Compositions for promoting membrane fusion and uses thereof

Abstract

To provide a composition for promoting membrane fusion and use thereof.SOLUTION: In some aspects, fusosome compositions and methods are described herein, including membrane-encapsulated preparations comprising a fusogen. In some embodiments, the fusosome is capable of fusing with the target cell, thereby delivering the conjugated biological agent to the cytoplasm of the target cell. The present disclosure provides, in some aspects, a fusosome comprising (a) a lipid bilayer, (b) a lumen (e.g., comprising a cytosol) surrounded by the lipid bilayer, and (c) an exogenous or overexpressed fusogen, e.g., wherein the fusogen is disposed within the lipid bilayer, wherein the fusosome is derived from a source cell, and wherein the fusosome has partial or complete nuclear inactivation (e.g., removal of the nucleus).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Patent Application No. 62 / 502,998, filed May 8, 2017, U.S. Patent Application No. 62 / 575,147, filed October 20, 2017, and U.S. Patent Application No. 62 / 595,862, filed December 7, 2017, each of which is incorporated by reference herein in its entirety. [Background technology]

[0002] Complex biological agents are promising therapeutic candidates for various diseases.However, because cell membrane acts as a barrier between cells and extracellular space, it is difficult to deliver large biological agents into cells.There is a need in the art for new methods to deliver complex biological agents into cells in subjects. Summary of the Invention [Means for solving the problem]

[0003] Membrane fusion is required for diverse biological processes such as fertilization, development, immune response, and tumorigenesis. The present disclosure provides a fusion-based method for delivering complex biological cargoes into cells.

[0004] Thus, in some embodiments, the present disclosure provides fusosomes comprising a lipid bilayer, a lumen surrounded by the lipid bilayer, and a fusogen. The fusosomes can be used, for example, to deliver cargo within the lumen or lipid bilayer to a target cell. The cargo can include, for example, therapeutic proteins, nucleic acids, and small molecules.

[0005] The present disclosure provides, in some aspects, (a) a lipid bilayer; (b) a lumen (e.g., containing the cytosol) surrounded by a lipid bilayer; (c) providing a fusosome comprising an exogenous or overexpressed fusogen, e.g., the fusogen is disposed within a lipid bilayer; Fusosomes are derived from source cells and Fusosomes have partial or complete nuclear inactivation (e.g., removal of the nucleus).

[0006] In some embodiments, one or more of the following exhibits: i) the fusosomes comprise or are comprised in a cell biological product; ii) the fusosomes contain enucleated cells; iii) fusosomes contain inactivated nuclei; iv) fusosomes fuse with target cells at, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher rates than non-target cells, e.g., in the assay of Example 54; v) the fusosomes fuse with target cells at, 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 rates than other fusosomes, e.g., in the assay of Example 54; vi) 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, e.g., in the assay of Example 54; vii) the fusogen is present in at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies or less, as measured, for example, by the assay of Example 29; viii) the fusosomes contain at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies or less of the therapeutic agent, e.g., as measured by the assay of Example 43 or 156; ix) The ratio of the copy number of the fusogen to the copy number of the therapeutic agent is 1,000,000:1 to 100,000:1, 100,000:1 to 10,000:1, 10,000:1 to 1,000:1, 1,000:1 to 100:1, 100:1 to 50:1, 50:1 to 20:1, 20:1 to 10:1, 10:1 to 5:1, 5:1 to 2: 1, 2:1 to 1:1, 1:1 to 1:2, 1:2 to 1:5, 1:5 to 1:10, 1:10 to 1:20, 1:20 to 1:50, 1:50 to 1:100, 1:100 to 1:1,000, 1:1,000 to 1:10,000, 1:10,000 to 1:100,000, or 1:100,000 to 1:1,000,000. x) the fusosomes contain a lipid composition substantially similar to that in the source cells, or one or more of CL, Cer, DAG, HexCer, LPA, LPC, LPE, LPG, LPI, LPS, PA, PC, PE, PG, PI, PS, CE, SM, and TAG are within 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the corresponding lipid levels in the source cells; xi) the fusosomes comprise a proteomic composition similar to that of the source cell, e.g., using the assays of Examples 42 or 155; xii) the fusosomes comprise a lipid to protein ratio that is within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cells, as measured, for example, using the assay of Example 49; xiii) the fusosomes comprise a ratio of protein to nucleic acid (e.g., DNA) that is within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cells, e.g., as measured using the assay of Example 50; xiv) the fusosomes comprise a lipid to nucleic acid (e.g., DNA) ratio that is within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cells, e.g., as measured using the assays of Examples 51 or 159; xv) the fusosomes have a half-life in a subject, e.g., a mouse, that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the half-life of a reference cell, e.g., a source cell, e.g., by the assay of Example 75; xvi) the fusosomes transport at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% more glucose (e.g., labeled glucose, e.g., 2-NBDG) across the membrane than other similar fusosomes, e.g., in the absence of a negative control, e.g., glucose, as measured, e.g., using the assay of Example 64; xvii) the fusosomes comprise an esterase activity in their lumen that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the esterase activity in a reference cell, e.g., a source cell or a mouse embryonic fibroblast, using, e.g., the assay of Example 66; xviii) the fusosomes comprise a metabolic activity level that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the citrate synthase activity in a reference cell, e.g., a source cell, e.g., as described in Example 68; xix) the fusosomes comprise a respiration level (e.g., oxygen consumption rate) that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the respiration level in a reference cell, e.g., a source cell, as described, e.g., in Example 69; xx) the fusosomes comprise an annexin-V staining level of an MFI of at most 18,000, 17,000, 16,000, 15,000, 14,000, 13,000, 12,000, 11,000, or 10,000, e.g., using the assay of Example 70; or the fusosomes comprise an annexin-V staining level that is at least 5%, 10%, 20%, 30%, 40%, or 50% lower than the annexin-V staining level of other similar fusosomes treated with menadione, e.g., in the assay of Example 70; or the fusosomes comprise an annexin-V staining level that is at least 5%, 10%, 20%, 30%, 40%, or 50% lower than the annexin-V staining level of macrophages treated with menadione in the assay of Example 70; xxi) the fusosomes have a miRNA content level that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more greater than the miRNA content level of the source cells, e.g., by the assay of Example 39; xxii) the fusosomes have a soluble protein:insoluble protein ratio that is 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than that of the source cells, e.g., within 1%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% of that of the source cells, e.g., by the assay of Example 47; xxiii) the fusosomes have LPS levels of less than 5%, 1%, 0.5%, 0.01%, 0.005%, 0.0001%, 0.00001% or less of the LPS content of the source cells, as measured, for example, by mass spectrometry, e.g., by the assay of Example 48; xxiv) the fusosomes are capable of transporting at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% more signaling, e.g., AKT phosphorylation in response to an extracellular signal, e.g., insulin, or glucose (e.g., labeled glucose, e.g., 2-NBDG) uptake in response to insulin, than other similar fusosomes, e.g., in the absence of a negative control, e.g., insulin, e.g., using the assay of Example 63; xxv) the fusosomes are targeted to a tissue, e.g., liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testis, ovary, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye, and when administered to a subject, e.g., a mouse, at least 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fusosomes in the population of administered fusosomes are present in the target tissue after 24, 48, or 72 hours, e.g., by the assay of Example 87 or 100. xxvi) the fusosomes have a level of juxtacrine signaling that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the level of juxtacrine signaling induced by a reference cell, e.g., a source cell or bone marrow stromal cell (BMSC), e.g., by the assay of Example 71; xxvii) the fusosomes have a level of paracrine signaling that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% greater than the level of paracrine signaling induced by a reference cell, e.g., a source cell or a macrophage, e.g., by the assay of Example 72; xxviii) the fusosomes polymerize actin at a level within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the level of polymerized actin in a reference cell, e.g., a source cell or a C2C12 cell, e.g., by the assay of Example 73; xxix) the fusosomes have a membrane potential within about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the membrane potential in a reference cell, e.g., a source cell or a C2C12 cell, e.g., by the assay of Example 74, or the fusosomes have a membrane potential of about -20 to -150 mV, -20 to -50 mV, -50 to -100 mV, or -100 to -150 mV; xxx) the fusosomes can extravasate from blood vessels at a rate that is at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the extravasation rate of source cells or cells of the same type as the source cells, e.g., by the assay of Example 57, wherein the source cells are neutrophils, lymphocytes, B cells, macrophages, or NK cells; xxxi) fusosomes are capable of crossing cell membranes, such as endothelial cell membranes or the blood-brain barrier; xxxii) the fusosomes are capable of secreting protein at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more than reference cells, e.g., mouse embryonic fibroblast cells, using, e.g., the assay of Example 62; xxxiii) the fusosomes meet pharmaceutical or Good Manufacturing Practice (GMP) standards; xxxiv) the fusosomes were produced in accordance with Good Manufacturing Practice (GMP); xxxv) the fusosomes have pathogen levels below a predetermined threshold, e.g., are substantially pathogen-free; xxxvi) the fusosomes have contaminant levels below a predetermined threshold, e.g., are substantially free of contaminants; xxxvii) the fusosomes have low immunogenicity, e.g., as described herein; xxxviii) the source cells are selected from neutrophils, granulocytes, mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells, myeloblasts, myoblasts, hepatocytes, or neurons, e.g., retinal neuronal cells; or xxxix) The source cells are other than 293 cells, HEK cells, human endothelial cells, or human epithelial cells, monocytes, macrophages, dendritic cells, or stem cells.

[0007] The present disclosure also provides, in some aspects, a) fusosomes derived from a source cell, a lipid bilayer, and a lumen that is miscible with aqueous solutions, e.g., water; b) exogenous or overexpressed fusogens positioned within the lipid bilayer; Also provided is a fusosome comprising: c) organelles disposed within a lumen, for example, a therapeutically effective number of organelles.

[0008] In some embodiments, one or more of the following indicates: i) the source cells are selected from endothelial cells, macrophages, neutrophils, granulocytes, leukocytes, stem cells (e.g., mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells), myeloblasts, myoblasts, hepatocytes, or neurons, e.g., retinal neuronal cells; ii) the organelle is selected from the group consisting of a Golgi apparatus, a lysosome, an endoplasmic reticulum, a mitochondrion, a vacuole, an endosome, an acrosome, an autophagosome, a centriole, a glycosome, a glyoxysome, a hydrogenosome, a melanosome, a mitosome, a nematocyst, a peroxisome, a proteasome, a vesicle, and a stress granule; iii) the fusosomes have a size greater than 5 um, 10 um, 20 um, 50 um, or 100 um; i) the fusosome, or a composition or preparation comprising a plurality of fusosomes, has a density other than 1.08 g / ml to 1.12 g / ml, e.g., the fusosome has a density of 1.25 g / ml + / - 0.05, e.g., as measured by the assay of Example 33; iv) fusosomes are not captured by the scavenger system in the circulation or by Kupffer cells in the liver sinus; v) the source cells are other than 293 cells; vi) the source cells are not transformed or immortalized; vii) the source cells are transformed or immortalized using methods other than adenovirus-mediated immortalization, e.g., immortalized by spontaneous mutation or immortalization by telomerase expression; viii) the fusogen is other than a VSVG, a SNARE protein, or a secretory granule protein; ix) the fusosomes do not contain Cre or GFP, e.g., EGFP; x) the fusosomes further contain an exogenous protein other than Cre or GFP, for example, EGFP; xi) the fusosome further comprises, for example, an exogenous nucleic acid (e.g., RNA, e.g., mRNA, miRNA, or siRNA) or an exogenous protein (e.g., an antibody, e.g., an antibody) within the lumen; xii) Fusosomes do not contain mitochondria.

[0009] The present disclosure also provides, in some aspects, (a) a lipid bilayer; (b) a lumen (e.g., containing the cytosol) surrounded by a lipid bilayer; (c) an exogenous or overexpressed fusogen, e.g., a fusogen disposed within a lipid bilayer; (d) a functional nucleus; and a fusosome, Fusosomes are derived from the source cell.

[0010] In some embodiments, one or more of the following indicates: i) the source cells are other than dendritic cells or tumor cells, and are selected from, for example, endothelial cells, macrophages, neutrophils, granulocytes, leukocytes, stem cells (e.g., mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells), myeloblasts, myoblasts, hepatocytes, or neurons, e.g., retinal neuronal cells; ii) the fusogen is other than a fusogenic glycoprotein; iii) the fusogen is a mammalian protein other than fertilin-beta; iv) fusosomes have low immunogenicity, e.g., as described herein; v) the fusosomes meet pharmaceutical or Good Manufacturing Practice (GMP) standards; vi) the fusosomes were produced according to good manufacturing practice (GMP); vii) the fusosomes have pathogen levels below a predetermined threshold, e.g., are substantially pathogen-free; viii) the fusosomes have contaminant levels below a predetermined threshold, e.g., are substantially free of contaminants;

[0011] The present disclosure also provides, in some embodiments, a purified fusosome composition comprising a plurality of fusosomes, at least one of which is a) a lipid bilayer and an aqueous lumen, wherein the fusosome is derived from a source cell; b) an exogenous or overexpressed fusogen disposed within the lipid bilayer; The fusosomes are at temperatures below 4, 0, -4, -10, -12, -16, -20, -80, or -160°C.

[0012] The present disclosure also provides, in some embodiments, a purified fusosome composition comprising a plurality of fusosomes, at least one of which is a) a lipid bilayer and an aqueous cavity; b) an exogenous or overexpressed protein fusogen disposed within the lipid bilayer; The fusosomes are at temperatures below 4, 0, -4, -10, -12, -16, -20, -80, or -160°C.

[0013] The present disclosure also provides, in some aspects, fusosome compositions comprising a plurality of fusosomes described herein.

[0014] The present disclosure also provides, in some aspects, a fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) a lumen containing the cytosol, the lumen being surrounded by a lipid bilayer; (c) exogenous or overexpressed fusogens positioned within the lipid bilayer; (d) cargo, including Fusosomes do not contain a nucleus the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein; The plurality of fusosomes, when contacted with a target cell population in the presence of an inhibitor of endocytosis, and when contacted with a reference target cell population that is not treated with an inhibitor of endocytosis, deliver cargo to at least 30% of the number of cells in the target cell population compared to the reference target cell population.

[0015] The present disclosure also provides, in some aspects, a fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) a lumen containing the cytosol, the lumen being surrounded by a lipid bilayer; (c) an exogenous or overexpressed retargeted fusogen placed within the lipid bilayer; and (d) cargo, including Fusosomes do not contain a nucleus the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein; (i) when the plurality of fusosomes contact a cell population comprising target cells and non-target cells, the cargo is present in at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold greater abundance in the target cells than in the non-target cells; or (ii) Multiple fusosomes fuse with target cells at a rate at least 50% higher than with non-target cells.

[0016] The present disclosure also provides, in some aspects, a fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) a lumen surrounded by a lipid bilayer; (c) an exogenous or overexpressed fusogen, the fusogen being disposed within the lipid bilayer; (d) cargo, including Fusosomes do not contain a nucleus i) the fusogen is present in a copy number of at least 1,000 copies; ii) the fusosomes contain the therapeutic agent in a copy number of at least 1,000 copies; iii) fusosomes contain lipids in which one or more of CL, Cer, DAG, HexCer, LPA, LPC, LPE, LPG, LPI, LPS, PA, PC, PE, PG, PI, PS, CE, SM, and TAG are within 75% of the corresponding lipid levels in the source cells; iv) fusosomes contain a proteome composition similar to that of the source cell; v) fusosomes are capable of signaling, e.g., transmitting extracellular signals, e.g., AKT phosphorylation in response to insulin, or uptake of glucose (e.g., labeled glucose, e.g., 2-NBDG) in response to insulin, e.g., at least 10% more than other similar fusosomes in the absence of a negative control, e.g., insulin; vi) the fusosomes are targeted to a tissue, e.g., liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testis, ovary, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye, and when administered to a subject, e.g., a mouse, at least 0.1% or 10% of the fusosomes in the population of administered fusosomes are present in the target tissue after 24 hours; or The source cells are one or more (e.g., at least two, three, four, or five) selected from neutrophils, granulocytes, mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells, myeloblasts, myoblasts, hepatocytes, or neurons, e.g., retinal neuronal cells.

[0017] The present disclosure also provides, in some aspects, pharmaceutical compositions comprising a fusosome composition described herein and a pharmaceutically acceptable carrier.

[0018] In certain aspects, the present disclosure also provides methods of administering a fusosome composition to a subject (e.g., a human subject), a target tissue, or a cell, comprising administering to the subject or contacting the target tissue or cell with a plurality of fusosomes described herein, a fusosome composition described herein, or a pharmaceutical composition described herein, thereby administering the fusosome composition to the subject.

[0019] The present disclosure also provides, in certain aspects, methods of delivering a therapeutic agent (e.g., a polypeptide, nucleic acid, metabolite, organelle, or subcellular structure) to a subject, target tissue, or cell, comprising administering to the subject or contacting the target tissue or cell with a plurality of fusosomes described herein, a fusosome composition comprising a plurality of fusosomes described herein, a fusosome composition described herein, or a pharmaceutical composition described herein, wherein the fusosome composition is administered in an amount and / or for a time such that the therapeutic agent is delivered.

[0020] The present disclosure also provides, in certain aspects, methods for delivering a function to a subject, target tissue, or cell, comprising administering to a subject or contacting a target tissue or cell with a plurality of fusosomes described herein, a fusosome composition comprising a plurality of fusosomes described herein, a fusosome composition described herein, or a pharmaceutical composition described herein, wherein the fusosome composition is administered in an amount and / or for a time such that the function is delivered.

[0021] The present disclosure also provides, in certain aspects, methods for targeting a function to a subject, target tissue, or cell, comprising administering to a subject or contacting a target tissue or cell with a plurality of fusosomes described herein, a fusosome composition comprising a plurality of fusosomes described herein, a fusosome composition described herein, or a pharmaceutical composition described herein, wherein the fusosome composition is administered in an amount and / or for a time such that the function is targeted.

[0022] The present disclosure also provides, in certain aspects, methods for modulating, e.g., enhancing, a biological function in a subject, target tissue, or cell, comprising administering to a subject or contacting a target tissue or cell with a plurality of fusosomes described herein, a fusosome composition comprising a plurality of fusosomes described herein, a fusosome composition described herein, or a pharmaceutical composition described herein, thereby modulating the biological function in the subject.

[0023] The present disclosure also provides, in certain aspects, methods for delivering or targeting a function to a subject, comprising administering to the subject a fusosome composition comprising a plurality of fusosomes described herein that contain the function, a fusosome composition described herein, or a pharmaceutical composition described herein, wherein the fusosome composition is administered in an amount and / or for a time such that the function in the subject is delivered or targeted. In embodiments, the subject has cancer, an inflammatory disorder, an autoimmune disease, a chronic disease, inflammation, impaired organ function, an infectious disease, a degenerative disease, a genetic disease, or an injury.

[0024] The present disclosure also provides, in some aspects, a method of producing a fusosome composition, comprising: a) providing a source cell containing, e.g., expressing, a fusogen; b) generating fusosomes from the source cells, the fusosomes comprising a lipid bilayer, a lumen, and a fusogen, thereby producing fusosomes; c) formulating the fusosomes, for example, as a pharmaceutical composition suitable for administration to a subject.

[0025] In embodiments, one or more of the following indicates: i) the source cells are other than 293 cells, HEK cells, human endothelial cells, or human epithelial cells; ii) the fusogen is other than a viral protein; iii) the fusosome, or a composition or preparation comprising a plurality of fusosomes, has a density other than, for example, 1.08 g / ml to 1.12 g / ml; iv) the fusosomes have a density of 1.25 g / ml + / - 0.05, e.g., as measured by the assay of Example 33; v) fusosomes are not captured by the scavenger system in the circulation or by Kupffer cells in the liver sinus; vi) the fusosomes are not captured by the reticuloendothelial system (RES) in the subject, e.g., by the assay of Example 76; vii) when a plurality of fusosomes are administered to a subject, less than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the plurality are or are not captured by the RES after 24, 48, or 72 hours, e.g., by the assay of Example 76; viii) the fusosomes have a diameter greater than 5 um, 6 um, 7 um, 8 um, 10 um, 20 um, 50 um, 100 um, 150 um, or 200 um; ix) fusosomes contain cell biological material; x) the fusosomes contain enucleated cells, or xi) Fusosomes contain an inactivated nucleus.

[0026] In some aspects, the present disclosure provides a method of producing a fusosome composition, comprising: a) providing a plurality of fusosomes described herein, a fusosome composition described herein, or a pharmaceutical composition described herein; b) formulating the fusosomes, for example, as a pharmaceutical composition suitable for administration to a subject.

[0027] In some aspects, the present disclosure provides a method of producing a fusosome composition, comprising: a) providing, e.g., generating, a plurality of fusosomes described herein or a fusosome composition described herein; b) assaying one or more fusosomes from the plurality to determine whether one or more (e.g., two, three, or more) criteria are met. In embodiments, the criterion(s) are selected from the following: i) fusosomes fuse with target cells at, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher rates than non-target cells, e.g., in the assay of Example 54; ii) the fusosomes fuse with target cells at a rate, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than other fusosomes, e.g., in the assay of Example 54; iii) the fusosomes fuse with the target cells at a rate such that the agent in the fusosomes is delivered to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the target cells after 24, 48, or 72 hours, e.g., in the assay of Example 54; iv) the fusogen is present in at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies or less, as measured, for example, by the assay of Example 29; v) the fusosomes contain at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies or less of the therapeutic agent, e.g., as measured by the assay of Example 43 or 156; vi) The ratio of the copy number of the fusogen to the copy number of the therapeutic agent is 1,000,000:1, 100,000:1, 10,000:1, 1,000:1, 100:1 to 50:1, 1,000,000:1 to 100,000:1, 100,000:1 to 10,000:1, 10,000:1 to 1,000:1, 1,000:1 to 100:1, 100:1 to 50:1, 50:1 to 20:1, 20:1 to 10:1, 10:1 to 5:1, 5:1 to 2:1, 1:1 to 2:1, 2:1 to 1:1, 1:1 to 1:2, 1:2 to 1:5, 1:5 to 1:10, 1:10 to 1:20, 1:20 to 1:50, 1:50 to 1:100, 1:100 to 1:1,000, 1:1,000 to 1:10,000, 1:10,000 to 1:100,000, or 1:100,000 to 1:1,000,000, or 1:20 to 1:50, 1:100, 1,000:1, 10,000:1, 100,000:1, and 1,000,000:1. vii) the fusosomes contain a lipid composition substantially similar to that of the source cells, or one or more of CL, Cer, DAG, HexCer, LPA, LPC, LPE, LPG, LPI, LPS, PA, PC, PE, PG, PI, PS, CE, SM, and TAG are within 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 75% of the corresponding lipid levels in the source cells; viii) the fusosomes comprise a proteomic composition similar to that of the source cells, e.g., using the assays of Examples 42 or 155; ix) the fusosomes comprise a lipid to protein ratio that is within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cells, e.g., as measured using the assay of Example 49; x) the fusosomes comprise a ratio of protein to nucleic acid (e.g., DNA) that is within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cells, as measured, for example, using the assay of Example 50; xi) the fusosomes comprise a ratio of lipid to nucleic acid (e.g., DNA) that is within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cells, as measured, for example, using the assays of Examples 51 or 159; xii) the fusosomes have a half-life in a subject, e.g., a mouse, that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the half-life of a reference cell, e.g., a source cell, e.g., by the assay of Example 75. xiii) the fusosomes transport at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% more glucose (e.g., labeled glucose, e.g., 2-NBDG) across the membrane than other similar fusosomes, e.g., in the absence of a negative control, e.g., glucose, as measured, e.g., using the assay of Example 64; xiv) the fusosomes comprise an esterase activity in their lumen that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the esterase activity in a reference cell, e.g., a source cell or a mouse embryonic fibroblast, using, e.g., the assay of Example 66; xv) the fusosomes comprise a metabolic activity level that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the metabolic activity (e.g., citrate synthase activity) in a reference cell, e.g., a source cell, e.g., as described in Example 68; xvi) the fusosomes comprise a respiration level (e.g., oxygen consumption rate) that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the respiration level in a reference cell, e.g., a source cell, as described, e.g., in Example 69; xvii) the fusosomes comprise an annexin-V staining level of an MFI of at most 18,000, 17,000, 16,000, 15,000, 14,000, 13,000, 12,000, 11,000, or 10,000, e.g., using the assay of Example 70; or the fusosomes comprise an annexin-V staining level that is at least 5%, 10%, 20%, 30%, 40%, or 50% lower than the annexin-V staining level of other similar fusosomes treated with menadione, e.g., in the assay of Example 70; or the fusosomes comprise an annexin-V staining level that is at least 5%, 10%, 20%, 30%, 40%, or 50% lower than the annexin-V staining level of macrophages treated with menadione in the assay of Example 70. xviii) the fusosomes have a miRNA content level that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more higher than the miRNA content level of the source cells, e.g., by the assay of Example 39; xix) the fusosomes have a soluble protein:insoluble protein ratio that is 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than that of the source cells, e.g., within 1%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% of that of the source cells, e.g., by the assay of Example 47; xx) the fusosomes have LPS levels of less than 5%, 1%, 0.5%, 0.01%, 0.005%, 0.0001%, 0.00001% or lower of the LPS content of the source cells or the lipid content of the fusosomes, as measured, for example, by mass spectrometry, e.g., by the assay of Example 48; xxi) the fusosomes are capable of transporting at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% more signaling, e.g., AKT phosphorylation in response to an extracellular signal, e.g., insulin, or glucose (e.g., labeled glucose, e.g., 2-NBDG) uptake in response to insulin, than other similar fusosomes, e.g., in the absence of a negative control, e.g., insulin, using, e.g., the assay of Example 63; xxii) the fusosomes have a level of juxtacrine signaling that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the level of juxtacrine signaling induced by a reference cell, e.g., a source cell or bone marrow stromal cell (BMSC), e.g., by the assay of Example 71; xxiii) the fusosomes have a level of paracrine signaling that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% greater than the level of paracrine signaling induced by a reference cell, e.g., a source cell or a macrophage, e.g., by the assay of Example 72; xxiv) the fusosomes polymerize actin at a level within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the level of polymerized actin in a reference cell, e.g., a source cell or a C2C12 cell, e.g., by the assay of Example 73; xxv) the fusosomes have a membrane potential within about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the membrane potential in a reference cell, e.g., a source cell or a C2C12 cell, e.g., by the assay of Example 74, or the fusosomes have a membrane potential of about -20 to -150 mV, -20 to -50 mV, -50 to -100 mV, or -100 to -150 mV; xxvi) the fusosomes are capable of secreting protein at a rate that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than a reference cell, e.g., a mouse embryonic fibroblast, using, e.g., the assay of Example 62; or xxvii) fusosomes have low immunogenicity, e.g., as described herein; and c) (Optionally) approve the release of a plurality of fusosomes or fusosome compositions if one or more of the criteria are met.

[0028] The present disclosure also provides, in some aspects, a method of producing a fusosome composition, comprising: a) providing, e.g., generating, a plurality of fusosomes described herein or a fusosome composition described herein; b) i) an immunogenic molecule, e.g., an immunogenic protein, e.g., as described herein; ii) a pathogen, e.g., a bacterium or virus, or iii) assaying one or more fusosomes from the plurality to determine the presence or level of one or more of the factors, c) (optionally) approving release of a plurality of fusosomes or fusosome compositions if one or more of the factors is below a reference value.

[0029] The present disclosure also provides, in some aspects, a method of administering a fusosome composition to a human subject, comprising: a) administering a first fusogen to a subject under conditions that allow for location of the first fusogen in one or more target cells in the subject; i) administering the first fusogen comprises administering a nucleic acid encoding the first fusogen under conditions that allow expression of the first fusogen in one or more target cells; or ii) the first fusogen does not contain a coiled-coil motif; b) administering to a human subject a fusosome composition comprising a plurality of fusosomes comprising a second fusogen, wherein the second fusogen is compatible with the first fusogen; and thereby administering the fusosome composition to a subject.

[0030] The present disclosure also provides, in some aspects, a method for delivering a therapeutic agent to a subject, comprising: a) administering a first fusogen to a subject under conditions that allow for location of the first fusogen in one or more target cells in the subject; i) administering the first fusogen comprises administering a nucleic acid encoding the first fusogen under conditions that allow expression of the first fusogen in one or more target cells; or ii) the first fusogen does not contain a coiled-coil motif; b) administering to a human subject a fusosome composition comprising a plurality of fusosomes containing a second fusogen and a therapeutic agent, wherein the second fusogen is compatible with the first fusogen; thereby delivering a therapeutic agent to the subject.

[0031] The present disclosure also provides, in some aspects, a method of modulating, e.g., enhancing, a biological function in a subject, comprising: a) administering a first fusogen to a subject under conditions that allow for location of the first fusogen in one or more target cells in the subject; i) administering the first fusogen comprises administering a nucleic acid encoding the first fusogen under conditions that allow expression of the first fusogen in one or more target cells; or ii) the first fusogen does not contain a coiled-coil motif; b) administering to a human subject a fusosome composition comprising a plurality of fusosomes comprising a second fusogen, wherein the second fusogen is compatible with the first fusogen; and thereby regulating a biological function in a subject.

[0032] In one aspect, the invention includes a fusosome comprising a chondrisome and a fusogen.

[0033] In one aspect, the invention includes a composition comprising a plurality of fusosomes, at least one fusosome comprising a chondrisome and a fusogen.

[0034] The present disclosure also provides, in some aspects, methods for assessing the fusosome content of target cells in a subject (e.g., fusosome fusion to target cells), comprising providing a biological sample from a subject receiving a fusosome composition (e.g., a fusosome composition described herein) and performing an assay to determine one or more characteristics of the biological sample resulting from fusion of fusosomes described herein with target cells in the biological sample. In some aspects, the present disclosure provides methods for measuring fusion with target cells, e.g., as described in Examples 54 or 124. In some embodiments, determining one or more characteristics of the biological sample comprises determining the presence of a fusogen, or the level of cargo or payload, or an activity associated with the cargo or payload.

[0035] In some aspects, the present disclosure provides methods for assessing fusosome content of target cells in a subject (e.g., fusosome fusion into target cells), the method comprising providing a biological sample from a subject receiving a fusosome composition, e.g., as described herein, and testing the biological sample for the presence of a fusogen, e.g., a fusogen described herein. In some cases, the level of fusogen detected is higher (e.g., at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000%, 10,000%, 50,000%, or 100,000% higher) than that observed in a corresponding biological sample from a subject not receiving the fusosome composition. In some embodiments, the subject is the same subject prior to administration of the fusosome composition; in some embodiments, the subject is a different subject.

