PBS-treated mice, e.g., when measured as described herein, e.g., as described in Example 17.

[0268] 261. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the measure of immunogenicity in recipient cells is a natural killer cell response.

[0269] 262. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the recipient cells do not induce a natural killer cell response, or induce a low natural killer cell response, e.g., below a control value.

[0270] 263. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the percentage of CD3+ / CMG+ cells is similar in recipient cells derived from retroviral vector-treated mice and PBS-treated mice, e.g., when measured as described herein, e.g., as described in Example 18.

[0271] 264. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the measure of immunogenicity in recipient cells is a CDS+ T cell response.

[0272] 265. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the recipient cells do not induce a CDS+ T cell response, or induce a low CDS+ T cell response, e.g., below control values.

[0273] 266. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the percentage of CD3+ / CMG+ cells is similar in recipient cells derived from retroviral vector-treated mice and PBS-treated mice, e.g., when measured as described herein, e.g., as described in Example 19.

[0274] 267. The retrovirus-like particle or retroviral vector of any of the above embodiments, wherein the fusogen is a retargeted fusogen.

[0275] 268. The retrovirus-like particle or retroviral vector of any of the above embodiments, comprising retroviral nucleic acid encoding one or both of: (i) a positive target cell-specific regulatory element operably linked to a nucleic acid encoding an exogenous agent, or (ii) a negative target cell-specific regulatory element operably linked to a nucleic acid encoding an exogenous agent.

[0276] 269. A pseudotyped retrovirus-like particle or retroviral vector (e.g., a particle or vector suitable for in vivo use in a human subject), comprising: a) a pseudotyped envelope comprising a fusogen, wherein the fusogen comprises a domain of at least 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen, e.g., a sequence in Table 4 or Table 5, and optionally, the wild-type paramyxovirus fusogen has a sequence of amino acids set forth in any one of SEQ ID NOs: 1-132; b) A pseudotyped retrovirus-like particle or retroviral vector comprising a retroviral nucleic acid encoding an exogenous agent (e.g., an exogenous polypeptide or an exogenous RNA), wherein the retroviral nucleic acid comprises one or more insulator elements.

[0277] 270. The pseudotyped retrovirus-like particle or retroviral vector of embodiment 269, wherein the retroviral nucleic acid comprises two insulator elements, e.g., a first insulator element upstream of the region encoding the exogenous agent, and a second insulator element downstream of the region encoding the exogenous agent, e.g., the first insulator element and the second insulator element comprise the same or different sequences.

[0278] 271. The pseudotyped retrovirus-like particle or retroviral vector of embodiment 269 or 270, wherein the variation in median exogenous agent level in samples of cells isolated after administration of the particle or vector to a subject at a first time point is at least about 10,000%, 5,000%, 2,000%, 1,000%, 500%, 200%, 100%, 50%, 20%, 10%, or 5%, or less, of the median exogenous agent level in samples of cells isolated after administration of the particle or vector to a subject at a second, later time point.

[0279] 272. The pseudotyped retrovirus-like particle or retroviral vector of embodiment 271, wherein the median expression level per cell is assessed only in cells having a retroviral genome copy number of at least 1.0.

[0280] 273. The pseudotyped retrovirus-like particle or retroviral vector of any of embodiments 269-272, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of target cells in a subject detectably contain the exogenous agent.

[0281] 274. The pseudotyped retrovirus-like particle or retroviral vector of any of embodiments 271-273, wherein the median payload gene expression level is assessed across cells isolated from the subject at 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, and 1095 days after administration of the retroviral composition to the subject.

[0282] 275. The pseudotyped retrovirus-like particle or retroviral vector of any of embodiments 269-274, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of target cells in a subject that detectably contain the exogenous agent at a first time point still detectably contain the exogenous agent at a later, second time point, wherein the first time point is 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days after administration of the retroviral composition to the subject.

[0283] 276. The pseudotyped retrovirus-like particle or retroviral vector of embodiment 275, wherein the second time point is 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days after the first time point.

[0284] 277. The pseudotyped retrovirus-like particle or retroviral vector of any of embodiments 269 to 276, which is not genotoxic or does not increase the rate of tumor formation in target cells compared to target cells not treated with the retrovirus-like particle or retroviral vector.

[0285] 278. The pseudotyped retrovirus-like particle or retroviral vector of any of embodiments 269 to 277, wherein the median exogenous drug level is assessed in a population of cells from a subject that received the retroviral vector or pharmaceutical composition.

[0286] 279. The pseudotyped retrovirus-like particle or retroviral vector of any of embodiments 269-278, wherein the median exogenous agent level assessed in a population of cells collected (e.g., isolated) from the subject on different days after administration differs from the median exogenous agent level in a population of cells assessed at 7 days, 14 days, 28 days, or 56 days by less than about 10,000%, 1000%, 100%, or 10%, e.g., 10,000% to 1000%, 1000% to 100%, or 100% to 10%, and wherein the cells within the population have a vector copy number of at least 1.0.

[0287] 280. The pseudotyped retrovirus-like particle or retroviral vector of any of embodiments 269-279, wherein exogenous agent levels are assessed across cells from a subject that has received the retroviral vector or pharmaceutical composition.

[0288] 281. The pseudotyped retrovirus-like particle or retroviral vector of any of embodiments 269-280, wherein the percentage of cells containing the exogenous agent is assessed in a plurality of cells collected (e.g., isolated) from the subject at 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, and 1095 days after administration of the retroviral vector or pharmaceutical composition.

[0289] 282. The pseudotyped retrovirus-like particle or retroviral vector of any of embodiments 269-281, wherein the difference in the percentage of cells containing the exogenous agent assessed in cells isolated on two different days after administration is less than 1%, 5%, 10%, 20%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 1500%, or 2000%.

[0290] 283. The pseudotyped retrovirus-like particle or retroviral vector of any of embodiments 269-282, wherein the percentage of target cells positive for the exogenous agent is similar across cells collected at 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days.

[0291] 284. The pseudotyped retrovirus-like particle or retroviral vector of any of embodiments 269-283, wherein at least as many target cells as at 7 days are positive for the exogenous agent at 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days, at least as many target cells as at 14 days are positive for the exogenous agent at 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days, and at least as many target cells as at 28 days are positive for the exogenous agent at 56 days, 112 days, 365 days, 730 days, or 1095 days. a pseudotyped retrovirus-like particle or retroviral vector, wherein at least as many target cells as on day 56 are positive for the exogenous agent at 112 days, 365 days, 730 days, or 1095 days, a number of target cells as on day 112 are positive for the exogenous agent at 365 days, 730 days, or 1095 days, a number of target cells as on day 365 are positive for the exogenous agent at 730 days or 1095 days, or a number of target cells as on day 730 are positive for the exogenous agent at 1095 days.

[0292] 285. The pseudotyped retrovirus-like particle or retroviral vector of any of embodiments 269-284, wherein the median exogenous agent level in target cells containing the exogenous agent is similar in cells collected at 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days, and the median exogenous agent level in target cells containing the exogenous agent at 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days is at least as high as at day 7, and the median exogenous agent level in target cells containing the exogenous agent at 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days is at least as high as at day 14 ...56 days, 112 days, 365 days, 730 days, or 1095 days is at least as high as at day 14. a pseudotyped retrovirus-like particle or retroviral vector in which the median exogenous agent level in target cells containing the exogenous agent at 112, 365, 730, or 1095 days is at least as high as 28 days, a median exogenous agent level in target cells containing the exogenous agent at 112, 365, 730, or 1095 days is at least as high as 56 days, a median exogenous agent level in target cells containing the exogenous agent at 365, 730, or 1095 days is at least as high as 112 days, a median exogenous agent level in target cells containing the exogenous agent at 730 or 1095 days is at least as high as 365 days, or a median exogenous agent level in target cells containing the exogenous agent at 1095 days is at least as high as 730 days.

[0293] 286. A method of delivering an exogenous agent to a subject (e.g., a human subject), comprising administering to the subject a retrovirus-like particle (e.g., a pseudotyped retrovirus-like particle) or retroviral vector (e.g., a pseudotyped retroviral vector) of any of the above embodiments, thereby delivering the exogenous agent to the subject.

[0294] 287. A method of modulating a function in a subject (e.g., a human subject), target tissue, or target cell, comprising contacting, e.g., administering to the subject, target tissue, or target cell a retrovirus-like particle (e.g., a pseudotyped retrovirus-like particle) or retroviral vector (e.g., a pseudotyped retroviral vector) of any of the above embodiments.

[0295] 288. The method of embodiment 287, wherein the target tissue or target cell is present in a subject.

[0296] 289. A method of treating or preventing a disorder, e.g., cancer, in a subject (e.g., a human subject), comprising administering to the subject a retrovirus-like particle (e.g., a pseudotyped retrovirus-like particle) or retroviral vector (e.g., a pseudotyped retroviral vector) of any of the above embodiments.

[0297] 290. A method of producing a retroviral vector or retrovirus-like particle of any of the above embodiments, comprising: a) providing source cells containing retroviral nucleic acid and a fusogen (e.g., a retargeted fusogen); b) culturing the source cells under conditions that allow for the production of retroviral vectors; c) isolating, enriching, or purifying the retroviral vector from the source cell, thereby producing the retroviral vector.

[0298] 291. A source cell for producing a retroviral vector, a) retroviral nucleic acid; b) viral structural proteins capable of packaging retroviral nucleic acid, wherein at least one structural protein comprises a fusogen that binds to a fusogen receptor; c) Source cells comprising fusogen receptors that are absent or present at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) compared to otherwise similar source cells.

[0299] 292. The source cell of embodiment 291, wherein binding of a fusogen to a fusogen receptor results in fusion of the retroviral vector with the target cell.

[0300] 293. The source cell of embodiment 291 or 292, which binds to a second similar source cell, e.g., the fusogens of the source cell bind to the fusogens on the second source cell.

[0301] 294. A population of source cells of any of embodiments 291-293.

[0302] 295. The population of source cells of embodiment 294, wherein less than 10%, 5%, 4%, 3%, 2%, or 1% of the cells in the population are multinucleated.

[0303] 296. The source cell or population of source cells of any of embodiments 291-295, wherein the source cell has been modified to reduce (e.g., not fuse with) other sources during production of the retrovirus described herein.

[0304] 297. A source cell or population of source cells of any of embodiments 291 to 296, wherein the fusogen (e.g., the retargeted fusogen) does not bind to a protein contained by the source cell, e.g., a protein on the surface of the source cell.

[0305] 298. A source cell or population of source cells of any of embodiments 291-297, wherein the fusogen (e.g., a retargeted fusogen) binds to a protein contained by the source cell but does not fuse with the cell.

[0306] 299. A source cell or population of source cells of any of embodiments 291-298, wherein the fusogen does not induce fusion with the source cells.

[0307] 300. The source cell or population of source cells of any of embodiments 291-299, wherein the source cell does not express a protein, e.g., an antigen, that binds to a fusogen.

[0308] 301. The source cell or population of source cells of any of embodiments 291-300, wherein a plurality of source cells do not form syncytia when expressing a fusogen, or wherein less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the cells in the population are multinucleated (e.g., containing two or more nuclei).

[0309] 302. The source cell or population of source cells of any of embodiments 291-301, wherein a plurality of the source cells do not form fusion cells when producing lentivirus, or less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the cells in the population are multinucleated.

[0310] 303. The source cell or population of source cells of any of embodiments 291-302, wherein less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the nuclei in the population are in syncytia.

[0311] 304. The source cell or population of source cells of any of embodiments 291-303, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of the nuclei in the population are in mononuclear cells.

[0312] 305. A source cell or population of source cells of any of embodiments 291-304, wherein the percentage of multinucleated cells is lower, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower, compared to a population of otherwise similar, unmodified source cells.

[0313] 306. A source cell or population of source cells of any of embodiments 291-305, wherein the percentage of nuclei present in syncytia is lower, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower, compared to a population of otherwise similar source cells that are unmodified.

[0314] 307. A source cell or population of source cells of any of embodiments 291-306, wherein multinucleated cells (e.g., cells with two or more nuclei) are detected using a microscopic assay, e.g., a DNA stain, e.g., by the assay of Example 20.

[0315] 308. The source cell or population of source cells of any of embodiments 291-307, wherein the number of functional viral particles obtained from the modified source cells is at least 10%, 20%, 40%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 5-fold, or 10-fold greater than the number of functional viral particles obtained from otherwise similar, unmodified source cells, e.g., using the assay of Example 20.

[0316] 309. A retroviral vector or retrovirus-like particle that lacks a fusogen receptor or that contains a fusogen receptor present at a reduced level (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) compared to an unmodified retroviral vector or retrovirus-like particle derived from an otherwise similar source cell.

[0317] 310. A method for producing a retroviral vector or retrovirus-like particle, comprising: a) providing source cells containing a fusogen (e.g., a retargeted fusogen), wherein the source cells lack fusogen receptors or contain fusogen receptors present at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) compared to otherwise similar source cells that are unmodified; b) culturing the source cells under conditions that allow for the production of retroviral vectors; c) isolating, enriching, or purifying the retroviral vector from the source cell, thereby producing the retroviral vector.

[0318] 311. The method of embodiment 310, wherein providing the source cells comprises knocking down or knocking out a fusogen receptor in the source cells or their precursors.

[0319] Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0320] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences mentioned herein, for example, in any table herein, are incorporated by reference. Unless otherwise specified, the sequence accession numbers specified herein, including any table herein, refer to database entries as of May 15, 2018. When a gene or protein refers to multiple sequence accession numbers, all sequence variants are encompassed. In addition, the materials, methods, and examples are merely illustrative and are not intended to be limiting. [Mode for Carrying Out the Invention]

[0321] The present disclosure provides, at least in part, fusosome methods and compositions for in vivo delivery. In some embodiments, the fusosomes contain a combination of elements that promote specificity for target cells, such as one or more of a retargeting fusogen, a positive target cell-specific regulatory element, and a non-target cell-specific regulatory element. In some embodiments, the fusosomes contain one or more modifications that reduce an immune response to the fusosomes.

[0322] definition Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0323] As used herein, "detectably present," when used in the context of an exogenous agent being detectably present, means that the exogenous agent itself is detectably present. For example, if the exogenous agent is a protein, the exogenous protein agent can be detectably present regardless of whether the nucleic acid encoding it is detectably present.

[0324] As used herein, "fusosome" refers to an amphiphilic lipid bilayer surrounding a lumen or cavity and a fusogen that interacts with the amphiphilic lipid bilayer. In embodiments, fusosomes contain nucleic acids. In some embodiments, fusosomes are membrane-enclosed preparations. In some embodiments, fusosomes are derived from source cells.

[0325] As used herein, a "fusosome composition" refers to a composition comprising one or more fusosomes.

[0326] As used herein, "fusogen" refers to an agent or molecule that creates an interaction between two membranes surrounding a lumen. In embodiments, a fusogen promotes membrane fusion. In other embodiments, a fusogen creates a connection, e.g., a pore, between two lumens (e.g., the lumen of a retroviral vector and the cytoplasm of a target cell). In some embodiments, a fusogen comprises a complex of two or more proteins, e.g., neither protein has fusogenic activity alone. In some embodiments, a fusogen comprises a targeting domain.

[0327] As used herein, "fusogen receptor" refers to an entity (e.g., a protein) contained by a target cell, and binding of a fusogen on a fusosome (e.g., a retrovirus) to a fusogen receptor on the target cell promotes delivery of a nucleic acid (e.g., a retroviral nucleic acid) (and optionally also an exogenous agent encoded therein) to the target cell.

[0328] As used herein, "insulator element" refers to a nucleotide sequence that blocks an enhancer or prevents heterochromatin spreading. Insulator elements can be wild-type or mutant.

[0329] As used herein, the term "effective amount" refers to that amount of a pharmaceutical composition that is sufficient to significantly and positively alter the symptoms and / or condition being treated (e.g., provide a positive clinical response). The effective amount of an active ingredient for use in a pharmaceutical composition will vary depending on such factors as the particular condition being treated, the severity of the condition, the duration of treatment, the nature of any concurrent therapy, the particular active ingredient(s) used, the particular pharmaceutically acceptable excipient(s) and / or carrier(s) used, and the knowledge and expertise of the attending physician.

[0330] As used herein, an "exogenous agent" in reference to a virus, VLP, or fusosome refers to an agent not contained in or encoded by the corresponding wild-type virus or fusogen produced from a corresponding wild-type source cell. In some embodiments, the exogenous agent is not naturally occurring, such as a protein or nucleic acid having a sequence that is mutated (e.g., by insertion, deletion, or substitution) compared to the naturally occurring protein. In some embodiments, the exogenous agent is not naturally occurring in the source cell. In some embodiments, the exogenous agent is naturally occurring in the source cell but exogenous to the virus. In some embodiments, the exogenous agent is not naturally occurring in the recipient cell. In some embodiments, the exogenous agent is naturally present in the recipient cell but is not present at a desired level or for a desired period of time. In some embodiments, the exogenous agent comprises RNA or a protein.

[0331] As used herein, the term "pharmaceutically acceptable" refers to excipients, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0332] As used herein, a "positive target cell-specific regulatory element" (or positive TCSSE) refers to a nucleic acid sequence that increases the level of an exogenous agent in a target cell compared to a non-target cell, wherein the nucleic acid encoding the exogenous agent is operably linked to the positive TCSSE. In some embodiments, the positive TCSSE is a functional nucleic acid sequence, e.g., the positive TCSSE can comprise a promoter or enhancer. In some embodiments, the positive TCSSE encodes a functional RNA sequence, e.g., the positive TCSSE can encode a splice site that promotes correct splicing of RNA in a target cell. In some embodiments, the positive TCSSE encodes a functional protein sequence, or the positive TCSSE can encode a protein sequence that promotes correct post-translational modification of a protein. In some embodiments, the positive TCSSE reduces the level or activity of a downregulator or inhibitor of the exogenous agent.

[0333] As used herein, a "negative target cell-specific regulatory element" (or negative TCSSE) refers to a nucleic acid sequence that reduces the level of an exogenous agent in non-target cells compared to target cells, wherein the nucleic acid encoding the exogenous agent is operably linked to the negative TCSSE. In some embodiments, the negative TCSSE is a functional nucleic acid sequence, e.g., an miRNA recognition site, that results in degradation or inhibition of a retroviral nucleic acid in non-target cells. In some embodiments, the nucleic acid sequence encodes a functional RNA sequence, e.g., the nucleic acid encodes an miRNA sequence present in an mRNA encoding an exogenous protein agent, thereby causing the mRNA to be degraded or inhibited in non-target cells. In some embodiments, the negative TCSSE increases the level or activity of a down-regulator or inhibitor of the exogenous agent.

[0334] As used herein, a "non-target cell-specific regulatory element" (or NTCSRE) refers to a nucleic acid sequence that reduces the level of an exogenous agent in non-target cells compared to target cells, wherein the nucleic acid encoding the exogenous agent is operably linked to the NTCSRE. In some embodiments, the NTCSRE is a functional nucleic acid sequence, e.g., an miRNA recognition site, that results in the degradation or inhibition of a retroviral nucleic acid in non-target cells. In some embodiments, the nucleic acid sequence encodes a functional RNA sequence, e.g., the nucleic acid encodes an miRNA sequence present in an mRNA encoding an exogenous protein agent, thereby causing the mRNA to be degraded or inhibited in non-target cells. In some embodiments, the NTCSRE increases the level or activity of a down-regulator or inhibitor of the exogenous agent. The terms "negative TCSRE" and "NTCSRE" are used interchangeably herein.

[0335] As used herein, a "retargeted fusogen" refers to a fusogen that includes a targeting moiety with a sequence that is not part of the naturally occurring form of the fusogen. In embodiments, the fusogen includes a different targeting moiety compared to the targeting moiety in the naturally occurring form of the fusogen. In embodiments, the naturally occurring form of the fusogen lacks a targeting domain, and the retargeted fusogen includes a targeting moiety that is not present in the naturally occurring form of the fusogen. In embodiments, the fusogen is modified to include a targeting moiety. In embodiments, the fusogen includes one or more sequence changes outside the targeting moiety compared to the naturally occurring form of the fusogen, e.g., in the transmembrane domain, fusogenically active domain, or cytoplasmic domain.

