Extracellular vesicles functionalized with ERV scintillins and their use for cargo delivery

Abstract

EVs have been recognized as vectors for drug delivery. In particular, loading EVs with targeting and therapeutic agents offers intriguing opportunities for converting EVs into biomimetic selective delivery systems. Indeed, EVs constitute physiological carriers that are potentially less immunogenic than artificial delivery vehicles. Here, we developed a novel method for on-demand controlled cargo loading into EVs. These EVs are equipped with nonviral fusogenic factors, as needed, thus facilitating the delivery of EV-cargo to acceptor cells. To sensitively measure this process, we tracked the fate of luciferase-tagged cargo. Cargo loading was enabled by a drug-reversible, inducible dimerization system. Briefly, donor cells were transfected with plasmids encoding FKBP-tagged CD63, a classical membrane EV marker, and FRB-Nanoluciferase (NLuc), a normally cytoplasmic protein. Upon addition of a dimerizer, FRB-Nluc interacts with FKBP-CD63 and is recruited to secreted EVs, which promotes delivery to acceptor cells. This phenomenon can be further enhanced if EVs are equipped with syncytin 1, a mammalian fusogenic protein that induces fusion between the EV membrane and the plasma membrane of acceptor cells. Using this novel method, we further demonstrated that the catalytic domain of diphtheria toxin (DTA), which is involved in protein synthesis inhibition and ultimately cell death, can be delivered to acceptor cells via functionalized EVs. This resulted in protein synthesis inhibition and death of the acceptor cells. This novel method and its resulting applications are expected to open new doors in precision medicine, especially when EVs are equipped with antibodies raised against cell-specific antigens.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention is in the field of medicine, in particular the field of cargo delivery to target cells.

[0002] Background of the Invention Extracellular vesicles (EVs) are now recognized as vectors of intercellular communication that can transfer nucleotides, lipids, and proteins from donor cells to acceptor cells (Skog, J. et al. Glioblastoma microvesicles transport RNA and proteins that promote tumor growth and provide diagnostic biomarkers. Nat. Cell Biol. 10, 1470-1476 (2008); Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654-659 (2007); Flaherty, SE et al. A lipase-independent pathway of lipid release and immune modulation by adipocytes. Science (80-. ). 363, 989-993 (2019); Al-Nedawi, K. et al. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumor cells). Nat. Cell Biol. 10, 619-24 (2008)). EV-mediated communication is associated with many physiological and pathophysiological functions, including cancer, immune response, cardiovascular disease, lipid homeostasis, regeneration, and stem cell-based therapy (Mathieu, M., Martin-Jaular, L., Lavieu, G. & Thery, C. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Nat. Cell Biol. 21, 9-17 (2019)).The range of tissues / cells that can release or capture EVs is broad, including neurons, adipocytes, and immune cells.

[0003] Therefore, EVs are generally recognized as physiologically important vectors and are considered promising candidates for transduction applications, such as targeted drug delivery. In particular, loading EVs with targeting and therapeutic agents offers intriguing opportunities for converting EVs into biomimetic selective delivery systems. Indeed, EVs constitute physiological carriers that are potentially less immunogenic than artificial delivery vehicles. EVs may advantageously alter the pharmacokinetics, biodistribution, and bioavailability of cargoes by (i) protecting them, (ii) directing them to the target site, and (iii) facilitating membrane transport (Murphy, DE et al. Extracellular vesicle-based therapeutics: natural versus engineered targeting and trafficking. Exp. Mol. Med. 51, 32 (2019)). Ultimately, the use of EVs or chemically formulated EV mimics to deliver therapeutic agents (including gene-editing toolboxes) to specific cells in the body will revolutionize cell and gene therapy.

[0004] Summary of the Invention The present invention is defined by the claims. In particular, the present invention relates to extracellular vesicles functionalized with ERV scintillin and their use for cargo delivery.

[0005] Detailed Description of the Invention Key Definitions As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. This term also encompasses amino acid polymers that have been modified, such as by disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling moiety. When discussed in the context of gene therapy, a polypeptide refers to an intact individual polypeptide or a fragment or genetically engineered derivative thereof that retains the desired biochemical function of the intact protein.

[0006] As used herein, the term "polynucleotide," as used herein, refers to a polymer of nucleotides of any length, comprising ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-, and single-stranded ribonucleic acid ("RNA"). The term also includes modified forms of polynucleotides, for example, by alkylation and / or capping, as well as unmodified forms of polynucleotides. In particular, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), spliced ​​or unspliced ​​polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA, any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing non-nucleotide backbones, such as polyamides (e.g., peptide nucleic acids, "PNAs") and polymorpholino polymers, and other sequence-specific synthetic nucleic acid polymers, provided that the polymer contains nucleobases in an arrangement that allows for base pairing and base stacking as found in DNA and RNA. In some embodiments, a polynucleotide includes an mRNA. In other aspects, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA contains at least one unnatural nucleobase. In some embodiments, all nucleobases of a particular class are replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein may be replaced with unnatural nucleobases, such as 5-methoxyuridine). In some embodiments, a polynucleotide (e.g., synthetic RNA or synthetic DNA) contains only natural nucleobases, i.e., A, C, T, and G in the case of synthetic DNA, or A, C, T, and U in the case of synthetic RNA.

[0007] As used herein, the term "encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for synthesizing other polymers and macromolecules in biological processes having either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing) and the non-coding strand (used as a template for transcription of the gene or cDNA) can be said to encode the protein or other product of that gene or cDNA. Unless otherwise specified, "polynucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "polynucleotide sequence encoding a protein or RNA" can also include introns, to the extent that some versions of nucleotide sequences that encode proteins may contain introns.

[0008] As used herein, the phrase "derived from" refers to a process used to isolate, derive, or generate a first component (e.g., a first polypeptide) or a second component that differs in information from the first component (e.g., a second polypeptide that differs from the first polypeptide).

[0009] As used herein, the "% identity" between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences (i.e., % identity = number of identical positions / total number of positions × 100). Comparison of sequences and determination of the % identity between two sequences can be achieved using a mathematical algorithm, as described below. The % identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53). The % identity between two nucleotide sequences or two amino acid sequences can also be determined using algorithms such as EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle can be used with a BLOSUM62 matrix, a "gap open penalty" of 10, a "gap extend penalty" of 0.5, a false "end gap penalty", an "end gap open penalty" of 10, and an "end gap extend penalty" of 0.5. Generally, "% identity" is a function of the number of matched positions divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 sequence positions are identical between two compared sequences after alignment, the identity is 60%. % identity is typically determined over the entire length of the query sequence being analyzed. Two molecules with the same primary amino acid or nucleic acid sequence are identical, regardless of any chemical and / or biological modifications.According to the present invention, a first amino acid sequence having at least 70% identity to a second amino acid sequence means that the first sequence has 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the second amino acid sequence.

[0010] As used herein, the term "mutation" has its general meaning in the art and refers to a substitution, deletion, or insertion. In particular, the term "substitution" means that a particular amino acid residue at a particular position is removed and another amino acid residue is inserted in the same position. Mutations herein are referred to in accordance with standard mutation nomenclature.

[0011] As used herein, the term "ERV syncytin" has its common meaning in the art and refers to highly fusogenic envelope glycoproteins from placental mammals, which belong to the endogenous retrovirus (ERV) family. These proteins are encoded by genes that are preferentially expressed in the placenta and induce syncytia formation when introduced into cultured cells (Cornelis G, Heidmann O, Degrelle SA, Vernochet C, Lavialle C, Letzelter C, et al (2013). Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants. PNAS 110(9): E828-E837.).

[0012] As used herein, the term "syncytin-1" or "SYN" has its common meaning in the art and refers to a protein found in humans and other primates that is encoded by the ERVW-1 gene (endogenous retrovirus group W envelope member 1). Syncytin-1 is a cell-cell fusion protein whose function is best characterized in placental development. This term is also known as endogenous retrovirus group W member 1, Env-W, envelope polyprotein gPr73, enbelin, HERV-7q envelope protein, HERV-W envelope protein, HERV-W_7q21.2 proviral ancestral Env polyprotein, and syncytin. An exemplary amino acid sequence for syncytin-1 is set forth in SEQ ID NO:1. The signal peptide spans from amino acid residue 1 to amino acid residue 20 in SEQ ID NO:1. The extracellular domain of syncytin-1 ranges from amino acid residue 21 to amino acid residue 443 in SEQ ID NO:1.

[0013] [ka]

[0014] As used herein, the term "ASCT1" refers to human neutral amino acid transporter A, encoded by the SLC1A4 gene. Syncytin-1 can bind to ASCT1 (Antony JM, Ellestad KK, Hammond R, Imaizumi K, Mallet F, Warren KG, Power C. The human endogenous retrovirus envelope glycoprotein, syncytin-1, regulates neuroinflammation and its receptor expression in multiple sclerosis: a role for endoplasmic reticulum chaperones in astrocytes. J Immunol. 2007 Jul 15;179(2):1210-24. doi: 10.4049 / jimmunol.179.2.1210. PMID: 17617614).

[0015] As used herein, the term "ASCT2" refers to the neutral amino acid transporter B(0) encoded by the SLC1A5 gene. ASCT2 has been described as a receptor for syncytin-1 (Blond JL, Lavillette D, Cheynet V, Bouton O, Oriol G, Chapel-Fernandes S, Mandland B, Mallet F, Cosset FL. An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor. J Virol. 2000;74:3321-3329. doi: 10.1128 / JVI.74.7.3321-3329.2000.).

[0016] As used herein, the term "syncytin-1 polypeptide" or "SYN polypeptide" refers to any polypeptide derived from syncytin-1 and containing the conserved motif SDGGGX2DX2R (SEQ ID NO: 19), which is essential for syncytin-1-hASCT2 interaction (see Cheynet V, Oriol G, Mallet F. Identification of the hASCT2-binding domain of the Env ERVWE1 / syncytin-1 fusogenic glycoprotein. Retrovirology. 2006 Jul 4;3:41. doi: 10.1186 / 1742-4690-3-41. PMID: 16820059; PMCID: PMC1524976). According to the present invention, the syncytin-1 polypeptide is capable of binding to the ASCT1 receptor, preferably the ASCT2 receptor, as determined by any assay known in the art (see, for example, Cheynet V. et al., supra).

[0017] As used herein, the term "extracellular vesicle" or "EV" has its common meaning in the art and refers to a cell-derived vesicle that contains a membrane surrounding an internal space. Extracellular vesicles include all membrane-bound vesicles that have a diameter smaller than the cell from which they are derived. Typically, extracellular vesicles range in diameter from 50 nm to 1000 nm and can contain a variety of macromolecular cargoes either within the internal space, on the outer surface of the extracellular vesicle, and / or across the membrane.

[0018] As used herein, the term "functionalized" refers to the fact that the EVs of the present invention include a polypeptide of interest (e.g., the ERV syncytin of the present invention) in their membrane.

[0019] As used herein, the terms "isolated," "to isolate," "purified," "to purify," "enriched," and "enriching," when used herein with respect to cells, mean that at a certain point in time, the EVs have been separated and purified and made available for therapeutic use. "Highly purified," "highly enriched," and "highly isolated," when used with respect to the extracellular vesicles, refer to cells of interest that are at least about 70%, about 75%, about 80%, about 85%, about 90%, or more of the cells, about 95%, at least 99% pure, at least 99.5% pure, or at least 99.9% or more pure, and preferably, the EVs can be about 95% or more.

[0020] As used herein, the term "donor cells" refers to cells suitable for producing EVs of the present invention.

[0021] As used herein, the term "target cell" refers to a cell that is desired to fuse with an EV of the present invention.

[0022] As used herein, the term "cargo" as used herein refers to any molecule, e.g., nucleic acid, polypeptide, pharmaceutical, etc., with a desired biological activity and suitable solubility profile, that is packaged into a viral EV.

[0023] As used herein, the term "loading" refers to the introduction or insertion of a substance or object into or onto an EV of the present invention. As used herein, the term "loading" refers to the introduction or insertion of a substance or object into or onto an EV of the present invention.

[0024] As used herein, the term "targeting moiety" refers to any molecule that specifically binds to a target.