[0036] In some aspects, the present disclosure provides methods for assessing fusosome content of target cells in a subject (e.g., fusosome fusion into target cells), the method comprising providing a biological sample from a subject receiving a fusosome composition, e.g., as described herein, and testing the biological sample for the presence of a fusogen, e.g., a fusogen described herein. In some cases, the detected cargo or payload level is higher (e.g., at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000%, 10,000%, 50,000%, or 100,000% higher) than that observed in a corresponding biological sample from a subject not receiving the fusosome composition. In some embodiments, the subject is the same subject prior to administration of the fusosome composition; in some embodiments, the subject is a different subject.

[0037] In some aspects, the present disclosure provides methods for assessing fusosome content of a target cell (e.g., fusosome fusion into a target cell in a subject), comprising providing a biological sample from a subject receiving a fusosome composition, e.g., as described herein, and testing the biological sample for a change in an activity associated with the fusosome composition, e.g., an activity associated with a cargo or payload delivered by the fusosome composition. In some cases, the level of activity detected is increased, for example, by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000%, 10,000%, 50,000%, or 100,000%, compared to that of a corresponding biological sample from a subject not receiving the fusosome composition (e.g., the same subject prior to administration of the fusosome composition). In some cases, the level of detected activity is reduced, e.g., by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000%, 10,000%, 50,000%, or 100,000%, compared to that of a corresponding biological sample from a subject not receiving the fusosome composition. In some embodiments, the subject is the same subject prior to administration of the fusosome composition; in some embodiments, the subject is a different subject.

[0038] In one aspect, the present disclosure provides a method of assessing fusosome fusion to a target cell in a subject, the method comprising providing a biological sample from a subject receiving a fusosome composition, e.g., as described herein, and assessing the level of unfused fusosomes in the biological sample.

[0039] Any of the aspects herein, eg, the fusosomes, fusosome compositions, and methods described above, can be combined with any of the embodiments herein, eg, one or more of the following embodiments.

[0040] In some embodiments, fusosomes can deliver (e.g., deliver) an agent, e.g., a protein, a nucleic acid (e.g., mRNA), an organelle, or a metabolite, to the cytosol of a target cell. Similarly, in some embodiments, the methods herein include delivering an agent to the cytosol of a target cell. In some embodiments, the agent is a protein (or a nucleic acid encoding a protein, e.g., an mRNA encoding a protein) that is absent, mutant, or at lower levels than wild-type in the target cell. In some embodiments, the target cell is from a subject with a genetic disease, e.g., a monogenic disease, e.g., a monogenic intracellular protein disease. In some embodiments, the agent comprises a transcription factor, e.g., an exogenous transcription factor or an endogenous transcription factor. In some aspects, the fusosome further comprises or delivers one or more (e.g., at least 2, 3, 4, 5, 10, 20, or 50) additional transcription factors, e.g., exogenous transcription factors, endogenous transcription factors, or a combination thereof.

[0041] In some embodiments, the fusosomes comprise (e.g., are capable of delivering to a target cell) a plurality of agents (e.g., at least two, three, four, five, 10, 20, or 50 agents), where each agent of the plurality acts on a step in a pathway within the target cell, e.g., the pathway is a biosynthetic pathway, a catabolic pathway, or a signaling cascade. In embodiments, each agent of the plurality upregulates a pathway or downregulates a pathway. In some embodiments, the fusosomes further comprise, or the method further comprises delivering, one or more additional agents (e.g., comprising a second plurality of agents) that do not act on a step in the pathway, e.g., that act on a step in a second pathway. In some embodiments, the fusosomes comprise (e.g., are capable of delivering to a target cell) a plurality of agents (e.g., at least two, three, four, five, 10, 20, or 50 agents), where each agent of the plurality is part of a single pathway, e.g., the pathway is a biosynthetic pathway, a catabolic pathway, or a signaling cascade. In some embodiments, the fusosome further comprises, or the method further comprises delivering, one or more additional agents (e.g., comprising multiple second agents) that are not part of a single pathway, e.g., are part of a second pathway.

[0042] In some embodiments, the target cells contain aggregated or misfolded proteins. In some embodiments, the fusosomes are capable of reducing (e.g., reducing) the level of aggregated or misfolded proteins in the target cells, or the methods herein include reducing the level of aggregated or misfolded proteins in the target cells.

[0043] In some embodiments, the agent is selected from a transcription factor, an enzyme (e.g., a nuclear or cytosolic enzyme), a reagent that mediates sequence-specific modifications to DNA (e.g., Cas9, ZFN, or TALEN), an mRNA (e.g., an mRNA encoding an intracellular protein), an organelle, or a metabolite.

[0044] In some embodiments, fusosomes can deliver (e.g., deliver) agents, such as proteins, to the plasma membrane of target cells. Similarly, in some embodiments, the methods herein include delivering agents to the plasma membrane of target cells. In some embodiments, delivering a protein includes delivering a nucleic acid (e.g., mRNA) encoding the protein to the target cell so that the target cell produces the protein and localizes it to its membrane. In some embodiments, the fusosomes include the protein, or the methods further include delivering the protein, and fusion of the fusosome with the target cell transfers the protein to the plasma membrane of the target cell. In some embodiments, the agent includes a cell surface ligand or antibody that binds to a cell surface receptor. In some embodiments, the fusosomes further include, or the methods further include delivering, a second agent that includes or encodes a second cell surface ligand or antibody that binds to the cell surface receptor, optionally further including or encoding one or more (e.g., 1, 2, 3, 4, 5, 10, 20, 50, or more) additional cell surface ligands or antibodies that bind to the cell surface receptor. In some embodiments, the first agent and the second agent form a complex, and optionally, the complex further comprises one or more additional cell surface ligands. In some embodiments, the agent comprises or encodes a cell surface receptor, e.g., an exogenous cell surface receptor. In some embodiments, the fusosome comprises or encodes the second cell surface receptor, and optionally further comprises or encodes one or more additional cell surface receptors (e.g., 1, 2, 3, 4, 5, 10, 20, 50, or more cell surface receptors).

[0045] In some embodiments, the first agent and the second agent form a complex, and optionally, the complex further comprises one or more additional cell surface receptors. In some embodiments, the agent comprises or encodes an antigen or antigen-presenting protein.

[0046] In some aspects, fusosomes can deliver (e.g., deliver) secreted agents, e.g., secreted proteins, to a target site (e.g., the extracellular space), e.g., by delivering a nucleic acid (e.g., mRNA) encoding the protein to a target cell under conditions that allow the target cell to produce and secrete the protein. Similarly, in some embodiments, the methods herein include delivering a secreted agent described herein. In embodiments, the secreted protein is endogenous or exogenous. In embodiments, the secreted protein comprises a therapeutic protein, e.g., an antibody molecule, a cytokine, or an enzyme. In embodiments, the secreted protein comprises an autocrine or paracrine signaling molecule. In embodiments, the secreted agent comprises a secretory granule.

[0047] In some aspects, the fusosomes can reprogram (e.g., reprogram) target cells (e.g., immune cells) by delivering an agent selected from, for example, a transcription factor or mRNA, or a plurality of such agents. Similarly, in some embodiments, the methods herein include reprogramming target cells. In embodiments, the reprogramming includes inducing pancreatic endocrine cells to exhibit one or more characteristics of pancreatic beta cells by inducing non-dopaminergic neurons to exhibit one or more characteristics of dopaminergic neurons, or by inducing exhausted T cells to exhibit one or more characteristics of non-exhausted T cells, e.g., killer T cells. In some embodiments, the agent comprises an antigen. In some embodiments, the fusosomes comprise a first agent comprising an antigen and a second agent comprising an antigen-presenting protein.

[0048] In some aspects, the fusosomes can provide (e.g., provide) one or more cell surface receptors to a target cell (e.g., an immune cell). Similarly, in some embodiments, the methods herein include providing one or more cell surface receptors.

[0049] In some embodiments, fusosomes can modify, e.g., modify, target tumor cells. Similarly, in some embodiments, the methods herein include modifying target tumor cells. In embodiments, fusosomes contain mRNA encoding an immunostimulatory ligand, an antigen-presenting protein, a tumor suppressor protein, or a pro-apoptotic protein. In some embodiments, fusosomes contain miRNA that can reduce target cell levels of an immunosuppressive ligand, a cell growth signal, or a growth factor.

[0050] In some embodiments, the fusosomes comprise an agent that is immunomodulatory, eg, immunostimulatory.

[0051] In some embodiments, the fusosomes are capable of presenting (e.g., causing) antigens to be presented to target cells. Similarly, in some embodiments, the methods herein include presenting antigens on target cells.

[0052] In some embodiments, the fusosomes promote regeneration of the target tissue. Similarly, in some embodiments, the methods herein include promoting regeneration of the target tissue. In embodiments, the target cells are cardiac cells, e.g., cardiomyocytes (e.g., quiescent cardiomyocytes), hepatoblasts (e.g., biliary hepatoblasts), epithelial cells, naive T cells, macrophages (e.g., tumor-infiltrating macrophages), or fibroblasts (e.g., cardiac fibroblasts). In embodiments, the source cells are T cells (e.g., T reg ), macrophages, or cardiomyocytes.

[0053] In some embodiments, fusosomes can deliver (e.g., deliver) nucleic acids to target cells, e.g., for gene therapy, e.g., to stably modify the genome of the target cell. Similarly, in some embodiments, methods herein include delivering nucleic acids to target cells. In some embodiments, the target cells have an enzyme deficiency, e.g., contain a mutation in an enzyme that results in reduced (e.g., no) activity of the enzyme.

[0054] In some embodiments, fusosomes can deliver (e.g., deliver) reagents that mediate sequence-specific modifications to DNA (e.g., Cas9, ZFN, or TALEN) within a target cell. Similarly, in some embodiments, the methods herein include delivering a reagent to a target cell. In embodiments, the target cell is a muscle cell (e.g., a skeletal muscle cell), a kidney cell, or a liver cell.

[0055] In some embodiments, fusosomes can deliver (eg, deliver) nucleic acids to target cells, for example, to transiently alter gene expression in the target cells.

[0056] In some embodiments, fusosomes can deliver (e.g., deliver) proteins to target cells, e.g., to temporarily rescue protein deficiency. Similarly, in some embodiments, the methods herein include delivering proteins to target cells. In embodiments, the protein is a membrane protein (e.g., a membrane transporter protein), a cytoplasmic protein (e.g., an enzyme), or a secreted protein (e.g., an immunosuppressive protein).

[0057] In some embodiments, fusosomes can deliver (e.g., deliver) organelles to target cells, e.g., the target cells have a defective organelle network. Similarly, in some embodiments, the methods herein include delivering organelles to target cells. In embodiments, the source cells are hepatocytes, skeletal muscle cells, or neurons.

[0058] In some embodiments, fusosomes can deliver (e.g., deliver) nuclei to target cells, for example, the target cells have genetic mutations. Similarly, in some embodiments, the methods herein include delivering nuclei to target cells. In some embodiments, the nuclei are autologous and contain one or more genetic changes related to the target cell, for example, the nuclei contain sequence-specific modifications to DNA (e.g., Cas9, ZFN, or TALEN), or artificial chromosomes, additional genetic sequences integrated into the genome, deletions, or any combination thereof. In embodiments, the source of the autologous nuclei is stem cells, for example, hematopoietic stem cells. In embodiments, the target cells are muscle cells (e.g., skeletal muscle cells or cardiac myocytes), hepatocytes, or neurons.

[0059] In some embodiments, fusosomes enable intracellular molecular delivery, for example, delivery of protein drugs to target cells. Similarly, in some embodiments, the methods herein include delivering molecules to the intracellular space of target cells. In embodiments, the protein drug is an inhibitor. In embodiments, the protein drug comprises a nanobody, scFv, camelid antibody, peptide, macrocycle, or small molecule.

[0060] In some embodiments, the fusosomes can cause the target cell to secrete (e.g., secrete) a protein, e.g., a therapeutic protein. Similarly, in some embodiments, the methods herein include causing the target cell to secrete a protein.

[0061] In some embodiments, the fusosomes are capable of secreting (e.g., secrete) an agent, e.g., a protein. In some embodiments, the agent, e.g., the secreted agent, is delivered to a target site in a subject. In some embodiments, the agent is a protein that cannot be or is difficult to produce recombinantly. In some embodiments, the fusosomes that secrete the protein are from a source cell selected from MSCs or chondrocytes.

[0062] In some embodiments, fusosomes comprise one or more cell surface ligands (e.g., 1, 2, 3, 4, 5, 10, 20, 50, or more cell surface ligands) on their membranes. Similarly, in some embodiments, the methods herein comprise presenting one or more cell surface ligands to target cells. In some embodiments, the fusosomes bearing the cell surface ligands are from a source cell selected from a neutrophil (e.g., the target cell is a tumor-infiltrating lymphocyte), a dendritic cell (e.g., the target cell is a naive T cell), or a neutrophil (e.g., the target is a tumor cell or a virus-infected cell). In some embodiments, the fusosomes comprise a membrane complex, e.g., a complex comprising at least two, three, four, or five proteins, e.g., a homodimer, heterodimer, homotrimer, heterotrimer, homotetramer, or heterotetramer. In some embodiments, the fusosomes comprise an antibody, e.g., a toxic antibody, and the fusosomes can deliver the antibody to a target site, e.g., by homing to the target site. In some embodiments, the source cells are NK cells or neutrophils.

[0063] In some embodiments, the methods herein include inducing secretion of a protein from a target cell or presentation of a ligand on the surface of the target cell. In some embodiments, the fusosomes can induce cell death of the target cell. In some embodiments, the fusosomes are from NK source cells.

[0064] In some embodiments, the fusosome or target cell is capable of phagocytosis (e.g., of a pathogen). Similarly, in some embodiments, the methods herein include inducing phagocytosis.

[0065] In some embodiments, fusosomes sense and respond to their local environment, hi some embodiments, fusosomes can sense levels of metabolites, interleukins, or antigens.

[0066] In some embodiments, the fusosomes are capable of chemotaxis, extravasation, or one or more metabolic activities. In some embodiments, the metabolic activity is selected from kynurinin, gluconeogenesis, prostaglandin fatty acid oxidation, adenosine metabolism, the urea cycle, and thermogenic respiration. In some embodiments, the source cells are neutrophils and the fusosomes are capable of homing to the site of injury. In some embodiments, the source cells are macrophages and the fusosomes are capable of phagocytosis. In some embodiments, the source cells are brown adipose tissue cells and the fusosomes are capable of lipolysis.

[0067] In some embodiments, the fusosomes comprise (e.g., are capable of delivering to a target cell) multiple agents (e.g., at least 2, 3, 4, 5, 10, 20, or 50 agents). In embodiments, the fusosomes comprise an inhibitory nucleic acid (e.g., siRNA or miRNA) and mRNA.

[0068] In some embodiments, the fusosomes comprise a membrane protein or a nucleic acid encoding a membrane protein (e.g., capable of being delivered to a target cell). In embodiments, the fusosomes are capable of reprogramming or transdifferentiating a target cell, e.g., the fusosomes comprise one or more agents that induce reprogramming or transdifferentiation of the target cell.

[0069] In some embodiments, the subject is in need of regeneration, hi some embodiments, the subject is suffering from cancer, an autoimmune disease, an infectious disease, a metabolic disease, a neurodegenerative disease, or a genetic disease (e.g., an enzyme deficiency).

[0070] In some embodiments (e.g., for assaying non-endocytic delivery of cargo), cargo delivery is assayed using one or more (e.g., all) of the following steps: (a) placing 30,000 HEK-293T target cells in a first well of a 96-well plate containing 100 nM bafilomycin A1 and placing a similar number of similar cells in a second well of a 96-well plate lacking bafilomycin A1; (b) culturing the target cells in DMEM medium at 37°C and 5% CO2 for 4 hours; (c) contacting the target cells with 10 μg of fusosomes containing the cargo; (d) incubating the target cells and fusosomes at 37°C and 5% CO2 for 24 hours; and (e) determining the percentage of cells in the first and second wells that contain the cargo. Step (e) may include detecting the cargo using microscopy, e.g., immunofluorescence. Step (e) may include indirectly detecting the cargo, e.g., detecting a downstream effect of the cargo, such as the presence of a reporter protein. In some embodiments, one or more of steps (a)-(e) above are performed as described in Example 135.

[0071] In some embodiments, an inhibitor of endocytosis (e.g., chloroquine or bafilomycin A1) inhibits endosomal acidification. In some embodiments, cargo delivery is independent of lysosomal acidification. In some embodiments, an inhibitor of endocytosis (e.g., dynasore) inhibits dynamin. In some embodiments, cargo delivery is independent of dynamin activity.

[0072] In some embodiments (e.g., for specific delivery of cargo to target cells versus non-target cells), cargo delivery is assayed using one or more (e.g., all) of the following steps: (a) placing 30,000 HEK-293T target cells overexpressing CD8a and CD8b into a first well of a 96-well plate and 30,000 HEK-293T non-target cells not overexpressing CD8a and CD8b into a second well of the 96-well plate; (b) culturing the target cells in DMEM medium at 37°C and 5% CO2 for 4 hours; (c) contacting the target cells with 10 μg of fusosomes containing the cargo; (d) incubating the target cells and fusosomes at 37°C and 5% CO2 for 24 hours; and (e) determining the percentage of cells in the first and second wells that contain the cargo. Step (e) may include detecting the cargo using microscopy, e.g., immunofluorescence. Step (e) may include indirectly detecting the cargo, e.g., detecting a downstream effect of the cargo, such as the presence of a reporter protein. In some embodiments, one or more of steps (a)-(e) above are performed as described in Example 124.

[0073] In some embodiments, ii) the source cells are other than 293 cells, HEK cells, human endothelial cells, or human epithelial cells; iii) the fusogen is other than a viral protein; iv) the fusosome, or a composition or preparation comprising a plurality of fusosomes, has a density other than, for example, 1.08 g / ml to 1.12 g / ml; v) the fusosomes have a density of 1.25 g / ml + / - 0.05, e.g., as measured by the assay of Example 33; vi) fusosomes are not captured by the scavenger system in the circulation or by Kupffer cells in the liver sinus; vii) the fusosomes are not captured by the reticuloendothelial system (RES) in the subject, e.g., by the assay of Example 76; viii) when a plurality of fusosomes are administered to a subject, less than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the plurality are captured by the RES after 24, 48, or 72 hours, e.g., by the assay of Example 76; ix) The fusosomes have a diameter greater than 5 um, 6 um, 7 um, 8 um, 10 um, 20 um, 50 um, 100 um, 150 um, or 200 um.

[0074] In some embodiments, the fusosomes comprise or are comprised in a cell biological preparation. In some embodiments, the fusosomes comprise enucleated cells. In some embodiments, the fusosomes comprise an inactivated nucleus. In some embodiments, the fusosomes do not comprise a functional nucleus.

[0075] In some embodiments, the fusosomes or fusosome compositions have or are identified as having one or more (e.g., at least two, three, four, or five) of the characteristics described herein, e.g., the following characteristics:

[0076] In some embodiments, fusosomes fuse with target cells at a rate that is, e.g., at least at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher than non-target cells, e.g., in the assay of Example 54. In some embodiments, fusosomes fuse with target cells at a rate that is, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% higher than other fusosomes, e.g., in the assay of Example 54. In some embodiments, fusosomes fuse with 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, e.g., in the assay of Example 54. In embodiments, the amount of targeted fusion is about 30% to 70%, 35% to 65%, 40% to 60%, 45% to 55%, or 45% to 50%, e.g., about 48.8%, e.g., in the assay of Example 54. In embodiments, the amount of targeted fusion is about 20% to 40%, 25% to 35%, or 30% to 35%, e.g., about 32.2%, e.g., in the assay of Example 55.

[0077] In some embodiments, the fusogen is present in at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies or less, e.g., as measured by the assay of Example 29. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the fusogens contained in fusosomes are located in the plasma membrane. In embodiments, fusosomes also contain fusogens internally, e.g., in the cytoplasm or organelles. In some embodiments, the fusogen comprises (or is identified as comprising) about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10%, 11%, 12%, 13%, 14%, 15%, 20% or more of the total protein in the fusosomes, or about 1-30%, 5-20%, 10-15%, 12-15%, 13-14%, or 13.6%, as determined, for example, according to the methods described in Example 162 and / or by mass spectrometry assays. In embodiments, the fusogen comprises (or is identified as comprising) about 13.6% of the total protein in the fusosomes. In some embodiments, the fusogen is (or is identified as) more or less abundant than one or more additional proteins of interest, as determined, for example, according to the methods described in Example 162. In one embodiment, the fusogen has (or is identified as having) a ratio to EGFP of about 140, 145, 150, 151, 152, 153, 154, 155, 156, 157 (e.g., 156.9), 158, 159, 160, 165, or 170.In another embodiment, the fusogen has (or is identified as having) a ratio to CD63 of about 2700, 2800, 2900, 2910 (e.g., 2912), 2920, 2930, 2940, 2950, ​​2960, 2970, 2980, 2990, or 3000, or about 1000-5000, 2000-4000, 2500-3500, 2900-2930, 2910-2915, or 2912.0, e.g., by mass spectrometry assay. In one embodiment, the fusogen has (or is identified as having) a ratio to ARRDC1 of about 600, 610, 620, 630, 640, 650, 660 (e.g., 664.9), 670, 680, 690, or 700. In another embodiment, the fusogen has (or is identified as having) a ratio to GAPDH of about 50, 55, 60, 65, 70 (e.g., 69), 75, 80, or 85, or about 1-30%, 5-20%, 10-15%, 12-15%, 13-14%, or 13.6%. In another embodiment, the fusogen has (or is identified as having) a ratio to CNX of about 500, 510, 520, 530, 540, 550, 560 (e.g., 558.4), 570, 580, 590, or 600, or about 300-800, 400-700, 500-600, 520-590, 530-580, 540-570, 550-560, or 558.4, e.g., by mass spectrometry assay.

[0078] In some embodiments, the fusosomes contain at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies or fewer of the therapeutic agent, e.g., as measured by the assay of Example 43 or 156. In some embodiments, the fusosomes comprise at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies of the protein therapeutic, e.g., as measured by the assay of Example 43 or 156. In some embodiments, the fusosomes comprise at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies of the nucleic acid therapeutic agent. In some embodiments, the fusosomes comprise at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies of the DNA therapeutic.In some embodiments, the fusosomes comprise at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies of the RNA therapeutic. In some embodiments, the fusosomes comprise at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies of the exogenous therapeutic agent. In some embodiments, the fusosomes comprise at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies of the exogenous protein therapeutic. In some embodiments, the fusosomes comprise at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies of the exogenous nucleic acid (e.g., DNA or RNA) therapeutic agent.In some embodiments, the ratio of the copy number of the fusogen to the copy number of the therapeutic agent is 1,000,000:1 to 100,000:1, 100,000:1 to 10,000:1, 10,000:1 to 1,000:1, 1,000:1 to 100:1, 100:1 to 50:1, 50:1 to 20:1, 20:1 to 10:1, 10:1 to 5:1, 50:1 to 50 ... :1 to 2:1, 2:1 to 1:1, 1:1 to 1:2, 1:2 to 1:5, 1:5 to 1:10, 1:10 to 1:20, 1:20 to 1:50, 1:50 to 1:100, 1:100 to 1:1,000, 1:1,000 to 1:10,000, 1:10,000 to 1:100,000, or 1:100,000 to 1:1,000,000.

[0079] In some embodiments, the fusosomes deliver at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies of a therapeutic agent to a target cell. In some embodiments, the fusosomes deliver at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies of the protein therapeutic to the target cell. In some embodiments, the fusosomes deliver at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies of the nucleic acid therapeutic agent to the target cell. In some embodiments, the fusosomes deliver at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies of the RNA therapeutic to the target cell.In some embodiments, the fusosomes deliver at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies of a DNA therapeutic to a target cell.

[0080] In some embodiments, fusosomes deliver at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the cargo (e.g., therapeutic agent, e.g., endogenous or exogenous therapeutic agent) contained within the fusosome to the target cell. In some embodiments, fusosomes that fuse with the target cell(s) deliver an average of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the cargo (e.g., therapeutic agent, e.g., endogenous or exogenous therapeutic agent) contained within the fusosomes that fuse with the target cell(s). In some embodiments, the fusosome composition delivers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the cargo (e.g., a therapeutic agent, e.g., an endogenous therapeutic agent or an exogenous therapeutic agent) contained in the fusosome to the target tissue.

[0081] In some embodiments, the fusosomes comprise 0.00000001 mg of fusogen per mg of total protein in the fusosomes, e.g., 0.00000001 to 0.0000001, 0.0000001 to 0.000001, 0.000001 to 0.00001, 0.00001 to 0.0001, 0.0001 to 0.001, 0.001 to 0.01, 0.01 to 0.1, or 0.1 to 1 mg of fusogen per mg of total protein in the fusosomes. In some embodiments, the fusosomes comprise 0.00000001 mg to 5 mg of fusogen per mg of lipid in the fusosomes, e.g., 0.00000001 to 0.0000001, 0.0000001 to 0.000001, 0.000001 to 0.0001, 0.0001 to 0.001, 0.001 to 0.01, 0.01 to 0.1, 0.1 to 1, or 1 to 5 mg of fusogen per mg of lipid in the fusosomes.

[0082] In some embodiments, the cargo is a protein cargo. In embodiments, the cargo is an endogenous or synthetic protein cargo. In some embodiments, the fusosomes have (or are identified as having) at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or more protein cargo molecules per fusosome. In one embodiment, the fusosomes have (or are identified as having) about 100, 110, 120, 130, 140, 150, 160, 166, 170, 180, 190, or 200 protein drug molecules per fusosome, as quantified, for example, according to the method described in Example 156. In some embodiments, the endogenous or synthetic protein cargo comprises (or is identified as comprising) about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, or more of the total protein in the fusosome. In one embodiment, the synthetic protein cargo comprises (or is identified as comprising) about 13.6% of the total protein in the fusosome. In some embodiments, the synthetic protein cargo comprises about 4×10 -3 , 5×10 -3 , 6×10-3 (For example, 6.37×10 -3 ), 7×10 -3 , or 8 × 10 -3 In embodiments, the synthetic protein cargo has (or is identified to have) a ratio to CD63 of about 10, 15, 16, 17, 18 (e.g., 18.6), 19, 20, 25, or 30, or about 10-30, 15-25, 16-19, 18-19, or 18.6. In embodiments, the synthetic protein cargo has (or is identified to have) a ratio to ARRDC1 of about 2, 3, 4 (e.g., 4.24), 5, 6, or 7. In embodiments, the synthetic protein cargo has (or is identified to have) a ratio to GAPDH of about 0.1, 0.2, 0.3, 0.4 (e.g., 0.44), 0.5, 0.6, or 0.7. In embodiments, the synthetic protein cargo has (or is identified to have) a ratio to CNX of about 1, 2, 3 (e.g., 3.56), 4, 5, or 6. In embodiments, the synthetic protein cargo has (or is identified to have) a ratio to TSG101 of about 10, 15, 16, 17, 18, 19 (e.g., 19.52), 20, 21, 22, 23, 24, 25, or 30.

[0083] In some embodiments, the fusogen comprises (or is identified as comprising) at least 0.5%, 1%, 5%, 10%, or more of the total protein in the fusosome, e.g., by mass spectrometry assay. In one embodiment, the fusogen comprises (or is identified as comprising) about 1-30%, 5-20%, 10-15%, 12-15%, 13-14%, or 13.6% of the total protein in the fusosome, e.g., by mass spectrometry assay. In some embodiments, the fusogen is more abundant than other proteins of interest. In embodiments, the fusogen has (or is identified as having) a ratio to payload protein, e.g., EGFP, of about 145-170, 150-165, 155-160, or 156.9, e.g., by mass spectrometry assay. In embodiments, the fusogen has (or is identified as having) a ratio to CD63 of about 1000-5000, 2000-4000, 2500-3500, 2900-2930, 2910-2915, or 2912.0, e.g., by mass spectrometry assay. In embodiments, the fusogen has a ratio to ARRDC1 of about 300-1000, 400-900, 500-800, 600-700, 640-690, 650-680, 660-670, or 664.9, e.g., by mass spectrometry assay. In embodiments, the fusogen has (or is identified as having) a ratio to GAPDH of about 20-120, 40-100, 50-90, 60-80, 65-75, 68-70, or 69.0, e.g., by mass spectrometry assay. In embodiments, the fusogen has a ratio to CNX of about 200-900, 300-800, 400-700, 500-600, 520-590, 530-580, 540-570, 550-560, or 558.4, e.g., by mass spectrometry assay. In embodiments, the mass spectrometry assay is the assay of Example 162.

[0084] In some embodiments, the number of lipid species present (e.g., shared) in both fusosomes and source cells is (or is identified to be) at least 300, 400, 500, 550, 560, or 569, e.g., using a mass spectrometry assay, or 500-700, 550-600, or 560-580. In embodiments, the number of lipid species present in fusosomes at levels at least 25% of the corresponding lipid levels in source cells (both normalized to total lipid levels in the sample) is (or is identified to be) at least 300, 400, 500, 530, 540, or 548, e.g., using a mass spectrometry assay, or 400-700, 500-600, 520-570, 530-560, or 540-550. In some embodiments, the ratio of lipid species present (e.g., shared) in both fusosomes and source cells to total lipid species in the source cells is (or is determined to be) about 0.4-1.0, 0.5-0.9, 0.6-0.8, or 0.7, or at least 0.4, 0.5, 0.6, or 0.7, e.g., using a mass spectrometry assay. In some embodiments, the mass spectrometry assay is the assay of Example 154.

[0085] In some embodiments, the number of protein species present (e.g., shared) in both fusosomes and source cells is (or is determined to be) at least 500, 1000, 1100, 1200, 1300, 1400, 1487, 1500, or 1600, or 1200-1700, 1300-1600, 1400-1500, 1450-1500, or 1480-1490, e.g., using a mass spectrometry assay. In embodiments, the number of protein species present in fusosomes at levels at least 25% of the corresponding protein levels in source cells (both normalized to total protein levels in the sample) is (or is determined to be) at least 500, 600, 700, 800, 900, 950, 957, 1000, or 1200, e.g., using a mass spectrometry assay. In some embodiments, the ratio of protein species present (e.g., shared) in both fusosomes and source cells to total protein species in the source cells is (or is determined to be) about 0.1-0.6, 0.2-0.5, 0.3-0.4, or 0.333, or at least about 0.1, 0.2, 0.3, 0.333, or 0.4, e.g., using a mass spectrometry assay. In embodiments, the mass spectrometry assay is the assay of Example 155.