[0336] As used herein, "retroviral nucleic acid" refers to a nucleic acid that comprises at least the minimum sequence requirements for packaging into a retrovirus or retroviral vector, alone or in combination with a helper cell, helper virus, or helper plasmid. In some embodiments, the retroviral nucleic acid further comprises or encodes an exogenous agent, a positive target cell-specific regulatory element, a non-target cell-specific regulatory element, or a negative TCSSE. In some embodiments, the retroviral nucleic acid comprises one or more (e.g., all) of the following: a 5'LTR (e.g., to facilitate integration), a U3 (e.g., to activate viral genome RNA transcription), an R (e.g., a Tat-binding region), a U5, a 3'LTR (e.g., to facilitate integration), a packaging site (e.g., psi (Ψ)), and an RRE (e.g., to bind Rev and facilitate nuclear export). Retroviral nucleic acid can comprise RNA (e.g., when part of a virion) or DNA (e.g., when introduced into a source cell or after reverse transcription in a recipient cell). In some embodiments, the retroviral nucleic acid is packaged using a helper cell, helper virus, or helper plasmid containing one or more (eg, all) of gag, pol, and env.

[0337] As used herein, "target cell" refers to a cell type to which it is desired that a fusosome (e.g., a lentiviral vector) deliver an exogenous agent. In embodiments, the target cell is a particular tissue type or class of cell, e.g., an immune effector cell, e.g., a T cell. In some embodiments, the target cell is a diseased cell, e.g., a cancer cell. In some embodiments, a fusogen, e.g., a retargeting fusogen (alone or in combination with a positive TCSRE, NTCSRE, negative TCSRE, or any combination thereof), results in preferential delivery of the exogenous agent to the target cell relative to non-target cells.

[0338] As used herein, "non-target cells" refers to cell types to which it is not desired that fusosomes (e.g., lentiviral vectors) deliver exogenous agents. In some embodiments, non-target cells are cells of a particular tissue type or class. In some embodiments, non-target cells are non-diseased cells, e.g., non-cancerous cells. In some embodiments, a fusogen, e.g., a retargeting fusogen (alone or in combination with a positive TCSRE, NTCSRE, negative TCSRE, or any combination thereof), results in reduced delivery of exogenous agents to non-target cells compared to target cells.

[0339] As used herein, the terms "treat," "treating," or "treatment" refer to ameliorating a disease or disorder, e.g., delaying or preventing or reducing the onset of a disease or disorder, e.g., the underlying cause of the disorder or at least one of its clinical symptoms.

[0340] 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 include, for example, extracellular vesicles, microvesicles, nanovesicles, exosomes, apoptotic bodies (derived from apoptotic cells), microparticles (e.g., derived from platelets), ectosomes (e.g., derived from neutrophils and monocytes in serum), prostatosomes (obtained from prostate cancer cells), cardiosomes (obtained from cardiac cells), or any combination thereof. In some embodiments, fusosomes are naturally released from source cells, and in some embodiments, the source cells are treated to enhance fusosome formation. In some embodiments, fusosomes are about 10-10,000 nm in diameter, e.g., about 30-100 nm in diameter. In some embodiments, fusosomes comprise one or more synthetic lipids.

[0341] In some embodiments, the fusosome is or comprises a virus, e.g., a retrovirus, e.g., a lentivirus. In some embodiments, the fusosome comprising a lipid bilayer comprises a retroviral vector comprising an envelope. For example, in some embodiments, the amphipathic lipid fusosome bilayer is or comprises a viral envelope. The viral envelope may comprise a fusogen, e.g., a fusogen endogenous to the virus or a pseudotyped fusogen. In some embodiments, the lumen or cavity of the fusosome comprises viral nucleic acid, e.g., a retroviral nucleic acid, e.g., a lentiviral nucleic acid. The viral nucleic acid may be a viral genome. In some embodiments, the fusosome further comprises, e.g., within its cavity or lumen, one or more viral nonstructural proteins.

[0342] Fusosomes can have various properties that facilitate delivery of a payload, such as a desired transgene or exogenous agent, to a target cell. For example, in some embodiments, the fusosome and source cell together contain sufficient nucleic acid(s) to create a particle capable of fusing with a target cell. In embodiments, these nucleic acid(s) encode a protein having one or more (e.g., all) of the following activities: gag polyprotein activity, polymerase activity, integrase activity, protease activity, and fusogenic activity.

[0343] Fusosomes can also contain various structures that facilitate delivery of a payload to a target cell. For example, in some embodiments, fusosomes (e.g., viruses, e.g., retroviruses, e.g., lentiviruses) contain one or more (e.g., all) of the following proteins: gag polyprotein, polymerase (e.g., pol), integrase (e.g., functional or non-functional mutants), protease, and fusogenic proteins. In some embodiments, fusosomes further contain rev. In some embodiments, one or more of the above proteins are encoded by the retroviral genome, and in some embodiments, one or more of the above proteins are provided upon introduction, for example, by a helper cell, helper virus, or helper plasmid. In some embodiments, the fusosomal nucleic acid (e.g., retroviral nucleic acid) comprises one or more (e.g., all) of the following nucleic acid sequences: a 5' LTR (e.g., comprising a U5 and lacking a functional U3 domain), a Psi packaging element (Psi), a central polypurine tract (cPPT) promoter operably linked to a payload gene, a payload gene (optionally comprising an intron before the open reading frame), a polyA tail sequence, a WPRE, and a 3' LTR (e.g., comprising a U5 and lacking a functional U3). In some embodiments, the fusosomal nucleic acid (e.g., retroviral nucleic acid) further comprises one or more insulator elements. In some embodiments, the fusosomal nucleic acid (e.g., retroviral nucleic acid) further comprises one or more miRNA recognition sites. In some embodiments, one or more of the miRNA recognition sites are located downstream of the polyA tail sequence, e.g., between the polyA tail sequence and the WPRE.

[0344] In some embodiments, the fusosomes provided herein are administered to a subject, e.g., a mammal, e.g., a human. In such embodiments, the subject may be at risk for, have symptoms of, or be diagnosed or identified as having a particular disease or condition (e.g., a disease or condition described herein). In one embodiment, the subject has cancer. In one embodiment, the subject has an infectious disease. In some embodiments, the fusosomes comprise a nucleic acid sequence encoding an exogenous agent for treating a disease or condition.

[0345] Lentiviral components and helper cells In some embodiments, the retroviral nucleic acid comprises one or more (e.g., all) of the following: a 5' promoter (e.g., to control expression of the entire packaged RNA), a 5' LTR (e.g., a U5 containing an R (polyadenylation tail signal) and / or a primer activation signal), a primer binding site, a psi packaging signal, an RRE element for nuclear export, a promoter immediately upstream of the transgene to control expression of the transgene, a transgene (or other exogenous drug element), a polypurine tract, and a 3' LTR (e.g., containing a mutated U3, R, and U5). In some embodiments, the retroviral nucleic acid further comprises one or more of a cPPT, a WPRE, and / or an insulator element.

[0346] Retroviruses typically replicate their genomic RNA into a linear, double-stranded DNA copy by reverse transcription, which then covalently integrates 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), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentiviruses.

[0347] In some embodiments, the retrovirus is a gum retrovirus. In some embodiments, the retrovirus is an epsilon retrovirus. In some embodiments, the retrovirus is an alpha retrovirus. In some embodiments, the retrovirus is a beta retrovirus. In some embodiments, the retrovirus is a delta retrovirus. In some embodiments, the retrovirus is a lentivirus. In some embodiments, the retrovirus is a spumaretrovirus. In some embodiments, the retrovirus is an endogenous retrovirus.

[0348] Exemplary lentiviruses include, but are not limited to, HIV (including human immunodeficiency viruses, HIV types 1 and 2), Visna-Maudi virus (VMV), Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV). In some embodiments, an HIV-based backbone (i.e., HIV cis-acting sequence elements) is used.

[0349] In some embodiments, a vector herein refers to a nucleic acid molecule that can transfer or transport another nucleic acid molecule. The transferred nucleic acid is generally linked to, for example, inserted into, the vector nucleic acid molecule. The vector may contain a sequence that directs autonomous replication in cells, or may contain a sequence sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-deficient retroviruses and lentiviruses.

[0350] Viral vectors can include, for example, nucleic acid molecules (e.g., transfer plasmids) containing virally derived nucleic acid elements that generally facilitate the transfer of nucleic acid molecules or their integration into a cellular genome or viral particles that mediate nucleic acid transfer. Viral particles typically contain various viral components, and often host cell components, in addition to the nucleic acid(s). Viral vectors can include, for example, viruses or viral particles that can transfer nucleic acids into cells or transferred nucleic acids (e.g., as naked DNA). Viral vectors and transfer plasmids can contain structural and / or functional genetic elements primarily derived from viruses. Retroviral vectors can include viral vectors or plasmids containing structural and functional genetic elements, or portions thereof, primarily derived from retroviruses. Lentiviral vectors can include viral vectors or plasmids containing structural and functional genetic elements, or portions thereof, primarily including LTRs derived from lentiviruses.

[0351] In embodiments, lentiviral vectors (e.g., lentiviral expression vectors) can comprise lentiviral transfer plasmids (e.g., as naked DNA) or infectious lentiviral particles. It should be understood that with respect to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., the sequences of these elements can be present in the lentiviral particle in RNA form, or in the DNA plasmid in DNA form.

[0352] In some vectors described herein, at least a portion of one or more protein coding regions that contribute to or are essential for replication may be absent compared to the corresponding wild-type virus. This results in a viral vector replication deficiency. In some embodiments, the vector is capable of transducing target non-dividing host cells and / or integrating its genome into the host genome.

[0353] The structure of wild-type retroviral genomes often includes a 5' long terminal repeat (LTR) and a 3' LTR, between which or within which are located a packaging signal that enables genome packaging, a primer binding site, an integration site that enables integration into the host genome, and the gag, pol, and env genes that encode packaging components that facilitate viral particle assembly. More complex retroviruses possess additional characteristics, such as the rev and RRE sequences in HIV, which enable efficient transport of integrated proviral RNA transcripts from the nucleus to the cytoplasm of infected target cells. In proviruses, viral genes are flanked on both ends by regions called long terminal repeats (LTRs). LTRs are involved in proviral integration and transcription. LTRs also function as enhancer-promoter sequences, allowing for the control of viral gene expression. Retroviral RNA encapsidation is mediated by the psi sequence located at the 5' end of the viral genome.

[0354] The LTRs themselves are usually similar (e.g., identical) sequences that can be divided into three elements called U3, R, and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably between different retroviruses.

[0355] In viral genomes, the transcription initiation site is usually at the boundary between U3 and R in one LTR, and the poly(A) addition (termination) site is at the boundary between R and U5 in the other LTR. U3 contains most of the proviral transcriptional control elements, including the promoter and numerous enhancer sequences that respond to cellular, and in some cases, viral, transcriptional activator proteins. Some retroviruses contain one or more of the following genes: tot, rev, tax, and rex, which encode proteins involved in the regulation of gene expression.

[0356] Regarding the structural genes gag, pol, and env themselves, gag encodes the internal structural protein of the virus. The Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid), and NC (nucleocapsid). The pol gene encodes DNA polymerase, associated RNase H, and reverse transcriptase (RT) including integrase (IN), which mediates genome replication. The env gene encodes the surface (SU) glycoprotein and transmembrane (TM) protein of the virion, which form a complex that interacts specifically with cellular receptor proteins. This interaction facilitates infection, for example, by fusion of the viral membrane with the cellular membrane.

[0357] In replication-defective retroviral vector genomes, gag, pol, and env may be absent or non-functional. The R regions at both ends of the RNA are usually repetitive sequences. U5 and U3 represent unique sequences at the 5' and 3' ends of the RNA genome, respectively.

[0358] Retroviruses may also contain additional genes encoding proteins other than gag, pol, and env. Examples of additional genes include one or more of vif, vpr, vpx, vpu, tat, rev, and nef (in HIV). EIAV (among others) has an additional gene, S2. Proteins encoded by additional genes perform various functions, some of which may overlap with functions provided by cellular proteins. In EIAV, for example, 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). It binds to a stable stem-loop RNA secondary structure called TAR. Rev regulates and coordinates viral gene expression via the rev response element (RRE) (Martarano et al. 1994 J. Virol. 68:3102-11). The mechanisms of action of these two proteins are thought to be largely similar to those of their counterparts in primate viruses. Furthermore, the EIAV protein Ttm has been identified as encoded by the first exon of tat, which is spliced ​​into the env coding sequence at the start of the transmembrane protein.

[0359] In addition to protease, reverse transcriptase, and integrase, non-primate lentiviruses contain a fourth pol gene product that encodes a dUTPase, which may play a role in the ability of these lentiviruses to infect certain non-dividing or slowly dividing cell types.

[0360] In embodiments, a recombinant lentiviral vector (RLV) is a vector that, in the presence of packaging components, has sufficient retroviral genetic information to allow packaging of an RNA genome into viral particles capable of infecting target cells. Infection of target cells can include reverse transcription and integration into the target cell genome. RLVs typically have non-viral coding sequences delivered to target cells by the vector. In embodiments, RLVs lack the independent replication capacity to generate infectious retroviral particles within target cells. RLVs typically lack functional gag-pol and / or env genes and / or other genes involved in replication. Vectors can be constructed as split-intron vectors, for example, as described in PCT Patent Application No. WO 99 / 15683, the entire contents of which are incorporated herein by reference.

[0361] In some embodiments, lentiviral vectors comprise a minimal viral genome, e.g., viral vectors that have been engineered to remove non-essential elements and retain essential elements to obtain the functionality required for infection, transduction, and delivery of a nucleotide sequence of interest to a target host cell, e.g., as described in WO98 / 17815, the entire contents of which are incorporated herein by reference.

[0362] A minimal lentiviral genome may contain, for example, (5')R-U5-one or more first nucleotide sequences-U3-R(3'). However, the plasmid vector used to generate the lentiviral genome in the source cell may also contain transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in the source cell. These regulatory sequences may include native sequences associated with transcribed retroviral sequences, such as the 5'U3 region, or they may include another viral promoter, such as a heterologous promoter such as the CMV promoter. Some lentiviral genomes contain additional sequences that promote efficient virus production. For example, in the case of HIV, rev and RRE sequences may be included. Alternatively, or in combination, codon optimization may be used; for example, genes encoding exogenous agents may be codon-optimized, as described, for example, in WO 01 / 79518, which is incorporated herein by reference in its entirety. Alternative sequences that perform similar or identical functions to the rev / RRE system may also be used. For example, a functional analog of the rev / RRE system is found in the Mason-Pfizer monkey virus. This is known as CTE, which contains an RRE-type sequence in the genome that is thought to interact with factors in infected cells. Cellular factors can be thought of as rev analogs. Therefore, CTE can be used as a substitute for the rev / RRE system. Furthermore, the Rex protein of HTLV-I can functionally replace the Rev protein of HIV-I. Rev and Rex have effects similar to those of IRE-BP.

[0363] In some embodiments, the retroviral nucleic acid (e.g., lentiviral nucleic acid, e.g., primate or non-primate lentiviral nucleic acid) (1) comprises a deleted gag gene, where the deletion in gag removes one or more nucleotides approximately 350-354 nucleotides downstream of the gag coding sequence; (2) has one or more accessory genes not present in the retroviral nucleic acid; (3) lacks the tat gene but includes a leader sequence between the end of the 5' LTR and the ATG of gag; and (4) is a combination of (1), (2), and (3). In one embodiment, the lentiviral vector comprises all of features (1), (2), and (3). This strategy is described in further detail in WO 99 / 32646, the entire contents of which are incorporated herein by reference.

[0364] In some embodiments, the primate lentiviral minimal system does not require any of the HIV / SIV additional genes vif, vpr, vpx, vpu, tat, rev, and nef for either vector production or transduction of dividing and non-dividing cells, hi some embodiments, the EIAV minimal vector system does not require S2 for either vector production or transduction of dividing and non-dividing cells.

[0365] Deleting additional genes can allow vectors to be generated without genes associated with disease in lentivirus (e.g., HIV) infections. Tat, in particular, is associated with disease. Second, deleting additional genes allows the vector to package more heterologous DNA. Third, genes of unknown function, such as S2, can be omitted, thereby reducing the risk of undesirable effects. Examples of minimal lentiviral vectors are disclosed in WO99 / 32646 and WO98 / 17815.

[0366] In some embodiments, the retroviral nucleic acid lacks at least tat and S2 (if an EIAV vector system), and possibly vif, vpr, vpx, vpu, and nef as well, hi some embodiments, the retroviral nucleic acid also lacks rev, RRE, or both.

[0367] In some embodiments, the retroviral nucleic acid comprises vpx. The Vpx polypeptide binds to and induces the degradation of SAMHD1, a restriction factor that degrades free dNTPs in the cytoplasm. Thus, Vpx degrades SAMHD1, increasing the concentration of free dNTPs in the cytoplasm, thereby increasing reverse transcription activity, thereby promoting the reverse transcription and integration of the retroviral genome into the target cell genome.

[0368] Different cells have different usage patterns for certain codons. This codon bias corresponds to the bias in the relative abundance of certain tRNAs in a cell type. By changing the codons in the sequence, they can be adjusted to match the relative abundance of the corresponding tRNAs, thereby increasing expression. Similarly, by intentionally selecting codons whose corresponding tRNAs are known to be rare in a particular cell type, expression can be reduced. Thus, an additional level of translational control is available. Further description of codon optimization can be found, for example, in WO99 / 41397, the entire contents of which are incorporated herein by reference.

[0369] Many viruses, including HIV and other lentiviruses, use a number of rare codons, and by altering these to correspond to commonly used mammalian codons, increased expression of packaging components in mammalian producer cells can be achieved.

[0370] Codon optimization has many other advantages. By altering their sequences, nucleotide sequences encoding packaging components can have RNA instability sequences (INS) reduced or eliminated from them. At the same time, the amino acid coding sequences of the packaging components are retained, thereby ensuring that the viral components encoded by the sequences remain identical or at least sufficiently similar so that the functionality of the packaging components is not impaired. In some embodiments, codon optimization also overcomes the Rev / RRE requirement for transport, making the optimized sequences Rev-independent. In some embodiments, codon optimization also reduces homologous recombination between different constructs within a vector system (e.g., between overlapping regions in the gag-pol and env open reading frames). In some embodiments, codon optimization results in increased viral titer and / or improved safety.

[0371] In some embodiments, only the codons associated with the INS are codon optimized, while in other embodiments, the sequences are codon optimized in their entirety, except for the sequence encompassing the gag-pol frameshift site.

[0372] The gag-pol gene contains two overlapping reading frames encoding gag-pol proteins. Expression of both proteins depends on a frameshift during translation. This frameshift occurs as a result of ribosome "slippage" during translation. This slippage is thought to be caused, at least in part, by secondary structures in the RNA that stall the ribosome. Such secondary structures are present downstream of the frameshift site in the gag-pol gene. In HIV, the overlapping region extends from nucleotide 1222 downstream of the start of gag (nucleotide 1 is the A of gag ATG) to the end of gag (nt 1503). As a result, the 281-bp fragment spanning the frameshift site and the overlapping region of the two reading frames is preferably not codon-optimized. In some embodiments, retaining this fragment allows for more efficient expression of the gag-pol proteins. In EIAV, the start of the overlap is at nt 1262 (nucleotide 1 is the A of gag ATG). The end of the overlap is at nt 1461. To ensure that the frameshift site and gag-pol overlap are maintained, the wild-type sequence can be retained from nt 1156 to 1465.

[0373] For example, optimal codon usage can be derived and conservative amino acid changes can be introduced into the gag-pol protein to accommodate convenient restriction sites.

[0374] In some embodiments, codon optimization is based on codons with low codon usage in mammalian systems. The third and sometimes the second and third bases can be changed.

[0375] Due to the degeneracy of the genetic code, it will be understood that many gag-pol sequences can be achieved by one skilled in the art. There are also many reported retroviral variants that can be used as a starting point for generating codon-optimized gag-pol sequences. Lentiviral genomes can be quite variable. For example, there are many quasispecies of HIV-I that are still functional. This is also true for EIAV. These variants can be used to enhance specific parts of the transduction process. Examples of HIV-I variants can be found in the HIV database maintained by Los Alamos National Laboratory. Details of EIAV clones can be found in the NCBI database maintained by the National Institutes of Health.

