Engineered cells and methods of use thereof in production of membrane-engineered extracellular vesicles

Abstract

Provided herein are cells comprising a deletion of PTDSS1, and expressing an additional transgene or transgene, e.g., for production of viruses including AAV comprising a therapeutic gene of interest, as well as methods for using the cells to make mEVs comprising the transgene products, e.g., nucleic acids, cytoplasmic or membrane proteins, or mEV-AAV, and as well as mEVs and mEV-associated products.

Description

[0001] ENGINEERED CELLS AND METHODS OF USE THEREOF IN PRODUCTION OF MEMBRANE-ENGINEERED EXTRACELLULAR VESICLES

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 644,199, filed on May 8, 2024. The entire contents of the foregoing are incorporated herein by reference.

[0004] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with Government support under Grant No.

[0006] DC017117 by the National Institutes of Health. The Government has certain rights in the invention.

[0007] TECHNICAL FIELD

[0008] Described herein are engineered cells that can be used to produce extracellular vesicles (EVs) with improved properties, as well as EVs produced thereby.

[0009] BACKGROUND

[0010] Bacterial, fungal, plant, and animal cells produce extracellular vesicles (EVs), which can include components of the membrane or cytoplasm of the producing cell. EVs have a number of uses including delivering therapeutic and diagnostic cargo, including proteins, lipids, viruses, and nucleic acids to target cells. See, e.g., Rogers et al., Front Physiol. 2020 May 21 : 11 :479; Stahl and Raposo, Essays Biochem. 2018 May 15;62(2):119-124.

[0011] SUMMARY

[0012] Provided herein are engineered cells comprising a mutation in a gene encoding phosphatidylserine synthase 1 (PTDSS1), wherein the mutation abrogates production of functional PTDSS1 protein, and preferably wherein the cell further comprises at least one additional transgene. In some embodiments, the at least one additional transgene is in a viral vector. In some embodiments, the viral vector is an adeno- associated vector (AAV), adenovirus, retrovirus, baculovirus, or lentivirus.

[0013] In some embodiments, an engineered cell of the present disclosure can comprise at least one phosphatidylcholine, at least one phosphatidylserine, at least one phosphatidylethanolamine, at least one phosphatidylinositol, at least one cholesterol, at least one sphingomyelin or other sphingolipid, at least one plasmalogen, or any combination thereof. In some embodiments, an engineered cell of the present disclosure can comprise: (i) at least one phosphatidylcholine in an amount ranging from about 15% to about 25% of total cell lipids; (ii) at least one phosphatidylserine in an amount ranging from about 5% to about 20% of total cell lipids; (iii) at least one phosphatidylethanolamine in an amount ranging from about 20% to about 30% of total cell lipids; (iv) at least one phosphatidylinositol in an amount ranging from about 15% to about 20% of total cell lipids; (v) at least one cholesterol in an amount ranging from about 1% to about 20% of total cell lipids; (vi) at least one sphingomyelin or other sphingolipid in an amount ranging from about 1% to about 10% of total cell lipids; and / or (vii) at least one plasmalogen in an amount ranging from about 15% to about 25% of total cell lipids.

[0014] Additionally provided herein are methods of producing a population of membrane-engineered extracellular vesicles (mEVs), the method comprising: maintaining an engineered cell comprising a mutation in a gene encoding phosphatidylserine synthase 1 (PTDSS1), wherein the mutation abrogates production of functional PTDSS1 protein, in culture media (e.g., for long enough for the cells to produce mEV, e.g., at least 6-24, 12-24, or 18-24 hours, 1 day, 2 days, 3 days, 4 days, or 5 days), and isolating extracellular vesicles from the media, thereby producing a population of mEVs. In some embodiments, the extracellular vesicles are isolated from the media without lysing the cell.

[0015] In some embodiments, the cell further comprises at least one additional transgene. In some embodiments, the at least one additional transgene is in a viral vector, optionally wherein the viral vector is an adeno-associated vector (AAV), adenovirus, retrovirus, baculovirus, or lentivirus. In some embodiments, the population of mEVs comprise the transgene or a protein product of the transgene. In some embodiments, the population of mEVs comprise the viral vector.

[0016] Further, provided herein are compositions comprising a population of membrane-engineered extracellular vesicles (mEVs) produced by a method described herein. In some embodiments, the mEVs disclosed herein have increased cholesterol as compared to EVs isolated from a parental cell expressing functional PTDSSl / without the mutation in PTDSS1. In some embodiments, the mEVs disclosed herein have a lipid profile as described herein, e.g., as shown in FIG. 6. In some embodiments, the mEVs disclosed herein mprise PS(16:0_23:6). In some embodiments, the mEVs disclosed herein are resistant to neutralization by a neutralizing antibody.

[0017] In some embodiments, the compositions comprise a population of mEV- enveloped viral vectors produced by a method described herein. In some embodiments, the viral vectors are AAV (mEV-AAV).

[0018] Also provided herein are methods of delivering a selected nucleic acid or protein to a target cell. The methods comprise providing an engineered cell comprising (i) a mutation in a gene encoding phosphatidylserine synthase 1 (PTDSS1), wherein the mutation abrogates production of functional PTDSS1 protein, and (ii) a transgene encoding the selected nucleic acid or protein; maintaining the cells in culture media (e.g., for long enough for the cells to produce mEV, e.g., at least 6-24, 12-24, or 18-24 hours, 1 day, 2 days, 3 days, 4 days, or 5 days), isolating extracellular vesicles (EVs) from the media comprising the selected nucleic acid or protein, and contacting the target cell with the EVs, thereby delivering the selected nucleic acid or protein to a target cell.

[0019] Additionally, provided herein are methods of delivering an AAV comprising a selected nucleic acid to a target cell. The methods comprise providing an engineered cell comprising (i) a mutation in a gene encoding phosphatidyl serine synthase 1 (PTDSS1), wherein the mutation abrogates production of functional PTDSS1 protein, and (ii) transgenes encoding the components of the AAV (e.g., rep and cap) and the selected nucleic acid; maintaining the cells in culture media (e.g., for long enough for the cells to produce mEV, e.g., at least 6-24, 12-24, or 18-24 hours, 1 day, 2 days, 3 days, 4 days, or 5 days), isolating extracellular vesicles (EVs) from the media comprising the AAV, and contacting the target cell with the EVs, thereby delivering the AAV comprising the selected nucleic acid to a target cell.

[0020] In some embodiments, a target cell is a brain cell. In some embodiments, a target cell is a brain cell in the brain of a living subject, preferably a mammal. In some embodiments, a target cell is a heart cell. In some embodiments, a target cell is a heart cell in the heart of a living subject, preferably a mammal. In some embodiments, the engineered cells are made from an HEK293T, HeLa, Sf9 / 21, CHO, BHK-21, Vero parental cell lines, or yeast cells.

[0021] Additionally, provided herein are pharmaceutical compositions. In some embodiments, a pharmaceutical composition comprises at least one of the membrane- engineered extracellular vesicles (mEVs) disclosed herein and at least one pharmaceutically accepted carrier. Also provided herein are kits for use in any of the methods disclosed herein, wherein the kits can comprise an engineered cell, an mEV, a viral vector, and / or a pharmaceutical composition as disclosed herein.

[0022] As used herein, the term “about” means plus or minus 10%.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0024] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0025] DESCRIPTION OF DRAWINGS

[0026] FIGS. 1A-1C: Deletion of phosphatidylserine synthase I produced significant membrane lipid alterations in APTDSS1 cell line. (FIG. 1 A) Heat map shows the fold-changes in the abundance of lipid species in the generated APTDSS1 cell line relative to conventional HEK293T. (FIG. IB) Volcano plot shows the significant (p<0.0005) changes (dots above the grey horizontal line) in lipid species when comparing engineered APTDSS1 and conventional HEK293T. (FIG. 1C) List of the top 10 most significantly altered lipid species depicted in volcano plot. Lipid species were analyzed in lipid extract of 20 million cells via LC-MS negative mode (n=3).

[0027] FIGS. 2A-2C: Engineered APTDSS1 cell line had a decreased cholesterol abundance. (FIG. 2A) Amount of cholesterol in lipids extracted from 20 million cells of HEK293T and APTDSS1 was significantly decreased. Cholesterol was significantly decreased in the lipid extract of APTDSS1 relative to HEK293T. (FIG. 2B) The abundance of fully saturated PS was decreased in the engineered cell line lacking PTDSS1. (FIG. 2C) The ratio of each lipid category relative to cholesterol was increased, demonstrating a decreased amount of cholesterol in the overall lipid composition of APTDSS1 (black) compared to HEK293T (grey). The total abundance of lipid categories for glycerophospholipids and sphingolipids (i.e., sphingomyelin) was similar in both analyzed cell lines.