[0086] In some embodiments, CD63 is present (or is determined to be present) in an amount less than 0.048%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10% of the total protein in fusosomes, e.g., by a mass spectrometry assay, e.g., the assay of Example 157.

[0087] In some embodiments, fusosomes are generated by extrusion through a filter, e.g., about 1-10, 2-8, 3-7, 4-6, or 5 um. In some embodiments, fusosomes have (or are identified as having) an average diameter of about 1-5, 2-5, 3-5, 4-5, or 5 um. In some embodiments, fusosomes have (or are identified as having) an average diameter of at least 1, 2, 3, 4, or 5 um.

[0088] In some embodiments, fusosomes are enriched (or identified as being enriched) in one or more (e.g., at least two, three, four, five, or all) of the following lipids compared to source cells: cholesteryl esters, free cholesterol, ether-linked lysophosphatidylethanolamine, lysophosphatidylserine, phosphatidate, ether-linked phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. In some embodiments, fusosomes are depleted (or identified as being depleted) in one or more (e.g., at least two, three, four, five, or all) of the following lipids compared to source cells: ceramide, cardiolipin, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, ether-linked phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, and triacylglycerol. In some embodiments, the fusosomes are enriched (or identified as being enriched) in one or more of the aforementioned enriched lipids and depleted in one or more of the aforementioned depleted lipids. In some embodiments, the fusosomes contain (or are identified as containing) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 5-fold, or 10-fold more enriched lipids as a percentage of total lipids than the corresponding level in the source cells. In some embodiments, the fusosomes contain (or are identified as containing) less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the depleted lipids as a percentage of total lipids than the corresponding level in the source cells. In embodiments, the lipid enrichment is measured by a mass spectrometry assay, e.g., the assay of Example 164.

[0089] In some embodiments, CE lipid levels are about 2-fold higher in fusosomes than in exosomes (compared to total lipids in the sample), and / or about 5, 6, 7, 8, 9, or 10-fold higher (or are identified as higher) in fusosomes than in parental cells. In some embodiments, ceramide lipid levels are about 2, 3, 4, or 5-fold higher (or are identified as higher) in parental cells than in fusosomes (compared to total lipids in the sample). In some embodiments, cholesterol levels are about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2-fold higher in exosomes than in fusosomes (compared to total lipids in the sample), and / or about 2-fold higher (or are identified as higher) in fusosomes than in parental cells. In some embodiments, CL lipid levels are at least about 5, 10, 20, 30, or 40-fold higher (or are identified as higher) in parental cells than in fusosomes (compared to total lipids in the sample). In some embodiments, DAG lipid levels are about 2 or 3 times higher in exosomes than in fusosomes (compared to total lipids in the sample), and / or about 1.5 or 2 times higher (or are identified as higher) in parental cells than in fusosomes. In some embodiments, PC lipid levels are about equal (or are identified as about equal) between exosomes and fusosomes (compared to total lipids in the sample), and / or about 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8 times higher (or are identified as higher) in parental cells than in fusosomes. In some embodiments, PC O-lipid levels are about equal (or are identified as about equal) between exosomes and fusosomes (compared to total lipids in the sample), and / or about 2 times higher (or are identified as higher) in parental cells than in fusosomes. In some embodiments, PE lipid levels are about 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8 times higher in fusosomes than in exosomes (compared to total lipids in the sample), and / or about 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8 times higher (or identified as higher) in parental cells than in fusosomes.In some embodiments, PE O-lipid levels are approximately equal (or are identified as approximately equal) between exosomes and fusosomes (compared to total lipids in the sample) and / or are about 1.5, 1.6, 1.7, 1.8, 1.9, or 2 times higher (or are identified as higher) in parental cells than in fusosomes. In some embodiments, PG lipid levels are approximately equal (or are identified as approximately equal) between exosomes and fusosomes (compared to total lipids in the sample) and / or are about 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher (or are identified as higher) in parental cells than in fusosomes. In some embodiments, PI lipid levels are approximately equal (or are identified as approximately equal) between exosomes and fusosomes (compared to total lipids in the sample) and / or are about 3, 4, 5, 6, or 7 times higher (or are identified as higher) in parental cells than in fusosomes. In some embodiments, PS lipid levels are approximately equal (or are identified as approximately equal) between exosomes and fusosomes (compared to total lipids in the sample) and / or are about 2-fold higher (or are identified as higher) in fusosomes than in parental cells. In some embodiments, SM lipid levels are approximately equal (or are identified as approximately equal) between exosomes and fusosomes (compared to total lipids in the sample) and / or are about 2-, 2.5-, or 3-fold higher (or are identified as higher) in fusosomes than in parental cells. In some embodiments, TAG lipid levels are approximately equal (or are identified as approximately equal) between exosomes and fusosomes (compared to total lipids in the sample) and / or are about 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, or more times higher (or are identified as higher) in parental cells than in fusosomes.

[0090] In some embodiments, fusosomes are enriched (or identified as being enriched) in one or more (e.g., at least two, three, four, five, or all) of the following lipids compared to exosomes: cholesteryl esters, ceramides, diacylglycerols, lysophosphatidates, and phosphatidylethanolamines, and triacylglycerols. In some embodiments, fusosomes are depleted (or identified as being depleted) in one or more (e.g., at least two, three, four, five, or all) of the following lipids compared to exosomes (e.g., compared to total lipids in the sample): free cholesterol, hexosylceramides, lysophosphatidylcholine, ether-linked lysophosphatidylcholine, lysophosphatidylethanolamine, ether-linked lysophosphatidylethanolamine, and lysophosphatidylserine. In some embodiments, fusosomes are enriched (or identified as being enriched) in one or more of the aforementioned enriched lipids and depleted in one or more of the aforementioned depleted lipids. In some embodiments, fusosomes comprise (or are identified as comprising) enriched lipids as a percentage of total lipids that are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 5-fold, or 10-fold greater than the corresponding levels in exosomes. In some embodiments, fusosomes comprise (or are identified as comprising) depleted lipids as a percentage of total lipids at levels that are less than 90%, 80%, 70%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the corresponding levels in exosomes. In embodiments, lipid enrichment is measured by a mass spectrometry assay, e.g., the assay of Example 164.

[0091] In some embodiments, ceramide lipid levels are about 2-fold higher in fusosomes than in exosomes (compared to total lipids in the sample), and / or are about 2-fold higher (or are identified as higher) in exosomes than in fusosomes. In some embodiments, HexCer lipid levels are about 1.5, 1.6, 1.7, 1.8, 1.9, or 2-fold higher (or are identified as higher) in exosomes than in fusosomes (compared to total lipids in the sample), and / or are about equal (or are identified as about equal) in parental cells and fusosomes. In some embodiments, LPA lipid levels are about 3 or 4-fold higher (or are identified as higher) in fusosomes than in exosomes (compared to total lipids in the sample), and / or are about 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8-fold higher (or are identified as higher) in fusosomes than in parental cells. In some embodiments, LPC lipid levels are about 2-fold higher in exosomes than in fusosomes (compared to total lipids in the sample), and / or about 1.5, 1.6, 1.7, 1.8, 1.9, or 2-fold higher (or are identified as higher) in parental cells than in fusosomes. In some embodiments, LPC O-lipid levels are about 3 or 4-fold higher in exosomes than in fusosomes (compared to total lipids in the sample), and / or about equal (or are identified as about equal) between parental cells and fusosomes. In some embodiments, LPE lipid levels are about 1.5, 1.6, 1.7, 1.8, 1.9, or 2-fold higher (or are identified as higher) in exosomes than in fusosomes (compared to total lipids in the sample), and / or about 1.5, 1.6, 1.7, 1.8, 1.9, or 2-fold higher (or are identified as higher) in parental cells than in fusosomes. In some embodiments, LPE O-lipid levels are (or are identified as) about 2- or 3-fold higher in exosomes than in fusosomes (compared to total lipids in the sample) and / or are (or are identified as) about equal between the parental cells and fusosomes. In some embodiments, LPS lipid levels are (or are identified as) about 3-fold higher in exosomes than in fusosomes (compared to total lipids in the sample).In some embodiments, PA lipid levels are about 1.5, 1.6, 1.7, 1.8, 1.9, or 2 times higher in fusosomes than in exosomes (relative to total lipids in the sample), and / or about 2 times higher (or identified as higher) in fusosomes than in parental cells. In some embodiments, PG lipid levels are about equal (or identified as about equal) between fusosomes and exosomes (relative to total lipids in the sample), and / or about 10, 11, 12, 13, 14, or 15 times higher (or identified as higher) in parental cells than in fusosomes.

[0092] In some embodiments, fusosomes contain lipid compositions substantially similar to that of the source cells, or one or more of CL, Cer, DAG, HexCer, LPA, LPC, LPE, LPG, LPI, LPS, PA, PC, PE, PG, PI, PS, CE, SM, and TAG are within 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the corresponding lipid levels in the source cells. In embodiments, the lipid composition of fusosomes is similar to the cells from which they are derived. In embodiments, fusosomes and parent cells have (or are identified as having) similar lipid compositions if about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% or more of the lipid species identified in any replicate sample of parent cells are present (or are identified as being present) in any replicate sample of fusosomes, e.g., as determined according to Example 154. In embodiments of identified lipids, the average level in fusosomes is about 10%, 15%, 20%, 25%, 30%, 35%, or 40% higher than the corresponding average lipid species level in parent cells (relative to the total lipids in the sample). In one embodiment, the lipid composition of fusosomes is enriched and / or depleted in specific lipids (relative to the total lipids in the sample) compared to parent cells.

[0093] In some embodiments, the lipid composition of the fusosomes is enriched and / or depleted (or identified as enriched and / or depleted) in specific lipids compared to parental cells, e.g., as determined according to the methods described in Example 164.

[0094] In some embodiments, fusosomes have (or are identified as having) a phosphatidylserine to total lipid ratio greater than that of parental cells. In embodiments, fusosomes have (or are identified as having) a phosphatidylserine to total lipid ratio of about 110%, 115%, 120%, 121%, 122%, 123%, 124%, 125%, 130%, 135%, 140%, or more compared to that of parental cells. In some embodiments, fusosomes are enriched (or are identified as being enriched) in cholesteryl esters, free cholesterol, ether-linked lysophosphatidylethanolamine, lysophosphatidylserine, phosphatidate, ether-linked phosphatidylethanolamine, phosphatidylserine, and / or sphingomyelin compared to parental cells. In some embodiments, fusosomes are depleted (or identified as being depleted) in ceramide, cardiolipin, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, ether-linked phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, and / or triacylglycerol compared to parent cells. In some embodiments, fusosomes are enriched (or identified as being enriched) in cholesteryl esters, ceramide, diacylglycerol, lysophosphatidate, phosphatidylethanolamine, and / or triacylglycerol compared to exosomes. In some embodiments, fusosomes are depleted (or identified as being depleted) in free cholesterol, hexosylceramide, lysophosphatidylcholine, ether-linked lysophosphatidylcholine, lysophosphatidylethanolamine, ether-linked lysophosphatidylethanolamine, and / or lysophosphatidyl compared to exosomes.

[0095] In some embodiments, the fusosomes have a cardiolipin:ceramide ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:ceramide ratio in the feeder cells, or a cardiolipin:diacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:diacylglycerol ratio of the cardiolipin in the feeder cells, or a cardiolipin:hexosylceramide ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:hexosylceramide ratio in the feeder cells. or having a cardiolipin:lysophosphatidate ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:lysophosphatidate ratio in the source cells, or having a cardiolipin:lysophosphatidylcholine ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:lysophosphatidylethanolamine ratio in the source cells. or 50% of the cardiolipin:lysophosphatidylethanolamine ratio in the source cells, or have a cardiolipin:lysophosphatidylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:lysophosphatidylglycerol ratio in the source cells, or have a cardiolipin:lysophosphatidylinositol ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:lysophosphatidylinositol ratio in the source cells, or have a cardiolipin:lysophosphatidylinositol ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:lysophosphatidylinositol ratio in the source cells. have a cardiolipin:lysophosphatidylserine ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:lysophosphatidylserine ratio, or have a cardiolipin:phosphatidate ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:phosphatidate ratio in the source cells, or have a cardiolipin:phosphatidylcholine ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:phosphatidylcholine ratio in the source cells;or have a cardiolipin:phosphatidylethanolamine ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:phosphatidylethanolamine ratio in the source cells, or have a cardiolipin:phosphatidylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:phosphatidylinositol ratio in the source cells. % of the cardiolipin:phosphatidyl inositol ratio in the source cells, or have a cardiolipin:phosphatidylserine ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:phosphatidylserine ratio in the source cells, or have a cardiolipin:cholesterol ester ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:sphingomyelin ratio in the source cells. , 20%, 30%, 40%, or 50% of the cardiolipin:triacylglycerol ratio in the source cells, or have a cardiolipin:triacylglycerol ratio that is within 10%, .... having a phosphatidylcholine:ceramide ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphorus:ceramide ratio, or having a phosphatidylcholine:diacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylcholine:diacylglycerol ratio in the source cells, or having a phosphatidylcholine:hexosylceramide ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylcholine:hexosylceramide ratio in the source cells;or having a phosphatidylcholine:lysophosphatidate ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylcholine:lysophosphatidate ratio in the source cells, or having a phosphatidylcholine:lysophosphatidylcholine ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylcholine:lysophosphatidylcholine ratio in the source cells, or 10%, 20%, 30% of the phosphatidylcholine:lysophosphatidylethanolamine ratio in the source cells, having a phosphatidylcholine:lysophosphatidylethanolamine ratio that is within 40%, or 50% of the phosphatidylcholine:lysophosphatidylglycerol ratio in the source cells, or having a phosphatidylcholine:lysophosphatidylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylcholine:lysophosphatidylinositol ratio in the source cells. or having a phosphatidylcholine:lysophosphatidylserine ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylcholine:lysophosphatidylserine ratio in the source cells, or having a phosphatidylcholine:phosphatidate ratio that is within 10%, 20%, 30%, 40%, or 50% of the cardiolipin:phosphatidate ratio in the source cells, or having a phosphatidylcholine:phosphatidate ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylcholine:phosphatidylethanolamine ratio in the source cells. %, 40%, or 50% of the phosphatidylcholine:phosphatidylethanolamine ratio in the source cells, or a cardiolipin:phosphatidylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylcholine:phosphatidylglycerol ratio in the source cells, or a phosphatidylcholine:phosphatidylinositol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylcholine:phosphatidylinositol ratio in the source cells,or having a phosphatidylcholine:phosphatidylserine ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylcholine:phosphatidylserine ratio in the source cells, or having a phosphatidylcholine:cholesterol ester ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylcholine:sphingomyelin ratio in the source cells. or having a phosphatidylcholine:sphingomyelin ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylcholine:sphingomyelin ratio in the source cells, or having a phosphatidylcholine:triacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylcholine:triacylglycerol ratio in the source cells, or 10% of the phosphatidylcholine:triacylglycerol ratio in the source cells, or having a phosphatidylcholine:triacylglycerol ratio that is within 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:ceramide ratio in the source cells, or having a phosphatidylethanolamine:diacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:diacylglycerol ratio in the source cells, or having a phosphatidylethanolamine:diacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:diacylglycerol ratio in the source cells, or having a phosphatidylethanolamine:hexosylceramide ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:hexosylceramide ratio in the source cells, or 10%, 20%, 30% of the phosphatidylethanolamine:lysophosphatidate ratio in the source cells;or having a phosphatidylethanolamine:lysophosphatidate ratio that is within 40%, or 50% of the phosphatidylethanolamine:lysophosphatidylcholine ratio in the source cells, or having a phosphatidylethanolamine:lysophosphatidylcholine ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:lysophosphatidylethanolamine ratio in the source cells. or having a phosphatidylethanolamine:lysophosphatidylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:lysophosphatidyl inositol ratio in the source cells, or having a phosphatidylethanolamine:lysophosphatidyl inositol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:lysophosphatidyl inositol ratio in the source cells. or having a phosphatidylethanolamine:lysophosphatidylserine ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:lysophosphatidylserine ratio in the source cells, or having a phosphatidylethanolamine:phosphatidate ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:phosphatidate ratio in the source cells, or having a phosphatidylethanolamine:phosphatidate ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:phosphatidate ratio in the source cells. or having a phosphatidylethanolamine:phosphatidylinositol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:phosphatidylinositol ratio in the source cells, or having a phosphatidylethanolamine:phosphatidylinositol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:phosphatidylinositol ratio in the source cells,or have a phosphatidylethanolamine:phosphatidylinositol ratio that is within 50%, or have a phosphatidylethanolamine:phosphatidylserine ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:phosphatidylserine ratio in the source cells, or have a phosphatidylethanolamine:phosphatidylserine ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:cholesterol ester ratio in the source cells, having a phosphatidylethanolamine:cholesterol ester ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:sphingomyelin ratio in the source cells, or having a phosphatidylethanolamine:sphingomyelin ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:triacylglycerol ratio in the source cells. or having a phosphatidylserine:triacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:ceramide ratio in the source cells, or having a phosphatidylserine:diacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:diacylglycerol ratio in the source cells, or 10%, 20%, 30% of the phosphatidylserine:diacylglycerol ratio in the source cells, or having a phosphatidylserine:diacylglycerol ratio that is within 40%, or 50% of the phosphatidylserine:hexosylceramide ratio in the source cells, or having a phosphatidylserine:lysophosphatidate ratio that is within 10%, 20%, 30%, ...lysophosphatidate ratio in the source cells, or having a phosphatidylserine:diacylglycerol ratio that is within 40%, 50%, or 50% of the phosphatidylserine:hexosylceramide ratio in the source cells. or having a phosphatidylserine:lysophosphatidylcholine ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:lysophosphatidylcholine ratio in the source cells, or having a phosphatidylserine:lysophosphatidylethanolamine ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:lysophosphatidylglycerol ratio in the source cells,or has a phosphatidylserine:lysophosphatidylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:lysophosphatidylinositol ratio in the source cells, or has a phosphatidylserine:lysophosphatidylserine ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:lysophosphatidylserine ratio in the source cells, or having a phosphatidylserine:phosphatidate ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:phosphatidate ratio in the source cells, or having a phosphatidylserine:phosphatidylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:phosphatidylglycerol ratio in the source cells, or 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:phosphatidylinositol ratio in the source cells or having a phosphatidylethanolamine:phosphatidyl inositol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:phosphatidylserine ratio in the source cells, or having a phosphatidylethanolamine:cholesterol ester ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:cholesterol ester ratio in the source cells, or having a phosphatidylethanolamine:sphingomyelin ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:sphingomyelin ratio in the source cells, or having a phosphatidylethanolamine:triacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylethanolamine:triacylglycerol ratio in the source cells, or 10%, 20%, 30%, 40% of the phosphatidylserine:ceramide ratio in the source cells,or has a phosphatidylserine:diacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:diacylglycerol ratio in the source cells, or has a phosphatidylserine:diacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:diacylglycerol ratio in the source cells, or has a phosphatidylserine:diacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:hexosylceramide ratio in the source cells. or having a phosphatidylserine: hexosylceramide ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine: lysophosphatidate ratio in the source cells, or having a phosphatidylserine: lysophosphatidate ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine: lysophosphatidylcholine ratio in the source cells. or having a phosphatidylserine:lysophosphatidylethanolamine ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:lysophosphatidylethanolamine ratio in the source cells, or having a phosphatidylserine:lysophosphatidylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:lysophosphatidylinositol ratio in the source cells. or having a phosphatidylserine:lysophosphatidyl inositol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:lysophosphatidylserine ratio in the source cells, or having a phosphatidylserine:lysophosphatidylserine ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:phosphatidate ratio in the source cells,or having a phosphatidylserine:phosphatidylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:phosphatidylglycerol ratio in the source cells, or having a phosphatidylserine:phosphatidylinositol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:cholesterol ester ratio in the source cells. or having a phosphatidylserine:cholesterol ester ratio that is within 0%, 40%, or 50% of the phosphatidylserine:sphingomyelin ratio in the source cells, or having a phosphatidylserine:sphingomyelin ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:sphingomyelin ratio in the source cells, or having a phosphatidylserine:sphingomyelin ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:sphingomyelin ratio in the source cells. having a phosphatidylserine:triacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the phosphatidylserine:triacylglycerol ratio, or having a sphingomyelin:ceramide ratio that is within 10%, 20%, 30%, 40%, or 50% of the sphingomyelin:ceramide ratio in the source cells, or a sphingomyelin:diacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the sphingomyelin:diacylglycerol ratio in the source cells or having a ratio of sphingomyelin:hexosylceramide that is within 10%, 20%, 30%, 40%, or 50% of the ratio of sphingomyelin:hexosylceramide in the source cells, or having a ratio of sphingomyelin:lysophosphatidate that is within 10%, 20%, 30%, 40%, or 50% of the ratio of sphingomyelin:lysophosphatidate in the source cells, or 10%, 20%, 30%, 40% of the ratio of sphingomyelin:lysophosphatidylcholine in the source cells,or have a ratio of sphingomyelin:lysophosphatidylcholine that is within 50%, or have a ratio of sphingomyelin:lysophosphatidylethanolamine that is within 10%, 20%, 30%, 40%, or 50% of the ratio of sphingomyelin:lysophosphatidylethanolamine in the source cells, or have a ratio of sphingomyelin:lysophosphatidylglycerol that is within 10%, 20%, 30%, 40%, or 50% of the ratio of sphingomyelin:lysophosphatidylglycerol in the source cells, or have a sphingomyelin:lysophosphatidylinositol ratio that is within 10%, 20%, 30%, 40%, or 50% of the sphingomyelin:lysophosphatidylinositol ratio in the source cells, or have a sphingomyelin:lysophosphatidylserine ratio that is within 10%, 20%, 30%, 40%, or 50% of the sphingomyelin:lysophosphatidylserine ratio in the source cells, or have a sphingomyelin:lysophosphatidylserine ratio that is within 10%, 20%, 30%, 40%, or 50% of the sphingomyelin:phosphatidate ratio in the source cells. or having a sphingomyelin:phosphatidate ratio that is within 10%, 20%, 30%, 40%, or 50% of the sphingomyelin:phosphatidylglycerol ratio in the source cells, or having a sphingomyelin:phosphatidylinositol ratio that is within 10%, 20%, 30%, 40%, or 50% of the sphingomyelin:phosphatidylinositol ratio in the source cells, or having a sphingomyelin:phosphatidylinositol ratio that is within 10%, 20%, 30%, 40%, or 50% of the sphingomyelin:phosphatidylinositol ratio in the source cells. or having a sphingomyelin:triacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the cholesterol ester ratio in the source cells, or having a cholesterol ester:ceramide ratio that is within 10%, 20%, 30%, 40%, or 50% of the cholesterol ester:ceramide ratio in the source cells,or having a cholesterol ester:diacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the cholesterol ester:diacylglycerol ratio in the source cells, or having a cholesterol ester:hexosylceramide ratio that is within 10%, 20%, 30%, 40%, or 50% of the cholesterol ester:hexosylceramide ratio in the source cells, or having a cholesterol ester:diacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the cholesterol ester:hexosylceramide ratio in the source cells, having a cholesterol ester:lysophosphatidate ratio that is within 10%, 20%, 30%, 40%, or 50% of the lysophosphatidate ratio, or having a cholesterol ester:lysophosphatidylcholine ratio that is within 10%, 20%, 30%, 40%, or 50% of the cholesterol ester:lysophosphatidylethanolamine ratio in the source cells; having a ratio of sterol esters:lysophosphatidylethanolamine, or having a ratio of cholesterol esters:lysophosphatidylglycerol that is within 10%, 20%, 30%, 40%, or 50% of the ratio of cholesterol esters:lysophosphatidylglycerol in the source cells, or having a ratio of cholesterol esters:lysophosphatidylinositol that is within 10%, 20%, 30%, 40%, or 50% of the ratio of cholesterol esters:lysophosphatidylinositol in the source cells; or having a cholesterol ester:lysophosphatidylserine ratio that is within 10%, 20%, 30%, 40%, or 50% of the cholesterol ester:lysophosphatidylserine ratio in the source cells, or having a cholesterol ester:lysophosphatidylserine ratio that is within 10%, 20%, 30%, 40%, or 50% of the cholesterol ester:lysophosphatidylserine ratio in the source cells, or having a cholesterol ester:lysophosphatidylserine ratio that is within 10%, 20%, 30%, 40%, or 50% of the cholesterol ester:phosphatidate ratio in the source cells. have a cholesterol ester:phosphatidate ratio that is within 0%, or 50%, of the cholesterol ester:phosphatidylglycerol ratio in the source cells, or have a cholesterol ester:phosphatidylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the cholesterol ester:phosphatidyl inositol ratio in the source cells,or having a cholesterol ester:triacylglycerol ratio that is within 10%, 20%, 30%, 40%, or 50% of the cholesterol ester:triacylglycerol ratio in the source cells.

[0096] In some embodiments, fusosomes comprise a proteome composition similar to that of source cells, e.g., using the assays of Examples 42 or 155. In some embodiments, the protein composition of fusosomes is similar to that of the parent cells from which they are derived. In some embodiments, the fractional content of each of multiple categories of proteins is determined as the sum of the intensity signals from each category divided by the sum of the intensity signals of all identified proteins in the sample, e.g., as described in Example 155. In some embodiments, fusosomes comprise (or are identified as comprising) varying amounts of compartment-specific proteins compared to parent cells and / or exosomes, e.g., as determined according to the method described in Example 165. In some embodiments, fusosomes are depleted (or are identified as being depleted) in endoplasmic reticulum proteins compared to parent cells and exosomes. In some embodiments, fusosomes are depleted (or are identified as being depleted) in exosomal proteins compared to exosomes. In some aspects, the fusosomes have (or are identified as having) less than 15%, 20%, or 25% of the proteins in the fusosomes as being exosomal proteins. In some embodiments, the fusosomes are (or are identified as being) depleted in mitochondrial proteins compared to the parent cell. In some embodiments, the fusosomes are (or are identified as being) enriched in nuclear proteins compared to the parent cell. In some embodiments, the fusosomes are (or are identified as being) enriched in ribosomal proteins compared to the parent cell and exosomes.In some embodiments, at least 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the protein in fusosomes is ribosomal protein, or about 0.025-0.2%, 0.05-0.15%, 0.06-1.4%, 0.07%-1.3%, 0.08%-1.2%, 0.09%-1.1%, 1%-20%, 3%-15%, 5%-12.5%, 7.5%-11%, 8.5%-10.5%, or 9%-10% of the protein in fusosomes is ribosomal protein.

[0097] In some embodiments, fusosomes comprise a lipid to protein ratio that is within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cells, as measured, for example, using the assay of Example 49. In embodiments, fusosomes comprise (or are identified as comprising) a lipid mass to protein ratio that is approximately equal to the lipid mass to protein ratio in nucleated cells. In embodiments, fusosomes comprise (or are identified as comprising) a lipid:protein ratio that is greater than that of the parent cells. In embodiments, fusosomes comprise (or are identified as comprising) a lipid:protein ratio that is about 110%, 115%, 120%, 125%, 130%, 131%, 132%, 132.5%, 133%, 134%, 135%, 140%, 145%, or 150% of the lipid:protein ratio of the parent cells. In some embodiments, fusosomes or fusosome compositions have (or are identified as having) a phospholipid:protein ratio of about 100-180, 110-170, 120-160, 130-150, 135-145, 140-142, or 141 μmol / g, e.g., in the assay of Example 150. In some embodiments, fusosomes or fusosome compositions have (or are identified as having) a phospholipid:protein ratio that is about 60-90%, 70-80%, or 75% of the corresponding ratio in the source cells, e.g., in the assay of Example 150.

[0098] In some embodiments, fusosomes comprise a protein to nucleic acid (e.g., DNA or RNA) ratio that is within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cells, as measured, for example, using the assay of Example 50. In embodiments, fusosomes comprise (or are identified as comprising) a protein mass to DNA mass ratio similar to that of the parent cells. In embodiments, fusosomes comprise (or are identified as comprising) a protein:DNA ratio that is about 85%, 90%, 95%, 96%, 97%, 98%, 98.2%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, or 110% of that of the parent cells. In some embodiments, the fusosomes comprise a protein to DNA ratio that is greater than the corresponding ratio in the source cells, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% greater, e.g., as measured using the assay of Example 50. In some embodiments, the fusosomes or fusomal compositions comprise (or are identified to comprise) a protein:DNA ratio that is about 20-35, 25-30, 26-29, 27-28, or 27.8 g / g, e.g., by the assay of Example 151. In some embodiments, the fusosomes or fusomal compositions comprise (or are identified to comprise) a protein:DNA ratio that is within about 1%, 2%, 5%, 10%, or 20% of the corresponding ratio in the source cells, e.g., by the assay of Example 151.

[0099] In some embodiments, fusosomes comprise a lipid to nucleic acid (e.g., DNA) ratio that is within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in source cells, e.g., as measured using the assay of Example 51 or 159. In some embodiments, fusosomes or fusosome compositions comprise (or are identified as comprising) a lipid:DNA ratio that is about 2.0-6.0, 3.0-5.0, 3.5-4.5, 3.8-4.0, or 3.92 μmol / mg, e.g., by the assay of Example 152. In some embodiments, fusosomes comprise a lipid to nucleic acid (e.g., DNA) ratio that is greater than the corresponding ratio in source cells, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% greater, e.g., as measured using the assay of Example 51 or 159. In embodiments, the fusosomes contain (or are identified as containing) a greater lipid:DNA ratio than the parent cell, hi embodiments, the fusosomes contain about 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150% or more lipid:DNA ratio compared to the parent cell.

[0100] In some embodiments, the fusosome composition has a half-life in a subject, e.g., a mouse, that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the half-life of a reference cell composition, e.g., a source cell, e.g., by the assay of Example 75. In some embodiments, the fusosome composition has a half-life in a subject, e.g., a mouse, that is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours, e.g., in a human subject or mouse, e.g., by the assay of Example 75. In embodiments, the fusosome composition has a half-life in a subject, e.g., a mouse, that is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours, e.g., in the assay of Example 134. In some embodiments, the therapeutic agent has a half-life in the subject that is, e.g., at least 10%, 20%, 50%, 2-fold, 5-fold, or 10-fold longer than the half-life of the fusosome composition. For example, the fusosomes may deliver the therapeutic agent to a target cell, and the therapeutic agent may be present after the fusosomes are no longer present or are no longer detectable.