[0025] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen. In natural rodent and primate antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. Two types of light chains exist: lambda (I) and kappa (K). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. In a typical IgG antibody, the light chain contains two domains: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine binding recognition and specificity to the antigen. The constant region domains of the light chain (CL) and heavy chain (CH) confer important biological properties, such as antibody chain assembly, secretion, placental transport, complement fixation, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of the variable portions of one light chain and one heavy chain. Antibody specificity resides in the structural complementarity between the antibody-combining site and an antigenic determinant. The antibody-combining site is primarily composed of residues from the hypervariable or complementarity-determining regions (CDRs). In some cases, residues from non-hypervariable or framework regions (FRs) may participate in the antibody-combining site or influence the overall domain structure and, consequently, the binding site. Complementarity-determining regions or CDRs refer to amino acid sequences that define both the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, and L-CDR3, and H-CDR1, H-CDR2, and H-CDR3, respectively. Thus, an antigen-binding site typically contains six CDRs, including a set of CDRs from each of the heavy chain V region and the light chain V region. The framework region (FR) refers to the amino acid sequence intervening between the CDRs.Thus, light and heavy chain variable regions typically comprise four framework regions and three CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Residues in antibody variable domains are conventionally numbered according to the system devised by Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (Kabat et al., 1992, hereinafter "Kabat et al."). The Kabat residue designations do not necessarily correspond directly to the linear numbering of amino acid residues in SEQ ID NO: sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, corresponding to shortening of or insertion into structural elements, whether framework or complementarity-determining region (CDR) of the basic variable domain structure. The correct Kabat numbering of residues can be determined for a given antibody by aligning the homologous residues in the antibody sequence with the "standard" Kabat numbering sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2), and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2), and residues 89-97 (L-CDR3) according to the Kabat numbering system. For the antibodies listed below, the CDRs were determined using the CDR search algorithm at www.bioinf.org.uk. See the section entitled "How to identify the CDRs by looking at a sequence" on the antibody page.

[0026] As used herein, the term "antibody fragment" refers to at least a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody, that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). A "fragment" comprises a portion of an intact antibody, generally the antigen-binding site or the variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabody; and any antibody fragment that is a polypeptide having a primary structure consisting of a single continuous sequence of consecutive amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"). These include, but are not limited to, (1) single-chain Fv molecules, (2) single-chain polypeptides containing only one light-chain variable domain or fragments thereof containing the three CDRs of that light-chain variable domain and no associated heavy chain portion, and (3) single-chain polypeptides containing only one heavy-chain variable region or fragments thereof containing the three CDRs of that heavy-chain variable region and no associated light chain portion, and multispecific antibodies formed from antibody fragments. Fragments of the present antibodies can be obtained using standard methods.

[0027] As used herein, the terms "single domain antibody", "sdAb" or "VHH" refer to a single heavy chain variable domain of an antibody type that can be found in camelid mammals, which do not naturally have light chains. Such VHHs are also called "nanobodies®". According to the present invention, the sdAb may in particular be a llama sdAb.

[0028] As used herein, the term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are contiguously linked, e.g., via a synthetic linker, e.g., a short variable polypeptide linker, and can be expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, an scFv can have the VL and VH variable regions in either order; e.g., with respect to the N- and C-termini of the polypeptide, an scFv can comprise a VL-linker-VH or a VH-linker-VL.

[0029] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to a target molecule (e.g., an epitope displayed on an antigen) while having relatively little detectable reactivity with other target molecules. As described elsewhere herein, specificity can be determined relatively, for example, by binding assays or competitive binding assays using a Biacore instrument. Specificity can be exhibited, for example, by a ratio of affinity / avidity for binding to a specific antigen relative to nonspecific binding to other unrelated molecules of about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1 or greater.

[0030] As used herein, the term "affinity" refers to the strength with which an antibody binds to a target molecule (e.g., an epitope). The affinity of a binding protein is given by the dissociation constant, Kd. For an antibody, Kd is defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant, Ka, is defined by 1 / Kd. Preferred methods for determining the affinity of binding proteins can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Association and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entirety. One preferred and standard method well known in the art for determining the affinity of binding proteins is the use of a Biacore instrument.

[0031] As used herein, the term "binding" refers to a direct association between two molecules, e.g., by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions (including interactions such as salt bridges and water bridges). In particular, as used herein, the term "binding" in the context of an antibody binding to a given target molecule (e.g., an antigen or epitope) typically refers to a direct association between two molecules, e.g., by covalent, electrostatic, hydrophobic, and ionic bonding and / or hydrogen bonding interactions (including interactions such as salt bridges and water bridges). -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9 M or less, about 10 -10 M or less or about 10 -11 K below M D The binding has an affinity corresponding to

[0032] As used herein, the terms "subject," "host," "individual," or "patient" refer to a mammal, preferably a human being, male or female, of any age, in need of treatment.

[0033] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventive treatment and curative or disease-modifying treatment, including treatment of patients at risk of or suspected of having a disease, as well as patients suffering from or diagnosed with a disease or medical condition, including the suppression of clinical recurrence. The treatment can be administered to a patient with a medical disorder or a patient at risk of ultimately developing a disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurrent disorder, or to prolong the patient's survival beyond that expected in the absence of such treatment. A "therapeutic regimen" refers to a pattern of disease treatment, e.g., a dosing pattern used during treatment. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used for the initial treatment of a disease. The general purpose of an induction regimen is to provide a high level of drug to the patient during the initial period of the treatment regimen. An induction regimen may utilize (in part or in whole) a "loading regimen." A "loading regimen" may involve administering a larger amount of drug than a physician would use during a maintenance regimen, administering a drug more frequently than a physician would use during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or portion of a treatment regimen) used to maintain a patient during disease treatment, e.g., to keep the patient in remission for an extended period of time (months or years). Maintenance regimens may utilize continuous treatment (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent treatment (e.g., interruption of treatment, intermittent treatment, treatment upon relapse, or treatment upon reaching certain predetermined criteria (e.g., pain, disease manifestation, etc.)).

[0034] As used herein, the term "pharmaceutical composition" refers to a composition described herein or a pharmaceutically acceptable salt thereof, and includes other agents, such as carriers and / or excipients. The pharmaceutical compositions provided herein typically include a pharmaceutically acceptable carrier.

[0035] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, diluents or other liquid media, dispersing or suspending aids, surfactants, isotonicity adjusting agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, that are appropriate for the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used to formulate pharmaceutical compositions and known techniques for their preparation.

[0036] The extracellular vesicles of the present invention A first object of the present invention relates to isolated extracellular vesicles functionalized with the ERV syncytin, loaded with one or more cargoes of interest, and optionally functionalized with a targeting moiety.

[0037] ERV syncytin The ERV syncytins of the present invention can be selected from human syncytins (e.g., HERV-W and HERV-FRD), mouse syncytins (e.g., syncytin-A and syncytin-B), syncytin-Ory1, syncytin-Car1, syncytin-Rum1, or their functional orthologs (Cornelis G, Heidmann O, Degrelle SA, Vernochet C, Lavialle C, Letzelter C, et al (2013). Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants PNAS 110(9): E828-E837; Dupressoir A, Marceau G, Vernochet C, Benit L, Kanellopoulos C, Sapin V et al (2005). Syncytin-A and syncytin-B, two fusogenic placenta-specific murine envelope genes of retroviral origin conserved in Muridae). Proceedings of the National Academy of Sciences of the United States of America 102: 725-730).

[0038] By functional ortholog is intended an orthologous protein encoded by an orthologous gene and exhibiting fusogenicity. Fusogenicity may be assessed using a fusion assay as described in Dupressoir A, Marceau G, Vernochet C, Benit L, Kanellopoulos C, Sapin V et al (2005). Syncytin-A and syncytin-B, two fusogenic placenta-specific murine envelope genes of retroviral origin conserved in Muridae. Proceedings of the National Academy of Sciences of the United States of America 102: 725-730. Briefly, cells are cultured in, for example, Lipofectamine (Invitrogen) and 5×10 5 Approximately 1-2 μg DNA corresponding to 5 x 10 cells or calcium phosphate precipitate (Invitrogen, 5 Transfection is performed using 5-20 μg DNA (corresponding to 1000 cells). Plates are typically examined for cell fusion 24-48 hours after transfection. Fused cells can be visualized using May-Grunwald staining and Giemsa staining (Sigma), and the fusion index can be calculated as [(NS) / T] × 100, where N is the number of nuclei in the fused cells, S is the number of fused cells, and T is the total number of nuclei counted.

[0039] Human syncytins include HERV-W and HERV-FRD. Functional orthologs of these proteins can be found in the Hominidae family. HERV-W refers to a highly fusogenic membrane glycoprotein belonging to the human endogenous retrovirus (HERV) family. HERV-W is an envelope glycoprotein and is also called syncytin-1. HERV-W has a sequence listed in the Ensembl database ENST00000493463, corresponding to the transcript ERVW-1-001. HERV-FRD also refers to a highly fusogenic membrane glycoprotein belonging to the human endogenous retrovirus (HERV) family. HERV-FRD is an envelope glycoprotein and is also called syncytin-2. HERV-FRD has a sequence listed in the Ensembl database ENSG00000244476, corresponding to the transcript ERVFRD-1.

[0040] Mouse syncytins include mouse syncytin-A (i.e., Mus musculus syncytin-A, synA) and mouse syncytin-B (i.e., Mus musculus syncytin-B, synB). Functional orthologs of these proteins can be found in the Muridae family. Mouse syncytin-A is encoded by the syncytin-A gene. Syncytin-A has a sequence listed in the Ensembl database Syna ENSMUSG00000085957. Mouse syncytin-B is encoded by the syncytin-B gene. Syncytin-B has a sequence listed in the Ensembl database Synb ENSMUSG00000047977.

[0041] Syncytin-Ory1 is encoded by the syncytin-Ory1 gene. Functional orthologues of syncytin-Ory1 can be found in the Leporidae family (typically rabbits and hares).

[0042] Syncytin-Car1 is encoded by the syncytin-Car1 gene, whose functional orthologues can be found in carnivorous mammals of the superorder Laurasiana (Cornelis et al., 2012; Lavialle et al., 2013).

[0043] Syncytin-Rum1 is encoded by the syncytin-Rum1 gene. Functional orthologues of syncytin-Rum1 can be found in ruminant mammals.

[0044] In some embodiments, the ERV syncytin of the present invention can typically be selected from the group consisting of HERV-W, HERV-FRD, syncytin-A, syncytin-B, syncytin-Ory1, syncytin-Car1, and syncytin-Rum1, and their functional orthologs. Preferably, the ERV syncytin is selected from the group consisting of HERV-W, HERV-FRD, mouse syncytin-A, and their functional orthologs. More preferably, the ERV syncytin is selected from the group consisting of HERV-W, HERV-FRD, and mouse syncytin-A. Even more preferably, the ERV syncytin is HERV-W or HERV-FRD.

[0045] In some embodiments, the ERV syncytin is a syncytin-1 polypeptide.

[0046] In some embodiments, the syncytin-1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2 (SDGGGX2DX2R) and is capable of binding to the ASCT1 receptor, preferably the ASCT2 receptor.

[0047] In some embodiments, the syncytin-1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3 (SDGGGVQDQAR).

[0048] In some embodiments, the syncytin-1 polypeptide of the invention comprises the amino acid sequence set forth in SEQ ID NO:3 (SDGGGVQDQAR), and includes at least 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400 or 450 consecutive amino acids of SEQ ID NO:1.

[0049] In some embodiments, the syncytin-1 polypeptide of the present invention comprises an amino acid sequence that is 70% identical to the amino acid sequence ranging from amino acid residue 21 to amino acid residue 538 in SEQ ID NO:1. In some embodiments, the syncytin-1 polypeptide of the invention comprises an amino acid sequence ranging from amino acid residue 21 to amino acid residue 538 of SEQ ID NO:1, wherein the arginine residue at position 393 (R) and the phenylalanine residue at position 399 (F) are mutated to confer immunosuppressive activity (Mangeney M, Renard M, Schlecht-Louf G, Bouallaga I, Heidmann O, Letzelter C, Richaud A, Ducos B, Heidmann T. Placental syncytins: Genetic disjunction between the fusogenic and immunosuppressive activity of retroviral envelope proteins. Proc Natl Acad Sci U S A. 2007 Dec 18;104(51):20534-9. doi: 10.1073 / pnas.0707873105. Epub 2007 Dec 12. PMID: 18077339; PMCID: PMC2154466. In some embodiments, the syncytin-1 polypeptides of the invention comprise an amino acid sequence ranging from amino acid residue 21 to amino acid residue 538 of SEQ ID NO:1, wherein the arginine residue (R) at position 393 is replaced by a glutamine residue (Q) and the phenylalanine residue (F) at position 399 is replaced by an alanine residue (A).

[0050] cargo Typically, the cargo can be of any nature that is compatible with loading into an EV.