[0376] The strategy for codon-optimizing gag-pol sequences can be used in connection with any retrovirus, such as EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-I, and HIV-2. Additionally, this method can be used to increase expression of genes from HTLV-I, HTLV-2, HFV, HSRV, and human endogenous retroviruses (HERVs), MLV, as well as other retroviruses.

[0377] As described above, packaging components for retroviral vectors can include the expression products of the gag, pol, and env genes. Additionally, packaging can use a short sequence of four stem-loops followed by partial sequences from gag and env as packaging signals. Thus, inclusion of deleted gag sequences in the retroviral vector genome (in addition to the complete gag sequence in the packaging construct) can be used. In embodiments, retroviral vectors contain packaging signals that include 255 to 360 nucleotides of gag in vectors that still retain the env sequence, or approximately 40 nucleotides of gag in specific combinations of gag and env deletions that are splice donor mutations. In some embodiments, retroviral vectors contain gag sequences that contain one or more deletions, for example, the gag sequence includes approximately 360 nucleotides from the N-terminus.

[0378] The 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, e.g., two, three, four, or more retroviruses.

[0379] The gag and pol coding sequences are generally organized as the Gag-Pol precursor in native lentiviruses. The gag sequence encodes the 55 kD Gag precursor protein, also known as p55. p55 is cleaved by virally encoded protease 4 (the product of the pol gene) during maturation into four smaller proteins called MA (matrix [p17]), CA (capsid [p24]), NC (nucleocapsid [p9]), and p6. The pol precursor protein is cleaved away from Gag by the virally encoded protease and further digested to separate the protease (p10), RT (p50), RNase H (p15), and integrase (p31) activities.

[0380] Native Gag-Pol sequences can be utilized in helper vectors (e.g., helper plasmids or helper viruses) and can be modified, including chimeric Gag-Pol sequences in which the Gag and Pol sequences are derived from different viruses (e.g., different species, subspecies, strains, clades, etc.) and / or sequences in which the sequences have been modified to improve transcription and / or translation and / or to reduce recombination.

[0381] In various examples, the retroviral nucleic acid comprises a polynucleotide encoding a 150-250 (e.g., 168) nucleotide portion of the gag protein that (i) contains a mutant INS1 inhibitory sequence that reduces restriction of RNA nuclear export compared to wild-type INS1, (ii) contains a two nucleotide insertion that results in a frameshift and premature termination, and / or (iii) does not contain the INS2, INS3, and INS4 inhibitory sequences of gag.

[0382] In some embodiments, the vectors described herein are hybrid vectors that contain both retroviral (e.g., lentiviral) and non-lentiviral sequences. In some embodiments, the hybrid vectors contain retroviral, e.g., lentiviral, sequences for reverse transcription, replication, integration, and / or packaging.

[0383] According to certain specific embodiments, most or all of the viral vector backbone sequence is derived from lentivirus, for example, HIV-1.However, it is understood that many different sources of retrovirus and / or lentivirus sequences can be used or combined, and many substitutions and changes in specific lentivirus sequences can be accommodated without impairing the ability of the transfer vector to perform the functions described herein.Various lentivirus 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, and many of them can be adapted to produce retroviral nucleic acid.

[0384] Long terminal repeats (LTRs) are typically found at both ends of a provirus. LTRs typically contain domains located at the ends of retroviral nucleic acids, which are direct repeats of their native sequence and include the U3, R, and U5 regions. LTRs generally promote retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. LTRs can contain multiple regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences for viral genome replication and integration. Viral LTRs are typically divided into three regions, designated U3, R, and U5. The U3 region typically contains enhancer and promoter elements. The U5 region typically lies between the primer binding site and the R region and can contain a polyadenylation sequence. The R (repeat) region may be flanked by U3 and U5 regions. LTRs are typically composed of U3, R, and U5 regions and can appear at both the 5' and 3' ends of the viral genome. In some embodiments, adjacent to the 5'LTR are sequences for reverse transcription of the genome (tRNA primer binding site) and efficient packaging of viral RNA into particles (Psi site).

[0385] The packaging signal can include a sequence located within the retroviral genome that mediates 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 encapsidation of the viral genome.

[0386] In various embodiments, the retroviral nucleic acid comprises a modified 5'LTR and / or 3'LTR. Either or both of the LTRs may comprise one or more modifications, including, but not limited to, one or more deletions, insertions, or substitutions. Modification of the 3'LTR is often performed to improve the safety of lentivirus or retrovirus systems by rendering the virus replication-deficient, for example, a virus that cannot replicate effectively and completely so that infectious virions are not produced (e.g., a progeny of a replication-deficient lentivirus).

[0387] In some embodiments, the vector is a self-inactivating (SIN) vector, e.g., a replication-deficient vector, e.g., a retroviral or lentiviral vector, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication, because during viral replication the right (3') LTR U3 region is replaced by the left (5') LTR The 3' LTR can be used as a template for the U3 region, and therefore, the absence of the U3 enhancer promoter inhibits viral replication. In embodiments, the 3' LTR is modified so that the U5 region is removed, altered, or replaced, for example, by an exogenous poly(A) sequence, and the 3' LTR, 5' LTR, or both the 3' and 5' LTRs can be modified LTRs.

[0388] In some embodiments, the U3 region of the 5'LTR is replaced with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include, for example, the viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate-early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. In some embodiments, the promoter can drive high levels of transcription in a Tat-independent manner. In certain embodiments, heterologous promoters have the additional advantage of controlling the manner in which the viral genome is transcribed. For example, heterologous promoters can be inducible, such that transcription of all or part of the viral genome occurs only in the presence of an inducing factor. Inducing factors include, but are not limited to, one or more compounds and physiological conditions, such as temperature or pH, under which the host cells are cultured.

[0389] In some embodiments, the viral vector contains a TAR (transactivation response) element, located, for example, in the R region of the lentiviral (e.g., HIV) LTR. This element interacts with the lentiviral transactivator (tat) gene element to enhance viral replication. However, this element is not required, for example, in embodiments in which the U3 region of the 5' LTR is replaced by a heterologous promoter.

[0390] The R region, e.g., the region within a retroviral LTR that begins at the start of the capping group (i.e., the start of transcription) and ends just before the start of the polyA tract, may be flanked by the U3 and U5 regions. The R region plays a role during reverse transcription in the transfer of nascent DNA from one end of the genome to the other.

[0391] Retroviral nucleic acids can also contain FLAP elements, e.g., nucleic acids whose sequences include the central polypurine tract and central termination sequence (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou et al., 2000, Cell, 101:173, which are incorporated herein by reference in their entireties. During HIV-1 reverse transcription, central initiation of positive-strand DNA at the central polypurine tract (cPPT) and central termination at the central termination sequence (CTS) can result in the formation of a triple-stranded DNA structure: the HIV-1 central DNA flap. In some embodiments, the retroviral or lentiviral vector backbone contains one or more FLAP elements upstream or downstream of the gene encoding the exogenous agent. For example, in some embodiments, the transfer plasmid contains a FLAP element, e.g., a FLAP element derived or isolated from HIV-1.

[0392] In embodiments, retroviral or lentiviral nucleic acid comprises one or more transport elements, such as cis-acting post-transcriptional regulatory elements that regulate the transport of RNA transcripts from the nucleus to the cytoplasm of cells.Examples of RNA transport elements include, but are not limited to, human immunodeficiency virus (HIV) rev response element (RRE) (see, for example, Cullen et al., 1991. J. Virol. 65:1053, and Cullen et al., 1991. Cell 58:423), and hepatitis B virus post-transcriptional regulatory element (HPRE), which are incorporated herein by reference in their entirety.Generally, RNA transport elements are located within the 3'UTR of genes, and can be inserted as one or multiple copies.

[0393] In some embodiments, expression of heterologous sequences in viral vectors is increased by incorporating one or more, for example, all of the following into the vector: a posttranscriptional regulatory element, a polyadenylation site, and a transcription termination signal. Various posttranscriptional regulatory elements can increase expression of heterologous nucleic acids in proteins, such as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886), the posttranscriptional regulatory element (HPRE) present in hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864), (Liu et al., 1995, Genes Dev., 9:1766), each of which is incorporated herein by reference in its entirety. In some embodiments, the retroviral nucleic acids described herein include a posttranscriptional regulatory element such as a WPRE or HPRE.

[0394] In some embodiments, the retroviral nucleic acids described herein lack or do not include post-transcriptional regulatory elements such as a WPRE or HPRE.

[0395] Elements directing the termination and polyadenylation of heterologous nucleic acid transcripts can be included, for example, to increase expression of exogenous agents. A transcription termination signal can be found downstream of the polyadenylation signal. In some embodiments, the vector contains a polyadenylation sequence 3' of the polynucleotide encoding the exogenous agent. A polyA site can contain a DNA sequence that directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. The polyadenylation sequence can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, contributing to increased 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.

[0396] In some embodiments, the retroviral or lentiviral vector further comprises one or more insulator elements, eg, an insulator element described herein.

[0397] In various embodiments, the vector comprises a promoter operably linked to a polynucleotide encoding an exogenous agent. The vector may have one or more LTRs, any of which may contain one or more modifications, such as nucleotide substitutions, additions, or deletions. The vector may further comprise one or more accessory elements that increase transduction efficiency (e.g., cPPT / FLAP), viral packaging (e.g., Psi (Ψ) packaging signal, RRE), and / or other elements that increase exogenous gene expression (e.g., poly(A) sequences), and may optionally include a WPRE or HPRE.

[0398] In some embodiments, the lentiviral nucleic acid includes one or more, e.g., from 5' to 3', one or more, e.g., all, of a promoter (e.g., CMV), an R sequence (e.g., including TAR), a U5 sequence (e.g., for integration), a PBS sequence (e.g., for reverse transcription), a DIS sequence (e.g., for genome dimerization), a psi packaging signal, a partial gag sequence, an RRE sequence (e.g., for nuclear export), a cPPT sequence (e.g., for nuclear export), a promoter driving expression of an exogenous agent, a gene encoding an exogenous agent, a WPRE sequence (e.g., for efficient transgene expression), a PPT sequence (e.g., for reverse transcription), an R sequence (e.g., for polyadenylation and termination), and a U5 signal (e.g., for integration).

[0399] Vectors engineered to remove splice sites Some lentiviral vectors integrate internal active genes and have strong splicing and polyadenylation signals that can result in the formation of transcripts that can be aberrantly truncated.

[0400] The mechanism of proto-oncogene activation may involve the generation of chimeric transcripts resulting from the interaction of promoter elements or splice sites contained in the genome of the insertional mutagen with cellular transcription units targeted by integration (Gabriel et al., 2009. Nat Med 15:1431-1436; Bokhoven et al., J Virol 83:283-29). Chimeric fusion transcripts containing vector sequences and cellular mRNAs can be generated either by read-through transcription, which initiates from the vector sequences and proceeds to adjacent cellular genes, or vice versa.

[0401] In some embodiments, the lentiviral nucleic acids described herein comprise 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 species and specific methods of such splice sites are described in WO2012 / 156839A2, all of which are incorporated by reference.

[0402] Retrovirus generation method Large-scale viral particle production is often useful to achieve a desired viral titer. Viral particles can be produced by transfecting transfer vectors into packaging cell lines containing viral structural and / or accessory genes, such as gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes or other retroviral genes.

[0403] In some embodiments, the packaging vector is an expression vector or viral vector lacking a packaging signal and containing a polynucleotide encoding one, two, three, four, or more viral structural and / or accessory genes. Typically, the packaging vector is contained in a packaging cell and introduced into cells via transfection, transduction, or infection. A retroviral, e.g., lentiviral, transfer vector can be introduced into a packaging cell line via transfection, transduction, or infection to generate 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 cells with a dominant selectable marker, such as neomycin, hygromycin, puromycin, blastocidin, zeocin, thymidine kinase, DHFR, Gln synthetase, or ADA, followed by selection in the presence of an appropriate drug and isolation of clones. The selectable marker gene can be physically linked to the packaging vector, for example, a gene encoded by an IRES or a self-cleaving viral peptide.

[0404] Packaging cell lines include cell lines that do not contain a packaging signal but stably or transiently express viral structural proteins and replication enzymes (e.g., gag, pol, and env) that can package viral particles. Any suitable cell line, for example, mammalian cells, such as human cells, can be used. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC23 cells, PA317 cells, WEHI cells, COS cells, BSC1 cells, BSC40 cells, BMT10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In embodiments, the packaging cells are 293 cells, 293T cells, or A549 cells.

[0405] Source cell lines include cell lines that can produce recombinant retroviral particles, including packaging cell lines and transfer vector constructs containing packaging signals.Methods for preparing virus stock solutions are described, for example, by Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633 and NRL Landau et al. (1992) J. Virol. 66:5110-5113, which are incorporated herein by reference.Infectious virus particles can be collected from packaging cells, for example, by cell lysis or by collecting the supernatant of cell culture.Optionally, the collected virus particles can be enriched or purified.

[0406] Packaging plasmids and cell lines In some embodiments, the source cells contain one or more plasmids encoding viral structural proteins and replication enzymes (e.g., gag, pol, and env) capable of packaging viral particles. In some embodiments, the sequences encoding at least two of the gag, pol, and env precursors are on the same plasmid. In some embodiments, the sequences encoding the gag, pol, and env precursors are on different plasmids. In some embodiments, the sequences encoding the gag, pol, and env precursors have the same expression signal, e.g., promoter. In some embodiments, the sequences encoding the gag, pol, and env precursors have different expression signals, e.g., different promoters. In some embodiments, expression of the gag, pol, and env precursors is inducible. In some embodiments, the plasmids encoding the viral structural proteins and replication enzymes are transfected simultaneously or at different times. In some embodiments, the plasmids encoding the viral structural proteins and replication enzymes are transfected simultaneously or at different times with the packaging vector.

[0407] In some embodiments, the source cell line contains one or more stably integrated viral structural genes. In some embodiments, expression of the stably integrated viral structural genes is inducible.

[0408] In some embodiments, expression of viral structural genes is regulated at the transcriptional level. In some embodiments, expression of viral structural genes is regulated at the translational level. In some embodiments, expression of viral structural genes is regulated at the post-translational level.

[0409] In some embodiments, expression of viral structural genes is regulated by a tetracycline (Tet)-dependent system, in which the Tet-regulated transcriptional repressor (Tet-R) binds to DNA sequences contained in the promoter and represses transcription through steric hindrance (Yao et al., 1998; Jones et al., 2005). Addition of doxycycline (dox) releases Tet-R, allowing transcription. Many other suitable transcriptionally regulated promoters, transcription factors, and small molecule inducers are suitable for regulating transcription of viral structural genes.

[0410] In some embodiments, the third generation lentiviral components, human immunodeficiency virus type 1 (HIV) Rev, Gag / Pol, and envelope, under the control of a Tet-regulated promoter and linked to an antibiotic resistance cassette, are separately integrated into the source cell genome. In some embodiments, the source cell only has one copy of each of the Rev, Gag / Pol, and envelope proteins integrated into its genome.

[0411] 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 with Rev, Gag / Pol, and envelope proteins stably integrated into the genome. See, for example, Milani et al., EMBO Molecular Medicine, 2017, the entire contents of which are incorporated herein by reference.

[0412] In some embodiments, the retroviral nucleic acid described herein is incapable of undergoing reverse transcription. Such nucleic acids, in embodiments, can transiently express an exogenous agent. The retrovirus or VLP may or may not contain a disabled reverse transcriptase protein. In embodiments, the retroviral nucleic acid contains a disabled primer binding site (PBS) and / or att site. In embodiments, one or more viral accessory genes, including rev, tat, vif, nef, vpr, vpu, vpx, and S2 or their functional equivalents, are disabled or absent from the retroviral nucleic acid. In embodiments, one or more accessory genes selected from S2, rev, and tat are disabled or absent from the retroviral nucleic acid.

[0413] Strategies for packaging retroviral nucleic acids Typically, modern retroviral vector systems consist of (1) a viral genome with cis-acting vector sequences for transcription, reverse transcription, integration, translation, and packaging of viral RNA into viral particles, and (2) a producer cell line expressing the trans-acting retroviral gene sequences (e.g., gag, pol, and env) required for the production of viral particles. By completely separating the cis- and trans-acting vector sequences, the virus cannot sustain replication beyond a single cycle of infection. The generation of live virus can be avoided by a number of strategies, such as minimizing overlap between cis- and trans-acting sequences to avoid recombination.

[0414] Viral vector particles containing sequences lacking or lacking viral RNA may be the result of removing or eliminating viral RNA from the sequence. In one embodiment, this can be achieved by using the endogenous packaging signal binding site in gag. Alternatively, the endogenous packaging signal binding site is located on pol. In this embodiment, the RNA to be delivered contains its cognate packaging signal. In another embodiment, a heterologous binding domain (heterologous to gag) located in the RNA to be delivered and a cognate binding site located in gag or pol can be used to ensure packaging of the RNA to be delivered. The heterologous sequence can be non-viral, or it can be viral, in which case it can be derived from a different virus. Vector particles can also be used to deliver therapeutic RNA, in which case functional integrase and / or reverse transcriptase are not required. These vector particles can also be used to deliver therapeutic genes of interest, in which case pol is typically included.

[0415] In one embodiment, gag-pol is mutated, and packaging signal is replaced with corresponding packaging signal.In this embodiment, particle can package RNA by new packaging signal.The advantage of this method is that it can package RNA sequence that lacks viral sequence, such as RNAi.

[0416] Another approach is to rely on overexpression of the RNA to be packaged. In one embodiment, the RNA to be packaged is overexpressed in the absence of any RNA containing a packaging signal. This can result in a significant level of therapeutic RNA being packaged, and this amount is sufficient to transduce cells and have a biological effect.

[0417] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a viral gag protein or a retroviral gag and pol protein, wherein the gag protein or pol protein comprises a heterologous RNA-binding domain that can recognize a corresponding sequence in an RNA sequence and facilitate packaging of the RNA sequence into a viral vector particle.

[0418] In some embodiments, the heterologous RNA binding domain comprises an RNA binding domain derived from a bacteriophage coat protein, a Rev protein, a protein of the U1 small nuclear ribonucleoprotein particle, a Nova protein, a TF111A protein, a TIS11 protein, a trpRNA-binding attenuation protein (TRAP), or a pseudouridine synthase.

[0419] In some embodiments, the methods herein include detecting or confirming the absence of replication-competent retrovirus. The methods may include assessing the RNA level of one or more target genes, such as viral genes (e.g., structural genes or packaging genes), whose gene products are expressed in certain cells infected with a replication-competent retrovirus, such as a gammaretrovirus or lentivirus, but are not present in the viral vector used to transduce the cells with heterologous nucleic acid, and which would not be present and / or expressed, or would not be expected, in cells that do not contain a replication-competent retrovirus. The presence of a replication-competent retrovirus may be determined when the RNA level of one or more target genes is higher than a control value, which can be measured directly or indirectly, for example, from a positive control sample containing the target genes. For a detailed disclosure, see WO2018 / 023094A1.

[0420] Repression of genes encoding exogenous drugs in source cells Proteins (over)expressed in source cells can have indirect or direct effects on vector virion assembly and / or infectivity. Incorporation of exogenous agents into vector virions can also affect downstream processing of vector particles.

[0421] In some embodiments, a tissue-specific promoter is used to restrict expression of the exogenous agent in the source cells. In some embodiments, a heterologous translational control system is used in eukaryotic cell culture to suppress translation of the exogenous agent in the source cells. More specifically, the retroviral nucleic acid may contain a binding site operably linked to a gene encoding the exogenous agent, where the binding site can interact with an RNA-binding protein such that translation of the exogenous agent is suppressed or prevented in the source cells.

[0422] In some embodiments, the RNA-binding protein is a tryptophan RNA-binding attenuation protein (TRAP), such as a bacterial tryptophan RNA-binding attenuation protein. The use of an RNA-binding protein (e.g., a bacterial trp operon regulator protein, tryptophan RNA-binding attenuation protein, TRAP) and its bound RNA target represses or prevents transgene translation in source cells. This system is called a transgene repression system in vector-producing cells, or a TRIP system.

[0423] In embodiments, placing a binding site for an RNA-binding protein (e.g., a TRAP binding sequence, tbs) upstream of the NOI translation start codon allows for specific repression of translation of mRNA derived from the internal expression cassette without adversely affecting the production or stability of the vector RNA. The number of nucleotides between the tbs and the translation start codon of the gene encoding the exogenous agent can vary from 0 to 12 nucleotides. The tbs can be placed downstream of an internal ribosome entry site (IRES) to repress translation of the gene encoding the exogenous agent in a multicistronic mRNA.