[0101] In some embodiments, the fusosomes transport glucose (e.g., labeled glucose, e.g., 2-NBDG) across the membrane at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than other similar fusosomes in the absence of a negative control, e.g., glucose, as measured, e.g., using the assay of Example 64. In some embodiments, the fusosomes transport (or are identified as transporting) glucose (e.g., labeled glucose, e.g., 2-NBDG) across the membrane at a level greater than other similar fusosomes treated with phloretin, e.g., in the assay of Example 126. In embodiments, fusosomes that have not been treated with phloretin are identified as transporting (or not transporting) glucose at a level at least 1%, 2%, 3%, 5%, or 10% higher (and optionally up to 15% higher) than other similar fusosomes that have been treated with phloretin, e.g., in the assay of Example 126. In some embodiments, the fusosomes comprise an esterase activity in their lumen that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the esterase activity in reference cells, e.g., source cells or mouse embryonic fibroblasts, using, e.g., the assay of Example 66. In some embodiments, the fusosomes comprise (or are identified as comprising) luminal esterase activity that is at least 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, or 5000-fold greater than unstained controls, e.g., by the assay of Example 127. In some aspects, the fusosomes comprise (or are identified as comprising) luminal esterase activity that is about 10-100-fold less than the esterase activity of the source cells, e.g., by the assay of Example 127. In some embodiments, the fusosomes comprise (or are identified as comprising) about 1E5-1E6, 6E5-8E5, 6.5E5-7E5, or 6.83E5 exosome equivalents of acetylcholinesterase activity, e.g., by the assay of Example 128.In some aspects, the fusosomes comprise a metabolic activity level (e.g., citrate synthase activity) that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the metabolic activity level in a reference cell, e.g., a source cell, e.g., as described in Example 68. In some embodiments, the fusosomes comprise a metabolic activity level (e.g., citrate synthase activity) that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the metabolic activity level in a reference cell, e.g., a source cell, e.g., as described in Example 68. In some embodiments, the fusosomes comprise (or are identified as comprising) a citrate synthase activity that is about 1E-2 to 2E-2, 1.3E-2 to 1.8E-2, 1.4E-2 to 1.7E-2, 1.5E-2 to 1.6E-2, or 1.57E-2 umol / ug fusosomes / min, e.g., by the assay of Example 129. In some embodiments, the fusosomes comprise a respiration level (e.g., oxygen consumption rate), e.g., a basal respiration level, an uncoupled respiration level, or a maximal respiration level, that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the respiration level in reference cells, e.g., source cells, e.g., as described in Example 69. In some embodiments, the fusosomes comprise a respiration level (e.g., oxygen consumption rate), e.g., a basal respiration level, an uncoupled respiration level, or a maximal respiration level, that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the respiration level in reference cells, e.g., source cells, as described, e.g., in Example 69. In embodiments, the fusosomes comprise (or are determined to comprise) a basal respiration rate of about 8-15, 9-14, 10-13, 11-12, or 11.3 pmol / min / 20 μg fusosomes, e.g., by the assay of Example 130.In embodiments, the fusosomes comprise (or are identified as comprising) an unbound respiration rate of about 8-13, 9-12, 10-11, 10-10.2, or 10.1 pmol / min / 20 μg fusosomes, e.g., by the assay of Example 130. In embodiments, the fusosomes comprise (or are identified as comprising) a maximal respiration rate of about 15-25, 16-24, 17-23, 18-22, 19-21, or 20 pmol / min / 20 μg fusosomes, e.g., by the assay of Example 130. In embodiments, the fusosomes have (or are identified as having) a basal respiration rate that is about 1%, 2%, 5%, or 10%, e.g., up to about 15%, higher than the unbound respiration rate, e.g., by the assay of Example 130. In embodiments, the fusosomes have (or are identified as having) a maximum respiration rate that is, e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than the basal respiration rate, e.g., by the assay of Example 130. In some embodiments, the fusosomes comprise an annexin-V staining level of an MFI of at most 18,000, 17,000, 16,000, 15,000, 14,000, 13,000, 12,000, 11,000, or 10,000, e.g., using the assay of Example 70, or the fusosomes have an annexin-V staining level that is at least about the same as that of other similar fusosomes treated with menadione, e.g., in the assay of Example 70. or the fusosomes comprise a level of annexin-V staining that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less than the level of annexin-V staining of macrophages treated with menadione in the assay of Example 70. In embodiments, the fusosomes comprise (or are identified as comprising) a level of annexin-V staining that is at least about 1%, 2%, 5%, or 10% less than the level of annexin-V staining of other similar fusosomes treated with antimycin A, e.g., in the assay of Example 131.In embodiments, the fusosomes comprise (or are identified as comprising) a level of annexin V staining that is within about 1%, 2%, 5%, or 10% of the annexin V staining level of other similar fusosomes treated with antimycin A, e.g., in the assay of Example 131.

[0102] In some embodiments, the fusosomes have a miRNA content level that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than the miRNA content level of the source cells, e.g., by the assay of Example 39. In some embodiments, the fusosomes have a miRNA content level that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more (e.g., up to 100% of the miRNA content level of the source cells), e.g., by the assay of Example 39. In some embodiments, the fusosomes have a total RNA content level that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more (e.g., up to 100% of the total RNA content level of the source cells) as measured, e.g., by the assay of Example 108.

[0103] In some embodiments, the fusosomes have a soluble protein:insoluble protein ratio that is 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than that of the source cells, e.g., within 1%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% of that of the source cells, e.g., by the assay of Example 47. In embodiments, the fusosomes have a soluble protein:insoluble protein ratio of about 0.3-0.8, 0.4-0.7, or 0.5-0.6, e.g., about 0.563, e.g., by the assay of Example 47. In some embodiments, the fusosome population has (or is identified as having) a soluble protein:insoluble protein mass ratio of about 0.3-0.8, 0.4-0.7, 0.5-0.6, or 0.563, or greater, than about 0.1, 0.2, 0.3, 0.4, or 0.5. In some embodiments, the fusosome population has (or is identified as having) a soluble protein:insoluble protein mass ratio that is greater than that of the source cells, e.g., at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or 20-fold greater. In embodiments, the soluble protein:insoluble protein mass ratio is determined by the assay of Example 123. In embodiments, the mass ratio of soluble:insoluble protein is lower (or is identified as lower) in the fusosome population than in the parental cells. In embodiments, when the fusosome to parental cell ratio is (or is identified as being) about 3%, 4%, 5%, 6%, 7%, or 8%, the soluble:insoluble ratio of the fusosome population is approximately equal (or is identified as approximately equal) to the soluble:insoluble ratio of the parental cells.

[0104] In some embodiments, the fusosomes have an LPS level of less than 5%, 1%, 0.5%, 0.01%, 0.005%, 0.0001%, 0.00001% or less of the LPS content of the source cells, as measured, e.g., by mass spectrometry, e.g., by the assay of Example 48. In some embodiments, the fusosomes enable signal transduction, e.g., AKT phosphorylation in response to an extracellular signal, e.g., insulin, or glucose (e.g., labeled glucose, e.g., 2-NBDG) uptake in response to insulin, to transport at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than other similar fusosomes, e.g., in the absence of a negative control, e.g., insulin, e.g., using the assay of Example 63. In some embodiments, the fusosomes are targeted to a tissue, e.g., liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testis, ovary, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye, and when administered to a subject, e.g., a mouse, at least 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90% of the fusosomes in the population of administered fusosomes are present in the target tissue after 24, 48, or 72 hours, e.g., by the assay of Example 87 or 100. In some embodiments, the fusosomes have a level of juxtacrine signaling that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the level of juxtacrine signaling induced by a reference cell, e.g., a source cell or bone marrow stromal cell (BMSC), e.g., by the assay of Example 71.In some embodiments, the fusosomes have a level of juxtacrine signaling that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (e.g., up to 100%) greater than the level of juxtacrine signaling induced by a reference cell, e.g., a source cell or bone marrow stromal cell (BMSC), e.g., by the assay of Example 71. In some embodiments, the fusosomes have a level of paracrine signaling that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the level of paracrine signaling induced by a reference cell, e.g., a source cell or macrophage, e.g., by the assay of Example 72. In some embodiments, the fusosomes have a level of paracrine signaling that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (e.g., up to 100%) higher than the level of paracrine signaling induced by a reference cell, e.g., a source cell or a macrophage, e.g., by the assay of Example 72. In some embodiments, the fusosomes polymerize actin at a level that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the level of polymerized actin in a reference cell, e.g., a source cell or a C2C12 cell, e.g., by the assay of Example 73. In some embodiments, fusosomes polymerize actin (or are identified as polymerizing actin) at a level that is constant over time, e.g., at least 3, 5, or 24 hours, e.g., by the assay of Example 147. In embodiments, the level of actin polymerization changes by less than 1%, 2%, 5%, 10%, or 20% over a 5-hour period, e.g., by the assay of Example 147.In some embodiments, the fusosomes have a membrane potential within about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the membrane potential of a reference cell, e.g., a source cell or a C2C12 cell, e.g., by the assay of Example 74; or the fusosomes have a membrane potential of about -20 to -150 mV, -20 to -50 mV, -50 to -100 mV, or -100 to -150 mV; or the fusosomes have a membrane potential of less than -1 mV, -5 mV, -10 mV, -20 mV, -30 mV, -40 mV, -50 mV, -60 mV, -70 mV, -80 mv, -90 mv, or -100 mV. In some embodiments, the fusosomes have (or are identified as having) a membrane potential of about -25 to -35, -27 to -32, -28 to -31, -29 to -30, or -29.6 millivolts, e.g., in the assay of Example 132. In some embodiments, the fusosomes can extravasate from blood vessels at a rate that is at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the extravasation rate of the source cells, e.g., using the assay of Example 57, where the source cells are neutrophils, lymphocytes, B cells, macrophages, or NK cells. In some embodiments, fusosomes allow for, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (e.g., up to 100%) chemotaxis compared to a reference cell, e.g., a macrophage, using, e.g., the assay of Example 58. In some embodiments, fusosomes allow for, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (e.g., up to 100%) phagocytosis compared to a reference cell, e.g., a macrophage, using, e.g., the assay of Example 60. In some embodiments, fusosomes are capable of crossing a cell membrane, e.g., an endothelial cell membrane or the blood-brain barrier.In some embodiments, fusosomes are capable of secreting protein at a rate that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than a reference cell, e.g., a mouse embryonic fibroblast, using, e.g., the assay of Example 62. In some embodiments, fusosomes are capable of secreting protein at a rate that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (e.g., up to 100%) greater than a reference cell, e.g., a mouse embryonic fibroblast, using, e.g., the assay of Example 62.

[0105] In some embodiments, the fusosomes are incapable of transcription of a reference cell, e.g., a source cell, or have less than 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the transcription activity of the reference cell, e.g., a source cell, e.g., using the assay of Example 19. In some embodiments, the fusosomes are incapable of nuclear DNA replication of a reference cell, e.g., a source cell, or have less than 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the nuclear DNA replication of the reference cell, e.g., a source cell, e.g., using the assay of Example 20. In some embodiments, the fusosomes lack chromatin or have a chromatin content that is less than 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the chromatin content of a reference cell, e.g., a source cell, using, e.g., the assay of Example 37.

[0106] In some embodiments, fusosome characteristics are described by comparison with a reference cell. In some embodiments, the reference cell is a source cell. In some embodiments, the reference cell is a HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR 90, IMR 91, PER.C6, HT-1080, or BJ cell. In some embodiments, fusosome population characteristics are described by comparison with a reference cell population, e.g., a source cell population or a HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR 90, IMR 91, PER.C6, HT-1080, or BJ cell population.

[0107] In some embodiments, the fusosomes meet pharmaceutical or Good Manufacturing Practice (GMP) standards. In some embodiments, the fusosomes were produced in accordance with Good Manufacturing Practice (GMP). In some embodiments, the fusosomes have pathogen levels below a predetermined standard, e.g., are substantially pathogen-free. In some embodiments, the fusosomes have contaminant levels below a predetermined standard, e.g., are substantially contaminant-free. In some embodiments, the fusosomes have low immunogenicity, e.g., as described herein.

[0108] In some embodiments, the immunogenicity of a fusosome composition is assayed by a serum inactivation assay (e.g., an assay detecting antibody-mediated neutralization or complement-mediated degradation). In some embodiments, fusosomes are not inactivated by serum or are inactivated at a level below a predetermined value. In some embodiments, serum from a fusosome-naive subject (e.g., a human or mouse) is contacted with the test fusosome composition. In some embodiments, serum from a subject that has received one or more administrations of fusosomes, e.g., at least two administrations of fusosomes, is contacted with the test fusosome composition. In embodiments, the serum-exposed fusosomes are then tested for their ability to deliver cargo to target cells. In some embodiments, the percentage of cells detectably containing cargo after treatment with serum-incubated fusosomes is at least 50%, 60%, 70%, 80%, 90%, or 95% of the percentage of cells detectably containing cargo after treatment with positive control fusosomes not contacted with serum. In some embodiments, serum inactivation is measured using the assay of Example 168.

[0109] In some embodiments, the immunogenicity of a fusosome composition is assayed by detecting complement activation in response to fusosomes. In some embodiments, fusosomes do not activate complement or activate complement at a level below a predetermined value. In some embodiments, serum from a fusosome-naive subject (e.g., a human or mouse) is contacted with the test fusosome composition. In some embodiments, serum from a subject that has received one or more administrations of fusosomes, for example, at least two administrations of fusosomes, is contacted with the test fusosome composition. In embodiments, the composition comprising serum and fusosomes is then tested for activated complement factors (e.g., C3a), for example, by ELISA. In some embodiments, fusosomes containing a modification described herein (e.g., having higher levels of a complement regulatory protein compared to a reference cell) have reduced complement activation, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%, compared to other similar fusosomes lacking the modification. In some embodiments, complement activation is measured using the assay of Example 169.

[0110] In some embodiments, the fusosomes or fusosome population will not be substantially inactivated by serum. In some embodiments, the fusosomes or fusosome population are resistant to serum inactivation, e.g., as quantified according to the methods described in Examples 167 or 168. In embodiments, the fusosomes or fusosome population are not substantially inactivated by serum or are resistant to serum inactivation after multiple administrations of the fusosomes or fusosome population to a subject, e.g., according to the methods described herein. In some embodiments, the fusosomes are modified to exhibit reduced serum inactivation after multiple administrations of the modified fusosomes, e.g., as compared to corresponding unmodified fusosomes, e.g., as quantified according to the methods described in Examples 167 or 168.

[0111] In some embodiments, the fusosomes do not substantially induce complement activity, e.g., as measured according to the method described in Example 169. In some embodiments, the fusosomes are modified to induce reduced complement activity compared to corresponding unmodified fusosomes. In embodiments, complement activity is measured by determining the expression or activity of a complement protein (e.g., DAF, a protein that binds decay-accelerating factor (DAF, CD55), e.g., factor H (FH)-like protein-1 (FHL-1), C4b-binding protein (C4BP), complement receptor 1 (CD35), membrane cofactor protein (MCP, CD46), profectin (CD59), a protein that inhibits classical and alternative complement pathway CD / C5 convertases, or a protein that regulates MAC assembly) in the cells.

[0112] In some embodiments, the source cells are endothelial cells, fibroblasts, blood cells (e.g., macrophages, neutrophils, granulocytes, leukocytes), stem cells (e.g., mesenchymal stem cells, umbilical cord stem cells, bone marrow stem cells, hematopoietic stem cells, induced pluripotent stem cells, e.g., induced pluripotent stem cells derived from cells of interest), embryonic stem cells (e.g., stem cells from embryonic yolk sac, placenta, umbilical cord, fetal skin, adolescent skin, blood, bone marrow, adipose tissue, erythropoietic tissue, hematopoietic tissue), myoblasts, parenchymal cells (e.g., hepatocytes). , alveolar cells, neuronal (e.g., retinal neuronal) progenitor cells (e.g., retinal progenitor cells, myeloblasts, myeloid progenitor cells, thymocytes, meiocytes, megakaryoblasts, promegakaryoblasts, melanoblasts, lymphoblasts, myeloid progenitor cells, normal cells, or hemangioblasts), progenitor cells (e.g., cardiac progenitor cells, satellite cells, radiation giardia cells, bone marrow stromal cells, pancreatic progenitor cells, endothelial progenitor cells, blast cells), or immortalized cells (e.g., HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR 90, IMR 91, PER.C6, HT-1080, or BJ cells). In some embodiments, the source cells are other than 293 cells, HEK cells, human endothelial cells, or human epithelial cells, monocytes, macrophages, dendritic cells, or stem cells.

[0113] In some embodiments, the source cells express (e.g., overexpress) ARRDC1 or an active fragment or variant thereof. In some embodiments, the fusosomes or fusosome compositions are measured, e.g., by mass spectrometry assay, at a concentration of about 1-3, 1-10, 1-100, 3-10, 4-9, 5-8, 6-7, 15-100, 60-200, 80-180, 100-160, 120-140, 3-100, 4-100, 5-100, 6-100, 15-100, 80-1 and a fusogen to ARRDC1 ratio of 00, 3-200, 4-200, 5-200, 6-200, 15-200, 80-200, 100-200, 120-200, 300-1000, 400-900, 500-800, 600-700, 640-690, 650-680, 660-670, 100-10,000, or about 664.9. In some embodiments, the level of ARRDC1 as a percentage of total protein content is at least about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, or the level of ARRDC1 as a percentage of total protein content is about 0.05-1.5%, 0.1%-0.3%, 0.05-0.2%, 0.1-0.2%, 0.25-7.5%, 0.5%-1.5%, 0.25-1%, 0.5-1%, 0.05-1.5%, 10%-30%, 5-20%, or 10-20%, e.g., as measured by mass spectrometry, e.g., according to the methods described in Example 166. In some embodiments, the fusosomes or fusosome compositions have a fusogen to TSG101 ratio of about 100 to 1,000, 100 to 400, 100 to 500, 200 to 400, 200 to 500, 200 to 1,000, 300 to 400, 1,000 to 10,000, 2,000 to 5,000, 3,000 to 4,000, 3,050 to 3,100, 3,060 to 3,070, or about 3,064, 10,000 to 100,000, 10,000 to 200,000, 10,000 to 500,000, 20,000 to 500,000, or 30,000 to 400,000, e.g., using a mass spectrometry assay, e.g., the assay of Example 162.In some embodiments, the fusosomes or fusosome compositions have a cargo to tsg101 ratio of about 1-3, 1-30, 1-20, 1-25, 1.5-30, 10-30, 15-25, 18-21, 19-20, 10-300, 10-200, 15-300, 15-200, 100-300, 100-200, 150-300, or about 19.5, e.g., using a mass spectrometry assay, e.g., the assay of Example 163. In some embodiments, the level of TSGlOl as a percentage of total protein content, e.g., as measured by mass spectrometry, e.g., according to the methods described in Example 166, is at least about 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.01%, 0.02% , 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, or the level of TSG101 as a percentage of total protein content is approximately 0.0001-0.001, 0.0001-0.002, 0.0001-0.01, 0.0001-0.1, 0.001-0.01, 0.002-0.006, 0.003-0.005, 0.001-0.1, 0.01-0.1, 0.02-0.06, 0.03-0.05, or 0.004.

[0114] In some embodiments, the fusosomes contain cargo, e.g., a therapeutic agent, e.g., an endogenous or exogenous therapeutic agent. In some embodiments, the therapeutic agent is selected from one or more of a protein, e.g., an enzyme, a transmembrane protein, a receptor, an antibody, a nucleic acid, e.g., DNA, a chromosome (e.g., a human artificial chromosome), RNA, mRNA, siRNA, miRNA, or a small molecule. In some embodiments, the therapeutic agent is an organelle other than a mitochondrion, e.g., an organelle selected from a nucleus, Golgi apparatus, lysosome, endoplasmic reticulum, vacuole, endosome, acrosome, autophagosome, centriole, glycosome, glyoxysome, hydrogenosome, melanosome, mitosome, nematocyst, peroxisome, proteasome, vesicle, and stress granule. In some embodiments, the organelle is a mitochondrion.

[0115] In some embodiments, the fusosomes enter target cells by endocytosis, e.g., the level of therapeutic agent delivered via the endocytic pathway is 0.01-0.6, 0.01-0.1, 0.1-0.3, or 0.3-0.6, e.g., by the assay of Example 91, or is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than chloroquine-treated reference cells contacted with similar fusosomes. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the fusosomes in the fusosome composition that enter target cells enter via a non-endocytic pathway, e.g., the fusosomes enter target cells via fusion with the cell surface. In some embodiments, the level of therapeutic agent delivered via a non-endocytic pathway in a given fusosome is: For example, using the assay of Example 90, the fusosome concentration is 0.1-0.95, 0.1-0.2, 0.2-0.3, 0.3-0.4, 0.4-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.9-0.95, or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than reference cells treated with chloroquine. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the fusosomes in the fusosome composition that enter the target cell enter the cytoplasm (e.g., do not enter endosomes or lysosomes). In some embodiments, less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the fusosomes in the fusosome composition that enter target cells enter endosomes or lysosomes. In some embodiments, the fusosomes enter target cells by a non-endocytic pathway, e.g., the level of delivered therapeutic agent is at least 90%, 95%, 98%, or 99% of the level in chloroquine-treated reference cells, e.g., using the assay of Example 91. In one embodiment, the fusosomes deliver the agent to the target cell via a dynamin-mediated pathway. In one embodiment, the level of agent delivered via the dynamin-mediated pathway ranges from 0.01 to 0.6, or is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than Dynasore-treated target cells contacted with similar fusosomes, e.g., as measured in the assay of Example 92. In one embodiment, fusosomes deliver agent to target cells via macropinocytosis.In one embodiment, the level of agent delivered via macropinocytosis ranges from 0.01 to 0.6, or is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than target cells treated with EIPA and contacted with similar fusosomes, as measured, for example, in the assay of Example 92. In one embodiment, fusosomes deliver agents to target cells via an actin-mediated pathway. In one embodiment, the level of agent delivered via the actin-mediated pathway ranges from 0.01 to 0.6, or is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than target cells treated with latrunculin B and contacted with similar fusosomes, as measured, for example, in the assay of Example 92.

[0116] In some embodiments, the fusosomes have a density of less than 1, 1-1.1, 1.05-1.15, 1.1-1.2, 1.15-1.25, 1.2-1.3, 1.25-1.35, or greater than 1.35 g / ml, e.g., by the assay of Example 33.

[0117] In some embodiments, the fusosome composition comprises less than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, or 10% of the source cells by protein mass, or less than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, or 10% of the cells have functional nuclei. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the fusosomes in the fusosome composition comprise organelles, e.g., mitochondria.

[0118] In some embodiments, the fusosome further comprises an exogenous therapeutic agent, which is selected from one or more of a protein, e.g., an enzyme, a transmembrane protein, a receptor, an antibody, a nucleic acid, e.g., DNA, a chromosome (e.g., a human artificial chromosome), RNA, mRNA, siRNA, miRNA, or a small molecule.

[0119] In embodiments, fusosomes enter cells by endocytosis or non-endocytosis pathways.

[0120] In some embodiments, the fusosomes or fusosome composition are refrigerated or frozen. In embodiments, the fusosomes do not contain a functional nucleus, or the fusosome composition comprises fusosomes without a functional nucleus. In embodiments, the fusosome composition contains less than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, or 10% of the source cells by protein mass, or less than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, or 10% of the cells have a functional nucleus. In embodiments, the fusosome composition is maintained at the temperature for at least 1, 2, 3, 6, or 12 hours, 1, 2, 3, 4, 5, or 6 days, 1, 2, 3, or 4 weeks, 1, 2, 3, or 6 months, or 1, 2, 3, 4, or 5 years. In embodiments, the fusosome composition has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the activity of the population prior to maintenance at the temperature, e.g., i) fusosomes fuse with target cells at, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher rates than non-target cells, e.g., in the assay of Example 54; ii) the fusosomes fuse with target cells at a rate, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than other fusosomes, e.g., in the assay of Example 54; 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, e.g., in the assay of Example 54; or iv) The fusogen is present in one or more of at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000 copies or less, as measured, for example, by the assay of Example 29.

[0121] In embodiments, the fusosome composition is stable at temperatures below 4° C. for at least 1, 2, 3, 6, or 12 hours, 1, 2, 3, 4, 5, or 6 days, 1, 2, 3, or 4 weeks, 1, 2, 3, or 6 months, or 1, 2, 3, 4, or 5 years. In embodiments, the fusosome composition is stable at temperatures below −20° C. for at least 1, 2, 3, 6, or 12 hours, 1, 2, 3, 4, 5, or 6 days, 1, 2, 3, or 4 weeks, 1, 2, 3, or 6 months, or 1, 2, 3, 4, or 5 years. In embodiments, the fusosome composition is stable at temperatures below −80° C. for at least 1, 2, 3, 6, or 12 hours, 1, 2, 3, 4, 5, or 6 days, 1, 2, 3, or 4 weeks, 1, 2, 3, or 6 months, or 1, 2, 3, 4, or 5 years.

[0122] In an embodiment, i) the source cells are other than 293 cells; ii) the source cells are not transformed or immortalized; iii) the source cells are transformed or immortalized using methods other than adenovirus-mediated immortalization, e.g., immortalized by spontaneous mutation or immortalization by telomerase expression; iv) the fusogen is other than a VSVG, a SNARE protein, or a secretory granule protein; v) the therapeutic agent is other than Cre or EGFP; vi) the therapeutic agent further comprises, for example, an exogenous nucleic acid (e.g., RNA, e.g., mRNA, miRNA, or siRNA) or an exogenous protein (e.g., an antibody, e.g., an antibody) in the lumen; vii) the fusosomes do not contain mitochondria.

[0123] In an embodiment, i) the source cells are other than 293 or HEK cells; ii) the source cells are not transformed or immortalized; iii) the source cells are transformed or immortalized using methods other than adenovirus-mediated immortalization, e.g., immortalized by spontaneous mutation or immortalization by telomerase expression; iv) the fusogen is not a viral fusogen, or v) The fusosomes have a size other than 40-150 nm, for example, greater than 150 nm, 200 nm, 300 nm, 400 nm, or 500 nm.

[0124] In an embodiment, i) the therapeutic agent is a soluble protein expressed by the source cell; ii) the fusogen is other than TAT, TAT-HA2, HA-2, gp41, Alzheimer's beta-amyloid peptide, Sendai virus protein, or amphipathic net-negative peptide (WAE 11); iii) the fusogen is a mammalian fusogen; iv) the fusosomes contain in their lumen a polypeptide selected from an enzyme, an antibody, or an antiviral polypeptide; v) the fusosomes do not contain an exogenous therapeutic transmembrane protein, or vi) the fusosomes do not contain CD63 or GLUT4, or the fusosomes contain less than 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10% (e.g., less than about 0.048%) of CD63, as determined, for example, according to the method described in Example 157.

[0125] In embodiments, the fusosomes are i) does not contain virus, is not infectious, or does not replicate in host cells; ii) is not a viral vector; iii) are not VLPs (virus-like particles); iv) does not contain a viral structural protein, e.g., a protein derived from gag, e.g., a viral capsid protein, e.g., a viral capsule protein, e.g., a viral nucleocapsid protein, or the amount of viral capsid protein is less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% of the total protein, e.g., by mass spectrometry, e.g., using the assay of Example 53 or 161; v) does not contain viral matrix proteins; vi) does not contain viral nonstructural proteins, such as pol or fragments or variants thereof, viral reverse transcriptase protein, viral integrase protein, or viral protease protein; vii) does not contain viral nucleic acid, e.g., viral RNA or viral DNA; viii) contains less than 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies of a viral structural protein per vesicle; or ix) Fusosomes are not virosomes.

[0126] In some embodiments, the fusosomes comprise (or are identified as comprising) less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the viral capsid protein (e.g., about 0.05% of the viral capsid protein). In embodiments, the viral capsid protein is a complex of rabbit endogenous lentivirus (RELIK) capsid and cyclophilin A. In embodiments, the ratio of viral capsid protein:total protein is (or is identified as being) about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1.

[0127] In some embodiments, the fusosomes do not contain (or are identified as not containing) gag protein or a fragment or variant thereof, or the amount of gag protein or a fragment or variant thereof is less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% of the total protein, e.g., by the assay of Example 53 or 161.

[0128] In embodiments, the ratio of fusosome copy number to viral structural protein copy number on fusosomes is at least 1,000,000:1, 100,000:1, 10,000:1, 1,000:1, 100:1, 50:1, 20:1, 10:1, 5:1, or 1:1, or is between 100:1 and 50:1, between 50:1 and 20:1, between 20:1 and 10:1, between 10:1 and 5:1, or 1:1. In embodiments, the ratio of fusosome copy number to viral matrix protein copy number on fusosomes is at least 1,000,000:1, 100,000:1, 10,000:1, 1,000:1, 100:1, 50:1, 20:1, 10:1, 5:1, or 1:1.

[0129] In an embodiment, i) fusosomes do not contain water-immiscible droplets; ii) fusosomes comprise an aqueous lumen and a hydrophilic exterior; iii) the fusogen is a protein fusogen, or iv) the organelle is one or more selected from mitochondria, Golgi apparatus, lysosomes, endoplasmic reticulum, vacuoles, endosomes, acrosomes, autophagosomes, centrioles, glycosomes, glyoxysomes, hydrogenosomes, melanosomes, mitosomes, nematocysts, peroxisomes, proteasomes, vesicles, and stress granules.

[0130] In an embodiment, i) the fusogen is a mammalian fusogen or a viral fusogen; ii) the fusosomes were not generated by loading the fusosomes with a therapeutic or diagnostic agent; iii) the source cells were not loaded with a therapeutic or diagnostic agent; iv) the fusosomes do not contain doxorubicin, dexamethasone, cyclodextrin, polyethylene glycol, microRNA, e.g., miR125, VEGF receptor, ICAM-1, E-selectin, iron oxide, fluorescent proteins, e.g., GFP or RFP, nanoparticles, or RNase, or exogenous forms of any of the foregoing; or v) the fusosomes further comprise an exogenous therapeutic agent having one or more post-translational modifications, e.g., glycosylation.

[0131] In embodiments, the fusosomes are unilamellar or multilamellar.