[0028] FIGS. 3A-3C: Engineered APTDSS1 cell line showed enrichment of enveloped-AAV relative to non-enveloped AAV in the supernatant. Size exclusion chromatography (SEC) was used to separate the fraction of EV- AAV (F1-F4) from the fractions of naked-free AAV (F5-F12) in the supernatant of AAV producer cell lines. Five days post-transfection for AAV production, 100 ml of supernatant for each cell line was collected. Concentrated samples containing EV- AAV and AAV released in the supernatant of each cell line were concentrated and processed as previously described (Cheng et al., Hum Gene Ther. (32)1457-1470 (2021)). SEC fractions 1 to 4 and 5 to 12 correspond to the EV- AAV and naked-free AAV fractions, respectively. After DNAse treatment and DNA viral extraction, AAV titer and yield in the fractions of each cell line were determined via qPCR. (FIG. 3 A) AAV yields in the supernatant of the AAV-producing HEK293T cell line showed a larger proportion of free-naked AAV relative to EV-AAV. (FIG. 3B) AAV yields in the supernatant of APTDSS1 indicated a reversed trend favoring the production of EV-AAV relative to free-naked AAV. (FIG. 3C) The engineered APTDSS1 cell line showed 8-fold increased abundance of EV-AAV compared to AAV and this ratio was significantly higher compared to conventional 293 T when producing EV-AAV.

[0029] FIGS. 4A-4C: Tetraspanin and cholesterol composition were increased in mEV-AAV9 relative to conventional EV-AAV9. (FIG. 4 A) Tetraspanin counts in EV-AAV9 (black) and mEV-AAV9 (white) were assessed via single-particle interferometric reflectance. Engineering strategy APTDSS1 produced mEV-AAV9 particles with an overall larger abundance of CD63, CD81, and CD9 tetraspanins relative to EV-AAV9. (FIG. 4B) The abundance of the CD9 tetraspanin was increased in the engineered mEV-AAV9 particles relative to conventional EV-AAV9. lodixanol gradients were used to purify AAV-enclosed vesicles from empty vesicles before assessment. (FIG. 4C) Cholesterol content was quantified in both types of enveloped vectors using fluorescent dyes for cholesterol and for the total lipids in the vesicles enclosing AAV. The engineered mEV-AAV9 vector showed a 2.4-fold greater relative abundance of cholesterol per particle compared to conventional EV-AAV9. Experiments were performed by triplicate.

[0030] FIGS. 5A and 5B: The membrane-engineered lipid envelope of AAV (mEV-AAV) improved human cell lines AC16 and HEK293T transduction. (FIG. 5 A) The human AC 16 cell line showed an 8.5 -fold greater percentage of transduction after mEV-AAV9 treatment compared to the AAV9 capsid. Transduction of engineered mEV-AAV9 was 1.7-fold higher compared to conventional EV-AAV9 (p=0.034). (FIG. 5B) The percentage of transduced human HEK293T cells indicated that enveloped vector variants mEV-AAV9 and EV-AAV9 transduced 13 - and 1.6-fold more cells compared to the non-enveloped conventional AAV9 capsid, respectively. Experiments were performed with three independent replicates.

[0031] FIGS. 6A and 6B: Membrane-engineered enveloped-AAV (mEV-AAV) had a different lipid composition than conventional enveloped AAV vectors (EV- AAV). (FIG. 6A) Lipidomic analyses of membrane-engineered extracellular vesicles when enclosing AAV (mEV-AAV). Heat map represents the fold changes of individual lipid species of the membrane-engineered vesicles enclosing AAV relative to the lipid composition of conventional vesicles enclosing AAV. (FIG. 6B) Lipid species found in the membrane-engineered vesicles enclosing AAV (mEV-AAV) with the highest abundance relative to conventional non-engineered EV-AAV particles.

[0032] FIG. 7: Membrane-engineered vesicles (mEV) increased the AAV resistance against neutralizing antibodies at high concentrations. Various concentrations of intravenous Immunoglobulins (IVIg) were incubated with AAV (empty square), EV-AAV (grey square) or mEV-AAV (solid circle) before transducing HeLa cells. All vectors harbor the AAV-CBA-firefly luciferase (FLuc) transgene expression cassette. Luminescence was measured to determine transduction for each treatment. Transduction of HeLa cells with the various concentrations of IVIg was normalized to the transduction with no IVIg. The statistical difference is indicated when examining the transduction of mEV-AAV or EV-AAV relative to the AAV condition at the same concentration of IVIg. **p=0.0032, *p = 0.036. Error bars indicate the standard error of the mean (SEM). Data represents the mean of three biological replicates.

[0033] FIGS. 8A-8C: Purified membrane-engineered extracellular vesicles enclosing AAV (mEV-AAV) showed functionality after intracranial injection in mice. (FIG. 8A) Coronal section showing cells in the cortical white matter (a’) and dorsal striatum (a”). (FIG. 8B) Coronal section showing cells in fimbria (b’) and internal capsule (b”). (FIG. 8C) An array of coronal sections of the brain injected with mEV-AAV. All representative images show mEV-AAV9 harboring AAV-CBA- GFP transgene expression cassette. Three C57BL / 6 mice were used.

[0034] DETAILED DESCRIPTION

[0035] Extracellular vesicles, including exosomes, are emerging as candidate drug delivery approaches for diagnostic and therapeutic molecules including proteins, nucleic acids, lipids, and viruses. Kar et al., ACS Biomater Sci Eng. 2023 Feb 13; 9(2): 577-594; Koh et al., Pharmaceutics. 2023 Aug; 15(8): 2042; Herrmann et al., Nature Nanotechnology 2021 July; 16:748-759; Zhu et al., J. Extracell. Vesicles 6, 1324730 (2017). A number of cells have been used to produce EVs to deliver drugs, including Human embryonic kidney 293 (HEK293) cells, which are commonly used based on their productivity, low immunogenicity and toxicity, simplicity of culture and east of transfection (Koh et al., Pharmaceutics. 2023 Aug; 15(8): 2042). HEK293T cells are also useful in the generation of therapeutic viruses including adeno-associated viruses (AAV), which are useful in gene therapy methods.

[0036] As described herein, HEK293T cells with a modification of a lipid metabolism gene encoding phosphatidylserine synthase 1 (PTDSS1) can be used as producer cells to generate exosomes with improved properties. Co-expressing one or more additional transgenes in the PTDSS1 KO cells (referred to herein as HEK293T-5PTDSS1) resulted in the production of a higher abundance of cell-released extracellular vesicles in the HEK293T-5PTDSS1 cell line as compared to the conventional HEK293T. The engineered cells showed several significant differences in the abundance of lipid species, including an increased abundance of the fully saturated form of the etherlipid of phosphatidylcholine linked to 17 and 16 carbon chains. The HEK293T- 5PTDSS1 cell line also contained a lower abundance of cholesterol.

[0037] When the cells were used for producing AAV, a higher proportion of extracellular vesicle-enveloped AAV (EV-AAV) was present in the supernatant relative to free-naked AAV when the viral particles were produced using the HEK293T-5PTDSS1 cell line, as compared to the conventional HEK293T cell line. Given the lower abundance of cholesterol in the novel cell line, the cholesterol content in the mEV-AAV variant was further analyzed and compared to conventional EV-AAV (produced with conventional HEK293T). The cholesterol content in mEV- AAV variants was increased by 2-fold relative to unaltered EV-AAV. Without wishing to be bound to any particular theory, it is hypothesized that the cholesterol is going to the vesicles instead of staying in the cell.

[0038] As used herein, the term “about” means plus or minus 10%. The terms “APTDSS1” and “5PTDSS1” are used interchangeably throughout the disclosure herein and are understood to both refer to an engineered cell comprising a mutation in a gene encoding phosphatidylserine synthase 1 (PTDSS1) wherein the mutation abrogates production of functional PTDSS1 protein.

[0039] Engineered Cells and Methods of Producing

[0040] Provided herein are genetically modified producer cells (and cell lines thereof, also referred to herein as “engineered cell” and “engineered producer cell” interchangeably) that have a mutation in a gene encoding phosphatidylserine synthase 1 (PTDSS1), wherein the mutation abrogates production of functional PTDSS1 protein, and membrane-engineered extracellular vesicles (these EVs are referred to herein as mEVs (for membrane-engineered EVs) obtained from those cells.

[0041] The engineered producer cells can be from any source, including mammals (e.g., HEK293T, HeLa, CHO, BEK-21, or Vero, or any other cell line), or from insects (e.g., Sf9 or Sf21 cells), or yeast. In some embodiments, a source of the engineered producer cells (e.g., the parental cell, or parental cell line) can be human cells. In some embodiments, a source of the engineered producer cells (e.g., the parental cell, or parental cell line) can be HEK293T cells.