[0051] In some embodiments, the cargo is selected from the group consisting of organic molecules, polymers, polypeptides, polynucleotides, and small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Cargos are also found among biomolecules, including peptides, saccharides, fatty acids, lipids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

[0052] In some embodiments, the cargo includes chemotherapeutic agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modulators, and neuroactive agents. Exemplary pharmaceutical agents suitable for the present invention are those described in "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, NY, (1996), Ninth edition, in the following sections: Drugs Acting at Synaptic and Neuroeffector Junctions; Drugs Acting on the Central Nervous System; Autacoids: Drug Therapy for Inflammation; Water, Salts, and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Cardiovascular Agents; Drugs Affecting Gastrointestinal Function; Drugs Affecting Uterine Motility; Chemotherapy of Parasitic Infections; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting on Hematopoietic Organs; Hormones and Hormone Antagonists; Vitamins; Dermatology; and Toxicology, all of which are incorporated herein by reference. Also included are toxins and biological and chemical warfare agents. See, e.g., Somani, SM (Ed.), "Chemical Warfare Agents," Academic Press, New York, 1992.

[0053] In some embodiments, the cargo is a polynucleotide. In some embodiments, the polynucleotide is an RNA or DNA molecule.

[0054] In some embodiments, a polynucleotide is introduced into target cells of a tissue or organ and can be expressed under appropriate conditions or can otherwise confer beneficial properties to the cells. Thus, the polynucleotide is selected based on the desired therapeutic outcome. For example, the polynucleotide encodes a polypeptide that confers beneficial properties or a desired therapeutic outcome to the cells. Examples of polynucleotides of interest include, but are not limited to, those that encode polypeptides selected from the group consisting of protective polypeptides (e.g., neuroprotective polypeptides, such as GDNF, CNTF, NT4, NGF, and NTN); anti-angiogenic polypeptides (e.g., soluble vascular endothelial growth factor (VEGF) receptors; VEGF-binding antibodies; VEGF-binding antibody fragments (e.g., single-chain anti-VEGF antibodies); and anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-Xl).

[0055] In some embodiments, the polynucleotide encodes an antigen. As used herein, the term "antigen" has its general meaning in the art and generally refers to a substance or fragment thereof that is recognized by and selectively binds to an antibody or T-cell antigen receptor, thereby eliciting an immune response. Antigens of the present invention are typically, but not exclusively, peptides and proteins. Antigens in the context of the present invention may include any subunit, fragment, or epitope of any proteinaceous molecule, including proteins or peptides of viral, bacterial, parasitic, fungal, protozoan, prion, cellular, or extracellular origin, which ideally elicit an immune response in a mammal, preferably resulting in protective immunity. In some embodiments, the antigen is a tumor antigen. In particular, antigens may be selected from the following virus families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae (e.g., Norovirus (also known as "Norwalk-like virus")), Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., Coronaviruses, e.g., Severe Acute Respiratory Syndrome (SARS) virus or SARS-CoV-2), Cortivirus, and the like. coviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire strain, Reston strain, Ivory Coast strain, or Sudan strain)), Flaviviridae (e.g., hepatitis C virus, dengue virus 1, dengue virus 2, dengue virus 3, and dengue virus 4), Hepadnaviridae (e.g., hepatitis B virus or hepatitis C virus), Herpesviridae (e.g., human herpesvirus (HSV) 1, 2, 3, 4, 5, and 6, cytomegalovirus, and Epstein-Barr virus (EBV)), Hypoviridae,The peptide may be isolated from any virus, including (but not limited to) viruses from any of the following families: Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., influenza viruses A and B), Papovaviridae, Papillomaviridae (e.g., human papillomaviruses (HPV)), Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus (RSV)), Parvoviridae, Picornaviridae (e.g., polioviruses, rhinoviruses, hepatoviruses, and aphthoviruses (e.g., foot-and-mouth disease virus)), Poxviridae (e.g., vaccinia virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentiviruses, e.g., human immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae, and Totiviridae.

[0056] In some embodiments, the polynucleotide of the present invention is an RNA molecule, particularly a messenger RNA (mRNA). In some embodiments, the EV encapsulates one or more RNA molecules capable of: i) transferring one or more desired endogenous or exogenous coding sequences in the target cell; ii) transferring one or more non-coding RNAs, such as shRNA, miRNA, sgRNA, lncRNA, or circRNA, capable of inducing an effect on gene expression; iii) transferring messenger RNA-type or other cellular RNAs (such as miRNAs), subgenomic replicons of RNA viruses (such as HCV), or the complete genome of an RNA virus; iv) co-expression of endogenous or exogenous coding or non-coding sequences in the target cell; or vi) contributing to genome modification of the target cell by genome engineering systems such as the CRISPR system.

[0057] In some embodiments, the polynucleotide is an antisense or siRNA sequence that acts to reduce the expression of a target sequence. Antisense or siRNA nucleic acids are designed to specifically bind to RNA, resulting in the formation of RNA-DNA or RNA-RNA hybrids, which halt DNA replication, reverse transcription, or messenger RNA translation. Gene expression is reduced by various mechanisms. Antisense nucleic acids based on a selected nucleic acid sequence can disrupt the expression of the corresponding gene. Antisense oligodeoxynucleotides (ODNs) include synthetic ODNs with chemical modifications from native nucleic acids or nucleic acid constructs that express such antisense molecules as RNA. One or a combination of antisense molecules can be administered, and the combination may contain multiple different sequences. Antisense oligonucleotides are generally at least about 7 nucleotides in length, usually at least about 12 nucleotides, more usually at least about 20 nucleotides, and no more than about 500 nucleotides in length, usually no more than about 50 nucleotides, more usually no more than about 35 nucleotides. Here, this length is governed by factors such as inhibitory efficiency, specificity including the absence of cross-reactivity, and the like.

[0058] Also of interest are RNAi agents. RNAi agents are small ribonucleic acid molecules (also referred to herein as interfering ribonucleic acids), i.e., oligoribonucleotides, which exist in a duplex structure, e.g., two separate oligoribonucleotides hybridized to each other, or a single ribonucleotide that assumes a small hairpin formation to produce a duplex structure. Oligoribonucleotide refers to a ribonucleic acid that does not exceed about 100 nt in length, typically not exceeding about 75 nt in length. Here, in certain embodiments, the length is less than about 70 nt. When the RNA agent is a duplex structure of two separate ribonucleic acids hybridized to each other, e.g., siRNA, the length of the duplex structure typically ranges from about 15 to 30 bp, usually from about 15 to 29 bp. Here, in certain embodiments, lengths of about 20 to 29 bp, e.g., 21 bp, 22 bp, are of particular interest. When the RNA agent is a single ribonucleic acid duplex structure present in a hairpin formation, i.e., shRNA, the length of the hybridizing portion of the hairpin is typically the same as that provided above for siRNA-type agents or 4-8 nucleotides longer.

[0059] In some embodiments, the cargo is a polynucleotide encoding an endonuclease, a base editing enzyme, an epigenome editor, or a prime editor, as described herein below.

[0060] In some embodiments, the cargo is a polypeptide. Polypeptides of interest include biologically active proteins, such as transcription factors, proteins involved in signal transduction pathways, cytokines, chemokines, toxins, etc. Such polypeptides may include proteins not found in the target cell, proteins from a different species, or cloned versions of proteins found in the target cell.

[0061] A preferred target protein of the present invention will be a protein with the same post-translational modifications as found in the target cell, expressed in a manner that results in the same post-translational modifications as those found in the target cell. Such modifications include glycosylation or lipid-modified addition of coenzyme groups or formation of quaternary structures. Wild-type proteins corresponding to proteins found in mutant or deleted forms in the target cell will be most preferred. In some embodiments, the polypeptide is a membrane protein or a non-membrane protein. Non-limiting examples of membrane proteins include ion channels, receptor tyrosine kinases, such as PDGF receptors and SCF-R receptors (stem cell factor receptors or c-kit or CD117), G protein-linked receptors, such as adrenergic receptors. Non-limiting examples of non-membrane proteins include cytoplasmic proteins, such as actin, Ras, ERK1 / 2, and nuclear proteins, such as steroid receptors, histone proteins, or transcription factors.

[0062] In some embodiments, the cargo is an endonuclease that provides site-specific knockdown of gene function. For example, if a dominant allele encodes a defective copy of a gene that is a structural protein and / or provides normal function in the wild type, a site-specific endonuclease can be targeted to the defective allele to knock out the defective allele. In addition to knocking out the defective allele, the site-specific nuclease can also be used to stimulate homologous recombination with donor DNA encoding a functional copy of the protein encoded by the defective allele. Thus, for example, the methods of the present invention can be used both to deliver a site-specific endonuclease that knocks out the defective allele and to deliver a functional copy of the defective allele, thereby repairing the defective allele and thereby providing production of a functional protein.

[0063] In some embodiments, the DNA targeting endonuclease is a transcription activator-like effector nuclease (TALEN). TALENs are artificially produced by fusing a TAL effector ("TALE") DNA binding domain, such as one or more TALEs, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 TALEs, to a DNA modification domain, such as a FokI nuclease domain. Transcription activator-like effectors (TALEs) can be engineered to bind any desired DNA sequence (Zhang (2011), Nature Biotech. 29: 149-153). By combining an engineered TALE with a DNA cleavage domain, a restriction enzyme specific to any desired DNA sequence can be produced. These can then be introduced into cells, where they can be used for genome editing (Boch (2011) Nature Biotech. 29: 135-6 and Boch et al. (2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501). TALEs are proteins secreted by Xanthomonas bacteria. The DNA-binding domain contains a highly conserved, repeated sequence of 33-34 amino acids, excluding the 12th and 13th amino acids. These two positions are highly variable and show a strong correlation with specific nucleotide recognition. Therefore, they can be engineered to bind to desired DNA sequences (Zhang (2011), Nature Biotech. 29: 149-153). To produce TALENs, TALE proteins are fused to a nuclease (N), such as a wild-type or mutant FokI endonuclease. Multiple mutations have been made to FokI for use in TALENs.These improve, for example, cleavage specificity or activity (Cermak et al. (2011) Nucl. Acids Res. 39: e82; Miller et al. (2011) Nature Biotech. 29: 143-8; Hockemeyer et al. (2011) Nature Biotech. 29: 731-734; Wood et al. (2011) Science 333: 307; Doyon et al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotech. 25: 786-793 and Guo et al. (2010) J. Mol. Biol. 200: 96). The FokI domain functions as a dimer, requiring two constructs with unique DNA-binding domains to be properly oriented and spaced apart for a site in the target genome. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between two individual TALEN binding sites are thought to be important parameters for achieving high levels of activity (Miller et al. (2011) Nature Biotech. 29: 143-8). TALENs can be used in cells to create double-strand breaks at sites in target nucleic acids, such as genes. If the repair mechanism improperly repairs the break via non-homologous end joining, mutations can be introduced at the break site (Huertas, P., Nat. Struct. Mol. Biol. (2010) 17: 11-16). For example, improper repair can introduce frameshift mutations. Alternatively, foreign DNA can be introduced into cells together with TALENs. Depending on the sequence of the foreign DNA and the chromosomal sequence, this process can be used to modify a target gene via a homologous direct repair pathway, e.g., to correct a defect in the target gene, thereby allowing expression of the repaired target gene, or, e.g., to introduce such a defect into a wt gene, thereby reducing expression of the target gene.

[0064] In some embodiments, the DNA targeting endonuclease is a zinc finger nuclease (ZFN). Similar to TALENs, ZFNs comprise a DNA-modifying domain, such as a nuclease domain, e.g., a FokI nuclease domain (or a derivative thereof), fused to a DNA-binding domain. In the case of ZFNs, the DNA-binding domain comprises one or more zinc fingers, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 zinc fingers (Carroll et al. (2011) Genetics Society of America 188: 773-782 and Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160). A zinc finger is a small protein structural motif stabilized by one or more zinc ions. A zinc finger may, for example, comprise Cys2His2 and can recognize a sequence of approximately 3 bp. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides that recognize sequences of approximately 6, 9, 12, 15, or 18 bp. Various selection and modular assembly techniques are available for generating zinc fingers (and combinations thereof) that recognize specific sequences, including phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells. Zinc fingers can be engineered to bind a specific nucleic acid sequence. Criteria for designing zinc fingers to bind to a specific nucleic acid sequence are known in the art (Sera (2002), Biochemistry, 41:7074-7081; Liu (2008) Bioinformatics, 24:1850-1857). ZFNs using the FokI nuclease domain or other dimeric nuclease domains function as dimers. Therefore, a pair of ZFNs is required to target a non-palindromic DNA site. Two individual ZFNs must bind their nucleases to opposite strands of DNA with the appropriate spacing (Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10570-5).Like TALENs, ZFNs can also create DSBs in DNA that, when improperly repaired, for example via non-homologous end joining, can result in frameshift mutations and reduce target gene expression in cells.