[0132] In embodiments, the fusosomes have a size within about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the size of the source cells, or the fusosome population has an average size within about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the average size of the source cells, e.g., as measured by the assay of Example 30. In embodiments, the fusosomes have a size that is about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the size of the source cells, or the fusosome population has an average size that is about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the average size of the source cells, e.g., as measured by the assay of Example 30. In embodiments, the fusosomes have (or are identified as having) a size smaller than the parent cells. In embodiments, fusosomes have (or are identified as having) a size that is within about 50%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 90% of that of a parent cell. In embodiments, fusosomes have (or are identified as having) less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less of the variance in size distribution of the parent cell, e.g., within about 90% of the sample. In embodiments, fusosomes have (or are identified as having) less than about 40%, 45%, 50%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, or 70% of the variance in size distribution of the parent cell, e.g., within about 90% of the sample. In some embodiments, fusosomes have (or are identified as having) an average size of greater than 30, 35, 40, 45, 50, 55, 60, 65, or 70 nm in diameter.In embodiments, fusosomes have an average size of about 100, 110, 120, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 140, or 150 nm in diameter. In embodiments, fusosomes are about 0.01% to 0.05%, 0.05% to 0.1%, 0.1% to 0.5%, 0.5% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, or 80% to 90% of the size of the source cells, as measured, for example, by the assay of Example 30. The population of fusosomes has a size within 90% or has an average size within about 0.01%-0.05%, 0.05%-0.1%, 0.1%-0.5%, 0.5%-1%, 1%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% of the size of the source cells. In embodiments, fusosomes are about 0.01% to 0.05%, 0.05% to 0.1%, 0.1% to 0.5%, 0.5% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, or 80% to 90% of the size of the source cells, e.g., as measured by the assay of Example 30. Alternatively, the population of fusosomes has an average size within about 0.01%-0.05%, 0.05%-0.1%, 0.1%-0.5%, 0.5%-1%, 1%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% of the size of the source cells.In embodiments, fusosomes have a diameter, or a population of fusosomes has an average diameter of less than about 500 nm (e.g., less than about 10, 50, 100, 150, 200, 250, 300, 350, 400, or 450 nm), as measured, e.g., by the assay of Examples 119, 120, or 121. In embodiments, fusosomes have a diameter, or a population of fusosomes has an average diameter of about 80-180, 90-170, 100-160, 110-150, 120-140, or 130 nm, as measured, e.g., by the assay of Examples 119, 120, or 121. In embodiments, the fusosomes have a diameter of about 11,000 nm to 21,000 nm, or the population of fusosomes has a mean diameter of about 11,000 nm to 21,000 nm, as measured, for example, by the assay of Examples 119, 120, or 121. In embodiments, the fusosomes have a diameter of about 10 to 22,000 nm, 12 to 20,000 nm, 14 to 18,720 nm, or 20 to 16,000 nm, as measured, for example, by the assay of Examples 119, 120, or 121. In embodiments, the fusosomes are about 0.01-0.1 μm, for example, as measured by the assay of Examples 119, 120, or 121. 3 , 0.02~1μm 3 , 0.03 to 1 μm 3 , 0.04~1μm 3 , 0.05~0.09μm 3 , 0.06~0.08μm 3 , 0.07 μm 3 or a population of fusosomes with a volume of approximately 0.01-0.1 μm 3 , 0.02~1μm 3 , 0.03 to 1 μm 3 , 0.04~1μm 3 , 0.05~0.09μm 3 , 0.06~0.08μm3 、0.07μm 3In embodiments, the fusosomes have a diameter of at least about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, or 250 nm, or a population of fusosomes has an average diameter of at least about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, or 250 nm, as measured, for example, by the assay of Example 32. In embodiments, e.g., as measured by the assay of Example 32, the fusosomes have a diameter of about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, or 250 nm (e.g., ±20%), or the population of fusosomes has a mean diameter of about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, or 250 nm (e.g., ±20%). In embodiments, the fusosomes have a diameter of at least about 500 nm, 750 nm, 1,000 nm, 1,500 nm, 2,000 nm, 2,500 nm, 3,000 nm, 5,000 nm, 10,000 nm, or 20,000 nm, or the population of fusosomes has an average diameter of at least about 500 nm, 750 nm, 1,000 nm, 1,500 nm, 2,000 nm, 2,500 nm, 3,000 nm, 5,000 nm, 10,000 nm, or 20,000 nm, as measured, for example, by the assay of Example 32. In embodiments, e.g., as measured by the assay of Example 32, the fusosomes have a diameter of about 500 nm, 750 nm, 1,000 nm, 1,500 nm, 2,000 nm, 2,500 nm, 3,000 nm, 5,000 nm, 10,000 nm, or 20,000 nm (e.g., ±20%), or the population of fusosomes has a mean diameter of about 500 nm, 750 nm, 1,000 nm, 1,500 nm, 2,000 nm, 2,500 nm, 3,000 nm, 5,000 nm, 10,000 nm, or 20,000 nm (e.g., ±20%).In embodiments, for example, by the assay of Example 120, the population of fusosomes has (or is identified as having) one or more of: a 10% interquartile diameter of about 40-90 nm, 45-60 nm, 50-55 nm, or 53 nm; a 25% interquartile diameter of about 70-100 nm, 80-95 nm, 85-90 nm, or 88 nm; a 75% interquartile diameter of about 200-250 nm, 210-240 nm, 220-230 nm, or 226 nm; or a 90% interquartile diameter of about 4000-5000 nm, 4300-4600 nm, 4400-4500 nm, or 4450 nm.

[0133] In embodiments, the fusosome composition comprises (or is identified as comprising) a GAPDH concentration of about 35-40, 36-39, 37-38, or 37.2 ng / mL, e.g., in the assay of Example 149. In embodiments, the GAPDH concentration of the fusosome composition is (or is identified as being) within about 1%, 2%, 5%, 10%, or 20% of the GAPDH concentration of the source cells, e.g., in the assay of Example 149. In embodiments, the GAPDH concentration of the fusosome composition is (or is identified as being) at least 1%, 2%, 5%, 10%, or 20% lower than the GAPDH concentration of the source cells, e.g., in the assay of Example 149. In embodiments, the fusosome composition comprises (or is identified as comprising) less than about 30, 35, 40, 45, 46, 47, 48, 49, 50, 55, 60, 65, or 70 μg of GAPDH per gram of total protein. In embodiments, the fusosome composition contains (or is identified as containing) less than about 500, 250, 100, or 50 μg of GAPDH per gram of total protein. In embodiments, the parent cells contain (or are identified as containing) at least 1%, 2.5%, 5%, 10%, 15%, 20%, 30%, 30%, 50% or more GAPDH per gram of total protein than the fusosome composition.

[0134] In an embodiment, i) fusosomes are not exosomes; ii) Fusosomes are microvesicles; iii) the fusosomes contain non-mammalian fusogens; iv) the fusosomes are engineered to incorporate fusogens; v) the fusosomes contain exogenous fusogens; vi) the fusosomes have a size of at least 80 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm, or the fusosome population has an average size of at least 80 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm; vii) fusosomes comprise one or more organelles, such as mitochondria, Golgi apparatus, lysosomes, endoplasmic reticulum, vacuoles, endosomes, acrosomes, autophagosomes, centrioles, glycosomes, glyoxysomes, hydrogenosomes, melanosomes, mitosomes, nematocysts, peroxisomes, proteasomes, vesicles, and stress granules; viii) the fusosome contains the cytoskeleton or a component thereof, such as actin, Arp2 / 3, formin, coronin, dystrophin, keratin, myosin, or tubulin; ix) Fusosomes, or compositions or preparations containing multiple fusosomes, can be isolated, for example, in a sucrose gradient centrifugation assay, as described, for example, in Thery et al., "Isolation and characterization of exosomes from have a flotation density of not more than 1.08-1.22 g / ml, or have a density of at least 1.18-1.25 g / ml, or 1.05-1.12 g / ml, as described in "Cell Culture Supernatants and Biological Fluids." Curr Protoc Cell Biol. 2006 Apr; Chapter 3: Unit 3.22 x) the lipid bilayer is enriched in ceramide or sphingomyelin, or a combination thereof, compared to the source cells, or the lipid bilayer is not enriched (e.g., depleted) in glycolipids, free fatty acids, or phosphatidylserine, or a combination thereof, compared to the source cells; xi) the fusosomes contain phosphatidylserine (PS) or CD40 ligand or both PS and CD40 ligand, e.g., as measured in the assay of Example 52 or 160; xii) the fusosomes are enriched for PS compared to the source cells, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fusosome population is positive for PS, e.g., by the assay of Kanada M, et al. (2015) Differential fates of biomolecules delivered to target cells via extracellular vesicles. Proc Natl Acad Sci USA 112:E1433-E1442; xiii) the fusosomes are substantially free of acetylcholinesterase (AChE) or contain less than 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, or 1000 AChE activity units / ug protein, e.g., by the assay of Example 67; xiv) the fusosomes are substantially free of tetraspanin family proteins (e.g., CD63, CD9, or CD81), ESCRT-related proteins (e.g., TSG101, CHMP4A-B, or VPS4B), Alix, TSG101, MHCI, MHCII, GP96, actinin-4, mitofilin, syntenin-1, TSG101, ADAM10, EHD4, syntenin-1, TSG101, EHD1, flotillin-1, heat shock 70 kDa proteins (HSC70 / HSP73, HSP70 / HSP72), or any combination thereof; or For example, the assay of Example 44 or 157 comprises less than 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 5%, or 10% of any individual exosome marker protein and / or less than 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% of the total exosome marker protein of any of said proteins, or any one or more of these proteins are de-enriched or not enriched in any one or more of these proteins compared to the source cells. xv) the fusosomes comprise a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) level below 500, 250, 100, 50, 20, 10, 5, or 1 ng of GAPDH / ug total protein, or below the GAPDH level in the source cells, e.g., less than 1%, 2.5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less than the level of GAPDH per ng / ug total protein in the source cells, e.g., using the assay of Example 45; xvi) the fusosomes are enriched in one or more endoplasmic reticulum proteins (e.g., calnexin), one or more proteasome proteins, or one or more mitochondrial proteins, or any combination thereof, or the amount of calnexin is less than 500, 250, 100, 50, 20, 10, 5, or 1 ng of calnexin / ug total protein, or the fusosomes contain 1%, 2.5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less calnexin (ng / ug) per total protein compared to the source cells, e.g., using the assay of Example 46 or 158, or the average fractional content of calnexin in fusosomes is about 1 x 10 -4 , 1.5×10 -4 , 2 × 10 -4 , 2.1×10 -4 , 2.2 × 10 -4 , 2.3 × 10 -4 , 2.4 × 10 -4 , 2.43 × 10 -4 , 2.5×10 -4 , 2.6×10 -4 , 2.7 × 10 -4 , 2.8×10 -4 , 2.9 × 10 -4 , 3×10 -4 , 3.5×10 -4 , or 4x10 -4 or the fusosomes contain an amount of calnexin per total protein that is about 70%, 75%, 80%, 85%, 88%, 90%, 95%, 99% or more less than the amount of calnexin in the parent cell. xvii) the fusosomes contain an exogenous agent (e.g., an exogenous protein, mRNA, or siRNA), as measured, for example, using the assays of Examples 39 or 40; or xviii) the fusosomes can be immobilized on a mica surface by atomic force microscopy for at least 30 minutes, e.g., by the assay of Kanada M, et al. (2015) Differential fates of biomolecules delivered to target cells via extracellular vesicles. Proc Natl Acad Sci USA 112:E1433-E1442.

[0135] In an embodiment, i) fusosomes are exosomes; ii) fusosomes are not microvesicles; iii) the fusosomes have a size of less than 80 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm, or the fusosome population has an average size of less than 80 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm; iv) fusosomes do not contain organelles; v) fusosomes do not contain the cytoskeleton or its components, such as actin, Arp2 / 3, formin, coronin, dystrophin, keratin, myosin, or tubulin; vi) Fusosomes, or compositions or preparations comprising a plurality of fusosomes, can be isolated, e.g., in a sucrose gradient centrifugation assay, as described, e.g., in Thery et al., "Isolation and characterization of exosomes from As described in "Cell Culture Supernatants and Biological Fluids." Curr Protoc Cell Biol. 2006 Apr; Chapter 3: Unit 3.22, it has a flotation density of 1.08 to 1.22 g / ml. vii) the lipid bilayer is not enriched in ceramide or sphingomyelin, or a combination thereof, compared to the source cells, or the lipid bilayer is enriched in glycolipids, free fatty acids, or phosphatidylserine, or a combination thereof, compared to the source cells; viii) the fusosomes do not contain or are depleted of source cells, phosphatidylserine (PS), or CD40 ligand, or both PS and CD40 ligand, as measured, for example, in the assays of Examples 52 or 160; ix) Fusosomes are not enriched (depleted) in PS compared to source cells, e.g., in less than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fusosome population, e.g., Kanada M, et al. (2015) The PS was positive by assaying differential fates of biomolecules delivered to target cells via extracellular vesicles. Proc Natl Acad Sci USA 112:E1433-E1442. x) the fusosomes contain acetylcholinesterase (AChE), e.g., at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, or 1000 AChE activity units / ug protein, e.g., by the assay of Example 67; xi) fusosomes express tetraspanin family proteins (e.g., CD63, CD9, or CD81), ESCRT-related proteins (e.g., TSG101, CHMP4A-B, or VPS4B), Alix, TSG101, MHCI, MHCII, GP96, actinin-4, mitofilin, syntenin-1, TSG101, ADAM10, EHD4, syntenin-1, TSG101, EHD1, flotillin-1, heat shock 70 kDa proteins (HSC70 / HSP73, HSP70 / HSP72), or any of these. or any combination thereof, e.g., by an assay of Example 44 or 157, e.g., less than 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 5%, or 10% of any individual exosome marker protein and / or less than 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% of the total exosome marker protein of any of said proteins, or any one or more of these proteins are enriched compared to the source cells. xii) the fusosomes comprise a level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) greater than 500, 250, 100, 50, 20, 10, 5, or 1 ng of GAPDH / ug total protein, or less than the level of GAPDH in the source cells, e.g., at least 1%, 2.5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% greater than the level of GAPDH per ng / ug total protein in the source cells, e.g., using the assay of Example 45; xiii) the fusosomes are not enriched (e.g., depleted) in one or more endoplasmic reticulum proteins (e.g., calnexin), one or more proteasome proteins, or one or more mitochondrial proteins, or any combination thereof, e.g., the amount of calnexin is less than 500, 250, 100, 50, 20, 10, 5, or 1 ng of calnexin / ug total protein, or the fusosomes contain 1%, 2.5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less calnexin (ng / ug) per total protein compared to the source cells, e.g., using the assay of Example 46 or 158, or the average fractional content of calnexin in fusosomes is about 1 x 10 -4 , 1.5×10 -4 , 2 × 10 -4 , 2.1×10 -4 , 2.2 × 10 -4 , 2.3 × 10 -4 , 2.4 × 10 -4 , 2.43 × 10 -4 , 2.5×10 -4 , 2.6×10 -4 , 2.7 × 10 -4 , 2.8×10 -4 , 2.9 × 10 -4 , 3×10 -4 , 3.5×10 -4 , or 4x10 -4 or the fusosomes contain an amount of calnexin per total protein that is about 70%, 75%, 80%, 85%, 88%, 90%, 95%, 99% or more less than the amount of calnexin in the parent cell; or xiv) the fusosomes cannot be immobilized on a mica surface by atomic force microscopy for at least 30 minutes, e.g., by the assay of Kanada M, et al. (2015) Differential fates of biomolecules delivered to target cells via extracellular vesicles. Proc Natl Acad Sci USA 112:E1433-E1442.

[0136] In embodiments, the average fractional content of calnexin in fusosomes is about 1×10 -4 , 1.5×10 -4 , 2 × 10 -4 , 2.1×10 -4 , 2.2 × 10 -4 , 2.3 × 10 -4 , 2.4 × 10 -4 , 2.43 × 10 -4 , 2.5×10 -4 , 2.6×10 -4 , 2.7 × 10 -4 , 2.8×10 -4 , 2.9 × 10 -4 , 3×10 -4 , 3.5×10 -4 , or 4 × 10 -4 In embodiments, the fusosomes contain an amount of calnexin that is about 70%, 75%, 80%, 85%, 88%, 90%, 95%, 99% or more less per total protein than the amount of calnexin in the parent cell.

[0137] In an embodiment, i) fusosomes do not contain VLPs; ii) fusosomes are virus-free; iii) fusosomes do not contain replication-competent virus; iv) fusosomes do not contain viral proteins, e.g., viral structural proteins, e.g., capsid proteins or viral matrix proteins; v) fusosomes do not contain capsid proteins of enveloped viruses; vi) the fusosomes do not contain nucleocapsid proteins, or vii) The fusogen is one or more of:

[0138] In an embodiment, the fusosome comprises cytosol.

[0139] In an embodiment, i) the fusosomes or source cells do not form teratomas when transplanted into a subject, e.g., by the assay of Example 102; ii) the fusosomes enable chemotaxis that is, for example, within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or greater than a reference cell, e.g., a macrophage, using, e.g., the assay of Example 58; iii) fusosomes can home at, for example, sites of injury, and fusosome or cell biology can be assessed using, for example, the assay of Example 59, e.g., from human cells, e.g., where the source cell is a neutrophil, or iv) the fusosomes are capable of phagocytosis, e.g., phagocytosis by fusosomes is detectable within 0.5, 1, 2, 3, 4, 5, or 6 hours, e.g., using the assay of Example 60, where the source cell is a macrophage.

[0140] In embodiments, the fusosomes or fusosome compositions retain any one, two, three, four, five, six or more of the properties for 5 days or less, e.g., 4 days or less, 3 days or less, 2 days or less, 1 day or less, e.g., about 12 to 72 hours, after administration to a subject, e.g., a human subject.

[0141] In embodiments, the fusosomes have one or more of the following characteristics: a) one or more endogenous proteins from the source cell, including, for example, membrane proteins or cytosolic proteins; b) comprises at least 10, 20, 50, 100, 200, 500, 1000, 2000, or 5000 different proteins; c) comprises at least 1, 2, 5, 10, 20, 50, or 100 different glycoproteins; d) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by mass of the protein in the fusosomes is naturally occurring protein; e) comprises at least 10, 20, 50, 100, 200, 500, 1000, 2000, or 5000 different RNAs; or f) For example, comprising at least 2, 3, 4, 5, 10, or 20 different lipids selected from CL, Cer, DAG, HexCer, LPA, LPC, LPE, LPG, LPI, LPS, PA, PC, PE, PG, PI, PS, CE, SM, and TAG.

[0142] In embodiments, the fusosomes are engineered to have one, two, three, four, five or more of the following properties, or the cell in which the fusosomes naturally occur or the nucleus does not naturally have one, two, three, four, five or more of the following properties: a) partial nuclear inactivation results in at least a 50%, 60%, 70%, 80%, 90% or more reduction in nuclear function, e.g., a reduction in transcription or DNA replication, or both, where transcription is measured by the assay of Example 19 and DNA replication is measured by the assay of Example 20; b) the fusosomes are unable to transcribe a reference cell, e.g., a source cell, or have less than 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the transcription activity of the reference cell, e.g., a source cell, e.g., using the assay of Example 19; c) the fusosomes are incapable of nuclear DNA replication of the reference cell, e.g., the source cell, or have nuclear DNA replication that is less than 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the nuclear DNA replication of the reference cell, e.g., the source cell, e.g., using the assay of Example 20. d) the fusosomes lack chromatin or have a chromatin content that is less than 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the chromatin content of a reference cell, e.g., a source cell, using, e.g., the assay of Example 37; e) the fusosomes lack a nuclear membrane or have less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the amount of nuclear membrane of a reference cell, e.g., a source cell or a Jurkat cell, e.g., by the assay of Example 36; f) the fusosomes lack functional nuclear pore complexes or have reduced nuclear import or export activity, e.g., by at least 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%, e.g., by the assay of Example 36, or the fusosomes lack one or more nuclear pore proteins, e.g., NUP98 or importin 7; g) the fusosomes are free of histones or have histone levels that are less than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the histone levels (e.g., H1, H2a, H2b, H3, or H4) of the source cells, e.g., by the assay of Example 37; h) the fusosomes contain less than 20, 10, 5, 4, 3, 2, or 1 chromosome; i) Nuclear functions are eliminated; j) fusosomes are enucleated mammalian cells; k) the nucleus is removed or inactivated, e.g., pushed out by mechanical force, radiation, or chemical ablation; or l) Fusosomes are, for example, from mammalian cells whose DNA has been completely or partially removed during interphase or mitosis.

[0143] In embodiments, the fusosomes contain mtDNA or vector DNA. In embodiments, the fusosomes do not contain DNA.

[0144] In embodiments, the source cells are primary cells, immortalized cells, or cell lines (e.g., myeloblast cell lines, e.g., C2C12). In embodiments, the fusosomes are from source cells with a modified genome having reduced immunogenicity (e.g., by genome editing, e.g., to remove MHC proteins or MHC complexes). In embodiments, the source cells are from cell cultures treated with anti-inflammatory signals. In embodiments, the source cells are from cell cultures treated with immunosuppressants. In embodiments, the source cells are substantially non-immunogenic, e.g., using the assays described herein. In embodiments, the source cells include an exogenous agent, e.g., a therapeutic agent. In embodiments, the source cells are recombinant cells.

[0145] In embodiments, the fusosomes further comprise an exogenous agent, e.g., a therapeutic agent, e.g., a protein or nucleic acid (e.g., DNA, a chromosome (e.g., a human artificial chromosome), RNA, e.g., mRNA or miRNA). In embodiments, the exogenous agent is, for example, at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500, ,000,000, or 1,000,000,000 copies or less, or are present at an average level of at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, or 1,000,000 copies or less per fusosome. In embodiments, the fusosomes are prepared by treating the mammalian cells with siRNA or a gene editing enzyme to alter, e.g., increase or decrease, the levels of one or more endogenous molecules, e.g., proteins or nucleic acids. In embodiments, the endogenous molecule is, for example, at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,00 In embodiments, the endogenous molecule (e.g., RNA or protein) is present at 0,000,000 copies (e.g., copies contained in fusosomes) or less, or is present at an average level of at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, or 1,000,000 copies per fusosome or less. In embodiments, the endogenous molecule (e.g., RNA or protein) is present at an average level of at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 500, 10, 100, 1500, 1 ...3 , 5.0×10 3 , 10 4 , 5.0×10 4 , 10 5 , 5.0×10 5 , 10 6 , 5.0×10 6 , 1.0×10 7 , 5.0×10 7 , or 1.0 × 10 8 It is present in high concentrations.

[0146] In embodiments, the active agent is selected from a protein, a protein complex (e.g., comprising at least 2, 3, 4, 5, 10, 20, or 50 proteins, e.g., comprising at least at least 2, 3, 4, 5, 10, 20, or 50 different proteins), a polypeptide, a nucleic acid (e.g., DNA, a chromosome, or RNA, e.g., mRNA, siRNA, or miRNA), or a small molecule. In embodiments, the exogenous agent comprises a site-specific nuclease, e.g., a Cas9 molecule, a TALEN, or a ZFN.

[0147] In embodiments, the fusogen is a viral fusogen, such as HA, HIV-1 ENV, HHV-4, gp120, or VSV-G. In embodiments, the fusogen is a mammalian fusogen, such as a SNARE, syncytin, myomaker, myomixer, myomerger, or FGFRL1. In embodiments, the fusogen is active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. In embodiments, the fusogen is not active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. In embodiments, fusosomes fuse with target cells at the surface of the target cells. In embodiments, the fusogen promotes fusion in a lysosome-independent manner. In embodiments, the fusogen is a protein fusogen. In embodiments, the fusogen is a lipid fusogen, such as oleic acid, glycerol monooleate, glycerides, diacylglycerol, or modified unsaturated fatty acids. In embodiments, the fusogen is a chemical fusogen, such as PEG. In embodiments, the fusogen is a small molecule fusogen, such as halothane, an NSAID, such as meloxicam, piroxicam, tenoxicam, and chlorpromazine. In embodiments, the fusogen is recombinant. In embodiments, the fusogen is biochemically incorporated, e.g., provided as a purified protein and contacted with the lipid bilayer under conditions that allow the fusogen to bind to the lipid bilayer. In embodiments, the fusogen is biosynthetically incorporated, e.g., expressed in source cells under conditions that allow the fusogen to bind to the lipid bilayer.

[0148] In embodiments, the fusosomes bind to a target cell. In embodiments, the target cell is other than a HeLa cell, or the target cell is not transformed or immortalized.

[0149] In some embodiments comprising a fusosome composition, the plurality of fusosomes are the same. In some embodiments, the plurality of fusosomes are different. In some embodiments, the plurality of fusosomes are from one or more source cells. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the plurality of fusosomes have a diameter within 10%, 20%, 30%, 40%, or 50% of the average diameter of the fusosomes in the fusosome composition. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the plurality of fusosomes have a volume within 10%, 20%, 30%, 40%, or 50% of the average volume of the fusosomes in the fusosome composition. In some embodiments, the fusosome composition has a size distribution variation of less than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, e.g., a size distribution variation within 10%, 50%, or 90% of the source cell population based on Example 31. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the plurality of fusosomes have a fusogen copy number within 10%, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, or 90% of the average fusogen copy number of fusosomes in the fusosome composition. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the plurality of fusosomes have a fusogen copy number within 10%, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, or 90% of the average therapeutic agent copy number of fusosomes in the fusosome composition. 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15In some embodiments, the fusosome composition is in a volume of at least 1 ul, 2 ul, 5 ul, 10 ul, 20 ul, 50 ul, 100 ul, 200 ul, 500 ul, 1 ml, 2 ml, 5 ml, or 10 ml.

[0150] In some embodiments, the fusosome composition delivers cargo to at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the number of cells in a target cell population compared to a reference target cell population.

[0151] In some embodiments, the fusosome composition delivers at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% more cargo to the target cell population compared to a reference target cell population or an unlabeled cell population. In some embodiments, the fusosome composition delivers at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% more cargo to the target cell population compared to a reference target cell population or an unlabeled cell population.

[0152] In some embodiments, less than 10% of the cargo enters the cell by endocytosis.

[0153] In some embodiments, the inhibitor of endocytosis is an inhibitor of lysosomal acidification, such as bafilomycin A1. In some embodiments, the inhibitor of endocytosis is a dynamin inhibitor, such as dynasore.

[0154] In some embodiments, the target cell population is at a physiological pH (eg, 7.3 to 7.5, eg, 7.38 to 7.42).

[0155] In some embodiments, the cargo delivered is determined using an endocytosis inhibition assay, eg, the assay of Examples 90, 92, or 135.

[0156] In some embodiments, the cargo enters the cell through a dynamin-independent pathway or a lysosomal acidification-independent pathway, a macropinocytosis-independent pathway (e.g., the inhibitor of endocytosis is an inhibitor of macropinocytosis, e.g., 5-(N-ethyl-N-isopropyl) amiloride (EIPA), e.g., at a concentration of 25 μM), or an actin-independent pathway (e.g., the inhibitor of endocytosis is an inhibitor of actin polymerization, e.g., latrunculin B, e.g., at a concentration of 6 μM).

[0157] In some embodiments, the plurality of fusosomes further comprises a targeting moiety, hi embodiments, the targeting moiety is comprised in the fusogen or in a separate molecule.

[0158] In some embodiments, when multiple fusosomes are contacted with a cell population comprising target cells and non-target cells, the cargo is present in at least 10 times more target cells than non-target cells.

[0159] In some embodiments, when a plurality of fusosomes are contacted with a cell population comprising target cells and non-target cells, the cargo is present in at least 2-fold, 5-fold, 10-fold, 20-fold, or 50-fold more target cells than non-target cells, and / or the cargo is present in at least 2-fold, 5-fold, 10-fold, 20-fold, or 50-fold more target cells than reference cells.

[0160] In some embodiments, the multiple fusosomes fuse with target cells at a rate at least 50% higher than with non-target cells.

[0161] In some embodiments, the presence of cargo is determined by microscopy, e.g., using the assay of Example 124. In some embodiments, fusion is determined by microscopy, e.g., using the assay of Example 54.

[0162] In some embodiments, the targeting moiety is specific for a cell surface marker on the target cell. In embodiments, the cell surface marker is a cell surface marker for skin cells, cardiac muscle cells, liver cells, intestinal cells (e.g., cells of the small intestine), pancreatic cells, brain cells, prostate cells, lung cells, colon cells, or bone marrow cells.

[0163] In some embodiments, the fusogen (e.g., a retargeted fusogen) comprises a Rhabdoviridae fusogen (e.g., VSV-G), a Filoviridae fusogen, an Arenaviridae fusogen, a Togaviridae fusogen, a Flaviviridae fusogen, a Bunyaviridae fusogen, or a Hepadnaviridae fusogen (e.g., Hep B), or a derivative thereof.

[0164] In some embodiments, the plurality of fusosomes, when contacted with a target cell population in the presence of an inhibitor of endocytosis, and when contacted with a reference target cell population that is not treated with an inhibitor of endocytosis, deliver cargo to at least 30% of the number of cells in the target cell population compared to the reference target cell population.

[0165] In some embodiments, the plurality of fusosomes, when contacted with a target cell population in the presence of an inhibitor of endocytosis, and when contacted with a reference target cell population that is not treated with an inhibitor of endocytosis, delivers at least 30% of the cargo in the target cell population compared to a reference target cell population.

[0166] In some embodiments, the fusosomes, when contacted with a target cell population, deliver cargo to a target cell location other than an endosome or lysosome, e.g., the cytosol, hi embodiments, less than 50%, 40%, 30%, 20%, or 10% of the cargo is delivered to an endosome or lysosome.

[0167] In some embodiments, the amount of viral capsid protein in the fusosome composition is determined using mass spectrometry, for example, using the assay of Examples 53 or 161.

[0168] In some embodiments, the plurality of fusosomes comprises exosomes, microvesicles, or a combination thereof.

[0169] In some embodiments, the plurality of fusosomes has an average size of at least 50 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm, while in other embodiments, the plurality of fusosomes has an average size of less than 100 nm, 80 nm, 60 nm, 40 nm, or 30 nm.

[0170] In some embodiments, the source cells are selected from neutrophils, HEK293 cells, granulocytes, mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells, myeloblasts, myoblasts, hepatocytes, or neurons, e.g., retinal neuronal cells.

[0171] In some embodiments, the plurality of fusosomes comprises a cell biology. In some embodiments, the plurality of fusosomes comprises enucleated cells.