[0042] Methods known in the art including CRISPR Cas editing can be used to introduce genomic alterations to knock out the PTDSS1 gene (including insertion of 1-2 bp in exon 1 to produce a frame shift, or deletion of all or part of the coding sequence resulting in no expression or expression of a non-functional product). The genomic sequence of human PTDSS1 can be found at RefSeqGene Accession ID No. NG_034054.2 (Range 5002-80095). Kits for using CRISPR to make knockouts in human cells are commercially available, e.g., from BioCat GmbH (KN201012-OR). Knockout PTDS SI cell lines can also be obtained commercially, e.g., HEK293T PTDSS1 knockout cells from abeam (ab255449) and Applied Biological Materials (38070141).

[0043] The 5PTDSS1 producer cells can also be transiently or stably engineered to express other transgenes, e.g., for nucleic acids, cytoplasmic or membrane proteins, or viruses including AAV, adenoviruses, retroviruses, baculoviruses, or lentiviruses, that will be packaged into the mEVs.

[0044] Methods for producing each of these types of viruses are known in the art. For example, for production of recombinant AAV (rAAV), a therapeutic gene of interest is encapsidated using the replication (rep) and capsid (cap) genes. The engineered cells can be used to produce extracellular vesicle-associated AAV, e.g., EV- AAV. EV- AAV (also referred to as exo-AAV) which has been shown to have enhanced transduction in vivo as well as the ability to evade neutralizing antibodies (US2013 / 0202559; Cheng et al., Hum Gene Then 2021 Dec;32(23-24): 1457-1470; Meliani A., et al., Blood Adv (2017) 1 (23): 2019-2031). Thus, for EV-AAV production the cell (referred to as a producer cell) needs to express the rep and cap genes specific to the serotype of interest, adenovirus (Ad) or herpes simplex virus (HSV) helper virus functions, and the proviral (vector DNA) construct comprising the therapeutic gene(s) (Grieger et al., Mol Then 2016 Feb; 24(2): 287-297; Naso et al., BioDrugs. 2017; 31(4): 317-334. Kimura et al., Scientific Reports 2019 Sept; 9: 13601; Su et al., Scientific Reports 2023 Dec; 13: 21670). Typically for rAAV production the producer cells transiently express all of the above, but one or more (e.g., the rep gene) can also be stably expressed in the cell (see, e.g., Jalsic et al., Mol Ther Methods Clin Dev. 2023 Sep 14; 30: 259-275. EV-AAV produced using the present methods is also referred to as mEV-AAV.

[0045] After expression of the transgene (e.g., after the cells are maintained in culture for long enough for the cells to produce mEV, e.g., at least 6-24, 12-24, or 18-24 hours, 1 day, 2 days, 3 days, 4 days, or 5 days), mEVs comprising the product can be isolated or purified. A number of methods can be used for mEV isolation and purification, such as differential ultracentrifugation (dUC), precipitation, size exclusion chromatography (SEC), affinity chromatography and tangential flow filtration. Preferably SEC is used to isolate mEV-AAV, e.g., from supernatant of intact cells rather than from cell lysates (see, e.g., Cheng et al., Hum Gene Ther. 2021 Dec;32(23-24): 1457-1470). The present methods and compositions can also be used to make “empty” mEV, e.g., in cells in which no additional transgene is expressed; in this case, the cells are maintained in culture long enough for the cells to produce mEV, e.g., at least 6-24, 12-24, or 18-24 hours, 1 day, 2 days, 3 days, 4 days, or 5 days.

[0046] The engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) can be either adherent cultures or suspension cultures. Adherent cultures refer to cells that are grown on a substrate surface, for example a plastic plate, dish or other suitable cell culture growth platform, and may be anchorage dependent. Suspension cultures refer to cells that can be maintained in, for example, culture flasks or large suspension vats, which allows for a large surface area for gas and nutrient exchange. Suspension cell cultures often utilize a stirring or agitation mechanism to provide appropriate mixing. Media and conditions for maintaining adherent cell cultures and / or cells in suspension are generally known in the art (see, e.g., Helgason, C. D., & Miller, C. L. (Eds.). (2016). BASIC CELL CULTURE PROTOCOLS (4th ed.) [Softcover], Springer).

[0047] Thus, provided herein are engineered producer cells comprising a deletion of PTDSS1, and optionally expressing an additional transgene or transgene, e.g., for production of AAV comprising a therapeutic gene of interest, as well as methods for using the cells to make mEVs comprising the transgene products, e.g., nucleic acids, cytoplasmic or membrane proteins, or mEV- AAV, and as well as mEVs and mEV- associated products. In some embodiments, an AAV produced by the engineered cells disclosed herein can be a variant of the AAV virus (i.e., an AAV serotype). Each AAV serotype comprises unique characteristics in terms of tissue tropism (preference for infecting certain tissues) and / or immunogenicity (how much they trigger an immune response). Non-limiting examples of AAV serotypes suitable for production by the engineered cells disclosed herein include ANC80, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. In some embodiments, an AAV produced by the engineered cells disclosed herein is AAV9. In some embodiments, an AAV produced by the engineered cells disclosed herein can be an AAV pseudotype. An AAV pseudotype contains the capsid of a first serotype and the genome of a second serotype (e.g., the pseudotype AAV2 / 5 would correspond to an AAV with the genome of serotype AAV2 and the capsid of AAV5). The engineered producer cells have the ability to produce a higher proportion of extracellular vesicles as compared to the parental cell line, including a higher proportion of EVs enveloping AAV relative to free-naked AAV in the supernatant, facilitating purification of EV-AAV variants, as well as lipid-modified variants of EVs including EV-AAV, with enhanced ability to deliver genetic material to target cell types. These enveloped AAV are referred to as mEV-AAV.

[0048] Engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) produce at least 2-fold more mEV-AAV compared to the amount of mEV-AAV produced by parental cells (e.g., native cells; cells that express PTDSS1). In some embodiments, engineered producer cells disclosed herein produce about 2-fold to about 10-fold, about 2-fold to about 9-fold, or about 2-fold to about 8-fold more mEV-AAV compared to the amount of mEV-AAV produced by parental cells. In some embodiments, engineered producer cells disclosed herein produce at least about or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold more mEV-AAV compared to the amount of mEV-AAV produced by parental cells. In some embodiments, engineered producer cells disclosed herein produce about 8-fold more mEV-AAV compared to the amount of mEV-AAV produced by parental cells.

[0049] Lipid Composition

[0050] The lipid composition of EVs dictates a number of their properties. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the main types of lipids in cellular membranes (plasma membrane and organelles). Cholesterol modulates fluidity, thickness, permeability, membrane curvature, and vesicle trafficking to maintain homeostasis (see, e.g., van Meer et al., Nat Rev Mol Cell Biol. 2008 Feb;9(2):l 12-24; Skotland et al., (2017) Progress in Lipid Research 30-41). Thus, mEVs produced as described herein have different physical properties than those produced using the parental HEK293T cell line, as do mEV-AAV.

[0051] Table 1 provides a summary of the lipid differences in a population of cells, on average 20 million cells (average of 2-3 15-cm dishes). In brief, Table 1 provides lipid extracts from 20 million cells that were analyzed through LC-MS where the internal standards C18:l-d7-cholesterol and 15:0-18:l-d7-PC were used to quantify the absolute amount of each lipid. All detected sphingolipids in Table 1 belong to the sub-category of sphingomyelins. STDEVA=standard deviation of a sample of values, AVG=average. TABLE 1.

[0052] Engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) have decreased cholesterol content compared to the cholesterol content of parental cells (e.g., native cells; cells that express PTDSS1). In some embodiments, the cholesterol content of engineered producer cells disclosed herein is decreased by about 1-fold to about 5-fold compared to the cholesterol content of parental cells. In some embodiments, the cholesterol content of engineered producer cells disclosed herein is decreased by at least about or about 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold compared to the cholesterol content of parental cells. In some embodiments, the cholesterol content of engineered producer cells disclosed herein is decreased by one-third compared to the cholesterol content of parental cells.

[0053] Table 2 provides the percentage of fully saturated lipids within each lipid category or class. As described herein, an increase in fully saturated phosphatidylserines was observed in the lipid extract from the 8PTDSS1 cell line relative to HEK293T (p=0.0144). STDEVA=standard deviation of a sample of values.

[0054] TABLE 2.

[0055] Engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) have increased total phosphatidylserine content compared to the total phosphatidylserine content of parental cells (e.g., native cells; cells that express PTDSS1). In some embodiments, the total phosphatidylserine content of engineered producer cells disclosed herein is increased by about 1-fold to about 5-fold compared to the total phosphatidylserine content of parental cells. In some embodiments, the total phosphatidylserine content of engineered producer cells disclosed herein is increased by at least about or about 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold compared to the total phosphatidylserine content of parental cells. In some embodiments, the total phosphatidylserine content of engineered producer cells disclosed herein is increased by one-third compared to the total phosphatidylserine content of parental cells.