[0065] In some embodiments, the DNA-targeting endonuclease is a CRISPR-associated endonuclease. In bacteria, the CRISPR / Cas locus encodes an RNA-guided adaptive immune system against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). Three types of CRISPR systems (I-VI) have been identified. CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element. CRISPR clusters are transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA). The CRISPR-associated endonucleases, Cas9 and Cpf1, belong to type II and type V CRISPR / Cas systems and have potent endonuclease activity that cleaves target DNA. Cas9 is guided by a transactivating small RNA (tracrRNA) that serves as a guide for RNase II processing of mature crRNA and pre-crRNA, which contain a unique target sequence of approximately 20 nucleotides (called the spacer). The crRNA:tracrRNA duplex guides Cas9 to the target DNA through complementary base pairing between the spacer on the crRNA and a complementary sequence on the target DNA (called the protospacer). Cas9 recognizes a three-nucleotide (NGG) protospacer adjacent motif (PAM) to identify the cleavage site (the third or fourth nucleotide from the PAM). The crRNA and tracrRNA can be expressed separately or engineered into artificial fusion small guide RNAs (sgRNAs) via synthetic stem-loops to mimic the native crRNA / tracrRNA duplex. Similar to shRNAs, such sgRNAs can be synthesized or in vitro transcribed for direct RNA transfection or expressed from U6- or H1-promoted RNA expression vectors.

[0066] In some embodiments, the CRISPR-associated endonuclease is a Cas9 nuclease. The Cas9 nuclease can have a nucleotide sequence identical to the wild-type sequence of Streptococcus pyrogenes. In some embodiments, the CRISPR-associated endonuclease can be a sequence from another species, such as another Streptococcus species, such as thermophilus; Pseudomonas aeruginosa; Escherichia coli; or other sequenced bacterial genomes, and archaea or other prokaryotic microorganisms. Alternatively, the wild-type Cas9 sequence of Streptococcus pyrogenes can be modified. The nucleic acid sequence can be codon-optimized, i.e., "humanized," for efficient expression in mammalian cells. The humanized Cas9 nuclease sequence can be, for example, a Cas9 nuclease sequence encoded by any of the expression vectors listed in Genbank Accession Numbers KM099231.1 GL669193757; KM099232.1 GL669193761, or KM099233.1 GL669193765. Alternatively, the Cas9 nuclease sequence can be, for example, a sequence contained within a commercially available vector, such as pX330, pX260, or pMJ920 from Addgene (Cambridge, Mass.). In some embodiments, the Cas9 endonuclease can have an amino acid sequence that is a variant or fragment of any of the Cas9 endonuclease sequences of Genbank Accession Numbers KM099231.1 GL669193757; KM099232.1 GL669193761, or KM099233.1 GL669193765, or the Cas9 amino acid sequence of pX330, pX260, or pMJ920 (Addgene, Cambridge, MA).

[0067] In some embodiments, the cargo is a base editing enzyme. As used herein, the term "base editing enzyme" refers to a fusion protein comprising a defective CRISPR / Cas nuclease linked to a deaminase polypeptide. This term is also known as a "base editor." As used herein, the term "deaminase" refers to an enzyme that catalyzes a deamination reaction. The term "deamination," as used herein, refers to the removal of an amine group from a molecule. In some embodiments, the deaminase is a cytidine deaminase, which catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine to inosine. Inosine is treated similarly to guanosine by cells, changing A to G (or T to C). Two classes of base editing enzymes, cytosine base editors (CBEs) and adenine base editors (ABEs), can be used to generate single base pair edits without double-strand breaks. Cytosine base editors are typically formed by fusing a defective CRISPR / Cas nuclease to a deaminase.

[0068] In some embodiments, the cargo is a prime editor consisting of a fusion protein in which a catalytically reduced Cas9 endonuclease is fused to an engineered reverse transcriptase. By complexing with a prime editing guide RNA (pegRNA), the prime editor can identify a target site and provide new genetic information to replace a target DNA nucleotide. Prime editors mediate targeted insertions, deletions, and base-to-base conversions without the need for double-strand breaks (DSBs) or donor DNA templates (Anzalone, Andrew V.; Randolph, Peyton B.; Davis, Jessie R.; Sousa, Alexander A.; Koblan, Luke W.; Levy, Jonathan M.; Chen, Peter J.; Wilson, Christopher; Newby, Gregory A.; Raguram, Aditya; Liu, David R. (21 October 2019). "Search-and-replace genome editing without double-strand breaks or donor DNA". Nature. 576 (7785): 149-157.).

[0069] In some embodiments, the EV is loaded with i) a polypeptide (or a polynucleotide encoding the same) selected from the group consisting of a CRISPR-associated endonuclease, a base editing enzyme, an epigenetic editing factor, and a primer editor, and ii) one or more guide RNA molecules.

[0070] As used herein, the term "guide RNA molecule" generally refers to an RNA molecule (or a collection of RNA molecules) that can bind to a Cas9 protein and target the Cas9 protein to a specific location within a target DNA. A guide RNA may contain two segments: a DNA-targeting guide segment and a protein-binding segment. The DNA-targeting segment contains a nucleotide sequence that is complementary to (or can hybridize under at least stringent conditions to) a target sequence. The protein-binding segment interacts with a CRISPR protein, e.g., Cas9 or a Cas9-associated polypeptide. These two segments may be located on the same RNA molecule or on two or more separate RNA molecules. When the two segments are on separate RNA molecules, the molecule containing the DNA-targeting guide segment may be referred to as a CRISPR RNA (crRNA), while the molecule containing the protein-binding segment is referred to as a trans-activating RNA (tracrRNA).

[0071] In some embodiments, the cargo is a toxin.

[0072] As used herein, the term "toxin" refers to a molecule or moiety that is generally lethal to cells. In some embodiments, the toxin is a bacterial toxin or a fragment thereof. As used herein, the term "bacterial toxin" refers to a polypeptide produced by pathogenic bacteria and involved in the pathogenic activity. A bacterial toxin may be a factor directly responsible for bacterial virulence or may be involved in its virulence. As used herein, the term "toxin fragment" refers to any portion of a toxin that retains toxic activity. In particular, bacterial toxins have often been described as exhibiting various distinct functional domains, particularly a domain responsible for toxic activity (catalytic site) distinct from other domains involved in site recognition or interaction with partners. Most bacterial toxins, such as diphtheria toxin, Pseudomonas exotoxin, and Clostridium perfringens enterotoxin, contain a receptor-binding portion that targets the toxin to a specific cell surface receptor and a portion of the toxin protein responsible for toxicity. For example, Clostridium perfringens enterotoxin binds to claudin-3 and claudin-4 on the cell surface. Clostridium perfringens enterotoxin (CPE) is a protein of 319 amino acid residues. A peptide consisting of residues 290-319 of CPE binds to claudin-3 and claudin-4 but is not toxic (Hanna, PC, et al., 1991, J. Biol. Chem. 266:11037-43).Approximately residues 45-116 of CPE form a large complex at the cell membrane and are responsible for cell lysis (Kokai-Kun, JF et al., 1996, Infect. Immun. 64:1020-25; Kokai-Kun, JF et al., 1997, Clin. Infect. Dis. 25 (Suppl. 2):S165-5167; Kokai-Kun, JF et al., Infect. Immun. 65:1014-1022; Kokai-Kun, JF et al., 1999, Infect. Immun. 67:5634-5641; Hanna, PC, et al., 1991, J. Biol. Chem. 266:11037-43). Deletion of residues 315-319 is sufficient to abolish receptor binding (Kokai-Kun, JF et al., 1999, Infect. Immun. 67:5634-5641). Thus, in some embodiments, the toxin is a fragment of CPE that contains residues 45-116 of CPE but lacks residues 315-319 of CPE. In some embodiments, the toxin is diphtheria toxin or a toxic fragment thereof. Diphtheria toxin is a 535 amino acid residue protein (SEQ ID NO: 4). The protein contains three domains: i) residues 1-193 (catalytic domain, with ADP-ribosyltransferase activity responsible for inactivating elongation factor-2 in cells, resulting in cell death (Choe, S. et al., 1992, Nature 357:216-222)), ii) residues 203-378 (responsible for translocation of the toxin across the cell membrane), and iii) residues 386-535 (responsible for binding to the receptor). Thus, in certain embodiments, the toxin of the present invention comprises the amino acid sequence set forth in SEQ ID NO:4.

[0073] [ka]

[0074] Other cargoes of interest include detectable markers such as luciferase, luciferin, green fluorescent protein, fluorescent dyes such as FITC, etc. Detectable markers may also include imaging entities such as metal nanoparticles such as gold, platinum, silver, etc. These may be provided as nanoparticles, typically less than 10 nm, less than about 5 nm, etc.

[0075] Loading System In some embodiments, an EV of the invention comprises a structural polypeptide capable of forming a dimer with a cargo polypeptide.

[0076] As used herein, the term "structural polypeptide" is a protein that is naturally incorporated into the membrane of an EV and contributes to the overall structure of said EV.

[0077] In some embodiments, the structural polypeptide is selected from among transmembrane proteins. As used herein, the term "transmembrane protein" has its general meaning in the art and refers to a membrane protein that spans the lipid bilayer of a membrane. In some embodiments, the transmembrane protein is a tetraspanin. As used herein, the term "tetraspanin" has its general meaning in the art and refers to a superfamily of small, four-transmembrane domain proteins that are involved in a wide variety of physiological processes. Tetraspanin members include, but are not limited to, CD9, CD37, CD53, CD63, CD81, and CD82. In some embodiments, the tetraspanin is CD63.

[0078] The means by which the structural polypeptide and cargo polypeptide dimerize is not particularly limited. In some embodiments, the structural polypeptide and cargo polypeptide (e.g., a toxin) are fused to their respective domains capable of dimerization in the presence of a compound, either directly or via a linker. For example, a system in which an FK506 binding protein ("FKBP domain") and an FKBP-rapamycin-related protein 1 (FRAP1 fragment) ("FRB domain") heterodimerize in the presence of rapamycin can be used. Thus, in some embodiments, a structural polypeptide is fused to the FKBP domain and a cargo polypeptide (e.g., a toxin) is fused to the FRB domain (or vice versa), and the FKBP and FRB domains dimerize in the presence of rapamycin during production of the EVs of the invention. In some embodiments, the FKBP domain consists of the amino acid sequence set forth in SEQ ID NO:5, and the FRB domain consists of the amino acid sequence set forth in SEQ ID NO:6.

[0079] [ka]

[0080] Alternatively, a system in which GAI (Gibberellin insensitive) and GID1 (Gibberellin insensitive dwarf1) form a heterodimer in the presence of gibberellin or GA3-AM (see, for example, Miyamoto T., et al., Rapid and Orthogonal Logic Gating with a Gibberellin-Induced Dimerization System, Nat Chem Biol., 8 (5), 465-470, 2012) or a system in which PyL (PYR1-like, consisting of amino acids 33 to 209) and ABI1 (consisting of amino acids 126 to 423) form a heterodimer in the presence of S-(+)-abscisic acid (ABA) (see, for example, Liang FS, et al., Engineering the ABA plant stress pathway for regulation of induced proximity, Sci Signal., 4 (164), rs2, 2011) can be used.

[0081] In some embodiments, the EVs of the present invention comprise a loading system in which the tetraspanin CD63 is fused to an FKBP2 domain. In some embodiments, the EVs of the present invention comprise a loading system consisting of the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, a cargo polypeptide (e.g., a toxin) is fused to the FRB domain, which allows dimerization with the CD63-FKBP2 fusion protein in the presence of rapamycin, allowing the cargo polypeptide to be loaded into the EV.

[0082] [ka]

[0083] Targeting site According to the present invention, a targeting moiety is a polypeptide having a binding domain. As used herein, the term "binding domain" refers to one or more regions of a polypeptide that mediate specific binding to a target molecule (e.g., an antigen, ligand, receptor, substrate, or inhibitor). Exemplary binding domains include an antibody variable domain, a receptor-binding domain of a ligand, a ligand-binding domain of a receptor, or an enzyme domain. As used herein, the term "ligand-binding domain" refers to any region or derivative thereof that retains at least a qualitative ligand-binding ability of any native receptor (e.g., a cell surface receptor) or the corresponding native receptor. As used herein, the term "receptor-binding domain" refers to any region or derivative thereof that retains at least a qualitative receptor-binding ability of any native ligand or the corresponding native ligand. In some embodiments, a polypeptide comprises at least one, two, three, four, or five binding sites. A polypeptide can be either a monomer or a multimer. For example, in some embodiments, a polypeptide is a dimer. In some embodiments, the dimer is a homodimer, comprising two identical monomeric subunits. In some embodiments, the dimer is a heterodimer, comprising two non-identical monomeric subunits. A subunit of the dimer may comprise one or more polypeptide chains. For example, in some embodiments, the dimer comprises at least two polypeptide chains. In some embodiments, the dimer comprises two polypeptide chains. In some embodiments, the dimer comprises four polypeptide chains (e.g., as in an antibody molecule).