[0424] Kill Switch System and Amplification In some embodiments, the polynucleotide or cell carrying the gene encoding the exogenous agent utilizes a suicide gene, such as an inducible suicide gene, to reduce the risk of direct toxicity and / or uncontrolled proliferation. In certain embodiments, the suicide gene is not immunogenic to the host cell carrying the exogenous agent. Examples of suicide genes include caspase-9, caspase-8, or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).

[0425] In certain embodiments, the vector contains a gene segment that renders target cells, e.g., immune effector cells, e.g., T cells, susceptible to negative selection in vivo. For example, transduced cells may be eliminated as a result of changes in an individual's in vivo conditions. A negatively selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered drug, e.g., a compound. Negatively selectable genes are known in the art and include, among others, the herpes simplex virus type I thymidine kinase (HSV-ITK) gene, which confers sensitivity to ganciclovir (Wigler et al., Cell 11:223, 1977), the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA 89:33 (1992)).

[0426] In some embodiments, the transduced cells, e.g., immune effector cells such as T cells, comprise a polynucleotide that further comprises a positive marker that allows for the selection of cells with a positively selectable phenotype in vitro. A positive selectable marker can be a gene that, when introduced into a target cell, expresses a dominant phenotype that allows for the positive selection of cells that carry the gene. Genes of this type include, among others, the hygromycin-B phosphotransferase gene (hph), which confers resistance to hygromycin B, the aminoglycoside phosphotransferase gene (neo or aph) from Tn5, which encodes resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multidrug resistance (MDR) gene.

[0427] In some embodiments, the positive selectable marker and negative selectable element are linked such that loss of the negative selectable element necessarily results in loss of the positive selectable marker. For example, positive and negative selectable markers can be fused so that loss of one necessarily results in loss of the other. An example of a fusion polynucleotide whose expression product produces a polypeptide that confers both the desired positive and negative selection functions is the hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene produces a polypeptide that confers hygromycin B resistance for in vitro positive selection and ganciclovir sensitivity for in vivo negative selection. See Lupton S.D. et al., Mol. and Cell. Biology 11:3374-3378, 1991. Further, in embodiments, the polynucleotide encoding the chimeric receptor is a retroviral vector containing a fusion gene, particularly one that confers hygromycin B resistance for in vitro positive selection and ganciclovir sensitivity for in vivo negative selection, such as the HyTK retroviral vector described in Lupton, SD et al. (1991), supra. Publications PCT / U591 / 08442 and PCT / U594 / 05601 also describe the use of bifunctional selectable fusion genes derived from the fusion of potent positive and negative selectable markers.

[0428] Suitable positive selectable markers are derived from genes selected from the group consisting of hph, nco, and gpt, and suitable negative selectable markers may be derived from genes selected from the group consisting of cytosine deaminase, HSV-ITK, VZVTK, HPRT, ARRT, and gpt. Other suitable markers are dual-functional selectable fusion genes, where the positive selectable marker is derived from hph or neo, and the negative selectable marker is derived from a cytosine deaminase or TK gene or selectable marker.

[0429] Strategies for modulating lentiviral integration In order to prevent retrovirus or lentivirus genome from integrating into target cell genome, disclosed retrovirus and lentivirus nucleic acid lacks or disables important protein / sequence.For example, viral nucleic acid lacking each amino acid that constitutes the highly conserved DDE motif of retrovirus integrase (Engelman and Craigie (1992) J.Virol.66:6361-6369, Johnson et al. (1986) Proc.Natl.Acad.Sci.USA 83:7648-7652, Khan et al. (1991) Nucleic Acids Res.19:851-860) can generate integration-defective retrovirus nucleic acid.

[0430] For example, in some embodiments, the 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. Illustrative, non-limiting examples of Type I mutations include DX1, DX2, DX3, DX4, DX5, DX6, DX7, DX8, DX9, DX10, DX11, DX12, DX13, DX14, DX15, DX16, DX17, DX18, DX19, DX20, DX21, DX22, DX23, DX24, DX25, DX36, DX37, DX38, DX40, DX51, DX52, DX53, DX54, DX55, DX66, DX77, DX78, DX89, DX90, DX91, DX92, DX93, DX94, DX95, DX96, DX97, DX98, DX99, DX100, DX110, DX111, DX120, DX130, DX140, DX151, DX161, DX172, DX183, DX194, DX195, DX196, DX197, DX198, DX199, DX111, DX112, DX113, DX114, DX115, DX115, DX116, DX117, DX118, DX119, DX120, DX121, 39~58 DX 35 These mutations affect either 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 of 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 the integrase protein.

[0431] In some embodiments, the retrovirus integrates an active transcription unit. In some embodiments, the retrovirus does not integrate near the 5' end of the gene, the transcription start site, or the DNAse 1 cleavage site. In some embodiments, the retrovirus does not integrate an active protooncogene or an inactive tumor suppressor gene. In some embodiments, the retrovirus is not genotoxic. In some embodiments, the lentivirus integrates an intron.

[0432] 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 group of cells, or an individual cell colony. Exemplary methods for determining copy number include polymerase chain reaction (PCR) and flow cytometry.

[0433] In some embodiments, the DNA encoding the exogenous agent is integrated into the genome. In some embodiments, the DNA encoding the exogenous agent is maintained episomally. In some embodiments, the integrated to episomal DNA encoding the exogenous agent is at least 0.01, 0.1, 0.5, 1.0, 2, 5, 10, 100.

[0434] In some embodiments, the DNA encoding the exogenous agent is linear. In some embodiments, the DNA encoding the exogenous agent is circular. In some embodiments, the ratio of linear to circular DNA copies encoding the exogenous agent is at least 0.01, 0.1, 0.5, 1.0, 2, 5, 10, or 100.

[0435] In some embodiments, the DNA encoding the exogenous agent is circular with one LTR. In some embodiments, the DNA encoding the exogenous agent is circular with two LTRs. In some embodiments, the ratio of the one LTR of the DNA encoding the exogenous agent to the two LTRs of the DNA encoding the exogenous agent is at least 0.1, 0.5, 1.0, 2, 5, 10, 20, 50, 100.

[0436] Episomal virus maintenance In integration-deficient retroviruses, circular cDNA by-products of retrotranscription (e.g., 1-LTR and 2-LTR) can accumulate in the cell nucleus without integrating into the host genome (see Yanez-Munoz RJ et al., Nat. Med. 2006, 12:348-353). Like other exogenous DNA, these intermediates are then ligated at the same frequency (e.g., 10 3 ~10 5 / cell) and can be integrated into cellular DNA.

[0437] In some embodiments, episomal retroviral nucleic acids do not replicate. Episomal viral DNA can be modified to be maintained in replicating cells by the inclusion of a eukaryotic origin of replication and a scaffold / matrix attachment region (S / MAR) for attachment to the nuclear matrix.

[0438] Therefore, in some embodiments, the retroviral nucleic acid described herein comprises a eukaryotic replication origin or a mutant thereof. Examples of eukaryotic replication origins of interest include the replication origin of the β-globin gene reported by Aladjem et al. (Science, 1995, 270:815-819), the consensus sequence derived from an autonomously replicating sequence combined with an alpha-satellite sequence previously isolated from monkey CV-1 cells and human skin fibroblasts reported by Price et al., Journal of Biological Chemistry, 2003, 278(22):19649-59, and the replication origin of the human c-myc promoter region reported by McWinney and Leffak (McWinney C. and Leffak M., Nucleic Acid Research 1990, 18(5):1233-42). In some embodiments, the mutant substantially maintains the ability to initiate replication in eukaryotes. The ability of a particular sequence to initiate replication can be determined by any suitable method, for example, autonomous replication assays based on bromodeoxyuridine incorporation and density shifts (Araujo FD et al., supra; Frappier L. et al., supra).

[0439] In some embodiments, the retroviral nucleic acid contains a scaffold / matrix attachment region (S / MAR) or a variant thereof, e.g., a non-consensus-like AT-rich DNA element several hundred base pairs in length, which organizes the nuclear DNA of eukaryotic genomes into chromatin domains by intermittent attachment to the protein scaffold or matrix of the cell nucleus. They are typically found in flanking regions, chromatin boundary regions, and non-coding regions such as introns. An example of an S / MAR region is the S / MAR region of the human IFN-γ gene (hIFN-γ) reported by Bode et al. (Bode J. et al., Science, 1992, 255:195-7). large ), a 1.8 kbp S / MAR of the human IFN-γ gene (hIFN-γ short) and the 0.2 Kbp minimal region of the S / MAR of the human dehydrofolate reductase gene (hDHFR) reported by Mesner LD et al., Proc Natl Acad Sci USA, 2003, 100:3281-86. In embodiments, functionally equivalent variants of S / MARs are sequences selected based on a set of six rules that have been suggested, jointly or individually, to contribute to S / MAR function (Kramer et al. (1996) Genomics 33, 305; Singh et al. (1997) Nucl. Acids Res 25, 1419). These rules have been incorporated into the MAR-Wiz computer program, freely available at genomecluster.secs.oakland.edu / MAR-Wiz. In embodiments, variants substantially maintain the same function of the S / MAR from which they are derived, in particular the ability to specifically bind to the nucleus or matrix. Those skilled in the art can determine whether a particular mutant can specifically bind to the nuclear matrix, for example, by using the in vitro or in vivo MAR assay reported by Mesner et al. (Mesner LD et al., supra). In some embodiments, a particular sequence is an S / MAR mutant if the mutant exhibits a tendency toward DNA strand separation. This property can be determined using specific programs based on equilibrium statistical mechanics methods. The stress-induced duplex destabilization (SIDD) analysis method is defined in Bode et al. (2005) J. Mol. Biol. 358, 597 as follows: "[...] calculates the degree to which an applied level of high-order helical stress reduces the free energy required to open the duplex at each position along each DNA sequence. The results are expressed as a SIDD profile, with sites of strong destabilization indicated by deep minima [...]" The SIDD algorithm and mathematical foundations (Bi and Benham (2004) Bioinformatics 20, 1477), as well as analysis of SIDD profiles, can be performed using the freely available internet resource WebSIDD (www.genomecenter.ucdavis.edu / benham).Thus, in some embodiments, a polynucleotide is considered a variant of an S / MAR sequence if it exhibits a similar SIDD profile as the S / MAR.

[0440] Fusogens and pseudotyping Fusogens contain viral envelope proteins (env) and generally determine the range of host cells that can be infected and transformed by fusosomes. For lentiviruses such as HIV-1, HIV-2, SIV, FIV, and EIV, native env proteins include gp41 and gp120. In some embodiments, the viral env proteins expressed by the source cells described herein are encoded on a vector separate from the viral gag and pol genes, as previously reported.

[0441] Examples of retroviral env genes that can be used include, but are not limited to, MLV envelope, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV (avian plague virus), and influenza virus envelope. Similarly, genes encoding envelopes from RNA viruses (e.g., Picoronavirus, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Birnaviridae, and Retroviridae RNA viruses) and DNA viruses (Hepadnaviridae, Circoviridae, Parvoviridae, Papoviridae, Adenoviridae, Herpesviridae, Poxyviridae, and Iridoviridae families) can be used. Representative examples include FeLV, VEE, HFVW, WDSV, SFV, rabies, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10, and EIAV.

[0442] In some embodiments, envelope proteins for presentation in 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 viruses, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, any virus of the Norwalk virus group, enteric adenovirus, parvovirus, dengue virus, monkey flu, or the like. Lyssaviruses such as rabies virus, Mononegavirus, and rabies virus, Lagos bat virus, Mokola virus, Dubenhage virus, European bat virus 1 and 2, Australian bat virus, ephemerovirus, vesiculovirus, vesicular stomatitis virus (VSV), herpesviruses such as herpes simplex virus types 1 and 2, varicella zoster, cytomegalovirus, Epstein-Barr virus (EBV), human herpesvirus (HHV), and human herpesvirus types 6 and 8. arenaviruses such as human immunodeficiency virus (HIV), papillomavirus, murine gammaherpesvirus, and Argentine hemorrhagic fever virus; Bunyaviridae such as Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphocytic choriomeningitis virus (LCMV), and Crimean-Congo hemorrhagic fever virus; Filoviridae (filoviruses) including hantaviruses, hemorrhagic fever viruses that cause renal syndrome, Rift Valley fever virus, Ebola hemorrhagic fever, and Marburg hemorrhagic fever; Flaviviridae including Kaisanur Forest disease virus; Paramyxoviridae such as Omsk hemorrhagic fever virus, viruses that cause tick-borne encephalitis, and Hendra and Nipah viruses; Alphaviridae such as Variola major and Variola minor (smallpox), Venezuelan equine encephalitis virus, and Eastern equine encephalitis virus; Western equine encephalitis virus; SARS-related coronavirus (SARS-CoV); West Nile virus; and any virus that causes encephalitis.

[0443] In some embodiments, the source cells described herein produce fusosomes, eg, recombinant retroviruses, eg, lentiviruses pseudotyped with VSV-G glycoprotein.

[0444] 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 the vesicular stomatitis virus G protein (VSV-G) envelope protein, allowing HIV to infect a wider range of cells, because the HIV envelope protein (encoded by the env gene) normally targets the virus to CD4+ presenting cells. In some embodiments, lentivirus envelope proteins are pseudotyped with VSV-G. In one embodiment, source cells produce recombinant retroviruses, e.g., lentiviruses, pseudotyped with VSV-G envelope glycoproteins.

[0445] Furthermore, fusogens or viral envelope proteins can be modified or engineered to contain polypeptide sequences that enable transduction vectors to target and infect host cells outside their normal range, or more specifically restrict transduction to cell or tissue types. For example, fusogens or envelope proteins can be linked in-frame to targeting sequences such as receptor ligands, antibodies (using recombinant antibody-type molecules such as antigen-binding portions of antibodies or single-chain antibodies), and the polypeptide portion or modifications thereof (e.g., if glycosylation sites are present in the targeting sequence) when presented on the transduction vector coat facilitate direct delivery of virion particles to the intended target cells. Furthermore, envelope proteins can further contain sequences that regulate cellular functions. By modulating cellular functions with transduction vectors, transduction efficiency can be increased or decreased for specific cell types in a mixed population of cells. For example, stem cells can be more specifically transduced with envelope sequences containing ligands or binding partners that specifically bind to stem cells rather than other cell types found in blood or bone marrow. Non-limiting examples include stem cell factor (SCF) and Flt-3 ligand. Other examples include, for example, antibodies (e.g., cell type-specific single chain antibodies) and essentially any antigen (including receptor) that binds to tissues such as lung, liver, pancreas, heart, endothelial, smooth, breast, prostate, epithelial, vascular cancers, etc.

[0446] Exemplary Fusogens In some embodiments, the retroviral vector or VLP comprises one or more fusogens, eg, to promote fusion of the retroviral vector or VLP with a membrane, eg, a cell membrane.

[0447] In some embodiments, retroviral vectors or VLPs contain one or more fusogens in their envelopes to target specific cell or tissue types. Fusogens include, but are not limited to, protein-based, lipid-based, and chemical-based fusogens. In some embodiments, retroviral vectors or VLPs contain a first fusogen that is a protein fusogen and a second fusogen that is a lipid fusogen or a chemical fusogen. The fusogens can bind to fusogen-binding partners on the surface of target cells. In some embodiments, retroviral vectors or VLPs containing fusogens integrate their membranes into the lipid bilayer of target cells.

[0448] In some embodiments, one or more of the fusogens described herein may be included in a retroviral vector or VLP.

[0449] Protein fusogens In some embodiments, the fusogen is a protein fusogen, e.g., a non-mammalian protein such as a mammalian protein or a homolog of a mammalian protein (e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity), a viral protein or a homolog of a viral protein (e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity), a naturally occurring protein or a derivative of a naturally occurring protein, a synthetic protein, a fragment thereof, a variant thereof, a protein comprising one or more of the fusogens or fragments, and any combination thereof.

[0450] In some embodiments, the fusogen causes mixing between lipids in the retroviral vector or VLP and lipids in the target cell, hi some embodiments, the fusogen causes the formation of one or more pores between the interior of the retroviral vector or VLP and the cytosol of the target cell.

[0451] mammalian proteins In some embodiments, fusogens may comprise mammalian proteins, see Table 1. Examples of mammalian fusogens include, but are not limited to, SNARE family proteins such as vSNAREs and tSNAREs, syncytin proteins such as syncytin-1 (DOI:10.1128 / JVI.76.13.6442-6452.2002) and syncytin-2, myomaker (biorxiv.org / content / early / 2017 / 04 / 02 / 123158, doi.org / 10.1101 / 123158, doi:10.1096 / fj .201600945R, doi:10.1038 / nature12343), myomixer (www.nature.com / nature / journal / v499 / n7458 / full / nature12343.html, doi:10.1038 / nature12343), myomerger (science.sciencemag.org / content / early / 2017 / 04 / 05 / science.aam9361 , DOI:10.1126 / science.aam9361), FGFRL1 (fibroblast growth factor receptor-like 1), minion (doi.org / 10.1101 / 122697), isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (disclosed, for example, in U.S. Pat. No. 6,099,857A), gag-linked proteins such as connexin 43, connexin 40, connexin 45, connexin 32, or connexin 37 (disclosed, for example, in U.S. Pat. No. 2007 / 024499). Examples of fusogens include Hap2, any protein capable of inducing syncytin formation between heterologous cells (see Table 2), any protein with fusogenic properties (see Table 3), their homologs, fragments, variants, and protein fusions comprising one or more of these proteins or fragments, as disclosed in US Pat. No. 2,4176. In some embodiments, the fusogen is encoded by a human endogenous retroviral element (hERV) found in the human genome.Additional exemplary fusogens are disclosed in US Pat. No. 6,099,857A and US Pat. No. 2007 / 0224176, the entire contents of which are incorporated herein by reference. [Table 1] [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2]

[0452] In some embodiments, the retroviral vector or VLP comprises a curvature-generating protein, such as epsin 1, dynamin, or a protein containing a BAR domain (see, e.g., Kozlov et al., CurrOp StrucBio 2015, Zimmerberg et al., Nat Rev 2006, Richard et al., Biochem J 2011).

[0453] Non-mammalian proteins viral proteins In some embodiments, the fusogen may comprise a non-mammalian protein, such as a viral protein. In some embodiments, the viral fusogen is a class I viral membrane fusion protein, a class II viral membrane protein, a class III viral membrane fusion protein, a viral membrane glycoprotein, or other viral fusion protein, or a homolog thereof, a fragment thereof, a mutant thereof, or a protein fusion comprising one or more proteins or fragments thereof.

[0454] In some embodiments, class I viral membrane fusion proteins include, but are not limited to, baculovirus F proteins, such as F proteins of the genus nuclear polyhedrosis virus (NPV), e.g., beet armyworm MNPV (SeMNPV) F protein and gypsy moth MNPV (LdMNPV), and paramyxovirus F proteins.

[0455] In some embodiments, class II viral membrane proteins include, but are not limited to, tick-borne osteoencephalitis E (TBEVE), Semliki Forest virus E1 / E2.

[0456] In some embodiments, class III viral membrane fusion proteins include, but are not limited to, rhabdovirus G (e.g., fusion protein G of vesicular gastritis virus (VSV-G)), herpesvirus glycoprotein B (e.g., herpes simplex virus 1 (HSV-1) gB)), Epstein-Barr virus glycoprotein B (EBVgB), Thogotovirus G, baculovirus gp64 (e.g., Autographa californica multiplex NPV (AcMNPV) gp64), and Borna disease virus (BDV) glycoprotein (BDVG).