[0172] In some embodiments, the fusogen (e.g., the retargeted fusogen) comprises a mammalian fusogen. In some embodiments, the fusogen (e.g., the retargeted fusogen) comprises a viral fusogen. In some embodiments, the fusogen (e.g., the retargeted fusogen) is a protein fusogen. In some embodiments, the fusogen (e.g., the retargeted fusogen) comprises Nipah virus protein F, measles virus F protein, Tupaia paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, Henipavirus F protein, Morbillivirus F protein, Respirovirus F protein, Sendai virus F protein, Rubulavirus F protein, or Avulavirus F protein, or a derivative thereof.

[0173] In some embodiments, the fusogen (e.g., a retargeted fusogen) is active at a pH of 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10. In some embodiments, the fusogen (e.g., a retargeted fusogen) is not active at a pH of 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.

[0174] In some embodiments, the fusogen is present in a copy number of at least 1, 2, 5, or 10 copies per fusosome.

[0175] In some embodiments, the fusogen (e.g., retargeted fusogen) comprises Nipah virus protein G, measles protein H, Tupaia paramyxovirus H protein, paramyxovirus G protein, paramyxovirus H protein, paramyxovirus HN protein, morbillivirus H protein, respirovirus HN protein, Sendai HN protein, rubulavirus HN protein, avulavirus HN protein, or a derivative thereof. In some embodiments, the fusogen (e.g., retargeted fusogen) comprises a sequence selected from Nipah virus F and G proteins, measles virus F and H proteins, Tupaia paramyxovirus F and H proteins, paramyxovirus F and G proteins or F and H proteins or F and HN proteins, Hendra virus F and G proteins, Henipavirus F and G proteins, morbillivirus F and H proteins, respirovirus F and HN proteins, Sendai virus F and HN proteins, rubulavirus F and HN proteins, or avulavirus F and HN proteins, or a derivative thereof, or any combination thereof.

[0176] In some embodiments, the cargo comprises an exogenous protein or exogenous nucleic acid. In some embodiments, the cargo comprises or encodes a cytoplasmic protein. In some embodiments, the cargo comprises or encodes a membrane protein. In some embodiments, the cargo comprises a therapeutic agent. In some embodiments, the cargo is present in a copy number of at least 1, 2, 5, 10, 20, 50, 100, or 200 copies per fusosome (e.g., up to about 1,000 copies per fusosome). In some embodiments, the ratio of copy number of the fusogen (e.g., the retargeted fusogen) to copy number of the cargo is 1000:1 to 1:1, or 500:1 to 1:1, or 250:1 to 1:1, or 150:1 to 1:1, or 100:1 to 1:1, or 75:1 to 1:1, or 50:1 to 1:1, or 25:1 to 1:1, or 20:1 to 1:1, or 15:1 to 1:1, or 10:1 to 1:1, or 5:1 to 1:1, or 2:1 to 1:1, or 1:1 to 1:2.

[0177] In some embodiments, the fusosome composition comprises: a) meets pharmaceutical or Good Manufacturing Practice (GMP) standards; b) Made in accordance with Good Manufacturing Practices (GMP); c) have pathogen levels below a predetermined threshold, e.g., are substantially pathogen-free; or d) have contaminant levels below predetermined standards, e.g., are substantially free of contaminants.

[0178] In some embodiments, the fusosome composition is at a temperature of less than 4, 0, -4, -10, -12, -16, -20, -80, or -160°C.

[0179] In some embodiments, the fusosome composition comprises a viral capsid protein or a DNA-integrating polypeptide. In some embodiments, the cargo comprises a viral genome.

[0180] In some embodiments, the fusosome compositions are capable of delivering nucleic acids to target cells, eg, for gene therapy, eg, to stably modify the genome of the target cell.

[0181] In some embodiments, the fusosome composition does not contain viral nucleocapsid protein, or the amount of viral nucleocapsid protein is less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% of the total protein, e.g., by mass spectrometry, e.g., using the assay of Example 53 or 161.

[0182] In embodiments, the pharmaceutical compositions described herein have one or more of the following characteristics: a) the pharmaceutical composition meets the standards of a drug product or Good Manufacturing Practice (GMP); b) the pharmaceutical composition is made in accordance with Good Manufacturing Practice (GMP); c) the pharmaceutical composition has a pathogen level below a predetermined threshold, e.g., is substantially pathogen-free; d) the pharmaceutical composition has contaminant levels below a predetermined standard, e.g., is substantially free of contaminants; or e) the pharmaceutical composition has low immunogenicity, e.g., as described herein.

[0183] In embodiments, the cargo of the pharmaceutical composition comprises a therapeutic agent.

[0184] In an embodiment, the biological function is a) modulating, e.g., inhibiting or stimulating, an enzyme; b) modulating, e.g., increasing or decreasing, the level of a molecule (e.g., a protein, nucleic acid, or metabolite, drug, or toxin) in a subject, e.g., by inhibiting or stimulating the synthesis of the factor or by inhibiting or stimulating its degradation; c) modulating, e.g., increasing or decreasing, the viability of target cells or tissues; or d) modulating the state of a protein, for example, increasing or decreasing the phosphorylation of a protein or modulating the conformation of a protein; e) promoting healing of injuries; f) modulating, e.g., increasing or decreasing, the interaction between two cells; g) modulating, e.g., promoting or inhibiting, cell differentiation; h) altering the distribution of a factor (e.g., a protein, nucleic acid, metabolite, drug, or toxin) in a subject; i) modulating, e.g., increasing or decreasing, the immune response; or j) modulating, e.g., increasing or decreasing, the recruitment of cells to target tissues.

[0185] In some embodiments of the methods of treatment herein, the plurality of fusosomes has a local effect. In some embodiments, the plurality of fusosomes has a distal effect.

[0186] In some embodiments, the subject has cancer, an inflammatory disorder, an autoimmune disease, a chronic disease, inflammation, impaired organ function, an infectious disease, a metabolic disease, a degenerative disorder, a genetic disease (e.g., a genetic defect, a recessive genetic disorder, or a dominant genetic disorder), or an injury. In some embodiments, the subject has an infectious disease, and the fusosomes comprise an antigen of the infectious disease. In some embodiments, the subject has a genetic defect, and the fusosomes comprise a protein that the subject is deficient in, or a nucleic acid (e.g., mRNA) encoding the protein, or a DNA encoding the protein, or a chromosome encoding the protein, or a nucleus comprising a nucleic acid encoding the protein. In some embodiments, the subject has a dominant genetic disorder, and the fusosomes comprise a nucleic acid inhibitor (e.g., siRNA or miRNA) of a dominant mutant allele. In some embodiments, the subject has a dominant genetic disorder, and / or the fusosomes comprise a nucleic acid inhibitor (e.g., siRNA or miRNA) of a dominant mutant allele, and / or the fusosomes also comprise mRNA encoding a non-mutated allele of the mutant gene that is not targeted by the nucleic acid inhibitor. In some embodiments, the subject is in need of vaccination. In some embodiments, the subject is in need of regeneration, for example, of an injured area.

[0187] In some embodiments, the fusosome composition is administered to the subject at least 1, 2, 3, 4, or 5 times.

[0188] In some embodiments, the fusosome composition is administered to a subject systemically (e.g., orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally) or locally. In some embodiments, the fusosome composition is administered to a subject so that it reaches a target tissue selected from the liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testes, ovaries, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye. In some embodiments (e.g., when the subject has an autoimmune disease), the fusosome composition is co-administered with an immunosuppressant, such as a glucocorticoid, a cytostatic agent, an antibody, or an immunophilin-modulating agent. In some embodiments (e.g., when the subject has cancer or an infectious disease), the fusosome composition is co-administered with an immunostimulant, such as an adjuvant, interleukin, cytokine, or chemokine. In some embodiments, administration of the fusosome composition results in the upregulation or downregulation of a gene in a target cell in a subject, e.g., the fusosome comprises a transcriptional activator or repressor, a translational activator or repressor, or an epigenetic activator or repressor.

[0189] In some embodiments of the methods of making herein, providing a source cell that expresses a fusogen comprises expressing an exogenous fusogen in the source cell or up-regulating expression of an endogenous fusogen in the source cell. In some embodiments, the method comprises inactivating the nucleus of the source cell.

[0190] In embodiments, the fusosome composition comprises at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15fusosomes. In embodiments, the fusosome composition comprises at least 10 ml, 20 ml, 50 ml, 100 ml, 200 ml, 500 ml, 1 L, 2 L, 5 L, 10 L, 20 L, or 50 L. In embodiments, the method comprises enucleating mammalian cells, e.g., by chemical enucleation, use of mechanical force, e.g., use of a filter or centrifuge, at least partial disruption of the cytoskeleton, or a combination thereof. In embodiments, the method comprises expressing a fusogen or other membrane protein in the source cells. In embodiments, the method comprises one or more of vesicle formation, hypotonic treatment, extrusion, or centrifugation. In embodiments, the method comprises genetically expressing an exogenous factor in the cells or loading the cells or fusosomes with an exogenous factor. In embodiments, the method comprises contacting the cells (e.g., source cells) with DNA encoding a polypeptide agent prior to enucleating the cells (e.g., source cells), e.g., prior to inactivating the nucleus. In embodiments, the method includes contacting the cell with RNA encoding a polypeptide agent, e.g., before or after inactivating the nucleus, e.g., enucleating the cell. In embodiments, the method includes introducing a therapeutic agent (e.g., a nucleic acid or protein) into fusosomes, e.g., by electroporation.

[0191] In embodiments, the fusosomes are from mammalian cells with modified genomes, e.g., to reduce immunogenicity (e.g., by genome editing, e.g., to remove MHC proteins or MHC complexes). In embodiments, the source cells are from cell cultures treated with anti-inflammatory signals. In embodiments, the method further comprises contacting the source cells of step a) with an immunosuppressant or anti-inflammatory signal, e.g., before or after inactivating the nucleus, e.g., enucleating the cells.

[0192] In some embodiments, if a detectable level, eg, a value above a reference value, is determined, the sample containing a plurality of fusosomes or fusosome compositions is discarded.

[0193] In some embodiments, the first fusogen is not a lipopeptide.

[0194] In some embodiments of the method for assessing the fusosome content of target cells (e.g., fusion of fusosomes into target cells) that results in the formation of recipient cells in a subject, the method further comprises collecting a biological sample from the subject. In embodiments, the biological sample comprises one or more recipient cells.

[0195] In some embodiments of methods for assessing the fusosome content of target cells in a subject (e.g., fusion of fusosomes into target cells), the method comprises separating recipient cells in the biological sample from unfused fusosomes in the biological sample, e.g., by centrifugation. In some embodiments, the method further comprises enriching the recipient cells relative to unfused fusosomes in the biological sample, e.g., by centrifugation. In some embodiments, the method further comprises enriching the target cells relative to non-target cells in the biological sample, e.g., by FACS.

[0196] In some embodiments of the method for assessing the fusosome content of a target cell in a subject (e.g., fusion of fusosomes into a target cell), the activity related to the fusosome composition is selected from the presence or level of a metabolite, the presence or level of a biomarker (e.g., protein level or post-translational modification, e.g., phosphorylation or cleavage).

[0197] In some embodiments of the method for assessing the fusosome content of target cells in a subject (e.g., fusion of fusosomes to target cells), the activity associated with the fusosome composition is immunogenic. In embodiments, the target cells are CD3+ cells, and the biological sample is a blood sample taken from the subject. In embodiments, the blood cells are enriched from the blood sample, e.g., using a buffered ammonium chloride solution. In embodiments, the enriched blood cells are incubated with an anti-CD3 antibody (e.g., a mouse anti-CD3-FITC antibody), and CD3+ cells are selected, e.g., by fluorescence-activated cell sorting. In embodiments, the cells, e.g., the sorted cells, e.g., CD3+ cells, are analyzed for the presence of antibodies on the cell surface, e.g., by staining with an anti-IgM antibody. In some embodiments, if the antibodies are present at a level above a baseline level, the subject is identified as having an immune response against the recipient cells.

[0198] In embodiments, immunogenicity is assessed by a cytolytic assay. In embodiments, recipient cells from a biological sample are incubated with immune effector cells capable of lysing other cells. In embodiments, the immune effector cells are from the subject or a subject not administered the fusosome composition. For example, in embodiments, immunogenicity is assessed by a PBMC cytolytic assay. In embodiments, recipient cells from a biological sample are incubated with peripheral blood mononuclear cells (PBMCs) from the subject or control PBMCs from a subject not administered the fusosome composition, and then lysis of the recipient cells by the PBMCs is assessed. In embodiments, immunogenicity is assessed by a natural killer (NK) cytolytic assay. In embodiments, recipient cells are co-incubated with NK cells from the subject or control NK cells from a subject not administered the fusosome composition, and then lysis of the recipient cells by the NK cells is assessed. In embodiments, immunogenicity is assessed by a CD8+ T cell lytic assay. In embodiments, recipient cells are co-incubated with CD8+ T cells from the subject or control CD8+ T cells from a subject not administered the fusosome composition, and then assessed for lysis of target cells by the CD8+ T cells. In some embodiments, if cell lysis occurs at a level above a baseline level, the subject is identified as having an immune response to the recipient cells.

[0199] In some embodiments, immunogenicity is assayed by phagocytosis of recipient cells, e.g., macrophages. In embodiments, the recipient cells are not targeted by macrophages for phagocytosis. In embodiments, the biological sample is a blood sample collected from a subject. In embodiments, blood cells are enriched from the blood sample, e.g., using a buffered ammonium chloride solution. In embodiments, the enriched blood cells are incubated with an anti-CD3 antibody (e.g., a mouse anti-CD3-FITC antibody), and CD3+ cells are selected, e.g., by fluorescence-activated cell sorting. In embodiments, fluorescently labeled CD3+ cells are incubated with macrophages and then examined for intracellular fluorescence within the macrophages, e.g., by flow cytometry. In some embodiments, if macrophage phagocytosis occurs at a level above a baseline level, the subject is identified as having an immune response to the recipient cells.

[0200] In some embodiments, the methods described herein include measuring or determining the fusomal content of target cells, e.g., fusion of fusosomes by target cells (e.g., determining whether fusion has occurred), e.g., as described in Examples 54 or 124. In embodiments, a detectable marker may be present in the fusosomes (e.g., conjugated to a cargo or payload molecule in the fusosome). In embodiments in which the cargo or payload comprises a protein, the cargo or payload may be detected directly, e.g., using a binding moiety (e.g., an antibody or antigen-binding fragment thereof). In certain embodiments, the protein payload is associated with (e.g., conjugated to) a detectable moiety, e.g., a moiety that can be specifically bound by an antibody molecule. In embodiments in which the cargo or payload comprises a nucleic acid (e.g., DNA or mRNA), the cargo or payload may be detected using a nucleic acid probe that can hybridize to the nucleic acid or using a binding moiety (e.g., an antibody or antigen-binding fragment thereof) that can specifically bind to a polypeptide encoded by the nucleic acid. In embodiments, fusion of fusosomes to target cells is determined by detecting a detectable marker. In embodiments, fusion of fusosomes into target cells is determined by measuring expression of cargo or payload (e.g., a polypeptide or non-coding RNA encoded by the nucleic acid cargo or payload). In embodiments, fusion of fusosomes into target cells is determined by measuring a downstream marker of cargo or payload activity. In some embodiments, the target or recipient cells are isolated from a subject before measuring or determining the fusogen content of the target or recipient cells, e.g., fusion of fusosomes by the target cells. In embodiments, the target or recipient cells are also stained with an endosomal or lysosomal dye or antibody to determine whether the payload is present in endosomes or lysosomes.In some embodiments, the payload does not co-localize with endosomes or lysosomes, or less than 50%, 40%, 30%, 20%, 10%, 5%, 2%, or 1% of the payload co-localizes with endosomes or lysosomes. In embodiments, the recipient cells are also stained with a cytoplasmic, nuclear, mitochondrial, or plasma membrane dye or antibody to determine whether the payload co-localizes with the target compartment, e.g., the cytoplasm, nucleus, mitochondria, or plasma membrane; in such embodiments, the payload may localize with the nucleus, mitochondria, or plasma membrane.

[0201] In embodiments, the method for producing fusosomes herein comprises expressing (e.g., overexpressing) ARRDC1 or an active fragment or variant thereof in source cells. In embodiments, the method further comprises isolating fusosomes from source cells expressing ARRDC1. In embodiments, the method comprises isolating at least 1.2 x 10 fusosomes per mL. 11 , 1.4×10 11 , 1.6×10 11 , 1.8×10 11 , 2.0×10 11 , 2.2 × 10 11 , 2.4 × 10 11 , 2.6×10 11 , or 2.8 × 10 11 particles, e.g., up to about 3 x 10 per mL 11particles per mL. In some embodiments, the method yields about 1.2, 1.4, 1.6, 1.8, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times more particles per mL than the same method performed with other similar source cells that do not express or overexpress ARRDC1 or an active fragment or variant thereof. In some embodiments, fusosomes generated from source cells that, when contacted with target cells, contain ARRDC1 or an active fragment or variant thereof (e.g., overexpress) produce detectable cargo delivery in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times more cells than fusosomes generated from similar source cells that do not express or overexpress ARRDC1 or an active fragment or variant thereof, e.g., using a microscopy assay, e.g., the assay of Example 170.

[0202] Enumerated embodiments 1. A fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) a lumen containing the cytosol, the lumen being surrounded by a lipid bilayer; (c) exogenous or overexpressed fusogens positioned within the lipid bilayer; (d) cargo, including Fusosomes do not contain a nucleus the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein; The plurality of fusosomes, when contacted with a target cell population in the presence of an inhibitor of endocytosis, and when contacted with a reference target cell population that is not treated with an inhibitor of endocytosis, deliver cargo to at least 30% of the number of cells in the target cell population compared to the reference target cell population.

[0203] 2. The fusosome composition of embodiment 1, which delivers cargo to at least 40%, 50%, 60%, 70%, or 80% of the number of cells in a target cell population compared to a reference target cell population or a non-target cell population, or delivers at least 40%, 50%, 60%, 70%, or 80% of the cargo to a target cell population compared to a reference target cell population or a non-target cell population.

[0204] 3. The fusosome composition of embodiment 1 or 2, wherein less than 10% of the cargo enters the cell by endocytosis.

[0205] 4. The fusosome composition of any of embodiments 1 to 3, wherein the inhibitor of endocytosis is an inhibitor of lysosomal acidification, such as bafilomycin A1.

[0206] 5. The fusosome composition of any of embodiments 1 to 4, wherein the cargo delivered is determined using an endocytosis inhibition assay, for example, the assay of Example 90 or 135.

[0207] 6. The fusomal composition of any of embodiments 1 to 5, wherein the cargo enters the cell through a dynamin-independent pathway or a lysosomal acidification-independent pathway, a macropinocytosis-independent pathway (e.g., the inhibitor of endocytosis is an inhibitor of macropinocytosis, such as 5-(N-ethyl-N-isopropyl) amiloride (EIPA), e.g., at a concentration of 25 μM), or an actin-independent pathway (e.g., the inhibitor of endocytosis is an inhibitor of actin polymerization, such as latrunculin B, e.g., at a concentration of 6 μM).

[0208] 7. The fusosome composition of any of embodiments 1-6, wherein the plurality of fusosomes further comprises a targeting moiety.

[0209] 8. The fusosome composition of embodiment 7, wherein the targeting moiety is included in the fusogen or in a separate molecule.

[0210] 9. When multiple fusosomes contact a cell population that includes target and non-target cells, (i) the cargo is present in at least 10 times more target cells than non-target cells, or (ii) The fusosome composition of any of embodiments 1 to 8, wherein the cargo is present in at least 2-fold, 5-fold, 10-fold, 20-fold, or 50-fold more target cells than non-target cells and / or reference cells.

[0211] 10. The fusosome composition of any of embodiments 1-9, wherein the plurality of fusosomes fuses with target cells at a rate at least 50% higher than with non-target cells.

[0212] 11. A fusosome composition comprising a plurality of fusosomes derived from a source cell, wherein the plurality of fusosomes are (a) a lipid bilayer; (b) a lumen containing the cytosol, the lumen being surrounded by a lipid bilayer; (c) an exogenous or overexpressed retargeted fusogen placed within the lipid bilayer; and (d) cargo, including Fusosomes do not contain a nucleus the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein; (i) when a plurality of fusosomes contact a cell population comprising target cells and non-target cells, cargo is present in at least 10 times more target cells than non-target cells, or at least 10 times more cargo is delivered to the cell population than to a reference cell population; or (ii) A fusosome composition in which a plurality of fusosomes fuse with target cells at a rate at least 50% higher than non-target cells, or in which at least 50% more cargo is delivered to a cell population compared to a reference cell population.

[0213] 12. The fusosome composition of embodiment 11, wherein the presence of cargo is measured by microscopy, for example, using the assay of Example 124.

[0214] 13. The fusosome composition of embodiment 11, wherein fusion is measured by microscopy, for example, using the assay of Example 54.

[0215] 14. The fusosome composition of any of embodiments 7 to 13, wherein the targeting moiety is specific for a cell surface marker on the target cell.

[0216] 15. The fusosome composition of embodiment 14, wherein the cell surface marker is a cell surface marker for skin cells, cardiac muscle cells, liver cells, intestinal cells (e.g., cells of the small intestine), pancreatic cells, brain cells, prostate cells, lung cells, colon cells, or bone marrow cells.

[0217] 16. The fusosome composition of any of embodiments 11-15, wherein the fusogen (e.g., retargeted fusogen) comprises a Rhabdoviridae fusogen (e.g., VSV-G), a Filoviridae fusogen, an Arenaviridae fusogen, a Togaviridae fusogen, a Flaviviridae fusogen, a Bunyaviridae fusogen, or a Hepadnaviridae fusogen (e.g., Hep B), or a derivative thereof.

[0218] 17. When multiple fusosomes are contacted with a target cell population in the presence of an inhibitor of endocytosis and with a reference target cell population that has not been treated with an inhibitor of endocytosis, (i) delivering cargo to at least 30% as many cells in a target cell population as compared to a reference target cell population; (ii) delivers at least 30% of the cargo to the target cell population compared to a reference target cell population; or (iii) A fusosome composition according to any one of embodiments 7 to 16, which delivers at least 30% more cargo to a target cell population compared to a reference target cell population.

[0219] 18. The fusosome composition of any of embodiments 1-17, which, when contacted with a target cell population, delivers cargo to a target cell location other than an endosome or lysosome, for example, the cytosol.

[0220] 19. The fusosome composition of embodiment 18, wherein less than 50%, 40%, 30%, 20%, or 10% of the cargo is delivered to endosomes or lysosomes.

[0221] 20. The amount of viral capsid protein in the fusosome composition is determined using mass spectrometry, for example, using the assay of Example 53 or 161, and / or 20. The fusosome composition of any of embodiments 1-19, wherein the fusosome composition does not contain viral nucleocapsid protein or the amount of viral nucleocapsid protein is less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% of total protein, e.g., by mass spectrometry, e.g., using the assay of Example 53 or 161.

[0222] 21. The fusosome composition of any of embodiments 1-20, wherein the plurality of fusosomes comprises exosomes, microvesicles, or a combination thereof.

[0223] 22. The fusosome composition of any of embodiments 1-21, wherein the plurality of fusosomes have an average size of at least 50 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm.

[0224] 23. A fusosome composition according to any one of embodiments 1 to 21, wherein the plurality of fusosomes have an average size of less than 100 nm, 80 nm, 60 nm, 40 nm, or 30 nm.

[0225] 24. The fusosome composition of any of embodiments 1-23, wherein the source cells are selected from neutrophils, HEK293 cells, granulocytes, mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells, myeloblasts, myoblasts, hepatocytes, or neurons, e.g., retinal neuronal cells.

[0226] 25. A fusosome composition according to any one of embodiments 1 to 24, wherein the plurality of fusosomes comprises a cell biological agent.

[0227] 26. The fusosome composition of any of embodiments 1-25, wherein the plurality of fusosomes comprises enucleated cells.

[0228] 27. The fusosome composition of any of embodiments 1-26, wherein the fusogen (e.g., the retargeted fusogen) comprises a mammalian fusogen.

[0229] 28. The fusosome composition of any of embodiments 1-27, wherein the fusogen (e.g., the retargeted fusogen) comprises a viral fusogen.

[0230] 29. A fusosome composition according to any of embodiments 1 to 28, wherein the fusogen (e.g., the retargeted fusogen) is active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10.

[0231] 30. A fusosome composition described in any of embodiments 1 to 29, wherein the fusogen (e.g., a retargeted fusogen) is not active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10.

[0232] 31. The fusosome composition of any of embodiments 1-30, wherein the fusogen (e.g., the retargeted fusogen) is a protein fusogen.

[0233] 32. The fusosome composition of any of embodiments 1-31, wherein the fusogen (e.g., a retargeted fusogen) comprises Nipah virus protein F, measles virus F protein, Tupaia paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, Henipavirus F protein, Morbillivirus F protein, Respirovirus F protein, Sendai virus F protein, Rubulavirus F protein, or Avulavirus F protein, or a derivative thereof.

[0234] 33. A fusosome composition according to any of embodiments 1-32, wherein the fusogen is present in a copy number of at least 2, 5, or 10 copies per fusosome.

[0235] 34. The fusosome composition of any of embodiments 1-33, wherein the fusogen (e.g., a retargeted fusogen) comprises Nipah virus protein G, measles protein H, Tupaia paramyxovirus H protein, paramyxovirus G protein, paramyxovirus H protein, paramyxovirus HN protein, morbillivirus H protein, respirovirus HN protein, Sendai HN protein, rubulavirus HN protein, abulavirus HN protein, or a derivative thereof.

[0236] 35. The fusosome composition of any of embodiments 1-34, wherein the fusogen (e.g., retargeted fusogen) comprises a sequence selected from Nipah virus F and G proteins, measles virus F and H proteins, Tupaia paramyxovirus F and H proteins, paramyxovirus F and G proteins or F and H proteins or F and HN proteins, Hendra virus F and G proteins, Henipavirus F and G proteins, Morbillivirus F and H proteins, Respirovirus F and HN proteins, Sendai virus F and HN proteins, Rubulavirus F and HN proteins, or Avulavirus F and HN proteins, or derivatives thereof, or any combination thereof.

[0237] 36. The fusosome composition of any of embodiments 1-35, wherein the cargo comprises an exogenous protein or an exogenous nucleic acid.

[0238] 37. The fusosome composition of any of embodiments 1-36, wherein the cargo comprises or encodes a cytoplasmic or membrane protein.

[0239] 38. The fusosome composition of any of embodiments 1-37, wherein the cargo comprises a therapeutic agent.

[0240] 39. The fusosome composition of any of embodiments 1-38, wherein the cargo is present in a copy number of at least 1, 2, 5, 10, 20, 50, 100, or 200 copies per fusosome (e.g., up to about 1,000 copies per fusosome).

[0241] 40. The fusosome composition of any of embodiments 1 to 39, wherein the ratio of the copy number of the fusogen (e.g., the retargeted fusogen) to the copy number of the cargo is 1000:1 to 1:1, 500:1 to 1:1, 250:1 to 1:1, 150:1 to 1:1, 100:1 to 1:1, 75:1 to 1:1, 50:1 to 1:1, 25:1 to 1:1, 20:1 to 1:1, 15:1 to 1:1, 10:1 to 1:1, 5:1 to 1:1, 2:1 to 1:1, or 1:1 to 1:2.

[0242] 41. a) the fusosome composition has a fusogen to CD63 ratio of about 100-10,000, 500-5,000, 1000-5000, 2000-4000, 2500-3500, 2900-2930, 2910-2915, or 2912.0, e.g., by mass spectrometry assay; or b) the fusosome composition has a protein cargo to CD63 ratio of about 5-35, 10-30, 15-25, 16-19, 18-19, or 18.6; or c) the fusosome composition of any one of embodiments 1 to 40, wherein less than 15%, 20%, or 25% of the proteins in the fusosomes are exosomal proteins.

[0243] 42. a) the fusogen comprises about 1-30%, 5-20%, 10-15%, 12-15%, 13-14%, or 13.6% of the total protein in the fusosome, e.g., by mass spectrometry assay; b) the fusogen has a ratio to GAPDH of about 20-120, 40-100, 50-90, 60-80, 65-75, 68-70, or 69, for example, by mass spectrometry assay; c) the fusogen has a ratio to CNX of about 200-900, 300-800, 400-700, 500-600, 520-590, 530-580, 540-570, 550-560, or 558.4, for example, by mass spectrometry assay; d) The fusosome composition of any one of embodiments 1 to 41, wherein 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the proteins in the fusosomes are ribosomal proteins, or approximately 1% to 20%, 3% to 15%, 5% to 12.5%, 7.5% to 11%, 8.5% to 10.5%, or 9% to 10% of the proteins in the fusosomes are ribosomal proteins.

[0244] 43. The fusosome composition of any of embodiments 1-42, wherein the source cells express (e.g., overexpress) ARRDC1 or an active fragment or variant thereof.

[0245] 44. For example, by mass spectrometry assay, approximately 1-3, 1-10, 1-100, 3-10, 4-9, 5-8, 6-7, 15-100, 60-200, 80-180, 100-160, 120-140, 3-100, 4-100, 5-100, 6-100, 15-100, 80-100, 3-200, 4-200, 5-200, 6-200, 44. The fusosome composition of any of embodiments 1-43, having a fusogen to ARRDC1 ratio of 15-200, 80-200, 100-200, 120-200, 300-1000, 400-900, 500-800, 600-700, 640-690, 650-680, 660-670, 100-10,000, or about 664.9.

[0246] 45. The fusosome composition of any of embodiments 1 to 44, wherein the level of ARRDC1 as a percentage of total protein content is at least about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, or 25%, or the level of ARRDC1 as a percentage of total protein content is about 0.01-25%, 0.5-20%, 2-15%, or 5-10%.

[0247] 46. ​​A fusosome composition according to any of embodiments 1 to 45, having a fusogen to tsg101 ratio of about 1,000 to 10,000, 2,000 to 5,000, 3,000 to 4,000, 3,050 to 3,100, 3,060 to 3,070, or about 3,064, for example, using a mass spectrometry assay, e.g., the assay of Example 162.

[0248] 47. A fusosome composition described in any of embodiments 1 to 46, having a fusogen to tsg101 ratio of about 10 to 30, 15 to 25, 18 to 21, 19 to 20, or 19.5, for example, using a mass spectrometry assay, for example, the assay of Example 163.

[0249] 48. The fusosome composition of any of embodiments 1 to 47, wherein the level of TSG101 as a percentage of total protein content is at least about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, or 0.007%, or the level of TSG101 as a percentage of total protein content is about 0.001-0.01, 0.002-0.006, 0.003-0.005, or 0.004.