[0056] Engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) have increased PC(0-16:0 / 17:0) content compared to the PC(0-16:0 / 17:0) content of parental cells (e.g., native cells; cells that express PTDSS1). PC(0-16:0 / 17:0) (1- hexadecyl-2-heptadecanoyl-sn-glycero-3-phosphocholine) is a fully saturated ether lipid detected in engineered APTDSS1 cells (see, e.g., FIG. 1A). In some embodiments, the PC(0-16:0 / 17:0) content of engineered producer cells disclosed herein is increased by at least about or about 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold,

[0057] 4.5-fold, 5-fold, 5.5-fold, or 6-fold compared to the PC(0-16:0 / 17:0) content of parental cells. In some embodiments, the PC(0-16:0 / 17:0) content of engineered producer cells disclosed herein is increased 4.2-fold, 4.3-fold, 4.4-fold, 4.5-fold, or

[0058] 4.6-fold compared to the PC(0-16:0 / 17:0) content of parental cells.

[0059] Other predicted lipid composition alterations include changes in the content of sphingolipids, phosphatidylserines, phosphatidylethanolamines, gangliosides and ceramides.

[0060] The following is a summary of the lipid composition of an exemplary cell line. Percentages vary depending upon culture conditions and growth phase. Lipid classes or categories (%> of total lipids)

[0061] • Phosphatidylcholines 15-25%

[0062] • Phosphatidylserines 5-20%

[0063] • Phosphatidylethanolamines 20-30%

[0064] • Phosphatidylinositols 15-20%

[0065] • Cholesterol 1-20%

[0066] • Sphingomyelin and other sphingolipids 1-10%

[0067] • Plasmalogens 15-25%

[0068] Specific lipid species made the engineered cell line lipid composition different from the conventional cell line. In the 8PTDSS1 cells,

[0069] • Cholesterol content was decreased by 2-fold

[0070] • Increased % saturated phosphatidylserines by 4-fold.

[0071] • Increased relative abundance of Phosphatidylcholine with ether-linked heptadecanoic acid and palmitic acid by 4-fold.

[0072] • Decreased abundance of specific lipid species with percentages varying from 8% to 40% relative to the content of the same lipid species in the parental HEK293T. Phosphatidylethanolamine with ether-linked palmitoleic acid and alpha-linolenic acid, Sphingomyelin with a total of 44 carbons and 2 double bonds in the sphingoid base and N-acyl chain, Phosphatidylethanolamine with ether-linked palmitoleic acid and linoleic acid, Phosphatidylserine with nonadecenoic acid and eicosenoic acid, Phosphatidylethanolamine with eicosatrienoic acid and a 17-carbon chain with one double bond and a carboxylic acid group, Sphingomyelin with a trihydroxy sphingoid base (total 35 carbons and 3 double bonds in the combined sphingoid and N-acyl chain), Phosphatidylcholine with stearidonic acid and docosahexaenoic acid, Phosphatidylserine with oleic acid and nonadecenoic acid, and Phosphatidylethanolamine with eicosatrienoic acid and 21 -carbon chain with one double bond and a carboxylic acid group. These lipids are shown in FIG. 1C.

[0073] Membrane-engineered extracellular vesicles (mEVs) produced by engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) can be comprised of lipids and / or fatty acids. In some embodiments, mEVs disclosed herein can be comprised of phospholipids, glycolipids, fatty acids, sphingolipids, phosphoglycerides, sterols, cholesterols, and / or phosphatidylserines.

[0074] The following provides some relevant characteristics of compositions in extracellular-vesicles produced by the engineered APTDSS1 cell line containing therapeutic cargo (e.g., AAV): 2.4-fold increased cholesterol content; and / or 2-fold increase in CD9 tetraspanin.

[0075] The membrane-engineered extracellular vesicles (mEVs) produced by engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) have increased cholesterol content per mEV compared to the cholesterol content per mEVs produced by parental cells (e.g., native cells; cells that express PTDSS1). In some embodiments, the cholesterol content per mEV produced by engineered producer cells disclosed herein is increased by about 1-fold to about 5-fold compared to the cholesterol content per mEVs produced by parental cells. In some embodiments, the cholesterol content per mEV produced by engineered producer cells disclosed herein is increased by at least about or about 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5- fold, 4-fold, 4.5-fold, or 5-fold compared to the cholesterol content per mEVs produced by parental cells. In some embodiments, the cholesterol content per mEV produced by engineered producer cells disclosed herein is increased by 2.1 -fold, 2.2- fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, or 2.7-fold compared to the cholesterol content per mEVs produced by parental cells.

[0076] The membrane-engineered extracellular vesicles (mEVs) produced by engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) have at least one lipid species per mEV that is not present in the mEVs produced by parental cells (e.g., native cells; cells that express PTDSS1). The mEVs produced by engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) have PS( 16 : 0 / 23 : 6) ( 1 -Palmitoyl-2-docosahexaenoyl-sn-glycero-3 -phosphocholine) whereas mEVs produced by parental cells (e.g., native cells; cells that express PTDSS1) have no PS(16:0 / 23 :6).

[0077] The membrane-engineered extracellular vesicles (mEVs) produced by engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) have increased content of at least one lipid species per mEV compared to the content of the at least one lipid species per mEVs produced by parental cells (e.g., native cells; cells that express PTDSS1). In some embodiments, the mEVs produced by engineered producer cells disclosed herein have increased content of at least two lipid species per mEV compared to the content of the at least one lipid species per mEVs produced by parental cells. In some embodiments, the mEVs produced by engineered producer cells disclosed herein have increased content of at least three lipid species per mEV compared to the content of the at least one lipid species per mEVs produced by parental cells.

[0078] The membrane-engineered extracellular vesicles (mEVs) produced by engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) have increased PS(18:0 / 22:5) (l -stearoyl-2-docosahexaenoyl-sn-glycero-3 -phospho-L- serine) content per mEV compared to PS(18:0 / 22:5) content per mEVs produced by parental cells (e.g., native cells; cells that express PTDSS1). In some embodiments, the PS(18:0 / 22:5) content per mEV produced by engineered producer cells disclosed herein is increased by at least 70-fold compared to the PS(18:0 / 22:5) content per mEVs produced by parental cells. In some embodiments, the PS(18:0 / 22:5) content per mEV produced by engineered producer cells disclosed herein is increased by about 70-fold to about 200-fold, about 80-fold to about 190-fold, or about 90-fold to about 180-fold compared to the PS(18:0 / 22:5) content per mEVs produced by parental cells. In some embodiments, the PS(18:0 / 22:5) content per mEV produced by engineered producer cells disclosed herein is increased by at least about or about 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 150-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, or 200-fold compared to the PS(18:0 / 22:5) content per mEVs produced by parental cells. In some embodiments, the PS(18:0 / 22:5) content per mEV produced by engineered producer cells disclosed herein is increased by 128-fold, 129-fold, 130-fold, 131-fold, 132-fold, or 133-fold compared to the PS(18:0 / 22:5) content per mEVs produced by parental cells. In some embodiments, the PS(18:0 / 22:5) content per mEV produced by engineered producer cells disclosed herein is increased by 130-fold compared to the PS(18:0 / 22:5) content per mEVs produced by parental cells.

[0079] The membrane-engineered extracellular vesicles (mEVs) produced by engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) have increased PC(16:0 / 19 : OCHO) ( 1 -hexadecanoyl-2-nonadecanoyl-glycero-3 - phosphocholine) content per mEV compared to PC(16:0 / 19: OCHO) content per mEVs produced by parental cells (e.g., native cells; cells that express PTDSS1). In some embodiments, the PC(16:0 / 19:0CHO) content per mEV produced by engineered producer cells disclosed herein is increased by at least 70-fold compared to the PC(16:0 / 19:OCHO) content per rnEVs produced by parental cells. In some embodiments, PC(16:0 / 19:OCHO) content per mEV produced by engineered producer cells disclosed herein is increased by about 70-fold to about 150-fold, about 80-fold to about 140-fold, or about 90-fold to about 130-fold compared to the PC(16:0 / 19:OCHO) content per mEVs produced by parental cells. In some embodiments, the PC(16:0 / 19:0CHO) content per mEV produced by engineered producer cells disclosed herein is increased by at least about or about 70-fold, 80-fold, 90-fold, or 100-fold compared to the PC(16:0 / 19:0CHO) content per mEVs produced by parental cells. In some embodiments, the PC(16:0 / 19:0CHO) content per mEV produced by engineered producer cells disclosed herein is increased by 102-fold, 103- fold, 104-fold, 105 -fold, or 106-fold compared to the PC(16:0 / 19:OCHO) content per mEVs produced by parental cells. In some embodiments, the PC(16:0 / 19:0CHO) content per mEV produced by engineered producer cells disclosed herein is increased by 104-fold compared to the PC(16:0 / 19:OCHO) content per mEVs produced by parental cells.