[0457] Examples of other viral fusogens, e.g., membrane glycoproteins and viral fusion proteins, include, but are not limited to, viral syncytial proteins such as influenza hemagglutinin (HA) or variants, or fusion proteins thereof, human immunodeficiency virus type 1 envelope protein (HIV-1 ENV), gp120 from HIV-associated LFA-1 that forms lymphocyte syncytia, HIV gp41, HIV gp160, or HIV transcriptional transactivator (TAT), viral glycoprotein VSV-G, viral glycoprotein from vesicular stomatitis virus of the Rhabdoviridae family, glycoproteins gB and gH-gL of varicella-zoster virus (VZV), murine leukemia virus (MLV)-10A1, gibbon ape leukemia virus glycoprotein (GaLV), G-type glycoproteins of rabies, mokola, vesicular stomatitis virus, and togavirus. Murine hepatitis virus JHM surface spike protein, porcine respiratory coronavirus spike and membrane glycoprotein, avian infectious bronchitis spike glycoprotein and its precursor, bovine enteric coronavirus spike protein, F and H, HN or G genes of measles virus, canine distemper virus, Newcastle disease virus, human parainfluenza virus 3, simian virus 41, Sendai virus and human respiratory syncytial virus, human herpesvirus type 1 with chaperone protein gL and gH of varicella-zoster virus, human, bovine, and cercopithecoid herpes Virus gB, Friend Murine Leukemia Virus and Mason-Pfizer Monkey Virus envelope glycoproteins, mumps virus hemagglutinin neuraminidase, and glioproteins F1 and F2, Venezuelan Equine Encephalomyelitis membrane glycoprotein, paramyxovirus F protein, SIV gp160 protein, Ebola virus G protein, or Sendai virus fusion protein, or homologs thereof, fragments thereof, mutants thereof, and protein fusions comprising one or more of these proteins or fragments thereof.

[0458] Non-mammalian fusogens include viral fusogens, their homologs, fragments thereof, and fusion proteins containing one or more of these proteins or 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 structure with a signature trimer of α-helical hairpins with a central coiled-coil structure. Class I viral fusion proteins include proteins with a central post-fusion six-helix bundle. Class I viral fusion proteins include influenza HA, parainfluenza F, HIV Env, Ebola GP, hemagglutinin from orthomyxoviruses, F proteins from paramyxoviruses (e.g., measles, (Katoh et al., BMC Biotechnology 2010, 10:37)), ENV proteins from retroviruses, and fusogens from filoviruses and coronaviruses. In embodiments, class II viral fusogens, such as dengue E glycoprotein, have a structural signature of a beta sheet that forms an elongated ectodomain that refolds to form a trimer of hairpins. In embodiments, class II viral fusogens lack a central coiled coil. Class II viral fusogens can be found in alphaviruses (e.g., E1 protein) and flaviviruses (e.g., E glycoprotein). Class II viral fusogens include fusogens from Semliki Forest virus, Sinbis virus, rubella virus, and dengue virus. In embodiments, class III viral fusogens, such as vesicular stomatitis virus G glycoprotein, combine the structural signatures found in classes I and II. In embodiments, class III viral fusogens contain an alpha helix (e.g., forming a six-helix bundle to fold the protein, similar to class I viral fusogens) and a beta sheet with an amphipathic fusion peptide at its terminus, reminiscent of class II viruses. Class III viral fusogens can be found in rhabdoviruses and herpesviruses.In embodiments, the Class IV viral fusogen is a fusion-associated small transmembrane (FAST) protein (doi:10.1038 / sj.emboj.7600767; Nesbitt, Rae L., "Targeted Intracellular Therapeutic Delivery Using Liposomes Formulated with Multifunctional FAST proteins" (2012) Electronic Thesis and Dissertation Repository. Paper 388), encoded by a non-enveloped reovirus. In embodiments, the Class IV viral fusogen is small enough not to form a hairpin (doi:10.1146 / annurev-cellbio-101512-122422; doi:10.1016 / j.devcel.2007.12.008).

[0459] 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.

[0460] In some embodiments, the fusogen is a Poxviridae fusogen.

[0461] Additional exemplary fusogens are disclosed in US9,695,446, US2004 / 0028687, US6,416,997, US7,329,807, US2017 / 0112773, US2009 / 0202622, WO2006 / 027202, and US2004 / 0009604, all of which are incorporated by reference in their entireties.

[0462] In some embodiments, a fusogen described herein comprises an amino acid sequence in Table 4, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion of a sequence, e.g., a portion 100, 200, 300, 400, 500, or 600 amino acids in length. For example, in some embodiments, a fusogen described herein comprises an amino acid sequence having at least 80% identity to any of the amino acid sequences in Table 4. In some embodiments, the nucleic acid sequences described herein encode an amino acid sequence in Table 4, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion of the sequence, e.g., a portion that is 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0463] In some embodiments, a fusogen described herein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-56, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion of the sequence, e.g., a portion 100, 200, 300, 400, 500, or 600 amino acids in length, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. For example, in some embodiments, a fusogen described herein comprises an amino acid sequence having at least 80% identity thereto. In some embodiments, the nucleic acid sequences described herein encode an amino acid sequence set forth in any one of SEQ ID NOs: 1-56, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion of the sequence, e.g., a portion that is 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] Table 4-8 Table 4-9 Table 4-10 Table 4-11 Table 4-12 Table 4-13 Table 4-14 Table 4-15 Table 4-16 Table 4-17 Table 4-18 Table 4-19 Table 4-20 Table 4-21 Table 4-22 Table 4-23 Table 4-24 [Table 4-25] [Table 4-26] [Table 4-27] [Table 4-28]

[0464] In some embodiments, a fusogen described herein comprises an amino acid sequence in Table 5, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion of a sequence, e.g., an amino acid sequence 100, 200, 300, 400, 500, or 600 amino acids in length, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. For example, in some embodiments, a fusogen described herein comprises an amino acid sequence having at least 80% identity to any of the amino acid sequences in Table 5. In some embodiments, the nucleic acid sequences described herein encode an amino acid sequence in Table 5, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion of the sequence, e.g., a portion that is 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0465] In some embodiments, a fusogen described herein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 57-132, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion of the sequence, e.g., a portion 100, 200, 300, 400, 500, or 600 amino acids in length, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. For example, in some embodiments, a fusogen described herein comprises an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 57-132. In some embodiments, the nucleic acid sequences described herein encode an amino acid sequence set forth in any one of SEQ ID NOs: 57-132, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion of the sequence, e.g., a portion that is 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] Table 5-8 Table 5-9 Table 5-10 Table 5-11 Table 5-12 Table 5-13 Table 5-14 Table 5-15 Table 5-16 Table 5-17 Table 5-18 Table 5-19 Table 5-20 Table 5-21 Table 5-22 Table 5-23 Table 5-24 [Table 5-25] [Table 5-26] [Table 5-27] [Table 5-28] [Table 5-29] [Table 5-30] [Table 5-31] [Table 5-32] [Table 5-33] [Table 5-34] [Table 5-35] [Table 5-36] [Table 5-37] [Table 5-38]

[0466] Other proteins In some embodiments, the fusogen may include a pH-dependent protein, a homolog thereof, a fragment thereof, or a protein fusion comprising one or more of the protein or fragment thereof. The fusogen may mediate membrane fusion at the cell surface or in an endosome or another membrane-bound space.

[0467] In some embodiments, fusogens include EFF-1, AFF-1, gap junction proteins such as connexins (Cn43, GAP43, CX43, etc.) (DOI: 10.1021 / jacs.6b05191), other tumor junction proteins, their homologs, their fragments, their mutants, and protein fusions comprising one or more of these proteins or fragments thereof.

[0468] Lipid fusogens In some embodiments, the retroviral vector or VLP can comprise one or more fusogenic lipids, such as saturated fatty acids. In some embodiments, the saturated fatty acids have 10-14 carbons. In some embodiments, the saturated fatty acids have longer chain carboxylic acids. In some embodiments, the saturated fatty acids are monoesters.

[0469] In some embodiments, the retroviral vector or VLP can comprise one or more unsaturated fatty acids. In some embodiments, the unsaturated fatty acids are C16-C18 unsaturated fatty acids. In some embodiments, the unsaturated fatty acids include oleic acid, glycerol monooleate, glycerides, diacylglycerols, modified unsaturated fatty acids, and any combination thereof.

[0470] Without wishing to be bound by theory, in some embodiments, negatively curved lipids promote membrane fusion. In some embodiments, a retroviral vector or VLP contains one or more negatively curved lipids, e.g., exogenous negatively curved lipids, within its membrane. In embodiments, negatively curved lipids or their precursors are added to media containing source cells, retroviral vectors, or VLPs. In embodiments, source cells are engineered to express or overexpress one or more lipid synthesis genes. The negatively curved lipids can be, for example, diacylglycerol (DAG), cholesterol, phosphatidic acid (PA), phosphatidylethanolamine (PE), or fatty acids (FA).

[0471] Without wishing to be bound by theory, in some embodiments, positively curved lipids inhibit membrane fusion. In some embodiments, the retroviral vector or VLP comprises a reduced level of one or more positively curved lipids, for example, exogenous positively curved lipids, in the membrane. In embodiments, the level is reduced by inhibiting lipid synthesis in the source cell, for example, by knocking out or knocking down a lipid synthesis gene. The positively curved lipid can be, for example, lysophosphatidylcholine (LPC), phosphatidylinositol (PtdIns), lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), or monoacylglycerol (MAG).

[0472] Chemical substance fusogen In some embodiments, the retroviral vector or VLP may be treated with a fusogenic chemical, hi some embodiments, the fusogenic chemical is polyethylene glycol (PEG) or a derivative thereof.

[0473] In some embodiments, the chemical fusogen induces localized dehydration between the two membranes, resulting in unfavorable molecular packing of the bilayer. In some embodiments, the chemical fusogen induces dehydration in the region near the lipid bilayer, causing the exclusion of aqueous molecules between the two membranes and allowing the two membranes to interact with each other.

[0474] In some embodiments, the chemical fusogen is a positive cation. Some non-limiting examples of positive cations include Ca, Mg, Mn, Zn, La, Sr, and H.

[0475] In some embodiments, chemical fusogens bind to target membranes by altering surface polarity, changing hydration-dependent intermembrane repulsion.

[0476] In some embodiments, the chemical fusogen is a soluble lipid that can be dissolved in. Some non-limiting examples include oleoylglycerol, dioleoylglycerol, trioleoylglycerol, and variants and derivatives thereof.

[0477] In some embodiments, the chemical fusogen is a water-soluble chemical. Some non-limiting examples include polyethylene glycol, dimethyl sulfoxide, and variants and derivatives thereof.

[0478] In some embodiments, the chemical fusogen is a small organic molecule. A non-limiting example includes n-hexyl bromide.

[0479] In some embodiments, the chemical fusogen does not alter the composition, cell viability, or ion transport properties of the fusogen or target membrane.

[0480] In some embodiments, the chemical fusogen is a hormone or vitamin. Some non-limiting examples include abscisic acid, retinol (vitamin A1), tocopherol (vitamin E), and variants and derivatives thereof.

[0481] In some embodiments, the retroviral vector or VLP comprises an agent that stabilizes actin and polymerized actin. Without wishing to be bound by theory, stabilized actin in the retroviral vector or VLP can promote fusion with target cells. In embodiments, the agent that stabilizes polymerized actin is selected from actin, myosin, biotin-streptavidin, ATP, neural Wiskott-Aldrich syndrome protein (N-WASP), or formin. See, e.g., Langmuir. 2011 Aug16;27(16):10061-71 and We et al., NatCommun. 2016 Aug31;7. In embodiments, the retroviral vector or VLP comprises exogenous actin, e.g., wild-type actin or actin containing a mutation that promotes polymerization. In embodiments, the retroviral vector or VLP comprises ATP or phosphocreatine, e.g., exogenous ATP or phosphocreatine.

[0482] Small molecule fusogens In some embodiments, retroviral vectors or VLPs can be treated with fusogenic small molecules, some non-limiting examples of which include halothane, the nonsteroidal anti-inflammatory drugs (NSAIDs) meloxicam, piroxicam, tenoxicam, and chlorpromazine.

[0483] In some embodiments, small molecule fusogens may exist in micelle-like aggregates or without aggregates.

[0484] Mutations in protein fusogens Protein fusogens or viral envelope proteins can be retargeted by mutating amino acid residues in the fusion protein or target protein (e.g., hemagglutinin protein). In some embodiments, the fusogens are randomly mutated. In some embodiments, the fusogens are rationally mutated. In some embodiments, the fusogens are subjected to directed evolution. In some embodiments, the fusogens are truncated and only a subset of the peptides are used in the retroviral vector or VLP. For example, amino acid residues in the measles hemagglutinin protein can be mutated to change the binding properties of the protein and redirect fusions (doi:10.1038 / nbt942, Molecular Therapy vol. 16 no. 8, 1427-1436 Aug. 2008, doi:10.1038 / nbt1060, DOI:10.1128 / JVI.76.7.3558-3563.2002, DOI:10.1128 / JVI.75.17.8016-8020.2001, doi:10.1073pnas.0604993103).

[0485] Protein fusogens can be retargeted by covalently linking a targeting moiety to a fusion protein or targeting protein (e.g., hemagglutinin protein). In some embodiments, the fusion and targeting moiety are covalently linked by expression of a chimeric protein comprising a fusogen linked to a targeting moiety. Targets include any peptide (e.g., receptor) that is presented on target cells. In some examples, the target is expressed at a higher level in target cells than in non-target cells. For example, single-chain variable fragments (scFv) can be conjugated to fusogens to redirect fusogenic activity to cells that present the scFv-binding target (doi:10.1038 / nbt1060, DOI10.1182 / blood-2012-11-468579, doi:10.1038 / nmeth.1514, doi:10.1006 / mthe.2002.0550, HUMAN GENETHERAPY11:817-826, doi:10.1038 / nbt942, doi:10.1371 / journal.pone.0026381, DOI10.1186 / s12896-015-0142-z). For example, engineered ankyrin repeat proteins (DARPins) can be conjugated to fusogens to redirect fusion activity to cells presenting DARPin-binding targets (doi:10.1038 / mt.2013.16, doi:10.1038 / mt.2010.298, doi:10.4049 / jimmunol.1500956), as well as combinations of different DARPins (doi:10.1038 / mto.2016.3). For example, receptor ligands and antigens can be conjugated to fusogens to redirect fusogenic activity to cells presenting target receptors (DOI:10.1089 / hgtb.2012.054, DOI:10.1128 / JVI.76.7.3558-3563.2002).Target proteins can also include, for example, antibodies or antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single-domain antibodies such as sdAbs (VL or VH), nanobodies, or camelid VHH domains), antigen-binding fibronectin type III (Fn3) scaffolds such as fibronectin polypeptide minibodies, ligands, cytokines, chemokines, or T-cell receptors (TCRs). Protein fusogens can be retargeted by non-covalently attaching a targeting moiety to the fusion protein or targeting protein (e.g., hemagglutinin protein). For example, fusion proteins can be engineered to bind to the Fc region of an antibody that targets an antigen on a target cell, redirecting fusion activity toward cells that present the antibody's target (DOI:10.1128 / JVI.75.17.8016-8020.2001, doi:10.1038 / nm1192). Modified and unmodified fusogens can be presented in the same retroviral vector or VLP (doi:10.1016 / j.biomaterials.2014.01.051).

[0486] Targeting moieties can be, for example, humanized antibody molecules, intact IgA, IgG, IgE or IgM antibodies, bispecific or multispecific antibodies (e.g., Zybodies®, etc.), antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof, single chain Fvs, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), camelid antibodies, masked antibodies (e.g., Probodies®), Small Modular Antibodies ImmunoPharmaceuticals ("SMIPs™"), single chain or tandem biantibodies (TandAb®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTEs®, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MocroProteins, Fynomers®, Centyrins®, and KALBITOR®.

[0487] In embodiments, the retargeted fusogen binds to a cell surface marker on the target cell, such as a protein, glycoprotein, receptor, cell surface ligand, agonist, lipid, sugar, class I transmembrane protein, class II transmembrane protein, or class III transmembrane protein.

[0488] Retroviral vectors or VLPs may display a targeting moiety that is not bound to a protein fusogen to redirect fusion activity to cells bound by the targeting moiety or to affect homing.

[0489] The targeting moiety added to a retroviral vector or VLP can be adjusted to have different binding strengths. For example, scFvs and antibodies with various binding strengths can be used to change the fusion activity of a retroviral vector or VLP for cells that present large or small amounts of target antigen (doi:10.1128 / JVI.01415-07, doi:10.1038 / cgt.2014.25, doi:10.1002 / jgm.1151). For example, DARPins with different affinities can be used to change the fusion activity of a retroviral vector or VLP for cells that present large or small amounts of target antigen (doi:10.1038 / mt.2010.298). The targeting moiety can also be adjusted to target different regions on the target ligand, affecting the fusion rate with cells that present the target (doi:10.1093 / protein / gzv005).

[0490] In some embodiments, protein fusogens can be altered to reduce immune reactivity, e.g., as described herein. For example, protein fusogens can be decorated with molecules that reduce immune interactions, such as PEG (DOI:10.1128 / JVI.78.2.912-921.2004). Thus, in some embodiments, the fusogen comprises PEG, e.g., a PEGylated polypeptide. Amino acid residues in fusogens that are targeted by the immune system can be altered so that they are not recognized by the immune system (doi:10.1016 / j.virol.2014.01.027, doi:10.1371 / journal.pone.0046667). In some embodiments, the protein sequence of the fusogen is altered to resemble an amino acid sequence found in humans (humanization). In some embodiments, the protein sequence of the fusogen is altered to a protein sequence that binds less strongly to the MHC complex. In some embodiments, the protein fusogen is derived from a virus or organism that does not infect humans (and to which humans have not been vaccinated), increasing the likelihood that the patient's immune system is naive to the protein fusogen (e.g., there is a negligible humoral or cell-mediated adaptive immune response to the fusogen) (doi:10.1006 / mthe.2002.0550, doi:10.1371 / journal.ppat.1005641, doi:10.1038 / gt.2011.209, DOI10.1182 / blood-2014-02-558163). In some embodiments, the glycosylation of the fusogen may be altered to alter immune interactions or reduce immune reactivity. While not wishing to be bound by theory, in some embodiments, a protein fusogen derived from a virus or organism that does not infect humans has no natural fusion targets in the patient and therefore has high specificity.

[0491] Positive target cell-specific regulatory elements In some embodiments, the retroviral nucleic acids described herein comprise positive target cell-specific regulatory elements, such as a tissue-specific promoter, a tissue-specific enhancer, a tissue-specific splice site, a tissue-specific site that extends the half-life of an RNA or protein, a tissue-specific mRNA export-facilitating site, a tissue-specific translation-enhancing site, or a tissue-specific post-translational modification site.

[0492] The retroviral nucleic acids described herein contain regions that interact with host cells to carry out transcription and translation, such as origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine-Dalgarno or Kozak sequences), introns, and untranslated regions such as 5' and 3' untranslated regions, which can direct, increase, regulate, or control the transcription or expression of operably linked polynucleotides. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters, can be used.

[0493] In certain embodiments, the control element is capable of directing, increasing, regulating, or controlling the transcription or expression of an operably linked polynucleotide in a cell-specific manner. In certain embodiments, the retroviral nucleic acid comprises one or more expression control sequences that are specific for a particular cell, cell type, or cell lineage, e.g., a target cell, i.e., expression of a polynucleotide operably linked to an expression control sequence that is specific for a particular cell, cell type, or cell lineage is expressed in the target cell and not (or at a lower level) in non-target cells.

[0494] In certain embodiments, the retroviral nucleic acid can include exogenous, endogenous, or heterologous regulatory sequences, such as promoters and / or enhancers.

[0495] In embodiments, a promoter contains a recognition site for binding of an RNA polymerase, which initiates and transcribes a polynucleotide operably linked to the promoter. In certain embodiments, a promoter that functions in mammalian cells contains an AT-rich region located approximately 25-30 bases upstream from the site where transcription begins, and / or another sequence, a CNCAAT region, where N can be any nucleotide, found approximately 70-80 bases upstream from the start of transcription.

[0496] In embodiments, an enhancer comprises a segment of DNA containing a sequence that can provide enhanced transcription and, in some instances, can function independently of the adaptation to another regulatory sequence. Enhancers can function cooperatively or additively with promoters and / or other enhancer elements. In some embodiments, a promoter / enhancer segment of DNA comprises a sequence that can provide both promoter and enhancer function.

[0497] Exemplary ubiquitous expression control sequences include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the viral simian virus 40 (SV40) (e.g., early or late), Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5, and P11 promoters from vaccinia virus, elongation factor 1-alpha (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), These include heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), beta-kinesin (β-KIN), human ROSA26 locus (Orions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken beta-actin (CAG) promoter, beta-actin promoter and myeloproliferative sarcoma virus enhancer, deleted negative control region, and d1587rev primer binding site substitution (MND) promoter (Challit et al., J Virol. 69(2):748-55 (1995)).