[0250] 49. e) meets pharmaceutical or Good Manufacturing Practice (GMP) standards; f) Made in accordance with Good Manufacturing Practices (GMP); g) have pathogen levels below a predetermined threshold, e.g., are substantially pathogen-free; or h) A fusosome composition described in any one of embodiments 1 to 48, having a contaminant level below a predetermined standard value, for example, being substantially free of contaminants.

[0251] 50. The fusosome composition of any one of embodiments 1 to 49, wherein the temperature is less than 4, 0, -4, -10, -12, -16, -20, -80, or -160°C.

[0252] 51. A pharmaceutical composition comprising the fusosome composition of any of the preceding embodiments and a pharmaceutically acceptable carrier.

[0253] 52. The pharmaceutical composition of embodiment 51, wherein the cargo comprises a therapeutic agent.

[0254] 53. A method for delivering a therapeutic agent to a subject, comprising administering to the subject a pharmaceutical composition described in embodiment 52, wherein the fusosome composition is administered in an amount and / or for a time such that the therapeutic agent is delivered.

[0255] 54. A method for producing a fusosome composition, comprising: a) providing a fusosome composition according to any one of embodiments 1 to 50; b) formulating the fusosomes as a pharmaceutical composition suitable for administration to a subject.

[0256] 55. A method for producing a fusosome composition, comprising: a) providing a fusosome composition according to any one of embodiments 1 to 50; b) assaying one or more fusosomes from the plurality to determine the presence or level of one or more of the following factors: (i) an immunogenic molecule, (ii) a pathogen, or (iii) a contaminant; c) approving the release of a plurality of fusosomes or fusosome compositions if one or more of the factors are below a reference value.

[0257] 56. A fusosome composition comprising a plurality of fusosomes derived from a source cell, wherein the plurality of fusosomes are (a) a lipid bilayer; (b) a lumen surrounded by a lipid bilayer; (c) an exogenous or overexpressed fusogen, the fusogen being disposed within the lipid bilayer; (d) cargo, including Fusosomes do not contain a nucleus, i) the fusogen is present in a copy number of at least 1,000 copies; ii) the fusosomes contain at least 1,000 copies of the therapeutic agent; iii) the fusosomes contain lipids in which one or more of CL, Cer, DAG, HexCer, LPA, LPC, LPE, LPG, LPI, LPS, PA, PC, PE, PG, PI, PS, CE, SM, and TAG are within 75% of the corresponding lipid levels in the source cells; iv) the fusosomes contain a proteome composition similar to that of the source cell; v) the fusosomes are capable of signaling, e.g., transmitting an extracellular signal, e.g., AKT phosphorylation in response to insulin, or uptake of glucose (e.g., labeled glucose, e.g., 2-NBDG) in response to insulin, e.g., at least 10% more than other similar fusosomes in the absence of a negative control, e.g., insulin; vi) when the fusosomes are targeted to a tissue, e.g., liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testis, ovary, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye, and administered to a subject, e.g., a mouse, at least 0.1% or 10% of the fusosomes in the population of administered fusosomes are present in the target tissue after 24 hours; or vii) The source cells are one or more (e.g., at least two, three, four, or five) selected from neutrophils, granulocytes, mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells, myeloblasts, myoblasts, hepatocytes, or neurons, e.g., retinal neuronal cells.

[0258] 57. The fusosome composition of embodiment 56, comprising a viral capsid protein or a DNA-incorporating polypeptide.

[0259] 58. The fusosome composition of embodiment 56, wherein the cargo comprises a viral genome.

[0260] 59. A fusosome composition according to embodiment 56, capable of delivering nucleic acids to target cells, for example for gene therapy, e.g., to stably modify the genome of the target cells.

[0261] Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0262] 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 in any table herein, refer to database entries as of May 8, 2017. When a gene or protein refers to multiple sequence accession numbers, all sequence variants are encompassed. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. In certain embodiments, for example, the following are provided: (Item 1) 1. A fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) a lumen containing a cytosol, the lumen being surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen disposed within the lipid bilayer; and (d) cargo, including the fusosome does not contain a nucleus, the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein; A fusosome composition, wherein the plurality of fusosomes, when contacted with a target cell population in the presence of an endocytosis inhibitor and when contacted with a reference target cell population that has not been treated with the endocytosis inhibitor, deliver the cargo to at least 30% more cells in the target cell population compared to the reference target cell population, or deliver at least 30% more of the cargo to the target cell population compared to the reference target cell population. (Item 2) 2. The fusosome composition of claim 1, wherein the cargo is delivered to at least 40%, 50%, 60%, 70%, or 80% more cells in the target cell population compared to the reference target cell population, or the fusosome composition delivers at least 40%, 50%, 60%, 70%, or 80% more cargo to the target cell population compared to the reference target cell population. (Item 3) 3. The fusosome composition of item 1 or 2, wherein less than 10% of the cargo enters the cell by endocytosis. (Item 4) 4. The fusosome composition according to any one of items 1 to 3, wherein the inhibitor of endocytosis is an inhibitor of lysosomal acidification, such as bafilomycin A1. (Item 5) 5. The fusosome composition of any one of items 1 to 4, wherein the delivered cargo is determined using an endocytosis inhibition assay, such as the assay of Example 135. (Item 6) 6. The fusosome composition of any one of items 1 to 5, wherein the cargo enters the cell through a dynamin-independent pathway or a lysosomal acidification-independent pathway, a macropinocytosis-independent pathway, or an actin-independent pathway. (Item 7) 7. The fusosome composition according to any one of items 1 to 6, wherein the plurality of fusosomes further comprises a targeting moiety. (Item 8) 8. The fusosome composition of claim 7, wherein the targeting moiety is contained in the fusogen or in a separate molecule. (Item 9) 9. The fusosome composition of any one of items 1 to 8, wherein when the plurality of fusosomes are contacted with a cell population comprising target cells and non-target cells, the cargo is present in at least 10 times more target cells than non-target cells, or at least 10 times more cargo is present in the target cell population compared to the reference target cell population. (Item 10) 10. The fusosome composition according to any one of items 1 to 9, wherein the plurality of fusosomes fuses with target cells at a rate at least 50% higher than non-target cells or reference cells. (Item 11) 1. A fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) a lumen comprising a cytosol, said lumen being surrounded by a lipid bilayer; (c) an exogenous or overexpressed retargeted fusogen disposed within the lipid bilayer; and (d) cargo, including the fusosome does not contain a nucleus, the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein; (i) when the plurality of fusosomes contacts a cell population comprising target cells and non-target cells, the cargo is present in at least 10 times more target cells than non-target cells or reference cells; or (ii) a fusosome composition, wherein said plurality of fusosomes fuse with target cells at a rate at least 50% higher than non-target cells or reference cells. (Item 12) 12. The fusosome composition of claim 11, wherein the presence of the cargo is determined by microscopy, for example, using the assay of Example 124. (Item 13) 12. The fusosome composition of claim 11, wherein fusion is measured by microscopy, for example, using the assay of Example 54. (Item 14) 14. The fusosome composition according to any one of items 7 to 13, wherein the targeting moiety is specific for a cell surface marker on the target cell. (Item 15) 15. The fusosome composition of item 14, wherein the cell surface marker is a cell surface marker for skin cells, cardiac muscle cells, liver cells, intestinal cells (e.g., cells of the small intestine), pancreatic cells, brain cells, prostate cells, lung cells, colon cells, or bone marrow cells. (Item 16) 16. The fusosome composition of any one of items 11 to 15, wherein the fusogen (e.g., retargeted fusogen) comprises a Rhabdoviridae fusogen (e.g., VSV-G), a Filoviridae fusogen, an Arenaviridae fusogen, a Togaviridae fusogen, a Flaviviridae fusogen, a Bunyaviridae fusogen, or a Hepadnaviridae fusogen (e.g., Hep B), or a derivative thereof. (Item 17) 17. The fusosome composition of any one of Items 7 to 16, wherein the plurality of fusosomes, when contacted with a target cell population in the presence of an endocytosis inhibitor and when contacted with a reference target cell population that has not been treated with the endocytosis inhibitor, deliver the cargo to at least 30% of the number of cells in the target cell population compared to the reference target cell population. (Item 18) 18. The fusosome composition of any one of items 1 to 17, which, when contacted with a target cell population, delivers cargo to a target cell location other than an endosome or lysosome, for example, the cytosol. (Item 19) 19. The fusosome composition of claim 18, wherein less than 50%, 40%, 30%, 20%, or 10% of the cargo is delivered to endosomes or lysosomes. (Item 20) 20. The fusosome composition of any one of items 1 to 19, wherein the amount of viral capsid protein in the fusosome composition is determined using mass spectrometry, for example, using the assay of Example 53. (Item 21) 21. The fusosome composition of any one of items 1 to 20, wherein the plurality of fusosomes comprises exosomes, microvesicles, or a combination thereof. (Item 22) 22. The fusosome composition of any one of items 1 to 21, wherein the plurality of fusosomes have an average size of at least 50 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm. (Item 23) 24. The fusosome composition according to any one of claims 1 to 21, wherein the plurality of fusosomes have an average size of less than 100 nm, 80 nm, 60 nm, 40 nm, or 30 nm. 24. The fusosome composition according to any one of items 1 to 23, wherein the source cells are selected from neutrophils, HEK293 cells, granulocytes, mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells, myeloblasts, myoblasts, hepatocytes, or neurons, e.g., retinal neuronal cells. (Item 25) 25. The fusosome composition according to any one of items 1 to 24, wherein the plurality of fusosomes comprises a cytobiological agent. (Item 26) 26. The fusosome composition according to any one of items 1 to 25, wherein the plurality of fusosomes comprises enucleated cells. (Item 27) 27. The fusosome composition of any one of items 1 to 26, wherein the fusogen (e.g., a retargeted fusogen) comprises a mammalian fusogen. (Item 28) 28. The fusosome composition of any one of items 1 to 27, wherein the fusogen (e.g., retargeted fusogen) comprises a viral fusogen. (Item 29) 29. The fusosome composition of any one of items 1 to 28, wherein the fusogen (e.g., retargeted fusogen) is active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. (Item 30) 30. The fusosome composition of any one of items 1 to 29, wherein the fusogen (e.g., retargeted fusogen) is not active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. (Item 31) 31. The fusosome composition of any one of items 1 to 30, wherein the fusogen (e.g., a retargeted fusogen) is a protein fusogen. (Item 32) 32. The fusosome composition of any one of items 1 to 31, wherein the fusogen (e.g., retargeted fusogen) comprises Nipah virus protein F, measles virus F protein, Tupaia paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, Henipavirus F protein, Morbillivirus F protein, Respirovirus F protein, Sendai virus F protein, Rubulavirus F protein, or Avulavirus F protein, or a derivative thereof. (Item 33) 33. The fusosome composition according to any one of items 1 to 32, wherein the fusogen is present in a copy number of at least 10 copies per fusosome. (Item 34) 34. The fusosome composition according to any one of items 1 to 33, wherein the plurality of fusosomes further comprise Nipah virus protein G, measles protein H, Tupaia paramyxovirus H protein, paramyxovirus G protein, paramyxovirus H protein, paramyxovirus HN protein, morbillivirus H protein, respirovirus HN protein, Sendai HN protein, rubulavirus HN protein, abulavirus HN protein, or a derivative thereof. (Item 35) 35. The fusosome composition of any one of items 1 to 34, wherein the fusogen (e.g., retargeted fusogen) comprises a sequence selected from Nipah virus F and G proteins, measles virus F and H proteins, Tupaia paramyxovirus F and H proteins, paramyxovirus F and G proteins or F and H proteins or F and HN proteins, Hendra virus F and G proteins, Henipavirus F and G proteins, Morbillivirus F and H proteins, Respirovirus F and HN proteins, Sendai virus F and HN proteins, Rubulavirus F and HN proteins, or Avulavirus F and HN proteins, or derivatives thereof, or any combination thereof. (Item 36) 36. The fusosome composition according to any one of items 1 to 35, wherein the cargo comprises an exogenous protein or an exogenous nucleic acid. (Item 37) 37. The fusosome composition of any one of items 1 to 36, wherein the cargo comprises or encodes a cytoplasmic or membrane protein. (Item 38) 38. The fusosome composition according to any one of items 1 to 37, wherein the cargo comprises a therapeutic agent. (Item 39) 39. The fusosome composition of any one of items 1 to 38, wherein the cargo is present in a copy number of at least 1, 2, 5, 10, 20, 50, 100, or 200 copies per fusosome (e.g., up to about 1,000 copies per fusosome). (Item 40) 40. The fusosome composition of any one of items 1 to 39, wherein the ratio of the copy number of the fusogen (e.g., retargeted fusogen) to the copy number of the cargo is 1000:1 to 1:1, 500:1 to 1:1, 250:1 to 1:1, 150:1 to 1:1, 100:1 to 1:1, 75:1 to 1:1, 50:1 to 1:1, 25:1 to 1:1, 20:1 to 1:1, 15:1 to 1:1, 10:1 to 1:1, 5:1 to 1:1, 2:1 to 1:1, or 1:1 to 1:2. (Item 41) a) the fusosome composition has a fusogen to CD63 ratio of about 100-10,000, 500-5,000, 1000-5000, 2000-4000, 2500-3500, 2900-2930, 2910-2915, or 2912.0, e.g., by mass spectrometry assay; or b) the fusosome composition has a protein cargo to CD63 ratio of about 5-35, 10-30, 15-25, 16-19, 18-19, or 18.6; or c) less than 15%, 20%, or 25% of the proteins in the fusosomes are exosomal proteins. (Item 42) a) the fusogen comprises about 1-30%, 5-20%, 10-15%, 12-15%, 13-14%, or 13.6% of the total protein in the fusosome, e.g., by mass spectrometry assay; b) the fusogen has a ratio to GAPDH of about 20-120, 40-100, 50-90, 60-80, 65-75, 68-70, or 69, for example, by mass spectrometry assay; c) the fusogen has a ratio to CNX of about 200-900, 300-800, 400-700, 500-600, 520-590, 530-580, 540-570, 550-560, or 558.4, for example, by mass spectrometry assay; d) The fusosome composition according to any one of Items 1 to 41, wherein 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the proteins in the fusosomes are ribosomal proteins, or approximately 1% to 20%, 3% to 15%, 5% to 12.5%, 7.5% to 11%, 8.5% to 10.5%, or 9% to 10% of the proteins in the fusosomes are ribosomal proteins. (Item 43) a) meets pharmaceutical or Good Manufacturing Practice (GMP) standards; b) Made in accordance with Good Manufacturing Practices (GMP); c) have pathogen levels below a predetermined threshold, e.g., are substantially pathogen-free; or d) A fusosome composition according to any one of items 1 to 42, having a contaminant level below a predetermined standard value, e.g., being substantially free of contaminants. (Item 44) 44. The fusosome composition according to any one of items 1 to 43, wherein the temperature is less than 4, 0, -4, -10, -12, -16, -20, -80, or -160°C. (Item 45) A pharmaceutical composition comprising the fusosome composition of any of the preceding items and a pharmaceutically acceptable carrier. (Item 46) 46. ​​The pharmaceutical composition of item 45, wherein the cargo comprises a therapeutic agent. (Item 47) 47. A method for delivering a therapeutic agent to a subject, comprising administering to the subject the pharmaceutical composition of claim 46, wherein the fusosome composition is administered in an amount and / or for a time such that the therapeutic agent is delivered. (Item 48) 1. A method of producing a fusosome composition, comprising: a) providing a fusosome composition according to any one of items 1 to 44; b) formulating the fusosomes as a pharmaceutical composition suitable for administration to a subject. (Item 49) 1. A method of producing a fusosome composition, comprising: a) providing a fusosome composition according to any one of items 1 to 44; b) assaying one or more fusosomes from said plurality to determine the presence or level of one or more of the following factors: (i) an immunogenic molecule, (ii) a pathogen, or (iii) a contaminant; c) approving release of the plurality of fusosomes or fusosome composition if one or more of the factors is below a reference value. (Item 50) 1. A fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) a lumen surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen, the fusogen being disposed within the lipid bilayer; (d) cargo, including the fusosome does not contain a nucleus, viii) the fusogen is present in a copy number of at least 1,000 copies; ix) the fusosomes contain the therapeutic agent in a copy number of at least 1,000 copies; x) the fusosomes comprise lipids in which one or more of CL, Cer, DAG, HexCer, LPA, LPC, LPE, LPG, LPI, LPS, PA, PC, PE, PG, PI, PS, CE, SM, and TAG are within 75% of the levels of the corresponding lipids in the source cells; xi) the fusosomes comprise a proteome composition similar to that of the source cell; xii) the fusosomes are capable of signaling, e.g., transmitting an extracellular signal, e.g., AKT phosphorylation in response to insulin, or uptake of glucose (e.g., labeled glucose, e.g., 2-NBDG) in response to insulin, e.g., at least 10% more than other similar fusosomes in the absence of a negative control, e.g., insulin; xiii) the fusosomes are targeted to a tissue, e.g., liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testis, ovary, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye, and when administered to a subject, e.g., a mouse, at least 0.1% or 10% of the fusosomes in the population of administered fusosomes are present in the target tissue after 24 hours; or xiv) The fusosome composition, wherein the source cells are one or more (e.g., at least two, three, four, or five) selected from neutrophils, granulocytes, mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells, myeloblasts, myoblasts, hepatocytes, or neurons, e.g., retinal neuronal cells. (Item 51) 51. The fusosome composition of item 50, comprising a viral capsid protein or a DNA-incorporating polypeptide. (Item 52) 51. The fusosome composition of claim 50, wherein the cargo comprises a viral genome. (Item 53) 51. The fusosome composition of item 50, capable of delivering nucleic acids to target cells, for example for gene therapy, e.g., to stably modify the genome of the target cells. [Brief explanation of the drawings]

[0263] The following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings specific embodiments presently illustrated. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0264] [Figure 1] The staining of fusosomes with the endoplasmic reticulum dye is quantified. [Figure 2] Staining of fusosomes with a mitochondrial dye is quantified. [Figure 3] The staining of fusosomes with the lysosomal dye is quantified. [Figure 4] Quantify the staining of fusosomes with a dye for F-actin. [Figure 5] 1 is a graph showing recovery of GFP fluorescence after photobleaching of cells contacted with fusogens expressing Cre and GFP. [Figure 6] Graph showing the percentage of target cells expressing RFP after contact with fusosomes or a negative control. [Figure 7] This image shows positive organelle delivery via fusion between donor and recipient HeLa cells. The white intracellular regions indicate overlap between donor and recipient mitochondria. The gray intracellular regions indicate areas where donor and recipient organelles do not overlap. [Figure 8] This image shows positive organelle delivery via fusion between donor and recipient HeLa cells. The white intracellular regions indicate overlap between donor and recipient mitochondria. The gray intracellular regions indicate areas where donor and recipient organelles do not overlap. [Figure 9] Figure 1 shows microscopic images of indicated tissues from mice injected with fusosomes. White represents RFP fluorescent cells, demonstrating delivery of protein cargo to cells in vivo. [Figure 10] 10 is a series of images showing successful delivery of fusosomes to mouse tissues in vivo by the indicated route of administration, resulting in luciferase expression by the targeted cells. [Figure 11] Shown are microscopic images of tdTomato fluorescence in mouse muscle tissue, demonstrating cell biology-driven delivery of protein cargo into muscle cells. [Figure 12] 1 is a graph showing the delivery of mitochondria into recipient HeLa Rho0 cells using protein-enhanced, enucleated VSV-G HeLa cells. [Figure 13] 1 is a series of images showing the generation and isolation of giant plasma membrane fusosomes. [Figure 14A]1 is a graph showing RFP expression in HEK293T cells generated by incubation with fusosomes carrying Cre recombinase and extrusion through membranes with pores of various sizes, as indicated. [Figure 14B] 1 is a series of graphs showing Eu:488 positive events (left panel) and median fluorescence intensity (MFI; right panel) of AF488 in parental cells and fusosomes. [Figure 14C] 10 is a series of graphs showing Edu:647 positive events and median fluorescence intensity of AF647 in parental cells and fusosomes. [Figure 14D] 1 is a graph showing the capacity of fusosomes and parental cells to polymerase actin over 3, 5, and 24 hours. [Figure 15] 1 is an electron microscope image showing fusosomes with a lipid bilayer structure. [Figure 16] 1 shows detection of VSV-G expression by Western blot. "+ control" represents 293T cells transfected with VSV-G. "- control" represents non-transfected 293T cells. [Figure 17A] 1 is a table showing measurement parameters and settings for submicron fusosomes. [Figure 17B] 1 is a table showing the measurement parameters and settings for ultramicron fusosomes. [Figure 17C] 1 is a series of graphs showing the size distribution of fusosomes and parent cells as measured by NTA and microscopy. [Figure 17D] 1 is a table showing the average diameter of fusosomes and parent cells as measured by NTA and microscopy. [Figure 18] 1 is a table showing size distribution statistics of fusosomes and parent cells as measured by NTA and microscopy. [Figure 19] 1 is a table showing the average size and volume of fusosomes and parent cells. [Figure 20]A series of graphs showing the detection of organelles within fusosomes: (A) endoplasmic reticulum, (B) mitochondria, (C) lysosomes. [Figure 21] FIG. 1 is a series of figures showing the soluble:insoluble ratios observed in fusosome or cell preparations. [Figure 22] FIG. 1 is a series of figures showing MvH(CD8)+F fusosome fusion to target or non-target cells, and the absolute amount of target fusion. [Figure 23] FIG. 1 shows the expression of hOx40L in PC3 cells treated with fusosomes. [Figure 24] FIG. 1 shows the mean fluorescence intensity of 2-NBDG in VSV-G fusosomes. [Figure 25] FIG. 1 shows esterase activity in the cytosol of VSV-G fusosomes. [Figure 26A] Figure 1 shows a series of figures showing the persistence of firefly luciferase signal in tissues from fusosome-injected mice. (A) Abdominal images and luminescence signals from fusosome-treated (right leg) and PBS-treated (left leg) FVB mice. The left side shows an overlay of images and luminescence signals, while the right side shows luminescence signals alone. (B) Total flux signals from fusosome-treated TA (black squares), PBS-treated TA (white circles), mouse background (black hexagons), and stage background (white hexagons). The y-scale is a log10 scale. The fusosome-treated leg had significantly greater signals 1 hour (p<0.0001), 6 hours (p<0.01), and 12 hours (p<0.01) after treatment. [Figure 26B] Same as above. [Figure 27A]Figure 1 shows a series of figures demonstrating fusosome-mediated Cre recombinase delivery in mice, as detected by bioluminescence imaging. (A) Ventral images and luminescence signal overlays of the exposed liver and spleen of mice treated with IV fusosomes (1x and 3x concentrations). The bottom shows the luminescence signal only. (B) Total flux signal of fusosome-targeted spleen and liver; y-scale is a log10 scale. Mice treated with 3x concentration fusosomes had significantly greater signal in the spleen than background 72 hours after treatment (p=0.0004). [Figure 27B] Same as above. [Figure 28A] A series of figures showing Cre recombinase delivery to mouse liver and spleen via fusosomes as detected by bioluminescence imaging. (A) From left to right: Dorsal images and luminescence signal overlays of excised liver, heart, lung, kidney, small intestine, pancreas, and spleen collected and imaged within 5 minutes of sacrifice. The bottom image shows luminescence signal only. (B) Total flux signal of fusosome-targeted spleen and liver and other tissues; y-scale is log10. Mice treated with 3x higher fusosome concentration had significantly greater signal in the spleen compared to the tissue with the lowest signal (heart) (p<0.0001). [Figure 28B] Same as above. [Figure 29] FIG. 10 is a table showing delivery of Cre cargo by NivG+F fusosomes via a non-endocytic pathway. [Figure 30] 1 is a series of images showing delivery of Cre cargo by VSV-G fusosomes via the endocytic pathway. [Figure 31] 1 is a graph showing the delivery of functional mitochondria into recipient HeLa Rho0 cells using Syn1 HeLa cell fusosomes. [Figure 32] 1 is a series of images showing in vitro delivery of DNA to recipient cells via fusosomes. [Figure 33] 1 is a series of images showing in vitro delivery of mRNA to recipient cells via fusosomes. [Figure 34A] Figure 1 shows a series of figures demonstrating the in vivo delivery of mRNA encoding firefly luciferase into mouse tissues using fusosomes. (A) Abdominal images and luminescence signals from fusosome-treated (right leg) and PBS-treated (left leg) FVB mice. The left side shows an overlay of images and luminescence signals, while the right side shows luminescence signals alone. (B) Total flux signals from fusosome-treated TA (black squares), PBS-treated TA (white circles), mouse background (black hexagons), and stage background (white hexagons). The y-scale is a log10 scale. The fusosome-treated leg had significantly greater signals 1 hour (p<0.0001), 6 hours (p<0.01), and 12 hours (p<0.01) after treatment. [Figure 34B] Same as above. [Figure 35] 1 is a series of images showing in vitro delivery of proteins to recipient cells via fusosomes. [Figure 36A] Figure 1 shows a series of figures demonstrating in vivo delivery of Cre recombinase protein into mouse tissues using fusosomes. (A) From left to right: luminescence signal of treated TA exposed to the abdomen and mouse image, and luminescence signal alone. (B) Total flux of treated vs. untreated leg, background (mouse chest), and stage background; y-scale is log10. [Figure 36B] Same as above. [Figure 37] FIG. 1 is a series of figures showing delivery of miRFP670 DNA to recipient cells via sonication-loaded fusosomes. [Figure 38] FIG. 1 is a series of figures showing delivery of BSA-AF647 protein to recipient cells via sonication-loaded fusosomes. [Figure 39] 1 is a histogram showing the size distribution and concentration of fusosome ghosts. [Figure 40] 1 is a series of graphs showing the median fluorescence intensity of Edu:647 positive events and AF647 in parental cells and fusosomes. [Figure 41] 1 is a graph showing the ratio of GAPDH:total protein measured by bicinchoninic acid assay in fusosomes and parental cells. [Figure 42] FIG. 1 is a graph showing lipid:protein ratios measured by bicinchoninic acid assay in fusosomes and parental cells. [Figure 43] 1 is a graph showing the protein:DNA ratio measured by bicinchoninic acid assay in fusosomes and parental cells. [Figure 44] 1 is a graph showing the lipid:DNA ratio measured by bicinchoninic acid assay in fusosomes and parental cells. [Figure 45] 1 is a series of images showing delivery of Cre into cells by VSV-G fusosomes in the presence or absence of the dynamin inhibitor Dynasore. [Figure 46] 1 is a graph showing protein levels of the exosomal marker CD63 in exosomes and fusosomes. [Figure 47] FIG. 1 is a graph showing the intensity of the calnexin signal detected in fusosomes and parental cells. [Figure 48] 1 is a graph showing the lipid:DNA ratio determined for fusosomes and parental cells. [Figure 49A] 1 is a series of graphs showing the ratio of lipid species as a percentage of total lipid in parental cells, exosomes, and fusosomes. [Figure 49B] Same as above. [Figure 50] FIG. 1 is a series of graphs showing the protein content of parental cells, exosomes, and fusosomes with respect to proteins associated with specific compartments, as indicated. [Figure 51] FIG. 1 is a series of graphs showing the levels of ARRDC1 (left panel) or TSG101 (right panel) as a percentage of the total protein content in parental cells, exosomes, and fusosomes. [Figure 52A]A series of graphs showing the effect of incorporating arrestin domain-containing protein 1 (ARRDC1) into the production of Cre-loaded fusosomes. (A) Percentage of RFP-positive cells detected after incubation with fusosomes generated in the presence or absence of ARRDC1. (B) Number of particles per mL detected using nanoparticle tracking analysis (fNTA) on fusosomes generated in the presence or absence of ARRDC1. [Figure 52B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0265] The present invention describes naturally occurring or engineered bilipid membranes containing fusogens.

[0266] definition As used herein, "cell membrane" refers to a membrane derived from a cell, e.g., a source cell or a target cell.

[0267] As used herein, a "chondrisome" is an intracellular device derived and isolated or purified from a mitochondrial network of natural cellular or tissue origin. A "chondrisome preparation" has biological activity (capable of interacting with or affecting a cell or tissue) and / or pharmaceutical activity.

[0268] As used herein, "biological preparation" refers to a portion of a cell, including the lumen and cell membrane, or a cell with partial or complete nuclear inactivation. In some embodiments, the biological preparation includes one or more of cytoskeletal components, organelles, and ribosomes. In some embodiments, the biological preparation is an enucleated cell, a microvesicle, or a cell ghost.

[0269] As used herein, "cytosol" refers to the aqueous component of the cytoplasm of a cell. The cytosol may contain proteins, RNA, metabolites, and ions.

[0270] As used herein, "exogenous agent" refers to an agent that i) does not occur in nature, such as a protein that has an altered sequence compared to an endogenous protein (e.g., by insertion, deletion, or substitution), or ii) does not occur naturally in the naturally occurring source cell of the fusosome in which the exogenous material is located.

[0271] As used herein, "fusion" refers to creating an interaction between two membrane-bound lumens, e.g., promoting the fusion of two membranes, or creating a connection, e.g., a pore, between two lumens.

[0272] As used herein, "fusogen" refers to an agent or molecule that creates an interaction between two membrane-bound lumens. In embodiments, a fusogen promotes membrane fusion. In other embodiments, a fusogen creates a connection, e.g., a pore, between two lumens (e.g., the lumen of a fusosome and the cytoplasm of a target cell). In some embodiments, a fusogen comprises a complex of two or more proteins, e.g., where neither protein has fusogenic activity alone. In some embodiments, a fusogen comprises a targeting domain.

[0273] As used herein, a "fusogen-binding partner" refers to an agent or molecule that interacts with a fusogen to promote fusion between two membranes. In some embodiments, a fusogen-binding partner may be or may include a surface feature of a cell.

[0274] As used herein, "fusosome" refers to a membrane-encapsulated preparation and fusogens that interact with an amphiphilic lipid bilayer.

[0275] As used herein, a "fusosome composition" refers to a composition comprising one or more fusosomes.

[0276] As used herein, "membrane-encapsulated preparation" refers to an amphipathic lipid bilayer surrounding a cargo within a lumen or cavity. In some embodiments, the cargo is exogenous to the lumen or cavity. In other embodiments, the cargo is endogenous to the lumen or cavity, e.g., endogenous to the source cell.

[0277] As used herein, "mitochondrial biogenesis" refers to the process of increasing mitochondrial biomass. Mitochondrial biogenesis includes increasing the number and / or size of mitochondria in a cell.