[0080] The membrane-engineered extracellular vesicles (mEVs) produced by engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) have increased PS(22:0 / 16: 1) (l-docosanoyl-2-(9Z-hexadecenoyl)-sn-glycero-3-phospho- L-serine) content per mEV compared to PS(22:0 / 16:l) content per mEVs produced by parental cells (e.g., native cells; cells that express PTDSS1). In some embodiments, the PS(22:0 / 16:l) content per mEV produced by engineered producer cells disclosed herein is increased by at least 50-fold compared to the PS(22:0 / 16:l) content per mEVs produced by parental cells. In some embodiments, PS(22:0 / 16:l) content per mEV produced by engineered producer cells disclosed herein is increased by about 50-fold to about 100-fold, about 60-fold to about 90-fold, or about 70-fold to about 80-fold compared to the PS(22:0 / 16:l) content per mEVs produced by parental cells. In some embodiments, the PS(22:0 / 16:l) content per mEV produced by engineered producer cells disclosed herein is increased by at least about or about 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold compared to the PS(22:0 / 16:l) content per mEVs produced by parental cells. In some embodiments, the PS(22:0 / 16:l) content per mEV produced by engineered producer cells disclosed herein is increased by 68- fold, 69-fold, 70-fold, 71-fold, or 72-fold compared to the PS(22:0 / l 6: 1) content per mEVs produced by parental cells. In some embodiments, the PS(22:0 / l 6: 1) content per mEV produced by engineered producer cells disclosed herein is increased by 70- fold compared to the PS(22:0 / l 6: 1) content per mEVs produced by parental cells.

[0081] The membrane-engineered extracellular vesicles (mEVs) produced by engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) have increased total tetraspanin counts per mEV compared to total tetraspanin counts per mEVs produced by parental cells (e.g., native cells; cells that express PTDSS1). In some embodiments, the mEVs produced by engineered producer cells disclosed herein have an increased abundance of CD63, CD81, and CD9 tetraspanins per mEV compared to the abundance of CD63, CD81, and CD9 tetraspanins per mEVs produced by parental cells. In some embodiments, the mEVs produced by engineered producer cells disclosed herein have an increased amount of CD63 tetraspanin per mEV compared to the amount of CD63 tetraspanin per mEVs produced by parental cells. In some embodiments, the mEVs produced by engineered producer cells disclosed herein have an increased amount of CD81 tetraspanin per mEV compared to the amount of CD81 tetraspanin per mEVs produced by parental cells. In some embodiments, the mEVs produced by engineered producer cells disclosed herein have an increased amount of CD63 tetraspanin per mEV compared to the amount of CD63 tetraspanin per mEVs produced by parental cells. In some embodiments, the mEVs produced by engineered producer cells disclosed herein have an increased amount of CD9 tetraspanin per mEV compared to the amount of CD9 tetraspanin per mEVs produced by parental cells. In some embodiments, the mEVs produced by engineered producer cells disclosed herein have an at least 2-fold increased amount of CD9 tetraspanin per mEV compared to the amount of CD9 tetraspanin per mEVs produced by parental cells. In some embodiments, the mEVs produced by engineered producer cells disclosed herein have about 2-fold to about 4-fold increased amount of CD9 tetraspanin per mEV compared to the amount of CD9 tetraspanin per mEVs produced by parental cells. In some embodiments, the mEVs produced by engineered producer cells disclosed herein have at least about or about 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, or 2.8-fold increased amount of CD9 tetraspanin per mEV compared to the amount of CD9 tetraspanin per mEVs produced by parental cells. Transduction and Resistance to Neutralization

[0082] In addition, as shown herein, the mEV-AAVs made using the engineered producer cells described herein have improved transduction in human cells, including in cardiomyocytes and kidney cells.

[0083] The mEV-AAVs produced by engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) can have at least an 8-fold greater percentage of transduction in human cells after mEV-AAV treatment compared to the percentage of transduction in human cells after AAV capsid treatment. In some embodiments, mEV-AAVs produced by engineered producer cells disclosed herein) can have at least about or about an 8-fold, 8.5-fold, or 9-fold greater percentage of transduction in human cells after mEV-AAV treatment compared to the percentage of transduction in human cells after AAV capsid treatment.

[0084] The mEV-AAVs produced by engineered producer cells disclosed herein (e.g., engineered APTDSS1 cells) have a greater percentage of in-vivo transduction in a tissue after local administration of the mEV-AAVs compared to the percentage of in- vivo transduction in a tissue after local administration of AAV capsids. In some embodiments, mEV-AAVs produced by engineered producer cells disclosed herein have a greater percentage of in-vivo transduction in a tissue after intraparenchymal injection of the mEV-AAVs compared to the percentage of in-vivo transduction in a tissue after intraparenchymal injection of AAV capsids.

[0085] Successful delivery of an AAV vector to the cell can be hampered by neutralizing antibodies present in the subject receiving the AAV vector and / or composition comprising thereof. As shown herein, the mEV-AAVs made using the engineered producer cells described herein (e.g., engineered APTDSS1 cells) have improved resistance against neutralizing antibodies AAV (i.e., improved resistance to neutralization). In some embodiments, mEV-AAVs produced by engineered producer cells disclosed herein have a higher percentage of resistance to neutralization compared that of conventional AAVs. In some embodiments, mEV-AAVs produced by engineered producer cells disclosed herein have at least 50% resistance to neutralization.

[0086] Compositions and Methods of Use

[0087] Provided herein are compositions comprising a population of membrane- engineered extracellular vesicles (mEVs) disclosed herein. The compositions described herein can thus include mEVs isolated from the 8PTDSS1 producer cells; the mEVs can envelop a number of different products, including viruses (e.g., viral vectors including adeno-associated vector (AAV), adenovirus, retrovirus, baculovirus, or lentivirus), proteins (including therapeutic antibodies), nucleic acids (including mRNA for use in gene therapy protocols), and other components present in the cytoplasm of the producer cells. In some embodiments, the compositions disclosed herein comprise a population of mEVs produced by at least one of the engineered cells disclosed herein (e.g., engineered APTDSS1 cells). In some embodiments, the compositions disclosed herein comprise a population of mEVs produced according to any of the methods disclosed herein. In some embodiments, the compositions disclosed herein comprise a population of mEV-enveloped viral vectors produced by the methods disclosed herein. In some embodiments, the compositions disclosed herein comprise at least one of the engineered cells disclosed herein (e.g., engineered APTDSS1 cells).

[0088] Compositions of the present disclosure can include pharmaceutical compositions. Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein, the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0089] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous (i.v.), intratumoral (i.t.), intradermal, subcutaneous, oral (e.g., inhalation), intraparenchymal, intracardiac, intrapericardial, transdermal (topical), transmucosal, and rectal administration. In some embodiments, a pharmaceutical composition of the present disclosure is formulated for intravenous (i.v.) delivery of at least one mEV disclosed herein. In some embodiments, a pharmaceutical composition of the present disclosure is formulated for intravenous (i.v.) delivery of a population of mEV-enveloped viral vectors. In some embodiments, a pharmaceutical composition of the present disclosure is formulated for intraparenchymal delivery of at least one mEV disclosed herein. In some embodiments, a pharmaceutical composition of the present disclosure is formulated for intraparenchymal delivery of a population of mEV-enveloped viral vectors. Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington, J.P. (2006) The Science and Practice of Pharmacy. 21st Edition, Lippincott Williams and Wilkins, Philadelphia, 772; United States Pharmacopeia 2024 / National Formulary (USP 47-NF 42); and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0090] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient (e.g., mEVs, mEV- enveloped viral vectors) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0091] The pharmaceutical compositions disclosed herein can be included in a container, pack, or dispenser together with instructions for administration. A kit comprising the engineered cells for use in the methods producing the mEVs and / or mEV-enveloped viral vectors of the present disclosure is also contemplated herein.

[0092] The compositions (e.g., pharmaceutical compositions) of the present disclosure can be used in the treatment, prevention, suppression or amelioration of a target disease and / or disorder that can benefit from gene therapy (e.g., AAV-based gene therapy). Provided herein are methods of using the mEVs to deliver at least one AAV-based gene therapy to a subject in need thereof. A subject in need thereof is intended to refer to subjects who could benefit from an AAV-based gene therapy, and / or are at risk for and / or have been diagnosed with one or more diseases and / or disorders that can be treated and / or ameliorated by administration of an AAV-based gene therapy. Preferably, the subject is human, but the methods disclosed herein can be used in other mammals, e.g., non-human veterinary subjects such as non-human primates, cats, dogs, horses, cows, goats, and rabbits.