[0084] In some embodiments, the targeting moiety is a ligand.

[0085] In some embodiments, the targeting moiety is an antibody or antibody fragment, such as an scFv or VHH or other functional fragment comprising an immunoglobulin minus the light chain, Fab, Fab', F(ab *)2, Fv, antibody fragment, diabody, scAB, single domain heavy chain antibody, single domain light chain antibody, Fd, CDR region, or any portion or peptide sequence of an antibody capable of binding to an antigen or epitope. Thus, in some embodiments, a polypeptide having a binding domain is a light chain immunoglobulin. In some embodiments, a polypeptide having a binding domain is a heavy chain immunoglobulin. In some embodiments, a polypeptide having a binding domain is a single heavy chain variable domain of an antibody type that can be found in camelid mammals and that naturally lacks light chains. Such single domain antibodies are also referred to as VHHs or "nanobodies®." For a general description of (single) domain antibodies, see also the above-cited prior art as well as EP 0368684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490 and WO 06 / 030220, WO 06 / 003388.

[0086] The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see, eg, Kohler and Milstein, Nature, 256:495, 1975).

[0087] In some embodiments, the antibody is a monoclonal antibody.

[0088] In some embodiments, the targeting moiety has binding affinity for a cell surface molecule of the target cell. In some embodiments, the cell surface molecule is a receptor. In some embodiments, the cell surface molecule is a transmembrane protein. In some embodiments, the targeting moiety is specific for a target protein antigen, carbohydrate antigen, or glycosylated protein. For example, an antibody can target glycosylated groups on antigens preferentially produced by transformed (neoplastic or cancerous) cells, infected cells, etc. (cells associated with other immune system-related disorders).

[0089] A partial list of suitable mammalian cells that may be targeted by the targeting moieties of the present invention includes, but is not limited to, blood cells, myoblasts, bone marrow cells, peripheral blood cells, umbilical cord blood cells, cardiomyocytes (and their precursors), chondrocytes (cartilage cells), dendritic cells, fetal neural tissue, fibroblasts, hepatocytes (liver cells), pancreatic islet cells, keratinocytes (skin cells), and stem cells.

[0090] In some embodiments, the targeting moiety is particularly suitable for targeting a population of malignant cells. Thus, in some embodiments, the targeting moiety is specific for a cancer antigen. Known cancer antigens include, but are not limited to, c-erbB-2 (erbB-2 is also known as c-neu or HER-2). c-erbB-2 is particularly associated with breast, ovarian, and colon tumor cells, as well as neuroblastoma, lung cancer, thyroid cancer, pancreatic cancer, prostate cancer, kidney cancer, and gastrointestinal cancer. Another class of cancer antigens are non-enzymatically functional oncofetal proteins. These antigens are found in a variety of neoplasms and are often referred to as "tumor-associated antigens." Carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) are two examples of such cancer antigens. AFP levels are elevated in patients with hepatocellular carcinoma: AFP is expressed at high levels in serum in 69% of patients with liver cancer. CEA is a 200 kDa serum glycoprotein that is found in adenocarcinoma of the colon and cancers of the lung and genitourinary tract. Yet another class of cancer antigens are antigens unique to particular tumors, sometimes called "tumor-specific antigens," such as heat shock proteins (e.g., hsp70 or hsp90 proteins) derived from specific types of tumors. Other targets include the MICA / B ligand of NKG2D. These molecules are expressed in many types of tumors but are not typically expressed in healthy cells.More specific examples of cancer antigens include epithelial cell adhesion molecule (Ep-CAM / TACSTD1), mesothelin, tumor-associated glycoprotein 72 (TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1, MDA7, cancer-associated virus vaccines (e.g., human papillomavirus antigens), prostate-specific antigens (PSA, PSMA), RAGE (renal antigen), CAMEL (CTL-recognized antigen on melanoma), CT antigens (e.g., MAGE-B5), and the like. , -B6, -C2, -C3 and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE and SAGE), mucin antigens (e.g., MUC1, mucin-CA125, etc.), cancer-associated ganglioside antigens, tyrosinase, gp75, C-myc, Mart1, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7, Ep-CAM, tumor-derived heat shock proteins, etc. (e.g., Acres et al., Curr Opin Mol Ther 2004 February, 6:40-7; Taylor-Papadimitriou et al., Biochim Biophys Acta. 1999 October 8; 1455(2-3):301-13; Emens et al., Cancer Biol Ther. 2003 July-August; 2(4 Suppl 1):S161-8 and Ohshima et al., Int J Cancer. 2001 July 1; 93(1):91-6. Other exemplary cancer antigen targets include CA195 tumor-associated antigen-like antigens (see, e.g., U.S. Pat. No. 5,324,822) and female urinary squamous cell carcinoma-like antigens (see, e.g., U.S. Pat. No. 5,306,811) and breast cell carcinoma antigens described in U.S. Pat. No. 4,960,716.

[0091] In some embodiments, the targeting moiety is selected from the group consisting of CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD5, CD6, CD7, CD8 alpha, CD8 beta, CD9, CD10, CD11a, CD11b, CD11c, CDw12, CD13, CD14, CD15u, CD16a, CD16b, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD 34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD45, CD46, CD47R, CD48, CD49a, CD49b, CD49c, CD49d, C D49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD 66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD 85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CDw93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD 107b, CD108, CD109, CD110, CD111, CD112, CDw113, CD114, CD115, CD116, CD117, CD118, CDw119, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD12 4, CDw125, CD126, CD127, CDw128a, CDw128b, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CDw136, CDw137, CD138, CD139, CD140a, CD140b, CD141,CD142、CD143、CD144、CDw145、CD146、CD147、CD148、CDw149、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CDw156C、CD157、CD158、CD159a、CD159c、CD160、CD161、CD162、CD162R、CD163、CD164、CD165、CD166、CD167a、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CDw186、CD191、CD192、CD193、CD195、CD196、CD197、CDw198、CDw199、CDw197、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CDw210、CD212、CD213a1、CD213a2、CDw217、CDw218a、CDw218b、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD235ab、CD236、CD236R、CD238、CD239、CD240CE、CD240D、CD240DCE、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CD248、CD249、CD252、CD253、CD254、CD256、CD257、CD258、CD261、CD262、CD263、CD264、CD265、CD266、CD267、CD268、CD269、CD271、CD272、CD273、CD274、CD275、CD276、CD277、CD278、CD279、CD280、CD281、CD282、CD283、CD284、CD289、CD292、CDw293、CD294、CD295、CD296、CD297、CD298、CD299、CD300a、CD300c、CD300e、CD301、CD302、CD303、CD304、CD305、It has binding affinity for a Cluster of Differentiation (CD) molecule selected from the group consisting of CD306, CD307, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CDw325, CD326, CDw327, CDw328, CDw329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CDw338, and CD339.

[0092] Methods for producing EVs of the present invention According to the present invention, EVs of the present invention are prepared from donors genetically engineered to express EV components, i.e., ERV syncytin, a cargo of interest, and optionally, a loading system and targeting moiety. Typically, donor cells are transduced to express one or more polynucleotides encoding various EV components. It is contemplated that polynucleotide constructs can be introduced into donor cells as naked DNA or in an appropriate vector. Naked DNA generally refers to DNA contained in a plasmid expression vector in an orientation suitable for expression. Physical methods for introducing polynucleotide constructs into donor cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Other means can be used, including colloidal dispersion systems, e.g., macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. In some embodiments, the polynucleotide construct is introduced into donor cells by a viral vector, such as an adeno-associated virus (AAV), retrovirus, lentivirus, bovine papilloma virus, adenovirus vector, vaccinia virus, polyoma virus, or an infectious virus. In some embodiments, the vector is a retrovirus. Retroviruses can be selected as gene delivery vectors due to their ability to integrate genes into the host genome, transfer large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specialized cell lines. To construct a retroviral vector, a polynucleotide is inserted into the viral genome in place of a specific viral sequence to produce a replication-deficient virus. To produce virions, a packaging cell line is constructed that contains the gag, pol, and / or env genes but does not contain the LTRs and / or packaging components.When a recombinant plasmid containing a cDNA with a retroviral long terminal repeat (LTR) and packaging sequence is introduced into this cell line (e.g., by calcium phosphate precipitation), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture medium. The medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are capable of infecting a wide variety of cell types. Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. Their increased complexity allows for the alteration of the viral life cycle, such as latent infection. Some examples of lentiviruses include human immunodeficiency viruses (HIV1, HIV2) and simian immunodeficiency virus (SIV). Lentiviral vectors have been generated by repeatedly attenuating the pathogenic genes of HIV. For example, deletion of the env, vif, vpr, vpu, and nef genes renders the vector biologically safe. Lentiviral vectors are known in the art. See, e.g., U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference. Typically, vectors are plasmid- or virus-based and are constructed to contain the necessary sequences for integration, selection, and transfer of foreign nucleic acid into host cells. The gag, pol, and env genes of a vector of interest are also known in the art. Thus, the relevant genes are cloned into a selected vector, which is then used to transform a target cell of interest. Recombinant lentiviruses capable of infecting non-dividing cells (in which a suitable host cell is transfected with two or more vectors containing packaging functions, i.e., gag, pol, and env, and rev and tat) are described in U.S. Pat. No. 5,994,133, which is incorporated herein by reference.This document describes a first vector capable of providing nucleic acid encoding the viral gag and pol genes and another vector capable of providing nucleic acid encoding the viral env gene for producing packaging cells. By introducing a vector providing a heterologous gene into the packaging cells, producer cells are obtained that release infectious viral particles carrying the foreign gene of interest. env is preferably an amphipathic envelope protein capable of transducing human and other cell types. Typically, the vectors of the invention contain "control sequences." Control sequences collectively refer to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like. Collectively, these sequences provide for the replication, transcription, and translation of the coding sequence in recipient cells. Not all of these control sequences need always be present, so long as the selected coding sequence is capable of replication, transcription, and translation in an appropriate host cell. Another nucleic acid sequence is a "promoter" sequence. Promoter sequence is used herein in its conventional sense to refer to a nucleotide region containing DNA regulatory sequences. Here, the regulatory sequence is derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. Transcriptional promoters may include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters." In some embodiments, the polynucleotide is encoded by a nucleic acid molecule whose sequence has been codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of synonymous codons (i.e., codons that encode the same amino acid) in coding DNA varies across species. This codon degeneracy allows identical polypeptides to be encoded by a variety of nucleotide sequences.Various codon optimization methods are known in the art, including, for example, those disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148. Various assays can be performed to confirm the presence of the polynucleotide in the donor cells. Such assays include well-known "molecular biological" assays, such as Southern and Northern blotting, RT-PCR and quantitative PCR, or "biochemical" assays, such as detecting the presence or absence of a specific peptide.

[0093] Donor cells include, but are not limited to, epithelial cells, circulating immune cells, hematopoietic cells, bone marrow cells, circulating vascular progenitor cells, cardiomyocytes, chondrocytes, osteocytes, beta cells, hepatocytes, and neural cells. Furthermore, donor cells include pluripotent stem cells. As intended herein, the term "pluripotent stem cells" refers to cells that are prone to differentiate into one or more cell types and have the ability to divide. Preferably, pluripotent stem cells are undifferentiated. Pluripotent stem cells encompass stem cells, particularly adult stem cells (e.g., mesenchymal stem cells (MSCs)) and embryonic stem cells. The term also encompasses induced pluripotent stem cells (IPS). In some embodiments, the donor cells are mesenchymal stem cells. As used herein, the term "mesenchymal stem cells" or "MSCs" has its common meaning in the art and refers to multipotent stromal cells that can differentiate into a variety of cells, including osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells), and adipocytes (fat cells) (see, e.g., Wang, Stem Cells 2004; 22(7); 1330-7; McElreavey; 1991 Biochem Soc Trans (1); 29s; Takechi, Placenta 1993 March / April; 14 (2); 235-45; Takechi, 1993; Kobayashi; Early Human Development; 1998; Jul. 10; 51 (3); 223-33; Yen; Stem Cells; 2005; 23 (1) 3-9). In some embodiments, donor cells include purified primary cells and immortalized cell lines. In some embodiments, the donor cells are cells in suspension (eg, circulating white blood cells (PBMCs)) or adherent cells (eg, endothelial cells).