[0278] As used herein, the term "purified" means altered or removed from its natural state. For example, a cell or cell fragment naturally present in a living animal is not "purified," but the same cell or cell fragment partially or completely separated from the coexisting materials of its natural state is "purified." A purified fusosome composition can exist in substantially pure form, or can exist in a non-native environment, such as, for example, a culture medium containing cells.

[0279] 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 mutations outside the targeting moiety, e.g., in the transmembrane domain, fusogenic domain, or cytoplasmic domain, compared to the naturally occurring form of the fusogen.

[0280] As used herein, "source cell" (used interchangeably with "parent cell") refers to the cell from which the fusosomes are derived.

[0281] fusosomes In some embodiments, the fusosome compositions and methods described herein include membrane-encapsulated preparations, e.g., naturally occurring or engineered lipid membranes, that contain fusogens. In some embodiments, the present disclosure provides portions of non-plant cells, e.g., mammalian cells, or derivatives thereof (e.g., mitochondria, chondrisomes, organelles, vesicles, or enucleated cells), that contain fusogens, e.g., proteins, lipids, and chemical fusogens.

[0282] Encapsulation Some embodiments of the compositions and methods described herein include fusosomes, e.g., bilayers of naturally occurring or engineered amphiphilic lipids bearing fusogens. Such compositions surprisingly find use in the methods of the invention. In some instances, the membrane regulates the function of Ahmad et al. 2014 Miro1. intercellular mitochondrial transport & Enhances mesenchymal stem cell rescue efficacy. EMBO Journal. 33(9):994-1010. The composition can take the form of autologous, allogeneic, xenogeneic, or engineered cells. In some embodiments, the composition comprises an engineered membrane, for example, as described in Orive et al. 2015. Cell encapsulation: technical and clinical advances. Trends in Pharmacology Sciences; 36(8):537-46, and Mishra. 2016. Handbook of Encapsulation and Controlled Release. CRC Press. In some embodiments, the composition comprises a naturally occurring membrane (McBride et al. 2012. A Vesicular Transport Pathway Shuttles Cargo from Mitochondria to Lysosomes. Current Biology 22:135-141).

[0283] In some embodiments, the compositions described herein comprise naturally occurring membranes, e.g., membrane vesicles prepared from cells or tissues. In one embodiment, the fusosomes are vesicles from MSCs or astrocytes.

[0284] In one embodiment, the fusosomes are exosomes.

[0285] Exemplary exosomes and other membrane inclusions are described, for example, in US2016137716, which is incorporated herein by reference in its entirety. In some embodiments, fusosomes include, for example, vesicles obtained from cells, such as microvesicles, exosomes, apoptotic bodies (from apoptotic cells), microparticles (which may be derived, for example, from platelets), ectosomes (which may be derived, for example, from neutrophils and monocytes in serum), prostatosomes (which may be obtained from prostate cancer cells), cardiosomes (which may be derived from cardiac cells), and the like.

[0286] Exemplary exosomes and other membrane inclusions are also described in WO / 2017 / 161010, WO / 2016 / 077639, US20160168572, US20150290343, and US20070298118, each of which is incorporated by reference in its entirety. In some embodiments, the fusosomes comprise extracellular vesicles, nanovesicles, or exosomes. In embodiments, the fusosomes comprise extracellular vesicles, such as cell-derived vesicles that comprise a membrane surrounding an interior space and having a smaller diameter than the cell from which they originate. In embodiments, the extracellular vesicles have a diameter of 20 nm to 1000 nm. In embodiments, fusosomes include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation, vesiculated organelles, and vesicles generated by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). In embodiments, extracellular vesicles are derived from living or dead organisms, explanted tissues or organs, or cultured cells. In embodiments, fusosomes include nanovesicles, e.g., small (e.g., 20-250 nm diameter, or 30-150 nm diameter) vesicles derived from cells containing a membrane surrounding an internal space, which are generated from the vesicles by direct or indirect manipulation. Generation of nanovesicles can, in some cases, cause destruction of the source cell. Nanovesicles may contain lipids or fatty acids and polypeptides. In embodiments, fusosomes include exosomes. In embodiments, exosomes are small (e.g., 20-300 nm in diameter, or 40-200 nm in diameter) vesicles derived from cells that contain a membrane surrounding an internal space and are generated from the cells by direct plasma membrane budding or fusion of late endosomes with the plasma membrane. In embodiments, the generation of exosomes does not result in the destruction of the source cell. In embodiments, exosomes contain lipids or fatty acids and polypeptides.

[0287] Exemplary exosomes and other membrane inclusions are also described in US20160354313, which is incorporated herein by reference in its entirety. In embodiments, the fusosome comprises a biocompatible delivery module, an exosome (e.g., about 30 nm to about 200 nm in diameter), a microvesicle (e.g., about 100 nm to about 2000 nm in diameter), an apoptotic body (e.g., about 300 nm to about 2000 nm in diameter), a membrane particle, a membrane vesicle, an exosome-like vesicle, an ectosome-like vesicle, an ectosome, or an exovesicle.

[0288] In one embodiment, the fusosomes are microvesicles. In some embodiments, the microvesicles are intracellular or extracellular vesicles between about 10 and 10,000 nm in diameter. In some embodiments, the microvesicles are naturally released from cells, and in some embodiments, the cells are treated to promote vesicle formation. In one embodiment, the fusosomes are exosomes. In some cases, the exosomes are about 30 to 100 nm in diameter. In some embodiments, the exosomes are generated from multivesicular bodies. In some embodiments, the cells are treated to promote exosome formation. In one embodiment, the fusosomes are cell ghosts. In one embodiment, the vesicles are plasma membrane vesicles, for example, giant plasma membrane vesicles.

[0289] Fusosomes can be made from several different types of lipids, for example, amphipathic lipids such as phospholipids. Fusosomes can comprise a lipid bilayer as the outermost surface. This bilayer can be composed of one or more lipids of the same or different types. Examples include, but are not limited to, phospholipids such as phosphocholine and phosphoinositol. Specific examples include, but are not limited to, DMPC, DOPC, and DSPC.

[0290] Fusosomes can be composed primarily of natural phospholipids and lipids such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, and monosialogangliosides. In embodiments, fusosomes contain only phospholipids and have low stability in plasma. However, manipulation of the lipid membrane with cholesterol can, in embodiments, increase stability and reduce the rapid release of encapsulated bioactive compounds into plasma. In some embodiments, fusosomes contain 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), for example, to increase stability (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679 for a review).

[0291] In some embodiments, fusosomes contain or are enriched in lipids that affect membrane curvature (see, e.g., Thiam et al., Nature Reviews (See Molecular Cell Biology, 14(12):775-785, 2013). Some lipids have small hydrophilic heads and large hydrophobic tails, which promote fusion pore formation by concentrating in localized regions. In some embodiments, fusosomes contain or are enriched in lipids with negative curvature, such as cholesterol, phosphatidylethanolamine (PE), diglycerides (DAG), phosphatidic acid (PA), and fatty acids (FA). In some embodiments, fusosomes are free of, depleted in, or largely depleted in lipids with positive curvature, such as lysophosphatidylcholine (LPC), phosphatidylinositol (Ptdlns), lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), and monoacylglycerol (MAG).

[0292] In some embodiments, lipids are added to fusosomes. In some embodiments, lipids are added to source cells in culture, which incorporate lipids into their membranes before or during fusosome formation. In some embodiments, lipids are added to cells or fusosomes in the form of liposomes. In some embodiments, methyl-beta cyclodextran (mβ-CD) is used to concentrate or deplete lipids (see, e.g., Kainu et al., Journal of Lipid Research, 51(12):3533-3541, 2010).

[0293] Fusosomes may contain, but are not limited to, DOPE (dioleoylphosphatidylethanolamine), DOTMA, DOTAP, DOTIM, DDAB, either alone or together with cholesterol, such as DOPE and cholesterol, DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol.Methods for the preparation of multilamellar vesicle lipids are known in the art (see, e.g., U.S. Patent No. 6,693,086, the teachings of which regarding the preparation of multilamellar vesicle lipids are incorporated herein by reference). When lipid membrane is mixed with aqueous solution, the formation of fusosomes can be spontaneous, but can also be promoted by applying force in the form of shaking by using homogenizer, sonicator or extrusion device (for review, see Spuch and Navarro, Journal of Drug Delivery, vol.2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679).Extruded lipid can be prepared by extruding through size-reducing filter, as described in Templeton et al., Nature Biotech, 15:647-652, 1997 (the teachings related to extruded lipid preparation are incorporated herein by reference).

[0294] In another embodiment, lipids can be used to form fusosomes. Lipids can be formulated using spontaneous vesicle formation procedures, including, but not limited to, DLin-KC2-DMA4, C12-200, the colipid disteaoylphosphatidylcholine, cholesterol, and PEG-DMG (see, e.g., Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4; doi:10.1038 / mtna.2011.3). Tekmira publications describe various aspects of lipid vesicles and lipid vesicle formulations (e.g., U.S. Patent Nos. 7,982,027, 7,799,565, 8,058,069, 8,283,333, 7,901,708, 7,745,651, 7,803,397, 8,101,741, 8, See, e.g., US Pat. Nos. 188,263, 7,915,399, 8,236,943, and 7,838,658, and European Patent Nos. 1766035, 1519714, 1781593, and 1664316, all of which are incorporated herein by reference and may be used and / or adapted to the present invention.

[0295] In some embodiments, the fusosomes described herein can comprise one or more polymers. The polymer can be biodegradable. Biodegradable polymer vesicles can be synthesized using methods known in the art. Exemplary methods for synthesizing polymer vesicles are described by Bershteyn et al., Soft Matter 4:1787-1787, 2008 and US2008 / 0014144 A1, and the specific teachings on microparticle synthesis are incorporated herein by reference.

[0296] Exemplary synthetic polymers that can be used include, but are not limited to, aliphatic polyesters, polyethylene glycol (PEG), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of lactic and glycolic acid (PLGA), polycarrolactone (PCL), polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone), as well as natural polymers such as albumin, alginate, and other polysaccharides (including dextran and cellulose), collagen, their chemical derivatives (substitution of chemical groups (e.g., alkyl, alkylene), addition, hydroxylation, oxidation, and other modifications commonly made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamins and hydrophobic proteins, copolymers and mixtures thereof. Generally, these materials degrade by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.

[0297] Fusogen In some embodiments, the fusosomes described herein (e.g., comprising vesicles or portions of cells) contain one or more fusogens, e.g., to facilitate fusion of the fusosome with a membrane, e.g., a cell membrane. These compositions may also include surface modifications made during or after synthesis to include one or more fusogens, e.g., fusogens that are complementary to the target cell. Surface modifications may include membrane modifications, e.g., insertion of lipids or proteins into the membrane.

[0298] In some embodiments, fusosomes contain one or more fusogens on their exterior surface (e.g., integrated into the cell membrane) to target specific cell or tissue types (e.g., cardiomyocytes). Fusogens include, but are not limited to, protein-based, lipid-based, and chemical-based fusogens. The fusogens can bind to partners on the surface of target cells. In some embodiments, fusogen-containing fusosomes will integrate their membranes into the lipid bilayer of the target cells.

[0299] In some embodiments, one or more fusogens described herein may be contained in a fusosome.

[0300] Protein fusogens In some embodiments, the fusogen includes a protein fusogen, e.g., 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 non-mammalian protein such as 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 fusogen comprising one or more of a fusogen or fragment, and any combination thereof.

[0301] In some embodiments, the fusogen causes intermixing between lipids in the fusosome and lipids in the target cell, hi some embodiments, the fusogen causes the formation of one or more pores between the lumen of the fusosome and the cytosol of the target cell, e.g., the fusosome is or comprises a connexin as described herein.

[0302] mammalian proteins In some embodiments, fusogens may comprise mammalian proteins (see Table 1). Examples of mammalian fusogens include proteins of the SNARE family, 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) (e.g., as disclosed in US 6,099,857A), gap junction proteins such as connexin 43, connexin 40, connexin 45, connexin 32, or connexin 37 (e.g., as disclosed in US 2007 / 0224176, Hap2, any protein capable of inducing syncytium formation between heterologous cells (see Table 2), fusogenic properties Fusogens may include, but are not limited to, any protein having the structure (see Table 3), a homolog thereof, a fragment thereof, a variant thereof, and a protein fusion comprising one or more proteins or fragments thereof. 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 6,099,857A and US 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] [Table 3-3]

[0303] In some embodiments, fusosomes comprise curvature-generating proteins, such as epsin 1, dynamin, or proteins containing a BAR domain. See, e.g., Kozlovet et al., CurrOp StrucBio 2015; Zimmerberg et al. Nat Rev 2006; Richard et al., Biochem J 2011.

[0304] 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 variant thereof, or a protein fusion comprising one or more proteins or fragments thereof.

[0305] 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., Spodoptera exigua MNPV (SeMNPV) F protein and Lymantria dispar MNPV (LdMNPV), and paramyxovirus F proteins.

[0306] In some embodiments, class II viral membrane proteins include, but are not limited to, tick-borne osteoencephalitis virus E (TBEV E), Semliki Forest virus E1 / E2.

[0307] In some embodiments, class III viral membrane fusion proteins include, but are not limited to, rhabdovirus G (e.g., membrane fusion protein G of vesicular stomatitis virus (VSV-G)), herpesvirus glycoprotein B (e.g., herpes simplex virus 1 (HSV-1) gB)), Epstein-Barr virus glycoprotein B (EBV gB), Thogotovirus G, baculovirus gp64 (e.g., Autographa California multiple NPV (AcMNPV) gp64), and Borna disease virus (BDV) glycoprotein (BDV G).

[0308] Examples of other viral fusogens, e.g., membrane glycoproteins and viral fusion proteins, include viral syncytial proteins or variants thereof, such as influenza hemagglutinin (HA), or fusion proteins thereof; human immunodeficiency virus type 1 envelope protein (HIV-1 ENV), gp120 from HIV binding LFA-1 to form lymphocyte syncytia, HIV gp41, HIV gp160, or transactivator of HIV transcription (TAT); viral glycoprotein VSV-G, a 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, and togaviruses; murine hepatitis virus JHM surface-projected protein; porcine respiratory coronavirus spike and membrane glycoproteins; avian infectious bronchitis spike glycoprotein and its precursor; bovine enteric coronavirus spike protein; 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 and simian varicella virus gH, including the chaperone protein gL; human, bovine, and cercopithecinous herpesvirus gB; envelope glycoproteins of Friend murine leukemia virus and Mason-Pfizer monkey virus; mumps virus hemagglutinin-neuraminidase, and glycoproteins F1 and F2; membrane glycoproteins from Venezuelan equine encephalomyelitis; paramyxovirus F protein; SIV gp160 protein; Ebola virus G protein; or Sendai virus fusion protein, or homologs thereof, fragments thereof, variants thereof, and protein fusions comprising one or more proteins or fragments thereof.

[0309] Non-mammalian fusogens include viral fusogens, their homologs, fragments thereof, and fusion proteins comprising one or more proteins or fragments thereof. Viral fusogens include class I fusogens, class II fusogens, class III fusogens, and class IV fusogens. In embodiments, class I fusogens, such as human immunodeficiency virus (HIV) gp41, have a characteristic post-fusion structure with a characteristic trimer of α-helical hairpins with a central coiled-coil structure. Class I viral fusion proteins include proteins with a central post-fusion six-helix bundle. Class I viral fusion proteins include influenza HA, parainfluenza F, HIV Env, Ebola GP, 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 the structural characteristics of a beta sheet that folds to form an elongated extracellular domain resulting in 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 features found in classes I and II. In embodiments, class III viral fusogens contain an alpha helix (e.g., forming a six-helix bundle that folds back on itself, similar to class I viral fusogens) and a beta sheet with an amphipathic fusion peptide at its end, reminiscent of class II viral fusogens. 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 encoded by non-enveloped reovirus (doi:10.1038 / sj.emboj.7600767, Nesbitt, Rae L., "Targeted Intracellular Therapeutic Delivery Using Liposomes Formulated with Multifunctional FAST proteins" (2012). Electronic Thesis and Dissertation Repository. Paper 388). In embodiments, the Class IV viral fusogen is small enough that it does not form hairpins (doi:10.1146 / annurev-cellbio-101512-122422, doi:10.1016 / j.devcel.2007.12.008).

[0310] 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.

[0311] In some embodiments, the fusogen is a Poxviridae fusogen.

[0312] Further 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, the entire contents of all of which are incorporated herein by reference.

[0313] Other proteins In some embodiments, fusogens may include pH-dependent (e.g., as in ischemic injury) proteins, their homologs, fragments thereof, and protein fusions containing one or more proteins or fragments thereof. Fusogens may mediate membrane fusion at the cell surface or in endosomes or other membrane-bound spaces.

[0314] 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, homologs thereof, fragments thereof, variants thereof, and protein fusions comprising one or more proteins or fragments thereof.

[0315] Modification of protein fusogens Protein fusogens can be retargeted by mutating amino acid residues in the fusion protein or targeting protein (e.g., hemagglutinin protein). In some embodiments, the fusogen is randomly mutated. In some embodiments, the fusogen is rationally mutated. In some embodiments, the fusogen is subjected to directed evolution. In some embodiments, the fusogen is truncated and only a subset of peptides are used in the fusosome. For example, amino acid residues in the measles hemagglutinin protein can be mutated to alter the protein's binding properties and change the direction of fusion (doi:10.1038 / nbt942, Molecular Therapy vol.16 no.8, 1427-1436 Aug.2008, doi:10.1038 / nbt1060, DOI:10.1128 / JVI.76.7.3558-3563.2002, DOI:10.1128 / JVI.75.17.8016-8020.2001, doi:10.1073pnas.0604993103).

[0316] Protein fusogens can be retargeted by covalently linking a targeting moiety to a fusion protein or targeting protein (such as a hemagglutinin protein). In some embodiments, the fusogen and targeting moiety are covalently linked by expression of a chimeric protein comprising the fusogen linked to the 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 (scFvs) can be conjugated to fusogens to redirect fusion activity to cells that present the scFv-binding target (doi:10.1038 / nbt1060, DOI 10.1182 / blood-2012-11-468579, doi:10.1038 / nmeth.1514, doi:10.1006 / mthe.2002.0550, HUMAN GENE THERAPY 11:817- 826, doi:10.1038 / nbt942, doi:10.1371 / journal.pone.0026381, DOI 10.1186 / s12896-015-0142-z). For example, engineered ankyrin repeat proteins (DARPins) can be conjugated to fusogens to redirect fusion activity to cells that present DARPin-binding targets (doi:10.1038 / mt.2013.16, doi:10.1038 / mt.2010.298, doi:10.4049 / jimmunol.1500956) and combinations of different DARPins (doi:10.1038 / mto.2016.3). For example, receptor ligands and antigens can be conjugated to fusogens to redirect fusion activity to cells that present target receptors (DOI:10.1089 / hgtb.2012.054, DOI:10.1128 / JVI.76.7.3558-3563.2002).Targeting 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 (sdFvs), Fd fragments consisting of VH and CH1 domains, linear antibodies, single-domain antibodies (either VL or VH) such as sdAbs, 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 to cells that present the antibody's target (DOI:10.1128 / JVI.75.17.8016-8020.2001, doi:10.1038 / nm1192). Altered and unaltered fusogens can be presented on the same fusosome (doi:10.1016 / j.biomaterials.2014.01.051).

[0317] Targeting moieties include, for example, humanized antibody molecules, intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fv; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small These may include Modular ImmunoPharmaceuticals ("SMIPs™"); single chain or tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies®; minibodies; BiTEs®; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®.

[0318] In embodiments, the retargeting 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.

[0319] Fusosomes may present targeting moieties that are not conjugated to protein fusogens to direct fusogenic activity to cells bound to the targeting moiety or to affect homing of the fusosome.

[0320] The targeting moieties added to fusosomes can be tailored to have different binding strengths. For example, scFvs and antibodies with varying binding strengths can be used to alter the fusion activity of fusosomes with cells presenting 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 alter the fusion activity of fusosomes with cells presenting large or small amounts of target antigen (doi:10.1038 / mt.2010.298). Targeting moieties can also be tailored to target different regions on the target ligand, which affects the fusion rate with cells presenting the target (doi:10.1093 / protein / gzv005).

[0321] In some embodiments, protein fusogens can be altered to reduce immune reactivity. 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 contains PEG, e.g., a PEGylated polypeptide. Amino acid residues of fusogens that are targeted by the immune system can be altered to prevent them from being 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, DOI 10.1182 / blood-2014-02-558163). In some embodiments, the glycosylation of the fusogen can be altered to alter immune interactions or reduce immune reactivity. Without wishing to be bound by theory, in some embodiments, the protein fusogen derived from a virus or organism that does not infect humans has no natural fusion target in the patient and therefore has high specificity.

[0322] Lipid fusogens In some embodiments, the fusosomes may be treated with a fusogenic lipid, such as a saturated fatty acid. In some embodiments, the saturated fatty acid has 10-14 carbons. In some embodiments, the saturated fatty acid has a long chain carboxylic acid. In some embodiments, the saturated fatty acid is a monoester.

[0323] In some embodiments, the fusosomes may be treated with unsaturated fatty acids. In some embodiments, the unsaturated fatty acids have C16 to 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.

[0324] Without wishing to be bound by theory, in some embodiments, negatively curvature lipids promote membrane fusion. In some embodiments, fusosomes contain one or more negatively curvature lipids in their membranes, e.g., exogenous negatively curvature lipids. In embodiments, negatively curvature lipids or their precursors are added to source cells or to culture media containing fusosomes. In embodiments, source cells are engineered to express or overexpress one or more lipid synthesis genes. The negatively curvature lipids can be, for example, diacylglycerol (DAG), cholesterol, phosphatidic acid (PA), phosphatidylethanolamine (PE), or fatty acids (FA).

[0325] Without wishing to be bound by theory, in some embodiments, positively curvature lipids inhibit membrane fusion. In some embodiments, fusosomes contain reduced levels of one or more positively curvature lipids in their membranes, for example, exogenous positively curvature lipids. In embodiments, this level is reduced by inhibiting lipid synthesis in source cells, for example, by knocking out or knocking down lipid synthesis genes. The positively curvature lipids can be, for example, lysophosphatidylcholine (LPC), phosphatidylinositol (PtdIns), lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), or monoacylglycerol (MAG).

[0326] Chemical Fusogens In some embodiments, the fusosomes may be treated with a fusogenic chemical, hi some embodiments, the fusogenic chemical is polyethylene glycol (PEG) or a derivative thereof.

[0327] In some embodiments, chemical fusogens induce local dehydration between the two membranes, resulting in unfavorable molecular packing of the bilayer. In some embodiments, chemical fusogens induce dehydration in the region near the lipid bilayer, causing displacement of aqueous molecules between the membranes and allowing interaction between the two membranes.

[0328] In some embodiments, the chemical fusogen is a positive ion. Some non-limiting examples of positive ions include Ca, Mg, Mn, Zn, La, Sr, and H.

[0329] In some embodiments, chemical fusogens bind to target membranes by modifying surface polarity, altering hydration-dependent intermembrane repulsion.

[0330] In some embodiments, the chemical fusogen is lipid soluble. Some non-limiting examples include oleoylglycerol, dioleoylglycerol, trioleoylglycerol, and variants and derivatives thereof.

[0331] 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.

[0332] In some embodiments, the chemical fusogen is a small organic molecule. A non-limiting example includes n-hexyl bromide.

[0333] In some embodiments, the chemical fusogen does not alter the composition, cell viability, or ion transport properties of the fusogen or target membrane.

[0334] 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.

[0335] In some embodiments, fusosomes contain actin and an agent that stabilizes polymerized actin. Without wishing to be bound by theory, stabilized actin within fusosomes may promote fusion with target cells. In embodiments, the agent that stabilizes polymerized actin is selected from actin, myosin, biotin-streptavidin, ATP, neuronal Wiskott-Aldrich syndrome protein (N-WASP), or formin. See, e.g., Langmuir. 2011 Aug 16;27(16):10061-71 and Wen et al., Nat Commun. 2016 Aug 31;7. In embodiments, fusosomes contain exogenous actin, e.g., wild-type actin or actin containing a mutation that promotes polymerization. In embodiments, fusosomes contain ATP or phosphocreatine, e.g., exogenous ATP or phosphocreatine.

[0336] Small molecule fusogens In some embodiments, fusosomes may be treated with fusogenic small molecules. Some non-limiting examples include halothane, nonsteroidal anti-inflammatory drugs (NSAIDs) such as meloxicam, piroxicam, tenoxicam, and chlorpromazine.

[0337] In some embodiments, small molecule fusogens may be present in or comprise micelle-like aggregates.

[0338] Fusogen modification In some embodiments, the fusogen is bound to a cleavable protein. In some cases, the cleavable protein can be cleaved by exposure to a protease. The engineered fusion protein can be bound to any domain of a transmembrane protein. The engineered fusion protein can be bound to a protein domain located in the intermembrane space by a cleavable peptide. The cleavable peptide can be cleaved by one or a combination of intermembrane proteases (e.g., HTRA2 / OMI, which requires a non-polar aliphatic amino acid—valine, isoleucine, or methionine—is preferred at the P1 position, and a hydrophilic residue—arginine—is preferred at the P2 and P3 positions).

[0339] In some embodiments, the fusogen is bound to an affinity tag. In some embodiments, the affinity tag aids in the separation and isolation of fusosomes. In some embodiments, the affinity tag is cleavable. In some embodiments, the affinity tag is non-covalently bound to the fusogen. In some embodiments, the affinity tag is present on the fusosome and separate from the fusogen.

[0340] In some embodiments, fusogenic proteins are engineered to include a proteolytic sequence, such as a mitochondrial or cytoplasmic proteolytic sequence, by any method known in the art or described herein. Fusogenic proteins may be engineered with proteolytic sequences, including, but not limited to, a caspase 2 protein sequence (e.g., Val-Asp-Val-Ala-Asp-|-) or other proteolytic sequences (see, e.g., Gasteiger et al., *The Proteomics Protocols Handbook*, 2005:571-607), a modified proteolytic sequence with at least 75%, 80%, 85%, 90%, 95% or more identity to a wild-type proteolytic sequence, such as a cytoplasmic proteolytic sequence, such as ubiquitin, or a modified cytoplasmic proteolytic sequence with at least 75%, 80%, 85%, 90%, 95% or more identity to a wild-type proteolytic sequence. In one embodiment, the invention includes compositions of mitochondria in a source or chondrosome that contain a protein modified with a proteolytic sequence that is at least 75%, 80%, 85%, 90%, 95% or more identical to the wild-type proteolytic sequence, e.g., a cytoplasmic proteolytic sequence, e.g., ubiquitin, or a modified cytoplasmic proteolytic sequence that is at least 75%, 80%, 85%, 90%, 95% or more identical to the wild-type proteolytic sequence.

[0341] In some embodiments, fusogens can be modified with a protease domain that recognizes a specific protein, e.g., overexpression of a protease, e.g., an engineered fusion protein with protease activity, such as a protease or a protease domain from a protease, e.g., MMP, mitochondrial processing peptidase, mitochondrial intermediate peptidase, inner membrane peptidase, etc.

[0342] Alfonzo, JD & Soll, D. Mitochondrial tRNA import - the challenge to understand has just begun.Biological Chemistry 390:717-722.2009, Langer,T.et al.Characterization of Peptides Released from Mitochondria.THE JOURNAL OF BIOLOGICAL CHEMISTRY.Vol.280,No.4.2691-2699,2005,Vliegh,P.et al.Synthetic therapeutic peptides:science and market.Drug Discovery Today.15(1 / 2).2010, Quiros PMm et al.,New roles for mitochondrial proteases in health, aging and disease.Nature Reviews Molecular Cell Biology.V16,2015, Weber-Lotfi,F.et al.DNA import competence and mitochondrial genetics.Biopolymers and Cell.Vol.30.N See 1.71-73,2014.

[0343] Fusosome generation Fusosomes produced by cells The fusosome composition can be produced from cultured cells, e.g., cultured mammalian cells, e.g., cultured human cells. The cells can be progenitor cells or non-progenitor (e.g., differentiated) cells. The cells can be primary cells or cell lines (e.g., mammalian, e.g., human, cell lines described herein). In embodiments, the cultured cells are progenitor cells, e.g., bone marrow stromal cells, bone marrow-derived adult progenitor cells (MAPCs), endothelial progenitor cells (EPCs), blast cells, intermediate progenitor cells formed in the subventricular zone, neural stem cells, muscle stem cells, satellite cells, liver stem cells, hematopoietic stem cells, bone marrow stromal cells, epidermal stem cells, embryonic stem cells, mesenchymal stem cells, umbilical cord stem cells, progenitor cells, muscle progenitor cells, myoblasts, cardiac myoblasts, neural progenitor cells, glial progenitor cells, neural progenitor cells, or hepatoblasts.

[0344] In some embodiments, the source cells are endothelial cells, fibroblasts, blood cells (e.g., macrophages, neutrophils, granulocytes, leukocytes), stem cells (e.g., mesenchymal stem cells, umbilical cord stem cells, bone marrow stem cells, hematopoietic stem cells, induced pluripotent stem cells, e.g., induced pluripotent stem cells derived from cells of interest), embryonic stem cells (e.g., stem cells from embryonic yolk sac, placenta, umbilical cord, fetal skin, adolescent skin, blood, bone marrow, adipose tissue, erythropoietic tissue, hematopoietic tissue), myoblasts, parenchymal cells (e.g., hepatocytes), ), alveolar cells, neurons (e.g., retinal neurons), progenitor cells (e.g., retinal progenitor cells, myeloblasts, myeloid progenitor cells, thymocytes, meiocytes, megakaryoblasts, promegakaryoblasts, melanoblasts, lymphoblasts, myeloid progenitor cells, normal cells, or hemangioblasts), progenitor cells (e.g., cardiac progenitor cells, satellite cells, radiation glial cells, bone marrow stromal cells, pancreatic progenitor cells, endothelial progenitor cells, blast cells), or immortalized cells (e.g., HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR 90, IMR 91, PER.C6, HT-1080, or BJ cells).

[0345] The cultured cells may be from epithelial, connective, muscle, or nervous tissue or cells, and combinations thereof. Fusosomes can be produced from cultured cells from any eukaryotic (e.g., mammalian) organ system, such as the cardiovascular system (heart, vasculature), digestive system (esophagus, stomach, liver, gallbladder, pancreas, intestine, colon, rectum, ...

Claims

[Claim 1] The invention as set forth in the drawings.

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