[0498] In some embodiments, the promoter is a tissue-specific promoter, e.g., a promoter that promotes expression in liver cells, e.g., hepatocytes, liver sinusoidal endothelial cells, bile duct cells, stellate cells, liver-resident antigen-presenting cells (e.g., Kupffer cells), liver-resident immune lymphocytes (e.g., T cells, B cells, or NK cells), or portal vein fibroblasts. Various suitable liver-specific promoters (e.g., hepatocyte-specific promoters and liver sinusoidal endothelial cell promoters) are listed in Table 6 below. Table 6 also lists several ubiquitous promoters that are not specific to liver cells. In some embodiments, the fusosomes (e.g., viral vectors) described herein comprise in their nucleic acid a promoter having a sequence in Table 6, 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, a fusosome (e.g., a viral vector) described herein comprises in its nucleic acid a promoter having a transcription factor binding site from a region within 3 kb of the transcription start site for a gene listed in Table 6. In some embodiments, a fusosome (e.g., a viral vector) described herein comprises 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 a gene listed in Table 6, or a transcriptionally active fragment thereof, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0499] In some embodiments, a fusosome (e.g., a viral vector) described herein comprises in its nucleic acid a promoter having a sequence set forth in any one of SEQ ID NOs: 133-142 or 161-168, or a transcriptionally active fragment thereof, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9] [Table 6-10] [Table 6-11]

[0500] Various consensus sequences within liver-specific cis-regulatory modules (e.g., promoters) have been described. In some embodiments, the liver-specific cis-regulatory module comprises binding sites for one or more of HNF1α, C / EBP, LEF1, FOX, IRF, LEF1 / TCF, Tal1β / E47, and MyoD. In some embodiments, the liver-specific cis-regulatory module is described in Chuah et al., "Liver-Specific Transcriptional Modules Identified by Genome-Wide In Silico Analysis Enable Efficient Gene Therapy in Mice and Non-Human Primates,” Mol Ther. 2014 Sep;22(9):1605-1613, the entire contents of which are incorporated herein by reference, including the sequences in FIG. 1 and Table 1 thereof. In some embodiments, the liver-specific cis-regulatory module comprises the human sequence of HS-CRM1, HS-CRM2, HS-CRM3, HS-CRM4, HS-CRM5, HS-CRM6, HS-CRM7, HS-CRM8, HS-CRM9, HS-CRM10, HS-CRM11, HS-CRM12, HS-CRM13, or HS-CRM14, as described in Dhuah et al., supra.

[0501] Internal ribosome entry sites (IRES) typically facilitate direct internal ribosome entry at an initiation codon, such as ATG, of a cistron (protein-coding region), thereby resulting in cap-independent translation of the gene. See, e.g., Jackson et al. (1990) Trends See 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 the multiple exogenous protein agents, the polynucleotide sequences can be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides.

[0502] The retroviral nucleic acid herein may also contain one or more Kozak sequences, e.g., short nucleotide sequences that promote initial binding of mRNA to the small ribosomal subunit and increase translation. The consensus Kozak sequence is (GCC)RCCATGG, where R is a purine (A or G) (Kozak, (1986) Cell. 44(2):283-92, and Kozak, (1987) Nucleic Acids Res. 15(20):8125-48).

[0503] Promoters that respond to heterologous transcription factors and inducers In some embodiments, the retroviral nucleic acid allows for conditional expression of the exogenous agent, for example, any type of conditional expression, including, but not limited to, inducible expression, repressible expression, cell type-specific expression, or tissue-specific expression. In some embodiments, to achieve conditional expression of the exogenous agent, expression is controlled by subjecting the cell, tissue, or organism to a treatment or condition that causes expression of the exogenous agent or results in increased or decreased expression of the exogenous agent.

[0504] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormones), metallothionine promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "gene switch" mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO2002 / 088346), tetracycline-dependent regulatory systems, and the like.

[0505] Transgene expression can be activated or repressed by the presence or absence of an inducer molecule. In some instances, the inducer molecule activates or represses gene expression in a stepwise manner, and in some instances, the inducer molecule activates or represses gene expression in an all-or-nothing manner.

[0506] A commonly used inducible promoter / system is the tetracycline (Tet) regulatory system. The Tet system is based on the coexpression of two elements in each target cell: (i) a tetracycline response element (TET) containing repeats of the Tet operator sequence (TetO) fused to a minimal promoter and connected to a gene of interest (e.g., a gene encoding an exogenous drug), and (ii) a fusion protein of a transcriptional transactivator (tTA), a Tet repressor (TetR), and the transactivation domain of the VP16 protein from herpes simplex virus. In the first-described system, transgene expression is active in the absence of tetracycline or its potent analog, doxycycline (Dox), and is referred to as the Tet-OFF system. However, modification of four amino acids within the transactivator protein results in a reverse tTA (rtTA), which binds to TetO only in the presence of Dox (Tet-ON system). In some embodiments, the VP16 domain in the transactivator has been replaced with a minimal activation domain, potential splice donor and acceptor sites have been removed, and the protein has been codon-optimized, resulting in an improved transactivator variant, rtTA2S-M2, with higher sensitivity to Dox and lower baseline activity. Additionally, various Tet-responsive promoter elements have been generated, including modifications to the TetO 36 nucleotides away from the adjacent operator to enhance regulation. Additional modifications may be useful to further reduce basal activity and increase the dynamic range of expression. For example, the pTet-T11 (simply T1I) variant exhibits a high dynamic range and low background activity.

[0507] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments, the retroviral nucleic acid contains at least one (typically two) site(s) for recombination mediated by the site-specific recombinase, including, for example, excision or integrative proteins, enzymes, cofactors, or related proteins involved in the recombination reaction, including one or more recombination sites (e.g., 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 50, etc.), which may be wild-type proteins (Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing the recombinant protein sequence or fragments thereof), fragments, and mutants thereof. Illustrative 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.

[0508] Riboswitches that regulate the expression of exogenous drugs Some of the compositions and methods provided herein include one or more riboswitches or polynucleotides containing one or more riboswitches. Riboswitches are a common feature in bacteria that regulate gene expression and are a means of achieving RNA control of biological functions. Riboswitches can reside in the 5' untranslated region of mRNA and can enable regulatory control of gene expression through the binding of small molecule ligands that trigger or inhibit riboswitch activity. In some embodiments, riboswitches control gene products that involve the production of small molecule ligands. While riboswitches typically act in a cis-like manner, riboswitches have been identified that act in a trans-like manner. Natural riboswitches consist of two domains: an aptamer domain that binds to a ligand through a three-dimensionally folded RNA structure, and a function-switching domain that triggers or inhibits riboswitch activity based on the absence or presence of a ligand. Thus, there are two ligand-sensitive conformations achieved by riboswitches, representing an on state and an off state (Garst et al., 2011). Function-switching domains can affect polynucleotide expression by regulating internal ribosome entry sites, pre-mRNA splice donor accessibility in retroviral gene constructs, translation, transcription termination, transcript degradation, miRNA expression, or shRNA expression (Dambach and Winkler 2009). Aptamers and function-switching domains can be used as modular components, allowing synthetic RNA devices to control gene expression as either native aptamers, mutated / evolved native aptamers, or fully synthetic aptamers identified from screening random RNA libraries (McKeague et al. 2016).

[0509] The purine riboswitch family represents one of the largest families, with over 500 recognized sequences (Mandal et al. 2003, US2008 / 0269258, and WO2006 / 055351). Purine riboswitches share a similar architecture consisting of three conserved helical / stem structures (PI, P2, P3) with intervening loop / junction elements (J1-2, L2, J2-3, L3, J3-1). The aptamer domains of the purine family of riboswitches naturally vary in their affinity / regulation with various purine compounds, including adenine, guanine, adenosine, guanosine, deoxyadenosine, and deoxyguanosine, due to sequence variation (Kim et al. 2007).

[0510] In some embodiments, the retroviral nucleic acid described herein comprises a promoter and a polynucleotide encoding an exogenous agent operably linked to a riboswitch. The riboswitch comprises one or more of: a.) an aptamer domain, e.g., an aptamer domain capable of binding to a nucleoside analog antiviral agent and exhibiting reduced binding to guanine or 2'-deoxyguanosine compared to the nucleoside analog antiviral agent; and b.) a function switching domain, e.g., a function switching domain capable of regulating expression of an exogenous agent, wherein binding of the nucleoside analog by the aptamer domain induces or represses the expression-regulating activity of the function switching domain, thereby regulating expression of the exogenous agent. In some embodiments, the exogenous agent can be a polypeptide, miRNA, or shRNA. For example, in one embodiment, the riboswitch is operably linked to a nucleic acid encoding a chimeric antigen receptor (CAR). In the non-limiting illustrative example provided herein, the exogenous gene encodes one or more engineered signaling polypeptides. For example, a target polynucleotide encoding a riboswitch and one or more engineered signaling polypeptides can be found in the genome of a source cell, in a replication-incompetent recombinant retroviral particle, in a T cell and / or a NK cell.

[0511] Aptamer domains, for example, can be used as modular building blocks and can be combined with any of the function switching domains to affect RNA transcripts. In any of the embodiments disclosed herein, the riboswitch can affect RNA transcripts by regulating the activity of an internal ribosome entry site (IRES), pre-mRNA splice donor accessibility, translation, transcription termination, transcript degradation, miRNA expression, or shRNA expression. In some embodiments, the function switching domain can control the binding of an anti-IRES to an IRES (see, e.g., Ogawa, RNA (2011), 17:478-488, the disclosure of which is incorporated herein by reference in its entirety). In any of the embodiments disclosed herein, the presence or absence of a small molecule ligand can cause the riboswitch to affect RNA transcripts. In some embodiments, the riboswitch can comprise a ribozyme. A riboswitch with a ribozyme can inhibit or enhance transcript degradation of a target polynucleotide in the presence of a small molecule ligand. In some embodiments, the ribozyme can be of the pistol class of ribozymes, the hammerhead class of ribozymes, the twist class of ribozymes, the hatchet class of ribozymes, or HDV (hepatitis delta virus).

[0512] Non-target cell-specific regulatory elements In some embodiments, the non-target cell-specific regulatory element or negative TCSSE comprises a tissue-specific miRNA recognition sequence, a tissue-specific protease recognition site, a tissue-specific ubiquitin ligase site, a tissue-specific transcriptional repression site, or a tissue-specific epigenetic repression site.

[0513] In some embodiments, non-target cells contain endogenous miRNA. Retroviral nucleic acid (e.g., a gene encoding an exogenous drug) can contain the recognition sequence of that miRNA. Therefore, when retroviral nucleic acid enters non-target cells, miRNA can downregulate the expression of exogenous substances. This helps achieve specificity between target cells and non-target cells.

[0514] In some embodiments, miRNAs are small, 20-22 nucleotide non-coding RNAs that are typically excised from approximately 70 nucleotide foldback RNA precursor structures known as pre-miRNAs. Generally, miRNAs negatively regulate their targets in one of two ways, depending on the degree of complementarity between the miRNA and the target. First, miRNAs that bind with perfect or near-perfect complementarity to protein-coding mRNA sequences typically induce the RNA-mediated interference (RNAi) pathway. miRNAs exert their regulatory effects by binding to imperfectly complementary sites within the 3' untranslated region (UTR) of their mRNA targets, typically suppressing target gene expression post-transcriptionally, apparently at the translational level, via a RISC complex that may be similar or identical to that used in the RNAi pathway. Consistent with translational control, miRNAs using this mechanism reduce the protein levels of their target genes, while the mRNA levels of these genes are only minimally affected. miRNAs (e.g., naturally occurring miRNAs or artificially designed miRNAs) can specifically target any mRNA sequence. For example, in one embodiment, one skilled in the art can design a short hairpin RNA construct that is expressed as a human miRNA (e.g., miR-30 or miR-21) primary transcript. This design adds a Drosha processing site to the hairpin construct, which has been shown to significantly increase knockdown efficiency (Pusch et al., 2004). The hairpin stem consists of a 22-nt dsRNA (e.g., antisense with perfect complementarity to the desired target) and a 15-19-nt loop from a human miR. By adding the miR loop and miR30 flanking sequences to one or both sides of the hairpin, Drosha and Dicer processing of the expressed hairpin increases by more than 10-fold compared to conventional shRNA designs without a microRNA. Increased Drosha and Dicer processing increases siRNA / miRNA generation and potency for the expressed hairpin.

[0515] Hundreds of different miRNA genes are differentially expressed during development and across tissue types. Several studies have suggested important regulatory roles for miRNAs in a wide range of biological processes, including developmental timing, cell differentiation, proliferation, apoptosis, carcinogenesis, insulin secretion, and cholesterol biosynthesis. (See Bartel 2004 Cell 116:281-97; Ambros 2004 Nature 431:350-55; Duf et al. 2005 Development 132:4645-52; Chen 2005 N. Engl. J. Med. 353:1768-71; Krutzfeldt et al. 2005 Nature 438:685-89.) Molecular analyses have shown that miRNAs have distinct expression profiles in different tissues. Computational methods have been used to analyze the expression of approximately 7,000 predicted human miRNA targets. The data suggest that miRNA expression broadly contributes to the tissue specificity of mRNA expression in many human tissues. (See Sood et al. 2006 PNAS USA 103(8):2746-51.)

[0516] Thus, miRNA-based methods can be used to restrict the expression of exogenous agents to target cell populations by using endogenous microRNA species to silence expression of the exogenous agent in non-target cell types. MicroRNAs induce sequence-specific post-transcriptional gene silencing in many organisms by inhibiting messenger RNA (mRNA) translation or by causing mRNA degradation. See, for example, Brown et al. 2006 Nature Med. 12(5):585-91 and WO 2007 / 000668, each of which is incorporated herein by reference in its entirety. In some embodiments, the retroviral nucleic acid comprises one or more (e.g., multiple) tissue-specific miRNA recognition sequences. In some embodiments, the tissue-specific miRNA recognition sequence is approximately 20-25, 21-24, or 23 nucleotides in length. In some embodiments, the tissue-specific miRNA recognition sequence has perfect complementarity to a miRNA present in non-target cells. In some embodiments, the exogenous agent does not include GFP, e.g., does not include a fluorescent protein, e.g., does not include a reporter protein. In some embodiments, the off-target cells are not hematopoietic cells and / or the miRNA is not present in hematopoietic cells.

[0517] In some embodiments, the methods herein include tissue-specific expression of an exogenous agent in a target cell, and include contacting a plurality of retroviral vectors comprising nucleotides encoding the exogenous agent and at least one tissue-specific microRNA (miRNA) target sequence with a plurality of cells, including target cells and non-target cells, wherein the exogenous agent is preferentially, e.g., restricted to, expression in the target cell.

[0518] For example, the retroviral nucleic acid can include at least one miRNA recognition sequence operably linked to a nucleotide sequence having a corresponding miRNA in non-target cells, e.g., hematopoietic progenitor cells (HSPCs) or hematopoietic stem cells (HSCs), thereby preventing or reducing expression of the nucleotide sequence in the non-target cells but not in target cells, e.g., differentiated cells. In some embodiments, the retroviral nucleic acid includes at least one miRNA sequence targeting an miRNA present in the non-target cells in an effective amount (e.g., the concentration of the endogenous miRNA is sufficient to reduce or prevent expression of the transgene) and contains a transgene. In embodiments, the miRNA used in this system is strongly expressed in non-target cells, such as HSPCs and HSCs, but not in differentiated progeny, e.g., myeloid and lymphoid lineages, preventing or reducing transgene expression in susceptible stem cell populations while maintaining expression and therapeutic efficacy in target cells.

[0519] In some embodiments, the negative TSCRE or NTSCRE comprises an miRNA recognition site, e.g., an miRNA recognition site bound by an miRNA endogenous to hematopoietic cells. For example, the negative TSCRE or NTSCRE is a sequence complementary to an miRNA endogenous to hematopoietic cells. Exemplary miRNAs are shown in Table 7 below. In some embodiments, the nucleic acid (e.g., a fusomal or retroviral nucleic acid) is complementary to or has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementarity to an miRNA in Table 7. In some embodiments, the nucleic acid (e.g., a fusomal or retroviral nucleic acid) comprises a sequence that is fully complementary to a seed sequence in an endogenous miRNA, e.g., an miRNA in Table 7. In embodiments, the seed sequence is at least 6, 7, 8, 9, or 10 nucleotides in length.

[0520] In some embodiments, the nucleic acid (e.g., a fusomal or retroviral nucleic acid) comprises a sequence complementary to, or having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementarity with, an miRNA set forth in any one of SEQ ID NOs: 143-160. In some embodiments, the nucleic acid (e.g., a fusomal or retroviral nucleic acid) comprises a TSCRE or NTSCRE that comprises a sequence that is fully complementary to a seed sequence within an endogenous miRNA set forth in any one of SEQ ID NOs: 143-160. In embodiments, the seed sequence is at least 6, 7, 8, 9, or 10 nucleotides in length. [Table 7]

[0521] In some embodiments, the negative TSCRE or NTSCRE comprises an miRNA recognition site for a miRNA described herein. Exemplary miRNAs are described in Griffiths-Jon 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.2002Apr30;12(9):735-9, Calin et al., Proc Natl Acad Sci USA.2004Mar2;101(9):2999-3004, Sempere et al. Genome Biol.2004;5(3):R13, Metzler et al., Genes Chromosomes Cancer.2004Feb;39(2):167-9, Calin et al., Proc Natl Acad Sci USA. 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.

[0522] In some embodiments, the negative TSCRE or NTSCRE is miR-1b, miR-189b, miR-93, miR-125b, miR-130, miR-32, miR-128, miR-22, miR124a, miR-296, miR-143, miR-15, miR-141, miR-143, miR-16, miR-127, miR99a, miR-1 83, miR-19b, miR-92, miR-9, miR-130b, miR-21, miR-30b, miR-16, miR-142-s, miR-99a, miR-212, miR -30c, miR-213, miR-20, miR-155, miR-152, miR-139, miR-30b, miR-7, miR-30c, miR-18, miR-137, miR- 219, miR-1d, miR-178, miR-24, miR-122a, miR-215, miR-142-a, miR-223, miR-142, miR-124a, miR-19 0, miR-149, miR-193, miR-181, let-7a, miR-132, miR-27a, miR-9*, miR-200b, miR-266, miR-153, miR- and 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.

[0523] In some embodiments, a nucleic acid (e.g., a fusomal nucleic acid or a retroviral nucleic acid) comprises two or more miRNA recognition sites. In some embodiments, each of the two or more miRNA recognition sites is recognized by any of the miRNAs described herein, e.g., listed in Table 7. In some embodiments, each of the two or more miRNA recognition sites is recognized by an miRNA set forth in any one of SEQ ID NOS: 143-160. In some embodiments, the two or more miRNA recognition sites can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more miRNA recognition sites. Two or more miRNA recognition sites can be arranged in tandem within the nucleic acid to provide multiple tandem binding sites for miRNAs.

[0524] In some embodiments, the two or more miRNA recognition sites can comprise at least one first miRNA recognition site, such as 1, 2, 3, 4, 5, 6, or more first miRNA recognition sites, and at least one second miRNA recognition site, such as 1, 2, 3, 4, 5, 6, or more second miRNA recognition sites. In some embodiments, the nucleic acid comprises two or more first miRNA recognition sites, each of which is present in tandem in the nucleic acid to provide multiple tandem binding sites for the first miRNA, and / or the nucleic acid further comprises two second miRNA recognition sites, each of which is present in tandem in the nucleic acid to provide multiple tandem binding sites for the second miRNA. In some embodiments, the first miRNA recognition site and the second miRNA recognition site are recognized by the same miRNA, and in some embodiments, the first miRNA recognition site and the second miRNA recognition site are recognized by different miRNAs. In some embodiments, the first miRNA recognition site and the second miRNA recognition site are recognized by miRNAs present in the same non-target cell, and in some embodiments, the first miRNA recognition site and the second miRNA recognition site are recognized by miRNAs present in different non-target cells. In some embodiments, one or both of the first miRNA recognition site and the second miRNA recognition site are recognized by an miRNA, such as any of those set forth in Table 7, as described herein. In some embodiments, one or both of the first miRNA recognition site and the second miRNA recognition site are recognized by an miRNA set forth in any one of SEQ ID NOS: 143-160. In some embodiments, one or more of the miRNA recognition sites on a fusosomal nucleic acid (e.g., an egletroviral nucleic acid) are transcribed in cis by an exogenous agent. In some embodiments, one or more of the miRNA recognition sites on a fusosomal nucleic acid (e.g., a retroviral nucleic acid) are located downstream of a polyA tail sequence, e.g., between the polyA tail sequence and the WPRE.In some embodiments, one or more of the miRNA recognition sites on the fusomal nucleic acid (eg, retroviral nucleic acid) are located downstream of the WPRE.