[0094] In some embodiments, the EVs of the present invention are prepared by any method known in the art. In some embodiments, the EVs of the present invention are prepared by methods known in the art for 3D culture, including, but not limited to, standard culture in 2D flasks, hanging drop culture, culture on a matrix, culture on microcarriers, culture on synthetic extracellular scaffolds, culture on chitosan membranes, culture under magnetic levitation, suspension culture in rotating bioreactors, or culture under non-contact inhibition conditions. See, for example, Haycock J W. (2011). "3D cell culture: a review of current approaches and techniques." Methods Mol Biol. 695: 1-15; Lee, J; Cuddihy MJ, Kotov N A. (14 Mar. 2008). Three-dimensional cell culture matrices: state of the art. doi:10.1089 / teb.2007.0150; Pampaloni, Francesco (October 2007). "The third dimension bridges the gap between cell culture and live tissue." Nature Reviews 8: 839-845 and Souza, Glauco (14 Mar. 2010). "Three-dimensional tissue culture based on magnetic cell levitation." Nature Nanotechnology: 291-296. The entire contents of each reference are incorporated by reference.

[0095] In some embodiments, the EVs of the present invention are prepared by the system culture described in WO 2019 / 002608. In particular, the EVs of the present invention are prepared according to the method described in the Examples. Specifically, the method requires a fluidic system containing at least one container, a liquid medium and producer cells contained in the container (including microcarriers suspended in the liquid medium, with the majority of the producer cells attached to the surface of the microcarriers), and a liquid medium stirring device, where the dimensions of the stirring device and the container make it possible to control the turbulence of the liquid medium in the container. Therefore, a further object of the present invention relates to a method for producing the EVs of the present invention, comprising the steps of: i) generating turbulence in the culture medium in the container (wherein the culture medium contains donor cells attached to the surface of the microcarriers, and the microcarriers are in a suspended state in the culture medium, optionally containing a dimerizer (e.g., rapamycin) in an amount for loading cargo polypeptides into the EVs); and ii) recovering the produced EVs from the liquid medium. Typically, the microcarriers are microbeads. Commercially available culture media can be used for the growth, cultivation, and maintenance of the donor cells. Such media include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM).

[0096] therapeutic use The present invention provides compositions and kits suitable for use in therapy (in vivo or ex vivo). The compositions and kits contain a certain amount of EVs of the present invention. According to the present invention, the therapeutic effect is mediated by one or more cargoes loaded onto the EVs of the present invention. For example, the EVs and compositions containing them can be used for gene therapy or vaccine purposes.

[0097] Therefore, a further object of the present invention relates to a method of treatment in a subject in need thereof, comprising administering to the subject a therapeutic amount of an EV of the present invention.

[0098] The types of diseases and disorders that can be treated by the methods of the present invention include, but are not limited to, infectious diseases, autoimmune diseases, inflammatory diseases, cancer, neurological diseases, cardiovascular diseases, eye diseases, ear diseases, blood diseases, bone diseases, congenital diseases, metabolic diseases, musculoskeletal diseases, gastrointestinal diseases, renal and genitourinary diseases, respiratory diseases, or skin diseases.

[0099] In particular, the EVs of the present invention, especially toxin-loaded EVs, are particularly suitable for the treatment of cancer.

[0100] As used herein, the term "cancer" has its common meaning in the art and refers to one or more cells that are growing or have proliferated in an uncontrolled manner to form cancerous tissue. This term includes, but is not limited to, solid tumors and blood-borne tumors. The terms "cancer" and "tumor" are used interchangeably throughout the subject specification. The term "cancer" is not limited in any way to the stage, grade, histomorphological characteristics, invasiveness, aggressiveness, or malignancy of the affected tissue or cell mass. Specifically, it includes stage 0 cancer, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, grade I cancer, grade II cancer, grade III cancer, malignant cancer, and primary cancer. As used herein, the term "solid cancer" includes, but is not limited to, "carcinoma," "adenocarcinoma," and "sarcoma." "Sarcoma" is a cancer of connective tissue, cartilage, bone, muscle, etc. "Carcinoma" is a cancer of epithelial (endothelial) cells. "Adenocarcinoma" refers to a carcinoma derived from cells of glandular origin.

[0101] Examples of cancers that can be treated by the methods and compositions of the present invention include, but are not limited to, cancer cells of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gingiva, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may be specifically of the following histological types, including, but not limited to: malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix. carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma in familial polyposis coli; solid cancer; malignant carcinoid tumor; bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; Papillary and follicular gland carcinoma; Non-encapsulated sclerosing carcinoma; Adrenal cortical carcinoma; Endometrioid carcinoma of the endometrium; Skin adnexal carcinoma; Apocrine gland carcinoma; Sebaceous gland carcinoma; Cerumen gland carcinoma; Mucoepidermoid carcinoma; Cystadenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinic cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous dysplasia; Malignant thymoma; Malignant ovarian interstitial carcinoma stromal tumor; malignant theca cell tumor; malignant granulosa cell tumor and malignant neuroblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; hemangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma of giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; Rhabdomyosarcoma;embryonic rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;malignant mixed tumor;Müllerian mixed tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;malignant mesenchymoma;malignant Brenner tumor;malignant Phyllodes tumor;synovial sarcoma;malignant mesothelioma;dysgerminoma;embryonic carcinoma;malignant teratoma;malignant ovarian goiter;choriocarcinoma;malignant mesonephroma;angiosarcoma;malignant hemangioendothelioma;Kaposi's sarcoma;malignant hemangiopericytoma;lymphangiosarcoma;osteosarcoma;parosteal osteosarcoma;chondrosarcoma;malignant chondroblastoma;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Malignant odontogenic tumor;Ameloblastic odontosarcoma;Malignant ameloblastoma;Ameloblastic fibrosarcoma;Malignant pinealoma;Chordoma;Malignant glioma;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrillar astrocytoma;Astrocytoblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal tumor;Cerebellar sarcoma;Ganglioneuroblastoma;Neurobioblastoma;Retinoblastoma;Olfactory neurogenic tumor;Malignant meningioma;Neurofibrillarcoma;Malignant neurilemmoma;Malignant granular cell tumor;Malignant phospholipid tumor Lymphoma; Hodgkin's disease; Hodgkin's lymphoma; granulomatous granuloma; malignant small lymphocytic lymphoma; diffuse large cell lymphoma; follicular lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma and hairy cell leukemia.

[0102] A further object of the present invention relates to a composition ("EV composition") comprising a certain amount of the EVs of the present invention. The compositions described herein encompass pharmaceutical compositions used for carrying out therapeutic methods in subjects in need thereof, including non-human mammals and human individuals in need thereof. The compositions of the present invention can be formulated for delivery to animals for veterinary purposes (e.g., livestock, e.g., cows, pigs, etc.) and other non-human mammalian subjects, as well as human subjects. For example, the EVs can be formulated with a physiologically acceptable carrier for use in gene transfer and gene therapy applications. In some embodiments, the compositions further comprise one or more transduction helper compounds. The transduction helper compounds are preferably selected from the group including cationic polymers, in particular, as described in Zuris et al. (2015, Nat Biotechnol, Vol. 33(no 1): 73-80).The transduction helper compounds used were polybrene (sometimes called hexadimethrine bromide), protamine sulfate, 12-myristate 13-acetate (also called phorbol myristate acetate or PMA, as described in Johnston et al., 2014, Gene Ther, Vol. 21(12): 1008-1020), vectofucin (described in Fenard et al., 2013, Molecular Therapy Nucleic Acids, Vol. 2: e90), poloxamer P338 (described in Anastasov et al., 2016, Lentiviral vectors and exosomes as gene and protein delivery tools, in Methods in Molecular Biology, Vol. 1448: 49-61), RetroNectin® reagent (commercialized by Clontech Laboratories Inc.), Viral Plus® transduction enhancer (Applied Biological Materials The cationic transduction helper compound may be selected from the group including EVs commercialized by TransPlus® viral transduction enhancer (commercialized by Clinisciences), Lentiboost® (commercialized by Sirion Biotech), or ExpressMag® transduction system (commercialized by Sigma-Aldrich). As shown in the examples herein, the cationic transduction helper compound may consist of polybrene. EVs can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. EVs can be formulated for parenteral administration by injection, e.g., bolus injection or continuous infusion. Injectable preparations can be provided in unit dosage form, e.g., ampoules or multi-dose containers, with added preservatives. EV compositions can take forms such as suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulatory agents, e.g., suspending agents, stabilizing agents, and / or dispersing agents.Liquid preparations of EV compositions can be prepared by conventional means using pharmaceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats and oils); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid). The preparations may also contain buffer salts. Alternatively, the compositions can be in powder form for constitution with a suitable vehicle, such as sterile, pyrogen-free water, before use.

[0103] The EV compositions of the present invention can be administered to a subject at a therapeutically effective dose to provide a therapeutic effect. In some embodiments, the amount of the EV compositions of the present invention is administered in a dosage unit ranging from about 0.1 to 5 micrograms (μg) per kilogram (kg). To this end, the EV compositions of the present invention can be formulated in a dose range of about 7 mg to about 350 mg to treat an average subject weighing 70 kg. The amount of the EV compositions of the present invention that can be administered can be selected from the group consisting of 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, or 5.0 mg / kg. The dose of EV in a unit dosage form of the composition may in particular be selected from the group comprising: 7mg, 8mg, 9mg, 10mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375mg, 400mg, 425mg, 450mg, 475mg, 500mg, 525mg, 550mg, 575mg, 600mg, 625mg, 650mg, 675mg, 700mg, 725mg or 750mg for treating an average subject weighing 70kg. These doses can be given once or repeatedly, for example, daily, every other day, weekly, biweekly, or monthly. In some embodiments, the composition can be administered to a subject in one dose, or two doses, or three doses, or four doses, or five doses, or six or more doses. The administration interval can be determined based on the physician's judgment that it is necessary.

[0104] The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. In some embodiments, the compositions may be in liquid or solid (e.g., lyophilized) form.

[0105] Administration of EVs to a human subject or animal in need thereof can be by any means known in the art for administering viral vectors. Exemplary modes of administration include rectal administration, transmucosal administration, topical administration, transdermal administration, inhalation administration, parenteral administration (e.g., intravenous administration, subcutaneous administration, intradermal administration, intramuscular administration, and intraarticular administration), and direct injection into a tissue or organ, or alternatively, intrathecal injection, direct intramuscular injection, intracerebroventricular injection, intravenous injection, intraperitoneal injection, intranasal injection, or intraocular injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for reconstitution into a liquid solution or suspension prior to injection, or as emulsions. Alternatively, the virus can be administered locally rather than systemically, for example, in a depot or sustained-release formulation.

[0106] The present invention will be further illustrated by the following figures and examples, which should not be construed as limiting the scope of the invention in any way. [Brief explanation of the drawings]