[0093] Also provided herein are methods of using the mEVs to deliver selected products to target cells. The target cells can be, e.g., brain cells, epithelial cells, endothelial cells, neurons (e.g., cerebral cortical pyramidal neurons), and / or glia (e.g., oligodendroglia, astrocytes, ependymal cells, and choroid plexus epithelium). The cells can be in vitro or in vivo. For example, the cells can be in a tissue, such as the brain or heart, of a living subject such as a mammal. The cells can also be harvested from a tissue and the mEVs then deliver selected products to target cells ex vivo. In some embodiments, the target cell is a brain cell (e.g., a neuron, glia). In some embodiments, the target cell is a heart cell (e.g., a cardiomyocyte).

[0094] EXAMPLES

[0095] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0096] Materials and Methods

[0097] The following materials and methods were used in the Examples below.

[0098] Cells and cell culture

[0099] Human HEK293T and HeLa cells were obtained from the American Type Culture Collection (Manassas, VA). HEK293T-APTDSS1 CRISPR / Cas9 knockout cell line was purchased from Abeam (ab266068). These cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and Penicillin-Streptomycin-Glutamine. HEK293T and HEK293T-APTDSS1 tested negative for mycoplasma using a Mycoplasma Detection Kit. AC 16 human cardiomyocyte cell line was purchased from Sigma Aldrich and cultured according to supplier specifications. All Adherent cell lines were cultured in a humidified atmosphere supplemented with 5% CO2 at 37°C.

[0100] Animals

[0101] All animal experiments were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care following guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Adult mice aged (8-10 weeks old) C57BL / 6 (strain # 000664) from The Jackson Laboratory (Bar Harbor, ME) were used. Intracranially injected animals were euthanized 4 weeks postinjection and perfused transcardially with phosphate-buffered saline (PBS).

[0102] Production and purification of AA V, EV-AA V and mEV-AA V vectors

[0103] AAV production was performed as described in Cheng et al., Hum Gene Ther. (32)1457-1470 (2021). Briefly, HEK293T cells were triple transfected using PEI MAX solution (Polysciences, Warrington, PA) with (1) rep / cap plasmid (pAR9), (2) an adenovirus helper plasmid, pAdAF6, and (3) ITR- flanked AAV transgene expression plasmid (either AAV-CAG-tdTomato or AAV-CBA- GFP or AAV-CBA- Fluc, all encoding single-stranded AAV genomes). Conventional AAV was purified from cell lysates 68-72 hours post-transfection by ultracentrifugation of an iodixanol density gradient. Iodixanol was removed, and buffer was exchanged to phosphate buffered saline (PBS) containing 0.001% v / v Pluronic F68 (Gibco, Grand Island, NY) using 7-kDa molecular weight cutoff Zeba desalting columns (Thermo Scientific). Vector was concentrated using Amicon Ultra-2 100-kDa MWCO ultrafiltration devices (Millipore Sigma). AAV vectors were stored at -80°C until use. For the production of extracellular vesicle enveloped-AAV (EV-AAV and mEV-AAV), supernatants from HEK293T and HEK293T-APTDSS1 cells were collected, and a series of differential-centrifugations were performed to separate cells, apoptotic bodies, and large microvesicles from the media. Sequential centrifugations were performed at 300 g for 5 min, 1,000 g for 10 min, and 20,000 g for 1 hour (h). For each step, the media was recovered and transferred to new tubes. Media was treated with Benzonase (25 U / mL with 2 mM MgCl ) for 1 h at 37°C as described in Gy orgy et al., Biomaterials. (35)7598-7609 (2014). Benzonase-treated media was concentrated using 100 kDA NMWL Amicon® Ultra- 15 columns (Millipore Sigma) for downstream purification using density gradients or size exclusion chromatography (see, Cheng et al., Hum Gene Ther. (32)1457-1470 (2021)).

[0104] Density gradient purification of EV-AA V vectors

[0105] 40%, 25%, and 10% v / v iodixanol solutions were dissolved in PBS using the 60%w / v Iodixanol OptiPrep TM (AXS-1114542 Cosmo Bio USA) as described in Cheng et al., Hum Gene Ther. (32)1457-1470 (2021). Gradients were prepared with increased density top to bottom as follows 15 ml of concentrated media containing enveloped-AAV, 9 ml 10% iodixanol, 6 ml 25% iodixanol, 5 ml 40% iodixanol, and 5 ml of 60 %w / v Iodixanol OptiPrep™. Enveloped-AAV were isolated in the 10% and 25% iodixanol phases after ultracentrifuge for 250,000 g at 4°C during 3 h. Iodixanol was exchanged for PBS using 7K MWCO Zeba™ spin columns. Enveloped-AAV vectors were stored at -80°C until use.

[0106] Size-exclusion purification of EV-AAV vectors

[0107] Size-exclusion column chromatography (SEC) was performed using qEVoriginal (35 nm) columns attached to the Automatic Fraction Collector (both from Izon Science, Ltd., Medford, MA) according to the manufacturer’s instructions. In brief, 0.5 mL of the concentrated media was applied to the PBS -equilibrated column, and 12, 0.5 mL fractions were collected. EVs eluted first in fractions 1 to 4 together with enveloped-AAV (see, Cheng et al., Hum Gene Ther. (32)1457-1470 (2021)). Smaller biomolecules, including free AAV, eluted later in fractions 5 to 12. All fractions 1 to 4 were pooled and used as the EV-AAV fraction as Characterized in Cheng et al., Hum Gene Ther. (32)1457-1470 (2021). EV-AAV fractions were stored at -80°C until use.

[0108] Vector quantitation

[0109] Enveloped-AAV vectors (EV-AAV and mEV-AAV) were treated with DNAse I for 1 h at 37°C. DNAse I was inactivated after treating the samples at 75°C for 15 min. AAV genomes were purified from the envelope-AAV samples using the High Pure Viral Nucleic Acid Kit (Roche, Indianapolis, IN). For all AAV-based vectors, the vector genomes were quantified using a Taqman qPCR in an ABI Fast 7500 Real-time PCR system (Applied Biosystems) using probes and primers to the ITR sequence and interpolated from a standard curve made with a restriction enzyme linearized AAV plasmid as described in Bennett et al., Sci Transl Med. 4, (2012).

[0110] Anti-AA V neutralization assay

[0111] In vitro neutralization assays were performed with Gamunex-C purified intravenous immunoglobulin (IVIg), (Grifols, Barcelona, Spain). HeLa cells were seeded at 10,000 cells per well in a 96-well plate the day before the assay. Next, 109vg of AAV9-Fluc, EV-AAV9-Fluc, and mEV-AAV9-Fluc vectors were mixed with serial dilutions of IVIg in FBS-free media. Vector samples mixed with media in the absence of IVIg served as control. The vectors were incubated with IVIg for 1 h at 37°C and then vector / IVIg complexes were incubated with the cells for 1.5 h at 37°C. After washing the cells once and replacing them with a complete medium, cells were incubated for 48 h before performing a luciferase assay using Bright-Glo™ Luciferase reagent (Promega, Madison, WI). A BioTek Synergy HTX multimode luminometer (BioTex, Winooski, VT) was used to detect luminescence. Relative light units (RLU) were normalized to the average RLU value in the no IVIg for each treatment. Values were expressed as the percentages of transduction relative to the no IVIg group.

[0112] Lipid extraction and lipidonucs

[0113] 20 million cells of HEK293T and HEK293T-APTDSS1 were cultured in 15 cm cell culture dishes. Three independent plates were utilized for each cell line. For each 15-cm dish, the spent media was aspirated and added 5 ml of lysis buffer 10 mM Tris ImM EDTA pH 7.4 (Millipore Sigma). Cells were lysed by centrifugation at 1,200 g for 10 min. Cell lysates were centrifuged at 25,000 g for 10 min. Pellets containing cellular membranes were resuspended in PBS. We added 18: 1 -d7- cholesterol and 15:0-18: l-d7-PC as internal standards for absolute quantification at a final concentration of 1 ng / pl. Lipids were extracted from membranes using the modified Bligh and Dyer Method (Bligh & Dyer, Can J Biochem Physiol. 37, (1959)), as previously reported for HEK293T cells in Dawaliby et al., J Biol Chem. 291, 3658- 3667 (2016). Briefly, 2 ml of a freshly prepared lipid extraction solution of chloroform and methanol (1 : 1 v / v) was added to the cell membranes pellet with 10 pl of 6N HC1. A Multi-Tube vortex was set to 2,500 rpm for 10 min and centrifuged the tubes 300 g for 10 min before recovering the chloroform phase. The extraction was performed the three times. LC-MS was used to determine the lipid profile and lipid abundance for each sample. LC-MS in the negative mode was run to determine the abundance of glycerophospholipids and sphingolipids. LC-MS in the positive mode was run to determine the abundance of cholesterol in lipid extracts from cells. For the enveloped-AAV (EV-AAV and mEV-AAV), we performed the lipid extraction as before and used the Amplex™ Red Cholesterol Assay Kit (Thermo Scientific) to quantify cholesterol abundance. The relative abundance of cholesterol in the samples was determined by normalizing the concentration of cholesterol in the analyzed samples by the relative abundance of extracellular vesicles determined with the exosome- membrane labeling green PKH67 fluorescent cell linker kit (Sigma Aldrich). Furthermore, LC-MS in the negative mode was run after performing the lipid extraction as previously described on the enveloped-AAV samples.