[0525] immunomodulation In some embodiments, the retroviral vectors or VLPs described herein comprise elevated CD47. See, e.g., US9,050,269, incorporated herein by reference in its entirety. In some embodiments, the retroviral vectors or VLPs described herein comprise elevated complement regulatory proteins. See, e.g., ES2627445T3 and US6790641, each of which is incorporated herein by reference in its entirety. In some embodiments, the retroviral vectors or VLPs described herein comprise an absence or reduced level of an MHC protein, e.g., MHC-1 class 1 or class II. See, e.g., US2017 / 0165348, incorporated herein by reference in its entirety.

[0526] Retroviral vectors or VLPs are sometimes recognized by the immune system of a subject.In the case of enveloped viral vector particles (such as retroviral vector particles), the membrane-associated proteins displayed on the surface of the viral envelope can be recognized, and the viral particles themselves can be neutralized.Furthermore, after infecting target cells, the viral envelope is integrated with the cell membrane, and as a result, the viral envelope proteins can be displayed on the surface of the cell or remain tightly bound to the surface of the cell.Therefore, the immune system can also target the cells infected by viral vector particles.Both effects can result in a decrease in the effectiveness of exogenous drug delivery by viral vectors.

[0527] The viral particle envelope is usually derived from the membrane of the source cell, and therefore membrane proteins expressed on the cell membrane from which the viral particle protrudes can be incorporated into the viral envelope.

[0528] immunoregulatory protein CD47 The internalization of extracellular material into cells is generally accomplished 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 falls into two general categories: phagocytosis, which involves the uptake of particles, and pinocytosis, which involves the uptake of fluids and solutes.

[0529] Studies using knockout mice lacking the membrane receptor CD47 have shown that professional phagocytes distinguish self from non-self (Oldenborg et al., 2000, Science 288(5473):2051-4). CD47 is a ubiquitous member of the Ig superfamily that interacts with the immune inhibitory receptor SIRPα (signal-regulatory protein α), found on macrophages (Fujioka et al., 1996, Mol. Cell. Biol. 16(12):6887-99; Veillette et al., 1998, J. Biol. Chem. 273(35):22719-28; Jiang et al., 1999, J. Biol. Chem. 274(2):559-62). Although CD47-SIRPα interactions appear to inactivate autologous macrophages in mice, a significant reduction in CD47 (perhaps 90%) is observed on human blood cells derived from several Rh genotypes, which show little or no evidence of anemia (Mouro-Chanteloup et al., 2003, Blood 101(1):338-344) and little or no evidence of enhanced cellular interactions with phagocytic monocytes (Arndt et al., 2004, Br. J. Haematol. 125(3):412-4).

[0530] In some embodiments, the retroviral vector or VLP (e.g., a viral particle having a radius of less than about 1 μm, less than about 400 nm, or less than about 150 nm) comprises at least a biologically active portion of CD47, e.g., on an exposed surface of the retroviral vector or VLP. In some embodiments, the retroviral vector (e.g., lentivirus) or VLP comprises a lipid coating. In embodiments, the amount of biologically active CD47 in the retroviral vector or VLP is about 20-250, 20-50, 50-100, 100-150, 150-200, or 200-250 molecules / μm 2 In some embodiments, the CD47 is human CD47.

[0531] The methods described herein can include avoiding phagocytosis of particles by phagocytes. The methods can include expressing at least one peptide comprising at least a biologically active portion of CD47 in a retroviral vector or VLP, such that when the CD47-containing retroviral vector or VLP is exposed to a phagocytic cell, the viral particle avoids phagocytosis by the phagocyte or exhibits reduced phagocytosis compared to an otherwise similar unmodified retroviral vector or VLP. In some embodiments, the half-life of the retroviral vector or VLP in a subject is extended compared to an otherwise similar unmodified retroviral vector or VLP.

[0532] MHC deletion Major histocompatibility complex class I (MHC-I) is a host cell membrane protein that can be incorporated into the viral envelope and is a major target of the body's immune response due to its inherently highly polymorphic nature (McDevitt HO (2000) Annu. Rev. Immunol. 18:1-17). MHC-I molecules exposed to the plasma membrane of source cells can be incorporated into viral particle envelopes during the process of vector budding. These MHC-I molecules derived from source cells and incorporated into viral particles can then be transferred to the plasma membrane of target cells. Alternatively, MHC-I molecules may remain tightly associated with the target cell membrane as a result of the tendency of viral particles to absorb and remain attached to the target cell membrane.

[0533] The presence of exogenous MHC-I molecules on or near the plasma membrane of transduced cells can induce an alloreactive immune response in a subject. This can lead to immune-mediated killing or phagocytosis of the transduced cells, either with ex vivo gene transfer followed by administration of the transduced cells to a subject, or with direct in vivo administration of viral particles. Furthermore, in the case of in vivo administration of MHC-I bearing viral particles into the bloodstream, the viral particles can be neutralized by pre-existing MHC-I-specific antibodies before reaching their target cells.

[0534] Thus, in some embodiments, source cells are modified (e.g., genetically engineered) to reduce MHC-I expression on the cell surface. In embodiments, the source comprises a genetically engineered disruption of the gene encoding β2-microglobulin (β2M). In embodiments, the source cells comprise genetically engineered disruptions of one or more genes encoding MHC-Iα chains. The cells may comprise genetically engineered disruptions in all copies of the gene encoding β2-microglobulin. The cells may comprise genetically engineered disruptions in all copies of the gene encoding the MHC-Iα chain. The cells may comprise both a genetically engineered disruption of the gene encoding β2-microglobulin and a genetically engineered disruption of the gene encoding the MHC-Iα chain. In some embodiments, the retroviral vector or VLP comprises a reduced number of surface-exposed MHC-I molecules. The number of surface-exposed MHC-I molecules can be reduced so that the immune response to MHC-I is reduced to a therapeutically relevant extent. In some embodiments, the enveloped viral vector particle is substantially devoid of surface-exposed MHC-I molecules.

[0535] HLA-G / E overexpression In some embodiments, the retroviral vector or VLP displays on its envelope a tolerogenic protein, e.g., an ILT-2 or ILT-4 agonist, e.g., HLA-E or HLA-G, or any other ILT-2 or ILT-4 agonist. In some embodiments, the retroviral vector or VLP has increased expression of HLA-E, HLA-G, ILT-2, or ILT-4 compared to a control retrovirus, e.g., an unmodified but similar retrovirus.

[0536] In some embodiments, the retroviral composition has reduced MHC class I compared to unmodified retrovirus and increased HLA-G compared to unmodified retrovirus.

[0537] In some embodiments, the retroviral vector or VLP has an increase in HLA-G or HLA-E expression, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more increase in HLA-G or HLA-E expression, compared to a control retrovirus, e.g., an unmodified but similar retrovirus, wherein HLA-G or HLA-E expression is assayed in vitro using flow cytometry, e.g., FACS.

[0538] In some embodiments, retroviruses with increased HLA-G expression exhibit reduced immunogenicity in a teratoma formation assay, for example, as measured by reduced immune cell infiltration.

[0539] Complement regulatory proteins Complement activity is normally controlled by numerous complement regulatory proteins (CRPs). These proteins prevent pseudoinflammation and host tissue damage. One group of proteins, including CD55 / decay-accelerating factor (DAF) and CD46 / membrane cofactor protein (MCP), inhibits the C3 / C5 convertases of the classical and alternative pathways. Another set of proteins, including CD59, regulates MAC assembly. CRPs have been used to prevent rejection of xenotransplanted tissues and have also been shown to protect viruses and viral vectors from complement inactivation.

[0540] Membrane attack complement regulators include, for example, decay accelerating factor (DAF) or CD55, factor H (FH)-like protein-1 (FHL-1), C4b-binding protein (C4BP), complement receptor 1 (CD35), membrane core protein (MCP) or CD46, and CD59 (protectin) (e.g., to prevent formation of the membrane attack complex (MAC) and protect cells from lysis).

[0541] Albumin-binding proteins In some embodiments, the lentivirus binds to albumin. In some embodiments, the lentivirus comprises a protein on its surface that binds to albumin. In some embodiments, the lentivirus comprises an albumin binding protein on its surface. In some embodiments, the albumin binding protein is a streptococcal albumin binding protein. In some embodiments, the albumin binding protein is a streptococcal albumin binding domain.

[0542] Expression of non-fusogenic proteins in lentiviral envelopes In some embodiments, the lentivirus is engineered to include one or more proteins on its surface. In some embodiments, the protein affects immune interactions with a subject. In some embodiments, the protein affects the pharmacology of the lentivirus in a subject. In some embodiments, the protein is a receptor. In some embodiments, the protein is an agonist. In some embodiments, the protein is a signaling molecule. In some embodiments, the protein on the lentivirus surface comprises an anti-CD3 antibody (e.g., OKT3) or IL7.

[0543] In some embodiments, mitogenic and / or cytokine-based transmembrane proteins may be present in the source cell and incorporated into the retrovirus as it buds from the source cell membrane. The mitogenic and / or cytokine-based transmembrane proteins may be expressed in the source cell as separate cell surface molecules rather than being part of the viral envelope glycoprotein.

[0544] In some embodiments of any of the aspects described herein, the retroviral vector, VLP, or pharmaceutical composition is substantially non-immunogenic. Immunogenicity can be quantified, for example, as described herein.

[0545] In some embodiments, the retroviral vector or VLP fuses with a target cell to generate a recipient cell. In some embodiments, recipient cells fused with one or more retroviral vectors or VLPs are evaluated for immunogenicity. In embodiments, recipient cells are analyzed for the presence of antibodies on the cell surface, for example, by staining with an anti-IgM antibody. In other embodiments, immunogenicity is evaluated by a PBMC cytolytic assay. In embodiments, recipient cells are incubated with peripheral blood mononuclear cells (PBMCs) and then evaluated for cell lysis by PBMCs. In other embodiments, immunogenicity is evaluated by a natural killer (NK) cytolytic assay. In embodiments, recipient cells are incubated with NK cells and then evaluated for cell lysis by NK cells. In other embodiments, immunogenicity is evaluated by a CD8+ T cell lytic assay. In embodiments, recipient cells are incubated with CD8+ T cells and then evaluated for cell lysis by CD8+ T cells.

[0546] In some embodiments, the retroviral vector or VLP comprises an elevated level of an immunosuppressant (e.g., an immunosuppressant protein) compared to a control retroviral vector or VLP, e.g., one produced from an otherwise similar source cell or HEK293 cell, but unmodified. In some embodiments, the elevated level is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold. In some embodiments, the retroviral vector or VLP comprises an immunosuppressant that is not present in the control cell. In some embodiments, the retroviral vector or VLP comprises a decreased level of an immunostimulatory substance (e.g., an immunostimulatory protein) compared to a control retroviral vector or VLP, e.g., one produced from an otherwise similar source cell or HEK293 cell, but unmodified. In some embodiments, the reduced level is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% compared to a control retroviral vector or VLP. In some embodiments, the immunostimulatory agent is substantially absent from the retroviral vector or VLP.

[0547] In some embodiments, the retroviral vector or VLP or the source cell from which the retroviral vector or VLP is derived has the following characteristics: a. 50%, 40%, 30%, 20%, 15%, 10%, or 5% or less expression of MHC class I or MHC class II compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from an otherwise similar source cell, or HeLa cells, or HEK293 cells; b. expression of less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% or less of one or more costimulatory proteins, including but not limited to, LAG3, ICOS-L, ICOS, Ox40L, OX40, CD28, B7, CD30, CD30L4-1BB, 4-1BBL, SLAM, CD27, CD70, HVEM, LIGHT, B7-H3, or B7-H4, compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from an otherwise similar source cell, or an HEK cell, or control cell as described herein; c. Expression of a surface protein that inhibits macrophage phagocytosis, e.g., CD47, that is greater than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more, as detectable, e.g., by a method described herein, compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from otherwise similar cells that are unmodified to the source cells, or Jurkat cells or HEK293 cells; d. Expression of a soluble immunosuppressive cytokine, e.g., IL-10, e.g., greater than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more, as detectable by, e.g., a method described herein, compared to a control retroviral vector or VLP, e.g., a cell that is similar to the source cell but unmodified, or an unmodified retroviral vector or VLP derived from HEK293 cells; e. expression of a soluble immunosuppressive protein, e.g., PD-L1, that is greater than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more, as detectable by, e.g., a method described herein, compared to a control retroviral vector or VLP, e.g., a cell that is similar to the source cell but unmodified, or an unmodified retroviral vector or VLP derived from HEK293 cells; f. expression of less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% or less of a soluble immunostimulatory cytokine, e.g., IFN-γ or TNF-α, compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cell, or HEK293 cells or U-266 cells; g. Less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% or less expression of an endogenous immunostimulatory antigen, e.g., Zg16 or Hormad1, compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cell, or HEK293 cells, A549 cells, or SK-BR-3 cells; h. Expression of HLA-E or HLA-G, e.g., expression detectable by the methods described herein, compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells similar to the source cell but unmodified, or HEK293 cells, or Jurkat cells; i. a surface glycosylation profile, e.g., comprising sialic acid, that acts to inhibit, e.g., NK cell activation; j. Less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% or less expression of TCR α / β compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cells, or HEK293 cells, or Jurkat cells; k. less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% or less expression of ABO blood groups compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cell, or HEK293 cells, or HeLa cells; l. Expression of less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% of a minor histocompatibility antigen (MHA), or less than that of a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cell, or HEK293 cells, or Jurkat cells; or m. Has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less mitochondrial MHA, or no detectable mitochondrial MHA, compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cells, or HEK293 cells, or Jurkat cells.

[0548] In some embodiments, the costimulatory protein is 4-1BB, B7, SLAM, LAG3, HVEM, or LIGHT, and the control cells are HDLM-2. In some embodiments, the costimulatory protein is BY-H3, and the control cells are HeLa. In some embodiments, the costimulatory protein is ICOSL or B7-H4, and the control cells are SK-BR-3. In some embodiments, the costimulatory protein is ICOS or OX40, and the control cells are MOLT-4. In some embodiments, the costimulatory protein is CD28, and the control cells are U-266. In some embodiments, the costimulatory protein is CD30L or CD27, and the control cells are Daudi.

[0549] In some embodiments, the retroviral vector, VLP, or pharmaceutical composition does not substantially elicit an immunogenic response by the immune system, e.g., the innate immune system. In embodiments, the immunogenic response can be quantified, e.g., as described herein. In some embodiments, the immunogenic response by the innate immune system includes a response by innate immune cells, including, but not limited to, NK cells, macrophages, neutrophils, basophils, eosinophils, dendritic cells, mast cells, or gamma / delta T cells. In some embodiments, the immunogenic response by the innate immune system includes a response by the complement system, including soluble blood components and membrane-bound components.

[0550] In some embodiments, the retroviral vector, VLP, or pharmaceutical composition does not substantially elicit an immunogenic response by the immune system, e.g., the adaptive immune system. In some embodiments, an immunogenic response by the adaptive immune system includes an immunogenic response by adaptive immune cells, including, but not limited to, a change, e.g., an increase, in the number or activity of T lymphocytes (e.g., CD4 T cells, CD8 T cells, and / or gamma-delta T cells) or B lymphocytes. In some embodiments, an immunogenic response by the adaptive immune system includes, but is not limited to, an increase in the level of a soluble blood component, including, but not limited to, a change, e.g., an increase, in the number or activity of cytokines or antibodies (e.g., IgG, IgM, IgE, IgA, or IgD).

[0551] In some embodiments, the retroviral vector, VLP, or pharmaceutical composition is modified to have reduced immunogenicity, ie, the retroviral vector, VLP, or pharmaceutical composition is less than 5%, 10%, 20%, 30%, 40%, or 50% less immunogenic than a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells that are otherwise identical to the source cell, HEK293 cell, or Jurkat cell.

[0552] In some embodiments of any of the aspects described herein, the retroviral vector, VLP, or pharmaceutical composition is derived from a source cell, e.g., a mammalian cell, having a modified genome, e.g., modified using a method described herein, e.g., to reduce, e.g., decrease, immunogenicity. Immunogenicity can be quantified, e.g., as described herein.

[0553] In some embodiments, the retroviral vector, VLP, or pharmaceutical composition comprises: a. MHC class I, MHC class II, or MHA; b. one or more costimulatory proteins, including but not limited to, LAG3, ICOS-L, ICOS, Ox40L, OX40, CD28, B7, CD30, CD30L4-1BB, 4-1BBL, SLAM, CD27, CD70, HVEM, LIGHT, B7-H3, or B7-H4; c. A soluble immune stimulating cytokine, e.g., IFN-gamma or TNF-a d. endogenous immune stimulatory antigens, such as Zg16 or Hormad1; eT cell receptor (TCR), f. ABO blood group coding genes, e.g., ABO genes; g. Transcription factors that promote immune activation, e.g., NFkB; h. Transcription factors that regulate MHC expression, such as class II transactivator (CIITA), regulator of Xbox 5 (RFX5), RFX-associated protein (RFXAP), or RFX ankyrin repeat (RFXANK, also known as RFXB), or i. Derived from a mammalian cell that has been depleted, e.g., has a knockout, of one, two, three, four, five, six, seven or more TAP proteins, e.g., TAP2, TAP1, TAPBP, that reduce MHC class I expression.

[0554] In some embodiments, the retroviral vector or VLP is derived from a source cell with a genetic modification that results in increased expression of an immunosuppressive agent, e.g., one, two, three or more of the following (e.g., prior to the genetic modification, the cell did not express the agent): a. increased expression of a surface protein that inhibits macrophage phagocytosis, e.g., CD47, compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from an otherwise similar source cell, HEK293 cell, or Jurkat cell; b. increased expression of a soluble immunosuppressive cytokine, e.g., IL-10, e.g., IL-10, compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cells, HEK293 cells, or Jurkat cells; c. increased expression of a soluble immunosuppressive protein, e.g., PD-1, PD-L1, CTLA4, or BTLA, e.g., compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cells, HEK293 cells, or Jurkat cells; d. Increased expression of a tolerogenic protein, e.g., an ILT-2 or ILT-4 agonist, e.g., HLA-E or HLA-G, or any other endogenous ILT-2 or ILT-4 agonist, e.g., HLA-E, HLA-G, ILT-2, or ILT-4, compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cells, HEK293 cells, or Jurkat cells; or e. A surface protein that inhibits complement activity, e.g., a complement regulatory protein, e.g., a protein that binds to decay accelerating factor (DAF, CD55), e.g., factor H (FH)-like protein-1 (FHL-1), e.g., C4b binding protein (C4BP), e.g., complement receptor 1 (CD35), e.g., a membrane cofactor protein (MCP, CD46), e.g., profectin (CD59), e.g., a protein that inhibits the classical and alternative complement pathway CD / C5 convertase enzyme, e.g., a protein that regulates MAC assembly, e.g., increased expression of a complement regulatory protein compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cells, HEK293 cells, or Jurkat cells.

[0555] In some embodiments, the increased expression level is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher compared to a control retroviral vector or VLP.