[0107] [Figure 1]EV Cargo Loading System. (A) A scheme illustrating the loading system. The EV membrane marker FKBP2-RFP-CD63 is coexpressed with the luminescent reporter FRB-tagged NanoLuciferase. Drug-induced FKBP2-FRB interaction is reversible, enabling EV-cargo loading. The fate of the luminescent cargo can be tracked in the extracellular medium and within recipient cells by luminometry. (B) FKBP2-RFP-CD63 (loader) was transiently expressed in HeLa WT cells and monitored by confocal microscopy. As expected, the loader exhibits an endosomal pattern. (C) The loader and FRB-NLuc cargo (also HA-tagged) were transiently expressed in HeLa WT cells. Transfected cells were incubated with or without a dimerizer for 1 h (dimerizer or no dimerizer). Cells were fixed, labeled, and monitored by confocal microscopy. The fluorescence intensities of both the loader and cargo were plotted. These results show that only in the presence of the drug did the two signals significantly colocalize, demonstrating the ability of the loading system to recruit the FRB fusion protein. [Figure 2]Drug-induced recruitment of cargo into EVs. (A) Loader and FRB-NLuc cargo were transiently expressed in HeLa WT cells, and EVs were produced by these cells in the presence (dimerizer+) or absence (dimerizer-) of a dimerizer. EVs were isolated by sequential centrifugation. Western blot analysis was performed to analyze the expression of the loader and cargo as well as classical positive and negative EV markers. Equal amounts of protein were loaded into each well. The dimerizer does not alter EV composition, as determined by Western blot analysis. (B) In parallel, the relative luminescence in isolated EVs was measured. The graph shows the luminescence activity in EVs emitted from cells treated or not with the dimerizer. NLuc specific activity was normalized to the "no drug" condition, which corresponds to nonspecific bulk loading of the overexpressed cargo, and plotted. Each dot represents the average of two technical duplicates. Treatment of donor cells with the drug significantly increased the specific NLuc activity of EVs by 3.5-fold. (C) A flotation assay was performed on drug-free and drug-treated EVs. The NLuc activity in each fraction was plotted. In both conditions, peak NLuc activity was observed in fraction 7, demonstrating that NLuc is associated with suspended EVs. A 4-fold increase in NLuc activity was also observed in fraction 7 under the drug-free condition compared to the drug-free condition (see Figure 2B). (D) Fractions obtained after flotation were analyzed by Western blot by monitoring cargo and various positive and negative EV markers. The results demonstrate that NLuc in fraction 7 is associated with EVs. (E-F) Particle size and concentration were measured by nanoparticle tracking analysis, and (G) EV protein concentration was measured by BCA. Each dot represents the average of technical duplicates. These last three parameters show no change when the donor cells are treated with drugs (dimerizers). [Figure 3]EV cargo loading increases uptake and delivery within recipient cells. (A) Uptake experiments were performed by incubating HeLa WT with loading EVs produced in the presence (drug) or absence (no drug) of a dimerizer for various time points. Note that the dimerizer was washed out during EV isolation, allowing for estimated delivery into recipient cells. The graph shows luminescence activity over time. Each point corresponds to the mean of technical duplicates and biological triplicates, and the error represents the SEM. When produced in the presence of a dimerizer, loading EVs can mediate uptake fourfold higher than in the "no drug" condition. (B) Content delivery assays were performed by incubating HeLa WT with loading EVs produced in the presence (dimerizer) or absence (no dimerizer) of a dimerizer for 24 hours. Briefly, acceptor cells were mechanically disrupted and the presence of luminescent cargo in membrane and cytoplasmic fractions after cell fractionation was examined. NLuc activity associated with membrane and cytoplasmic fractions in the "drug" condition was normalized to the "no drug" (no dimerizer) condition. Results confirm a 4-fold increase in overall uptake (membrane + cytoplasm). [Figure 4] Engineering virus-free fusogenic EVs. EVs from HeLa cells transiently expressing GFP ("Mock condition"), VSV-G, or syncytin-1 (Syn1) were characterized according to various parameters. Note that the donor cells stably express the common EV cargo, NLuc-HSP70. (A) Particle concentration, (B) EV protein concentration, (C) particle size, and (D) EV NLuc specific activity were measured and plotted. All parameters except particle size were normalized to the "Mock" condition. Results show increased particle and protein concentrations in the VSV-G and Syn1 conditions. Nevertheless, EV size and NLuc specific activity remained unchanged, suggesting that EV loading capacity was unchanged. [Figure 5]SYN1-Positive Fusogenic EVs Increase EV Cargo Delivery. (A) NLuc-Hsp70 activity was measured by incubating HeLa WT cells with EVs carrying NLuc-Hsp70 and either GFP ("Mock" condition), VSV-G, or Syn1 at various time points. Syn1-EV-mediated uptake demonstrates a significant increase compared to the "Mock" condition. (B) Cellular content delivery assays were performed by incubating HeLa WT cells with fusogenic EVs carrying GFP ("Mock" condition), VSV-G, or Syn1 for 24 hours. Results show a 5-fold increase in content delivery for fusogenic EVs compared to the control, and comparable EV delivery for both VSV-G and Syn1. [Figure 6] DTA-resistant donor cells. (A) Parental HeLa cells were infected with lentivirus-encoded shRNA targeting the DPH2 gene to generate DTA-resistant donor cells (DPH2KD). DPH2 knockdown in donor cells was confirmed by qRT-PCR. (B) Plasmids encoding DTA-HA and / or mCherry were transfected into parental or DPH2KD cells. Equal amounts of protein were analyzed by Western blot. Expression of DTA-HA inhibits protein synthesis in parental cells, including the synthesis of detectable amounts of DTA-HA itself (mCherry is not detected). However, DPH2KD allows not only the expression of DTA-HA but also the coexpression of mCherry. This indicates that DPH2KD cells are resistant to DTA-HA-induced disruption of protein synthesis. (C) Quantitative protein synthesis assays show that DPH2KD cells maintain approximately 80% of de novo protein synthesis with DTA-HA expression. Under these conditions, parental cells exhibit less than 3% de novo protein synthesis. [Figure 7]Heterodimerization-dependent DTA loading into EVs. (A) Scheme of heterodimerization-dependent DTA loading into EVs. FRB-DTA ​​and FKBP-CD63 are coexpressed in donor cells. Addition of a dimerizer binds the two proteins, recruiting FRB-DTA ​​to the EV membrane during biogenesis and enabling efficient loading of FRB-DTA ​​into EVs. After washing away the dimerizer, FRB-DTA-loaded EVs can deliver their contents into the cytoplasm of acceptor cells. (B) Western blot analysis shows that FRB-DTA ​​is efficiently loaded into EVs by adding a dimerizer in DPH2KD donor cells. The same amount of protein was loaded for each sample. [Figure 8] Palm-DTA Loading into EVs. (A) Scheme of Palm-DTA association with membranes. (B) In contrast to DTA-HA, which is found only in a soluble form, cell fractionation shows that Palm-DTA is mostly membrane-associated, with only a small amount of soluble protein remaining in the cytoplasm. (C) Quantitative protein synthesis assays show that Palm-DTA is highly potent in parental cells, while DPH2KD cells are partially resistant to its activity. (D) Western blot analysis reveals that Palm-DTA is efficiently loaded into EVs when expressed in DPH2KD donor cells. [Figure 9]Killer EVs are potent in vitro. (A) Western blot characterization of EVs generated from DPH2KD donor cells expressing either Palm-DTA, Palm-DTA + VSV-G (killer EVs), or nothing (mock). (B) Particle measurements obtained for the EVs in A. (C) The indicated EVs were incubated with GFP-PEST-expressing HT1080 acceptor cells for 24 hours. After incubation, GFP fluorescence quantification by FACS demonstrated that killer EVs efficiently inhibited protein synthesis in the acceptor cells. (D) Quantification of the data in panel C demonstrates that coexpression of VSV-G and Palm-DTA significantly improved the activity of Palm-DTA-containing EVs in both the level of protein synthesis inhibition (GFP MFI) and the level of cell death induction (cell count). (E) Experiments similar to those in C and D demonstrate that the effect of killer EVs is dose-dependent, as protein synthesis inhibition increases with increasing EV dose. Killer EVs are five times more efficient than Palm-DTA EVs. (F) Microscopic observation of GFP-PEST-expressing HT1080 acceptors incubated with killer EVs shows complete cell disappearance after 3 days. [Figure 10] Virus-free killer EVs are potent in vitro. (A) DPH2KD donor cells expressing FKBP2-RFP-CD63, FRB-DTA-HA, and syncytin 1 were treated with or without a dimerizer for 24 hours, after which EVs were isolated. GFP-PEST-expressing HT1080 acceptor cells were treated with or without syncytin 1-positive EVs, with or without DTA loading, using a drug-induced loading system. GFP fluorescence quantification by FACS demonstrates that virus-free killer EVs (DT-loaded and decorated with syncytin 1) efficiently disrupt protein synthesis in acceptor cells. (B-C) Quantification of the data in panel A demonstrates that syncytin 1 + DTA + EVs exhibit superior efficiency in terms of protein synthesis inhibition (GFP MFI) and cell death induction (cell count). [Figure 11] Generation of edited EVs. (A) HeLa cells stably expressing FRB-Cas9-HA (FC9H) and CXCR4 gRNA and the resulting EVs were characterized by Western blot analysis using various positive markers (Alix, CD63, Hsp70, CD9) and negative markers (calnexin). Expression of FRB-Cas9-HA was also analyzed using an antibody recognizing the HA tag. Equal amounts of protein were loaded for both "cell" and "EV" conditions. (B) Wild-type HeLa, stable FC9H+ HeLa, and stable RNP HeLa (FC9H+ / CXCR4 gRNA+) were labeled with α-CXCR4 antibody conjugated to APC (blue) or unlabeled (red) and analyzed by FACS using the RL1 laser. Results for "labeled cells" are plotted against those for "unlabeled cells." Fluorescence intensity is plotted on the x-axis, and cell number is normalized to the mode on the y-axis. (C-D) Wild-type HeLa cells were incubated with EVs containing only Cas9 and gRNA (RNP+EVs) or EVs additionally decorated with syncytin-1 (Syn1+ / RNP+EVs). After 48 hours, acceptor cells were harvested, labeled with α-CXCR4 antibodies conjugated to APCs, and analyzed by FACS using the RL1 laser. Results for "RNP+EVs" or "Syn1+ / RNP+EVs" are plotted against the "no EVs" condition. Fluorescence intensity is plotted on the x-axis, and cell number is normalized to the mode on the y-axis.

[0108] Example 1: Method for loading-fusion EV system Cell culture. HeLa cells—wild-type (ATCC, Virginia, USA) and genetically modified forms—were grown in DMEM GlutaMAX (Gibco, Illinois, USA) supplemented with 10% FBS at 37°C and 5% CO2. HeLa expressing NanoLuciferase-Hsp70 were generated according to Bonsergent et al. Nat Comm. 2021. HeLa CD8-GFP or FRB-NanoLuciferase-HA were transfected with Lipofectamine 2000 and selected with hygromycin B (50 mg / mL, Invitrogen, Massachusetts, USA). HeLa NLuc-CD63 were transfected with Lipofectamine 2000 and selected with Geneticin (50 mg / mL, Gibco, Illinois, USA).

[0109] Transfection. Cells were transfected with Lipofectamine 2000 (Invitrogen) for 20 minutes by mixing 10 μg DNA in 10 μL of Lipofectamine 2000 (total volume: 2 mL per 10 cm dish) and 1 μg DNA in 1 μL of Lipofectamine 2000 (total volume: 100 μL per 24-well plate well). Cells were incubated with the transfection mixture for 6 hours at 37°C and 5% CO2, and the medium was replaced with serum-free DMEM GlutaMAX (Gibco, Illinois, USA). A / C heterodimerization agent (Takara Bio Inc., Shiga, Japan) was added at this stage for loading experiments.

[0110] EV isolation. Donor cells were transfected according to the transfection section. EVs were produced in serum-starved conditions in 5 mL of DMEM GlutaMAX per 10 cm dish. After 36 h of production, the medium was collected and centrifuged at 2,000 g for 20 min at 4°C to remove dead cells and debris, then at 10,000 g for 30 min at 4°C to remove large vesicles and apoptotic bodies (45 Ti rotor), and then at 100,000 g for 1 h 30 min at 4°C to isolate EVs (45 Ti rotor, Optima™ XE-90 Ultracentrifuge, Beckman Coulter, California, USA). Finally, the 100 kg pellet was collected and centrifuged again in PBS at 100,000 g for 1 h 10 min at 4°C to wash out the medium (SW55 rotor). The final pellet was resuspended in PBS and either used immediately or stored at 4°C.

[0111] Floating assay. EV isolation was performed without a washing step. The 100 kg pellet was resuspended in 1 mL of 60% sucrose in PBS (prepared according to M.M. Temoche-Diaz, BioProtoc. 2020) and allowed to settle to the bottom of an SW55 tube. On top of the 60% fraction, 1 mL of the 30% fraction was layered, followed by 1 mL of PBS. The sample was then centrifuged (SW55 rotor) at 4°C for at least 15 hours, and nine 300 μL fractions were collected. The luminescence activity of each fraction was directly analyzed. Each fraction was then diluted to a total of 4 mL with PBS, the sucrose was washed out, and the fractions were centrifuged at 100,000 g for 1 hour at 4°C for Western blotting (MLA-50 rotor, Optima™ MAX-XP Ultracentrifuge, Beckman Coulter, California, USA).

[0112] NLuc-based uptake and content delivery assays were performed using EVs carrying FRB-NanoLuciferase-HA or NanoLuciferase-CD63 as donor EVs according to Bonsergent et al. 2021. Luminescence was read using the Nano-Glo Luciferase Assay System (Promega, Wisconsin, USA) on an iD3 SpectraMax microplate reader (Molecular Devices, California, USA).

[0113] Recruitment assay. Cells were seeded onto glass coverslips on day 0 and cotransfected with pC4-FKBP2-RFP-CD63 and pC4-FRB-NLuc-HA (30% / 70%, respectively) the following day. On day 3, cells were treated with or without an A / C heterodimerizer (Takara) at 37°C for 1 h and then prepared for confocal microscopy by labeling FRB-NLuc-HA green.

[0114] Cloning. PCR oligonucleotides were ordered from Eurofins Genomics (Luxembourg, Luxembourg). PCR reactions were performed according to Thermo Fisher or NEB protocols, and digestion and ligation (vector:insert molar ratio 1:3) were performed according to NEB protocols and software. 2 μL of the ligation product was used to transform 20 μL of competent bacteria (Library Efficiency™ DH5α Competent Cells, Thermo Fisher Scientific, Massachusetts, USA) at 42°C for 30 seconds. The cells were harvested in 200 μL of SOC medium and incubated at 37°C for 1 hour with agitation. They were then spread onto ampicillin or kanamycin agar plates and grown overnight at 37°C.