[0114] In vitro transduction assays

[0115] 3.5xl06AC16 or HEK293T cells were plated in 12-well plates. One day later, we used a dose of 5.4xl04vg / cell of AAV9-CAG-tdTomato, or the density-gradient purified enveloped-AAV variants (EV-AAV and mEV-AAV). Three days after transduction, we recovered the cells and immediately performed flow cytometry to quantify the percentage of transduced cells per treatment. We used cells with no vector to set the tdTomato fluorescence threshold. Stereotaxic injection in mice

[0116] Adult mice were anesthetized using isoflurane, and analgesia was achieved with buprenorphine (0.15 mg / kg) and local scalp administration of lidocaine (5 mg / kg). Once deeply anesthetized, mice were placed into a Just For Mouse™ Stereotaxic Frame with an integrated animal warming base (Stoelting, Wood Dale, IL). Adult C57BL / 6 mice (n = 3 / group) were bilaterally injected into the striatum using 1.3xl09vg / hemisphere (2.6xl09vg / animal). We used the following coordinates from bregma in mm: anterior / posterior, AP +0.5; medial / lateral, ML +2.0; dorsal / ventral, DV -2.5. Vectors were infused at a rate of 0.2 mL / min using a Quintessential Stereotaxic Injector pump (Stoelting) to drive a gas-tight Hamilton Syringe (Hamilton, NV) attached to a 10 mL 33G NEUROS model syringe (Hamilton, NV). After injection, the needle was left in place for 2 min to allow the vector solution to disperse and not backflow up the cannula. Buprenorphine (0.15 mg / kg) was injected subcutaneously twice a day for 2 days after the surgery for pain control.

[0117] Tissue harvest and analyses

[0118] Four weeks after brain surgeries, mice were deeply anesthetized with an overdose of ketamine / xylazine and transcardially perfused with PBS. Brains were harvested and cut sagittally into two hemispheres. For each brain, one hemisphere was postfixed in 4% formaldehyde in PBS for 2 days, 30% w / v sucrose solution was used as a cryoprotectant, and the second hemisphere was dissociated using 2 ml screw-cap tubes (BioExpress, cat no H-6110-10) containing 1.4 mm diameter ceramic beads (Mo Bio, cat 13113-325). Post-fixed brains were embedded, cryo-sectioned, and imaged. Briefly, coronal floating sections (50 pm) were cut using a Microm HM430 (Thermo Fisher Scientific, Waltham, MA). All sections were collected in rostral-to- caudal serial order in phosphate buffer (0.1 M, pH 7.2). A stereotaxic mouse brain atlas (Paxinos and Franklin, 2001) was used to identify the structures within sections. The most rostral section corresponded to stereotaxic coordinates interaural = 5.58 mm and bregma = 1.78 mm, and the most caudal section to stereotaxic coordinates interaural = 1.00 mm and bregma = - 2.80 mm in the atlas. Each section was stained with DAPI (Thermo Fisher Scientific) and mounted with Vestashield mounting medium (Vector Laboratories, Burlingame, CA). Imaging was performed with a Keyence BZ-X800 microscope (KEYENCE Corporation of America, Itasca, IL). Statistics

[0119] GraphPad Prism 9.0 was used for PC for statistical analysis. We performed unpaired two-tailed t-tests to compare differences between the mean values of HEK293T and HEK293T-APTDSS1 datasets, with p values < 0.05 considered significant. We used ANOVA and a post hoc Tukey test to compare vector treatments.

[0120] Example 1. Metabolic engineering targeting the lipid composition of the plasma membrane of 293T cells.

[0121] It was hypothesized that metabolic engineering targeting the lipid composition of the plasma membrane of 293T cells would generate a cell line with the ability to produce membrane-engineered EV (mEV), including EV- AAV (mEV-AAV). We rationally chose the gene encoding for the enzyme phosphatidyl serine synthase I, and deleted it via CRISPR-Cas9. The engineered cell line HEK293T Aptdssl was confirmed via sequencing.

[0122] Lipidomics showed several significant alterations of membrane lipids in the knock-out cell line relative to the conventional HEK293T cell line. FIG. 1A. shows a heat map with the fold-change of lipid species abundance, and FIG. IB shows a volcano plot with significant (p<0.0005) changes in lipid species. FIG. 1C lists the top 10 most significantly altered lipid species in KO-PTDSS1 relative to conventional 293T cells. As shown in FIG. 2A, cholesterol was significantly decreased in KO- PTDSS1. On the other hand, the abundance of phospholipids classes did not change, producing an increase in the ratio of Phospholipids / Cholesterol (FIG. 2B). The abundance of phospholipids relative to cholesterol, or the ratio PL / CHOL (see FIG. 2C) can produce membranes with different properties (e.g., fluidity, thickness, permeability, membrane curvature) within the cell.

[0123] Example 2. Production and characterization of mEV-AAV compared to EV-AAV.

[0124] Membrane-engineered EV (mEV)-associated AAV (mEV-AAV) were produced from the engineered HEK293T cells described in Example 1 using methods as described in US2013 / 0202559; Cheng et al., Hum Gene Ther. 2021 Dec;32(23- 24): 1457-1470. 5 days after transfection of AAV plasmids the mEV-AAV were collected. However, the membrane-engineered vesicles themselves (mEV) were produced within the first day as long as the cells were alive. Fractions 1-4 (F1-F4) from the SEC column corresponds to the EV-AAV fraction, while F5-F12 from the SEC column corresponds to the free AAV fraction. When the supernatant of 293 T cell line producing AAV was processed via SEC, as shown in FIG. 3A the amount of EV-AAV (SEC Fl to F4) was significantly lower compared to free AAV (SEC F5 to Fl 2). As shown in FIG. 3B, when the supernatant of 293 T KO-PTDSS1 cell line producing AAV was processed via SEC and the amount of EV-AAV (SEC Fl to F4) was significantly higher compared to free AAV (SEC F5 to F12). As shown in FIG. 3C, the engineered AAV producer KO-PTDSS1 cell line produced 8-times more EV- AAV than free AAV in the supernatant, a significant difference from the parental 293T cells.

[0125] Tetraspanin counts in EV-AAV9 and mEV-AAV9 were assessed via singleparticle interferometric reflectance. As shown in FIG. 4A, APTDSS1 produced mEV- AAV9 particles with an overall larger abundance of CD63, CD81, and CD9 tetraspanins relative to EV-AAV9. As shown in FIG. 4B, the abundance of the CD9 tetraspanin was increased in the engineered mEV-AAV9 particles relative to conventional EV-AAV9. lodixanol gradients were used to purify AAV-enclosed vesicles from empty vesicles before assessment. Finally, cholesterol content was quantified in both types of enveloped vectors using fluorescent dyes for cholesterol and for the total lipids in the vesicles enclosing AAV. The engineered mEV-AAV9 vector showed a 2.4-fold greater relative abundance of cholesterol per particle compared to conventional EV-AAV9 (FIG. 4C).

[0126] Example 3. Transduction properties of mEV-AAV in vitro

[0127] 10,000 cells were seeded in 96-well plates and treated with 5.4xl04vg / cell of AAV-CAG-tdTomato packaged in non-enveloped AAV9 capsid, conventional enveloped EV-AAV9 and membrane-engineered enveloped AAV9 (mEV-AAV9). Four days post-transduction, the percentage of transduced cells was determined using flow cytometry. As shown in FIG. 5A, the human AC 16 cardiomyocyte cell line showed an 8.5-fold greater percentage of transduction after mEV-AAV9 treatment compared to the AAV9 capsid. Transduction of engineered mEV-AAV9 was 1.7-fold higher compared to conventional EV-AAV9 (p=0.034). The percentage of transduced human HEK293T cells indicated that enveloped vector variants mEV-AAV9 and EV- AAV9 transduced 13- and 1.6-fold more cells compared to the non-enveloped conventional AAV9 capsid, respectively, as shown in FIG. 5B. Example 4. EV-AAV derived from PTDSS1 mutant showed enrichment of cholesterol and other membrane lipids.