[0556] In some embodiments, the retroviral vector or VLP is derived from a source cell that has been modified to have reduced expression of an immunostimulatory substance, for example, one, two, three, four, five, six, seven, eight or more of the following: a. expression of less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% or less of MHC class I or MHC class II compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cell, HEK293 cells, or HeLa cells; b. 50%, 40%, 30%, 20%, 15%, 10%, or 5% or less expression of one or more costimulatory proteins, including but not limited to, LAG3, ICOS-L, ICOS, Ox40L, OX40, CD28, B7, CD30, CD30L4-1BB, 4-1BBL, SLAM, CD27, CD70, HVEM, LIGHT, B7-H3, or B7-H4, compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cell, HEK293 cells, or a reference cell described herein; c. Expression of soluble immunostimulatory cytokines, e.g., IFN-gamma or TNF-a, less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% or less, compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cell, HEK293 cells, or U-266 cells; d. expression of less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% or less of an endogenous immunostimulatory antigen, e.g., Zg16 or Hormad1, compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cell, such as HEK293 cells, A549 cells, or SK-BR-3 cells; e. expression of less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% or less of the T cell receptor (TCR) compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cell, HEK293 cells, or Jurkat cells; f. expression of less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% or less of the ABO blood group compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cell, HEK293 cells, or HeLa cells; g. expression of less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% or less of a transcription factor that promotes immune activation, e.g., NFkB, compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cell, e.g., HEK293 cells or Jurkat cells; h. Less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% or less expression of transcription factors controlling MHC expression, e.g., class II transactivator of transcription (CIITA), regulator of Xbox 5 (RFX5), RFX-related protein (RFXAP), or RFX ankyrin repeat (RFXANK; also known as RFXB), compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cell, HEK293 cells, or Jurkat cells; or i. Expression of a TAP protein, e.g., TAP2, TAP1, or TAPBP, that reduces MHC class I expression by less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% or less compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells otherwise similar to the source cell, HEK293 cells, or HeLa cells.

[0557] In some embodiments, a retroviral vector, VLP, or pharmaceutical composition derived from a mammalian cell, e.g., HEK293, that has been modified using an shRNA-expressing lentivirus to reduce MHC class I expression has reduced expression of MHC class I compared to an unmodified retroviral vector or VLP, e.g., a unmodified retroviral vector or VLP derived from an unmodified cell (e.g., a mesenchymal stem cell). In some embodiments, a retroviral vector or VLP derived from a mammalian cell, e.g., HEK293, that has been modified using an HLA-G-expressing lentivirus to increase HLA-G expression has increased expression of HLA-G compared to an unmodified retroviral vector or VLP derived from an unmodified cell (e.g., HEK293).

[0558] In some embodiments, the retroviral vector, VLP, or pharmaceutical composition is derived from a source cell, e.g., a mammalian cell that is substantially non-immunogenic, and the source cell stimulates, e.g., induces, T cell IFN-gamma secretion, e.g., at levels of 0 pg / mL to >0 pg / mL, when assayed, e.g., in vitro, by an IFN-γ ELISPOT assay.

[0559] In some embodiments, the retroviral vector, VLP, or pharmaceutical composition is derived from a source cell, e.g., a mammalian cell, and the mammalian cell is from a cell culture that has been treated with an immunosuppressant, e.g., a glucocorticoid (e.g., dexamethasone), a cytostatic agent (e.g., methotrexate), an antibody (e.g., muromonab (OKT3)-CD3), or an immunophilin modulator (e.g., cyclosporine or rapamycin).

[0560] In some embodiments, the retroviral vector, VLP, or pharmaceutical composition is derived from a source cell, eg, a mammalian cell, and the mammalian cell comprises an exogenous agent, eg, a therapeutic agent.

[0561] In some embodiments, the retroviral vector, VLP, or pharmaceutical composition is derived from a source cell, eg, a mammalian cell, and the mammalian cell is a recombinant cell.

[0562] In some embodiments, the retroviral vector, VLP, or agent is derived from mammalian cells genetically modified to express a viral immunoevasin, eg, hCMV US2, or US11.

[0563] In some embodiments, the surface of the retroviral vector or VLP, or the surface of the source cell, is modified by covalent or non-covalent attachment with a polymer, e.g., a biocompatible polymer that reduces immunogenicity and immune-mediated clearance, e.g., PEG.

[0564] In some embodiments, the surface of the retroviral vector or VLP, or the surface of the source cell, is modified by covalent or non-covalent attachment with sialic acid, eg, a sialic acid containing sugar polymer that includes an NK inhibitory glycan epitope.

[0565] In some embodiments, the surface of the retroviral vector or VLP, or the surface of the source cell, is enzymatically treated, for example with a glycosidase enzyme, such as α-N-acetylgalactosaminidase, to remove ABO blood groups.

[0566] In some embodiments, the surface of the retroviral vector or VLP, or the surface of the source cell, is enzymatically treated to elevate, eg, induce expression of, an ABO blood group that matches the recipient's blood type.

[0567] Parameters for assessing immunogenicity In some embodiments, the retroviral vector or VLP is derived from a source cell, e.g., a mammalian cell, that has been modified, e.g., modified using the methods described herein, to be substantially non-immunogenic or have reduced immunogenicity. The immunogenicity of the source cell and retroviral vector or VLP can be determined by any of the assays described herein.

[0568] In some embodiments, the retroviral vector or VLP has an increase in in vivo graft survival, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more increase, compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells that are otherwise similar to the source cell.

[0569] In some embodiments, the retroviral vectors or VLPs have reduced immunogenicity as measured by a reduction in the humoral response following transplantation of one or more of the retroviral vectors or VLPs into a suitable animal model, e.g., an animal model described herein, compared to the humoral response following transplantation of one or more of a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells similar to the source cells but unmodified, into a suitable animal model, e.g., an animal model described herein. In some embodiments, the reduction in the humoral response is measured by anti-cellular antibody titers, e.g., anti-retroviral or anti-VLP antibody titers, e.g., by ELISA, in serum samples. In some embodiments, serum samples from animals administered the retroviral vectors or VLPs have a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in anti-retroviral or anti-VLP antibody titers compared to samples from animals administered the unmodified retroviral vector or VLP. In some embodiments, serum samples from animals administered the retroviral vector or VLP have increased anti-retroviral or anti-VLP antibody titers, e.g., an increase of 1%, 2%, 5%, 10%, 20%, 30%, or 40% from baseline, where baseline is referenced to a serum sample from the same animal prior to administration of the retroviral vector or VLP.

[0570] In some embodiments, the retroviral vector or VLP has a reduction in macrophage phagocytosis, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in macrophage phagocytosis, compared to a control retroviral vector or VLP, e.g., an unmodified retroviral vector or VLP derived from cells that are otherwise similar to the source cell, where the reduction in macrophage phagocytosis is determined by assaying an in vitro phagocytic index, e.g., as described in Example 8. In some embodiments, the retroviral vector or VLP has a phagocytic index of 0, ...

Claims

1. A fusosome, a) a lipid bilayer comprising a paramyxoviral retargeting fusogen, said paramyxoviral retargeting fusogen linked to a targeting moiety, and said paramyxoviral retargeting fusogen resulting in preferential delivery of an exogenous agent to target cells relative to non-target cells; b) a nucleic acid encoding the exogenous agent, (i) a positive target cell-specific regulatory element operably linked to the nucleic acid encoding the exogenous agent, wherein the positive target cell-specific regulatory element increases expression of the exogenous agent in the target cell relative to an otherwise similar fusosome lacking the positive target cell-specific regulatory element, and the positive target cell-specific regulatory element is a tissue-specific promoter; or (ii) a non-target cell-specific regulatory element operably linked to the nucleic acid encoding the exogenous agent, wherein the non-target cell-specific regulatory element reduces expression of the exogenous agent in the non-target cell relative to an otherwise similar fusosome lacking the non-target cell-specific regulatory element. and a nucleic acid further comprising or encoding: Claim 2: i) the fusosomes fuse with target cells at a higher rate than with non-target cells, and optionally, the higher rate is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2x, 3x, 4x, 5x, 10x, 20x, 50x, or 100x; ii) the fusosomes fuse with target cells at a higher rate than with other fusosomes, and optionally the higher rate is 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; iii) the fusosomes fuse with target cells at a rate such that the exogenous agent in the fusosomes is delivered to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the target cells after 24, 48, or 72 hours; iv) the fusosomes deliver the nucleic acid to target cells at a higher rate than non-target cells, and optionally the higher rate is 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; v) the fusosome delivers the nucleic acid to a target cell at a higher rate than another fusosome, and optionally the higher rate is 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; or vi) the fusosomes of claim 1 deliver the nucleic acid to target cells at a rate such that the exogenous agent in the fusosomes is delivered to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of target cells after 24, 48, or 72 hours.

3. When the fusosomes are administered to a subject, i) less than 10%, 5%, 4%, 3%, 2%, or 1% of the exogenous agent detectably present in the subject is in non-target cells; ii) at least 90%, 95%, 96%, 97%, 98%, or 99% of the cells of the subject detectably containing the exogenous agent are target cells, optionally the target cells are of a single cell type, and optionally the target cells are T cells; iii) fewer than 1,000,000, 500,000, 200,000, 100,000, 50,000, 20,000, or 10,000 of the cells of the subject detectably containing the exogenous agent are non-target cells; iv) the average level of the exogenous agent in all target cells of the subject is at least 100-fold, 200-fold, 500-fold, or 1,000-fold higher than the average level of the exogenous agent in all non-target cells of the subject; or v) the exogenous agent is not detectable in any non-target cells in the subject.

4. A fusosome according to any one of claims 1 to 3, wherein the retargeted fusogen comprises a sequence selected from Nipah virus F and G proteins, measles virus F and H proteins, Tupaia paramyxovirus F and H proteins, paramyxovirus F and G proteins or F and H proteins or F and HN proteins, Hendra virus F and G proteins, Henipavirus F and G proteins, Morbillivirus F and H proteins, Respirovirus F and HN proteins, Sendai virus F and HN proteins, Rubulavirus F and HN proteins, Canine distemper virus F and H proteins, or Abulavirus F and HN proteins, or derivatives thereof, or any combination thereof.

5. A fusosome described in any of claims 1 to 4, wherein the fusogen comprises a domain of at least 100 amino acids in length having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen, optionally wherein the wild-type paramyxovirus fusogen is set forth in any one of SEQ ID NOs: 1 to 132, optionally wherein the wild-type paramyxovirus is a Nipah virus, and further optionally wherein the Nipah virus is a Henipa virus.

6. The positive target cell-specific regulatory element comprises a tissue-specific promoter, a tissue-specific enhancer, a tissue-specific splice site, a tissue-specific site that extends the half-life of an RNA or protein, a tissue-specific mRNA export promoting site, a tissue-specific translation enhancing site, or a tissue-specific post-translational modification site; 6. The fusosome of any one of claims 1 to 5, wherein optionally the tissue-specific promoter is a hepatocyte-specific promoter, and further optionally the hepatocyte-specific promoter is selected from the group consisting of hAAT, ApoE, HCR-hAAT, enhanced transthyretin, TTR, Alb, Apoa2, Cyp3a4, LP1B, MIR122, hemopexin, and HLP.

7. The non-target cell-specific regulatory element comprises a tissue-specific miRNA recognition sequence, a tissue-specific protease recognition site, a tissue-specific ubiquitin ligase site, a tissue-specific transcriptional repression site, or a tissue-specific epigenetic repression site; and / or The fusosome of any one of claims 1 to 6, wherein the non-target cell-specific regulatory element is located or encoded within a transcribed region encoding the exogenous agent, and optionally, the RNA produced by the transcribed region comprises the tissue-specific miRNA recognition sequence within a UTR or coding region.

8. A fusosome described in any one of claims 1 to 7, wherein the target cells are cancer cells and the non-target cells are non-cancerous cells.

9. A fusosome described in any one of claims 1 to 8, wherein the exogenous agent is an exogenous polypeptide or exogenous RNA, and optionally, the exogenous agent is a therapeutic agent.

10. The fusosome further comprises: a) a first exogenous or overexpressed immunosuppressive protein in said lipid bilayer, and a second exogenous or overexpressed immunosuppressive protein in said lipid bilayer; b) a first exogenous or overexpressed immunosuppressive protein and a second immunostimulatory protein present in said lipid bilayer, said second immunostimulatory protein being absent or at reduced levels, optionally said reduced levels being at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to fusosomes produced from an unmodified but similar source cell; or c) a first immunostimulatory protein that is absent or present at reduced levels, optionally said reduced levels being at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to fusosomes produced from an otherwise similar source cell that is unmodified, and a second immunostimulatory protein that is absent or present at reduced levels, optionally said reduced levels being at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to fusosomes produced from an otherwise similar source cell that is unmodified; Optionally, when the fusosomes are administered to a subject: i) the fusosomes do not produce a detectable antibody response or antibodies to the fusosomes are present at levels less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels; ii) the fusosomes do not produce a detectable cellular immune response, or a cellular immune response to the fusosomes is present at a level less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels; iii) the fusosomes do not elicit a detectable innate immune response, or the innate immune response to the fusosomes is present at a level less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels; iv) less than 10%, 5%, 4%, 3%, 2%, or 1% of the fusosomes are inactivated by serum; v) target cells that receive the exogenous agent from the fusosomes do not develop a detectable antibody response, or antibodies to the target cells are present at levels less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels; or vi) target cells that receive the exogenous agent from the fusosomes do not develop a detectable cellular immune response, or a cellular response against the target cells is present at a level less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels; Optionally, the background level is the corresponding level in the same subject prior to administration of the fusosomes.

11. The fusosome described in claim 10, wherein the immunosuppressive protein is a complement regulatory protein, CD47, or MHC, and optionally, the MHC is an HLA protein.

12. A fusosome described in any of claims 1 to 11, wherein the nucleic acid comprises one or more insulator elements, optionally wherein the nucleic acid comprises two insulator elements, optionally wherein the two insulator elements comprise a first insulator element upstream of the region encoding the exogenous drug and a second insulator element downstream of the region encoding the exogenous drug, and optionally wherein the first insulator element and the second insulator element comprise the same or different sequences. (i) the variability in the median level of the exogenous agent in a sample of cells isolated after administering the fusosomes to a subject at a first time point is at least 10,000%, 5,000%, 2,000%, 1,000%, 500%, 200%, 10%, 200%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 1000%, 1000%, 1500%, 2000%, 3000%, 4000%, 5000%, 1000%, 1500%, 2000%, 3000%, 4000%, 5000%, 6 ... 0%, 50%, 20%, 10%, or 5%, or less than 10,000%, 5,000%, 2,000%, 1,000%, 500%, 200%, 100%, 50%, 20%, 10%, or 5%, or about 10,000%, 5,000%, 2,000%, 1,000%, 500%, 200%, 100%, 50%, 20%, 10%, or 5%; (ii) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of target cells in the subject detectably contain the exogenous agent; (iii) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of target cells in a subject that detectably contain the exogenous agent at a first time point still detectably contain the exogenous agent at a later second time point; and / or (iv) The fusosomes of claim 12, wherein the fusosomes are not genotoxic or increase the rate of tumor formation in target cells.

14. A fusosome described in any of claims 1 to 13, wherein the exogenous drug is a membrane protein or the nucleic acid encoding the exogenous drug encodes a membrane protein.

15. The membrane protein is selected from the group comprising a chimeric antigen receptor (CAR), a T-cell receptor, an integrin, an ion channel, a pore-forming protein, a toll-like receptor, an interleukin receptor, a cell adhesion protein or a transport protein; and / or The membrane protein is a chimeric antigen receptor comprising at least one antigen-binding domain, and optionally, the at least one antigen-binding domain is selected from the group consisting of epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), RET receptor, Eph receptor, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CFTR, CIC-1, CIC-2, CIC-4, CIC-5, CIC-7, CIC-Ka, CIC-Kb, bestrophin, TMEM16A, GABA receptor, glycine receptor, ABC transporter, NAV1.1, NAV1.2, NAV1.3, 25 NAV1.4, NAV1.5, NAV1.6, NAV1.7, NAV1.8, NAV1.9, sphingosine-1-phosphate receptor (S1P1R), NMDA channel, transmembrane protein, multispanning transmembrane protein, T cell receptor motif, T cell alpha chain, T cell beta chain, T cell gamma chain, T cell delta chain, CCR7, CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62L, CD68, CD80, CD95, CD117, CD127, 30CD133, CD137 (4-1BB), CD163, F4 / 80, IL-4Ra, Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, NKp46, perforin, CD4+, Th1, Th2, Th17, Th40, Th22, Th9, Tfh, classical Treg. FoxP3+, Tr1, Th3, Treg17, TREG, CDCP1, NT5E, EpCAM, CEA, gpA33, mucin, TAG-72, carbonic anhydrase IX, PSMA, folate-binding protein, ganglioside, CD2, CD3, GM2, Lewis-γ2, VEGF, VEGFR1 / 2 / 3, αVβ3, α5β1, ErbB1 / EGFR, ErbB1 / HER2, ErB3, MET, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANKL, FAP, tenascin, PDL-1, BAFF, HDAC, ABL, F LT3, KIT, MET, RET, IL-1β, ALK, RANKL, mTOR, CTLA-4, IL-6, IL-6R, JAK3, BRAF, PTCH, Smoothened, PIGF, ANPEP, TIMP1, PLAUR, PTPRJ, LTBR, ​​or ANTXR1, folate receptor alpha (FRa), ERBB2 (Her2 / neu), EphA2, IL-13Ra2, epidermal growth factor receptor (EGFR), mesothelin, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, 10 EGFRvIII, GD2, GD3, BCMA, MUC16 (CA125), L1CAM, LeY, MSLN, IL13Rα1, L1-CAM, TnAg, prostate-specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, interleukin-11 receptor a (IL-11Ra), PSCA, PRSS21, VEGFR2, Lewis Y, CD24, platelet-derived growth factor receptor beta (PDGFR beta), SSEA-4, CD20, MUC1, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-1 receptor, CAIX, LMP2, gplOO, bcr- abl, tyrosinase, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLACl, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2) ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYPIBI, BORIS, SART3, PAX5, OYTES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, 2515. The fusosome of claim 14, which targets an antigen selected from the group comprising CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, neoantigen, CD133, CD15, CD184, CD24, CD56, CD26, CD29, CD44, HLA-A, HLA-B, HLA-C, (HLA-A, B, C)CD49f, CD151, CD340, CD200, tkrA, trkB, or trkC, or an antigenic fragment or portion thereof, and optionally wherein the at least one antigen-binding domain targets CD19.

16. (i) the targeting moiety comprises an scFv. (ii) the fusosome is a retroviral vector; (iii) the fusosome delivers the nucleic acid to a target cell; and / or (iv) the target cells are selected from the group consisting of T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, hepatocytes, hematopoietic stem cells, CD34+ hematopoietic stem cells, CD105+ hematopoietic stem cells, CD117+ hematopoietic stem cells, CD105+ endothelial cells, B cells, CD20+ B cells, CD19+ B cells, cancer cells, CD133+ cancer cells, EpCAM+ cancer cells, CD19+ cancer cells, Her2 / Neu+ cancer cells, GluA2+ neurons, GluA4+ neurons, NKG2D+ natural killer cells, SLC1A3+ astrocytes, SLC7A10+ adipocytes, and CD30+ lung epithelial cells; A fusosome according to any one of claims 1 to 15.

17. Use of fusosomes described in any of claims 1 to 16 in the manufacture of a pharmaceutical for delivering an exogenous agent to a subject, wherein the fusosomes are administered to the subject to deliver the exogenous agent, and optionally the subject is a human subject.

18. Use of a fusosome described in any of claims 1 to 16 in the manufacture of a pharmaceutical for regulating a function in a subject, target tissue, or target cell, wherein the subject, target tissue, or target cell is contacted with the fusosome, and optionally the subject is a human subject.

19. Use of fusosomes described in any of claims 1 to 16 in the manufacture of a pharmaceutical for the treatment of a disease or disorder in a subject, wherein the fusosomes are administered to the subject, and optionally the subject is a human subject.

20. A method for producing a cell comprising: a) providing said nucleic acid and a retargeting fusogen of said paramyxovirus; and b) culturing said cells under conditions that allow the production of said fusosomes; c) isolating, enriching, or purifying the fusosomes from the cells, thereby producing the fusosomes.

21. A composition for delivering an exogenous drug to a subject, comprising a fusosome described in any one of claims 1 to 16, and characterized in that the composition is administered to the subject to deliver the exogenous drug, and optionally the subject is a human subject.

22. A composition for regulating a function in a subject, target tissue, or target cell, comprising a fusosome described in any one of claims 1 to 16, contacting the subject, target tissue, or target cell with the fusosome, and optionally, the subject is a human subject.

23. A composition for treating a disease or disorder in a subject, comprising a fusosome described in any one of claims 1 to 16, and characterized in that the composition is administered to the subject, and optionally the subject is a human subject.

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