[0128] We characterized the lipid composition differences between the EV-AAV particles released from HEK293T and HEK293T-APTDSS1. Lipidomics revealed an enrichment of several lipid species in the EV-AAV particles derived from the APTDSS1 cell line (FIG. 6A). Specifically, four lipid species had over 70-fold enrichment compared to conventional EV-AAV derived from HEK293T. Given the remarkable differences in the composition of the membrane lipids in EV-AAV derived from EEK-293 T-APTDSS1, we refer hereafter to these particles as mEV- AAV.

[0129] Among the several alterations in the relative abundance of lipid species, we detected phosphatidylserine (16:0 / 23:6) only in the mEV-AAV vectors. Other heterogenous PS species, with one saturated and one unsaturated fatty acid, were enriched more than 70-fold (FIG. 6B), suggesting that the deletion of PTDSS1 produced alterations in the flux of adjacent PS biosynthesis pathways.

[0130] Example 5. Membrane-engineered EV-AAV showed resistance against AAV-neutralizing antibodies.

[0131] To determine the resistance of mEV-AAV9 against neutralizing antibodies, we used an in-vitro neutralization assay using methods as described in Cheng et al., Hum Gene Ther. 2021 Dec;32(23-24): 1457-1470. We tested the ability of AAV9, EV- AAV9, and mEV-AAV9 to transduce cells in the presence of different concentrations of intravenous immunoglobulins (IVIg). Both EV-AAV9 and mEV-AAV9 showed resistance against the highest tested concentration of IVIg relative to naked AAV (FIG. 7). For example, at a 1 :250 dilution of IVIg, conventional AAV9 displayed only 13.4% resistance to neutralization, whereas mEV-AAV9 displayed 50% resistance (FIG. 7).

[0132] Example 6. Intracranial injection of membrane-engineered EV-AAV resulted in robust transduction in various brain regions of adult mice.

[0133] We performed intraparenchymal bilateral injections into the striatum of adult C57BL / 6 mice using the lipid-modified mEV-AAV9-CBA-GFP vector. In brief, adult mice were anesthetized using isoflurane, and analgesia was achieved with buprenorphine (0.15 mg / kg) and local scalp administration of lidocaine (5 mg / kg). Once deeply anesthetized, mice were placed into a Just For Mouse™ Stereotaxic Frame with an integrated animal warming base (Stoelting, Wood Dale, IL). Adult C57BL / 6 mice (n =3 / group) were bilaterally injected into the striatum using 1.3xl09vg / hemisphere (2.6xl09vg / animal). We used the following coordinates from bregma in mm: anterior / posterior, AP +0.5; medial / lateral, ML +2.0; dorsal / ventral, DV -2.5. Vectors were infused at a rate of 0.2 mL / min using a Quintessential Stereotaxic Injector pump (Stoelting) to drive a gas-tight Hamilton Syringe (Hamilton, NV) attached to a 10 mL 33G NEUROS model syringe (Hamilton, NV). After injection, the needle was left in place for 2 min to allow the vector solution to disperse and not backflow up the cannula. Buprenorphine (0.15 mg / kg) was injected subcutaneously twice a day for 2 days after the surgery for pain control.

[0134] Robust transduction in the brain parenchyma was observed four weeks postinjection. Transduction of cells with the morphology of neurons was observed in different brain areas, including cortical white matter, dorsal striatum (FIG. 8A), fimbria, and internal capsule (FIG. 8B). Furthermore, mEV-AAV9 transduction was present across various coronal sections spanning the most rostral (interaural = 5.58 mm, bregma = 1.78 mm) and most caudal (interaural = 1.00 mm, bregma = -2.80 mm) sections (FIG. 8C).

[0135] OTHER EMBODIMENTS

[0136] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. An engineered cell comprising a mutation in a gene encoding phosphatidylserine synthase 1 (PTDSS1), wherein the mutation abrogates production of functional PTDSS1 protein, and wherein the cell further comprises at least one additional transgene.

2. The engineered cell of claim 1, wherein the at least one additional transgene is in a viral vector.

3. The engineered cell of claim 2, wherein the viral vector is an adeno-associated vector (AAV), adenovirus, retrovirus, baculovirus, or lentivirus.

4. The engineered cell of any one of claims 1-3, wherein the engineered cell comprises at least one phosphatidylcholine, at least one phosphatidylserine, at least one phosphatidylethanolamine, at least one phosphatidylinositol, at least one cholesterol, at least one sphingomyelin or other sphingolipid, at least one plasmalogen, or any combination thereof.

5. The engineered of any one of claims 1-4, wherein the engineered cell comprises:(i) at least one phosphatidylcholine in an amount ranging from about 15% to about 25% of total cell lipids;(ii) at least one phosphatidyl serine in an amount ranging from about 5% to about 20% of total cell lipids;(iii) at least one phosphatidylethanolamine in an amount ranging from about 20% to about 30% of total cell lipids;(iv) at least one phosphatidylinositol in an amount ranging from about 15% to about 20% of total cell lipids;(v) at least one cholesterol in an amount ranging from about 1% to about 20% of total cell lipids;(vi) at least one sphingomyelin or other sphingolipid in an amount ranging from about 1% to about 10% of total cell lipids; and / or(vii) at least one plasmalogen in an amount ranging from about 15% to about 25% of total cell lipids.

6. A method of producing a population of membrane-engineered extracellular vesicles (mEVs), the method comprising: maintaining an engineered cell comprising a mutation in a gene encoding phosphatidylserine synthase 1 (PTDSS1), wherein the mutation abrogates production of functional PTDSS1 protein, in culture media, and isolating extracellular vesicles from the media, thereby producing a population of mEVs.

7. The method of claim 6, wherein the extracellular vesicles are isolated from the media without lysing the cell.

8. The method of claim 6, wherein the cell further comprises at least one additional transgene.

9. The method of claim 8, wherein the at least one additional transgene is in a viral vector, optionally wherein the viral vector is an adeno-associated vector (AAV), adenovirus, retrovirus, baculovirus, or lentivirus.

10. The method of claim 9, wherein the population of mEVs comprise the transgene or a protein product of the transgene.

11. The method of claim 10, wherein the population of mEVs comprise the viral vector.

12. A composition comprising a population of membrane-engineered extracellular vesicles (mEVs) produced by the method of any one of claims 6-11.

13. The composition of claim 12, wherein the mEVs have increased cholesterol as compared to EVs isolated from a parental cell expressing functional PTDSSl / without the mutation in PTDSS1.

14. The composition of claim 12, wherein the mEVs have a lipid profile as shown in FIG. 6.

15. The composition of any one of claims 12-14, wherein the mEVs comprise PS(16:0_23:6).

16. The composition of any one of claims 12-15, comprising a population of mEV-enveloped viral vectors produced by the method of claims 6-11.

17. The composition of claim 16, wherein the viral vectors are AAV (mEV-AAV).

18. The composition of claim 17, wherein at least one mEV-AAV is resistant to neutralization by a neutralizing antibody.

19. A method of delivering a selected nucleic acid or protein to a target cell, the method comprising: providing an engineered cell comprising (i) a mutation in a gene encoding phosphatidylserine synthase 1 (PTDSS1), wherein the mutation abrogates production of functional PTDSS1 protein, and (ii) a transgene encoding the selected nucleic acid or protein; maintaining the cells in culture media, isolating extracellular vesicles (EVs) from the media comprising the selected nucleic acid or protein, and contacting the target cell with the EVs thereby delivering the selected nucleic acid or protein to a target cell.

20. A method of delivering an AAV comprising a selected nucleic acid to a target cell, the method comprising: providing an engineered cell comprising (i) a mutation in a gene encoding phosphatidylserine synthase 1 (PTDSS1), wherein the mutation abrogates production of functional PTDSS1 protein, and (ii) transgenes encoding at least one component of the AAV, wherein the at least one component of the AAV comprises rep and / or cap, and the selected nucleic acid; maintaining the cells in culture media, isolating extracellular vesicles (EVs) from the media comprising the AAV, and contacting the target cell with the EVs, thereby delivering the AAV comprising the selected nucleic acid to a target cell.

21. The method of claim 19 or 20, wherein the target cell is a brain cell.

22. The method of claim 21, wherein the brain cell is in the brain of a living subject, preferably a mammal.

23. The method of claim 19 or 20, wherein the target cell is a heart cell, optionally wherein the heart cell is a cardiomyocyte.

24. The method of claim 21, wherein the heart cell is in the heart of a living subject, preferably a mammal.

25. The cell, method, or composition of any of the preceding claims, wherein the engineered cell is made from an HEK293T, HeLa, Sf9 / 21 , CHO, BHK-21, Vero parental cell lines, and yeast cells.

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