[0115] The plasmid pC4-GFP-HA was generated by Gregory Lavieu. VSV-G was purchased from AddGene (#8454). Syncytin-1 was provided by Thierry Heidmann. pC4-FRB-HA was generated using pC4-R H E (ARIAD manufactured by Takara Bio). pC4-FKBP2-HA was transformed into pC4-R H pC4M-F2E and pC4M-F2E(ARIAD) were digested with XbaI and SpeI, and FKBP2 was ligated to empty pC4-R H pC4-FKBP2-RFP-CD63 was generated by amplifying RFP-CD63 (kindly provided by Walther Mothes) and inserting it into pC4-FKBP2-HA digested with EcoRI and BamHI. pC4-FRB-NLuc-HA was generated by amplifying NLuc (from NLuc-Hsp70, Bonsergent et al. 2021) and inserting it into pC4-R using the SpeI restriction site. H It was generated by inserting into E.

[0116] Antibodies. Primary antibodies: anti-TGN46 (PA5-23068, Invitrogen), anti-hCD9 (clone MM2-57, Millipore), anti-hCD63 (556019, BD Pharmingen), anti-HA (for IF, 66006-2-Ig, Proteintech; for WB, C29F4, Cell Anti-Cherry (5993-100, BioVision), Anti-Calnexin (ab133615, Abcam), Anti-ALIX (Clone 3A9, 2171S, Cell Signaling), Anti-HSP70 / HSP72 (Clone C92F3A-5, ADI-SPA-810F, Enzo Life Sciences), anti-actin (clone C4, MAB1501, Millipore). Secondary antibodies for Western blotting: goat anti-rabbit IgG (H+L)-HRP conjugate (1706515, Bio-Rad) and goat anti-mouse IgG (H+L)-HRP conjugate (1706516, Bio-Rad). Secondary antibodies for immunofluorescence: goat anti-mouse IgG (H+L) highly cross-absorbing secondary antibody, Alexa Fluor™ 488 (A11029, Thermo Fisher Scientific).

[0117] Western blotting. Cells were harvested and washed with PBS. The pellet was resuspended in lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, protease / phosphatase inhibitor cocktail (Roche, Switzerland), pH 8) on ice for 20 min. Then, the cells were centrifuged at 20,000 g for 15 min to pellet the membrane, and the supernatant was collected. The protein concentrations of cell lysates and EVs were estimated using a Micro-BCA™ Protein Assay Kit (Thermo Scientific, Illinois, USA). Samples were mixed with 4X Laemmli buffer (Bio-Rad, California, USA) supplemented with 10% β-mercaptoethanol (BME) for a final concentration (except for CD63 protein, which cannot be detected in the presence of BME). Electrophoresis was performed on a 4-20% polyacrylamide gel (Bio-Rad, California, USA) in Tris / glycine / SDS buffer (Bio-Rad). Proteins were transferred to an Immun-Blot PVDF membrane (0.2 μm, Bio-Rad) using the TransBlot Turbo system (Bio-Rad). Precision Plus Protein™ Standards (Bio-Rad) were used as a ladder. The membrane was then blocked in 0.05% Tween 5% milk in PBS for 1 hour at room temperature and incubated overnight with primary antibodies diluted 1 / 1000 in 0.05% Tween 5% milk in PBS. The membrane was then washed for 1 hour with 0.05% Tween in PBS, incubated with secondary antibodies diluted 1 / 10,000 in 0.05% Tween in PBS, and washed for 1 hour with 0.05% Tween in PBS. Membranes were revealed using Clarity™ Western ECL substrate (Bio-Rad) and an ImageQuant™ LAS 400 (GE Healthcare Life Sciences, Chicago, USA). Image analysis and quantification were performed using Fiji software.

[0118] Confocal microscopy Stable cell lines were seeded onto coverslips either one day before fixation or two days before transfection. The following day, cells were transfected for transient protein expression. Cells were then rinsed three times with cold PBS and incubated in 4% PFA for 15 minutes at room temperature. For antibody labeling, cells were permeabilized with Triton-X100 (Sigma-Aldrich, Massachusetts, USA) for 15 minutes at room temperature, incubated with primary antibodies at a 1 / 500 dilution for 2 hours at room temperature, followed by secondary antibodies at a 1 / 2,000 dilution for 1 hour at room temperature. Finally, DAPI staining was performed at a 1 / 10,000 dilution, if desired. Coverslips were mounted with ProLong™ Diamond Antifade Mountant (Invitrogen).

[0119] Images were acquired using an LSM 880 confocal microscope (ZEISS, Baden-Württemberg, Germany). Image analysis and quantification were performed using Fiji software.

[0120] Nanoparticle tracking analysis was performed using a ZetaView x20 (Particle Metrix, Ammersee, Germany) with the following parameters: laser 488 nm, scattering, 11 positions, 1 cycle, sensitivity 80, shutter 100, pH 7 input, T °C sensing. All samples were diluted in 1x filtered PBS.

[0121] Example 2: Method for "Killer EV" Cell culture. HeLa and HT1080 cells (ATCC, Virginia, USA) and their transgenic derivatives were grown in DMEM medium (Gibco, Illinois, USA) supplemented with 10% heat-inactivated fetal bovine serum (Biowest, France) at 37°C, 5% CO2, and high humidity. HT1080 cell medium was further supplemented with MEM NEAA (Gibco, Illinois, USA).

[0122] Stable DPH2KD HeLa cells were obtained by lentiviral transfection with shRNA targeting DPH2 (Horizon Discovery, Cat # VGH5518-200302258, UK) and selected with 4 μg / mL puromycin (Gibco, Illinois, USA). Stable GFP-PEST HT1080 clones were obtained by transfection with a plasmid encoding GFP-PEST (Addgene, Cat # 26821, Massachusetts, USA) followed by cell selection with 0.5 mg / mL Geneticin (Gibco, Illinois, USA).

[0123] Transient transfection was performed using Lipofectamine 2000 (Invitrogen, Massachusetts, USA) according to the manufacturer's instructions.

[0124] Plasmid construction. To construct a plasmid encoding DTA-HA, the sequence of DTA (obtained from Addgene, Cat # 42521, Massachusetts, USA) was cloned into pC4R HThe DTA-HA construct was fused to an HA tag sequence using an infusion cloning strategy (Takara Bio Europe, France) using the XbaI / SpeI cloning sites in the E backbone (ARIAD Pharmaceuticals, Massachusetts, USA). The DTA-HA construct was then subcloned into the pCDNA3.1 backbone (Invitrogen, Massachusetts, USA) using the NheI / BamHI cloning sites.

[0125] To construct a plasmid encoding Palm-DTA-HA, the SNAP25 palmitoylation sequence ( Greaves et al., JBC 2000 ) was inserted into the N-terminus of DTA-HA using infusion cloning (Takara Bio Europe, France).

[0126] To construct a plasmid encoding FRB-DTA-HA, the FRB sequence was first inserted into the pcDNA3.1 backbone (Invitrogen, Massachusetts, USA) using the NheI / BamHI cloning sites to create the plasmid pC4R H E was used as a FRB template for cloning, and the DTA-HA sequence was then cloned into the BamHI / XbaI sites of this plasmid.

[0127] qRT-PCR. Total RNA was extracted from cells using the Nucleospin RNA kit (Macherey Nagel, France) according to the manufacturer's instructions. Equal amounts of total RNA were reverse transcribed using the iScript cDNA synthesis kit and subjected to qPCR using the iTaq SYBR green kit (Bio-Rad, France), all according to the manufacturer's instructions. qPCR was performed on a CFX96 system (Bio-Rad, France) at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. DPH2 gene expression was normalized to the PGK housekeeping gene according to the 2-ΔΔCt formula.

[0128] Protein synthesis assay. Parental HeLa cells or DPH2KD HeLa cells were seeded in 24-well plates and then cotransfected with a plasmid encoding NanoLuc-Hsp70 and either a mock or DTA-HA or Palm-DTA-HA. Six hours after transfection, cells were detached and split into three wells of a 96-well plate. After 24 hours, cells were washed with DPBS, and NanoLuc activity was measured in each well using the Nano-Glo Live Cell Assay System (Promega, Wisconsin, USA) according to the manufacturer's instructions, using an iD3 SpectraMax microplate reader (Molecular Devices, California, USA). Percent protein synthesis was calculated for each cell type tested relative to mock-transfected cells (mock set at 100%).

[0129] EV preparation. EV donor cells were transfected with the indicated plasmids for 16 hours and then incubated in serum-free DMEM for 24 hours. The conditioned medium was collected and centrifuged at 2000 × g for 20 minutes at 4°C to remove cell debris. EVs were then pelleted by ultracentrifugation at 100,000 × g for 1 hour and 30 minutes at 4°C (45Ti rotor and Optima™ XE-90 ultracentrifuge, Beckman Coulter, California, USA). The EV pellet was washed with DPBS and centrifuged at 100,000 × g for 1 hour and 30 minutes at 4°C (MLA50 rotor with dedicated adapter and Optima MAX-XP ultracentrifuge, Beckman Coulter, California, USA). The washed pellet was resuspended in DPBS, and the EVs were either stored at -20°C (if intended for Western blot analysis) or immediately applied to acceptor cells.

[0130] Western blot. Cells to be analyzed were scraped in DPBS on ice and pelleted at 1,000 × g for 5 min at 4 °C. The cell pellet was resuspended in PBX lysis buffer (DPBS, 1% Triton-X-100, EDTA-free protease / phosphatase inhibitor cocktail (Roche, Switzerland)) and incubated on ice for 10 min with intermittent vortexing. Samples were then centrifuged at 15,000 × g for 10 min at 4 °C to pellet nuclei and unbroken cells. The supernatant (cell lysate, CL) was collected. Protein concentrations of the cell lysate and EVs were obtained using a Micro BCA Protein Assay Kit (Thermo Scientific, Illinois, USA). Samples were mixed with Laemmli buffer (Bio-Rad, France) containing 10% β-mercaptoethanol, except for CD63 and CD9 detection (without β-mercaptoethanol), and loaded onto a 4-15% polyacrylamide gel (Bio-Rad, France). After electrophoresis, proteins were transferred onto a PVDF membrane using the TransBlot Turbo system (Bio-Rad, France).The membrane was incubated in DPBS containing 0.05% Tween 20 and 5% non-fat milk (blocking buffer), and then incubated with primary antibodies (α-actin (Cat# MAB1501, Millipore, Germany), α-ALIX (Cat# 2171, Cell Signaling, Massachusetts, USA), α-calnexin (Cat# ab133615, Abcam, UK), α-CD63 (Cat# 556019, BD Bioscience, New Jersey, USA), α-CD9 (Cat# cbl162, Millipore, Germany), α-Hsp70 (Cat# ADI-SPA-810-D, Enzo LifeScience, New York, USA), α-HA (Cat# 3724, Cell Signaling, Massachusetts, USA) diluted 1 / 1000 in blocking buffer. The membranes were incubated overnight at 4°C with either α-mouse or α-rabbit (Cat# 115-035-003, Jackson ImmunoResearch, UK) or α-mCherry (Cat# 5993, BioVision, California, USA). The membranes were then washed and finally incubated with HRP-conjugated secondary antibodies (α-mouse or α-rabbit, Cat# 115-035-003, Jackson ImmunoResearch, UK) diluted 1 / 5000 in DPBS containing 0.05% Tween 20. The HRP signal on the membrane was developed using Clarity Western ECL substrate (Bio-Rad, France) and imaged using an ImageQuant LAS 4000 (GE Healthcare Life Sciences, France).

[0131] Cytoplasmic / Membrane Fractionation. Cells to be analyzed were scraped in DPBS on ice and pelleted at 1000 × g for 5 min at 4 °C. The cell pellet was resuspended in 5 volumes of hypotonic lysis buffer (10 mM Tris-HCl pH 8, 0.5 mM MgCl2, and EDTA-free protease / phosphatase inhibitor cocktail (Roche, Switzerland)), incubated on ice for 10 min, and then homogenized by passing the cells up and down 10 times with a 26 g needle. Osmolarity was restored by adding 0.25 volumes of hypotonic buffer containing 0.6 M NaCl. Nuclei and unbroken cells were pelleted at 500 × g for 5 min at 4 °C. EDTA was added to the supernatant to a final concentration of 0.05 M, and the sample was then ultracentrifuged at 100,000 × g for 30 minutes at 4 °C (MLA50 rotor with dedicated adapter and Optima MAX-XP ultracentrifuge, Beckman Coulter, California, USA). The resulting supernatant was used as the cytoplasmic fraction. The pellet was resuspended in PBX and centrifuged at 10,000 × g for 15 minutes at 4 °C to pellet insoluble material. The supernatant was used as the membrane fraction.

[0132] Particle Measurement. Nanoparticle tracking analysis was performed using ZetaView® QUATT (Particle Metrix, Meerbusch, Germany) and the corresponding software (ZetaView 8.02.28). For size measurements, a 448 nm laser was used in scattering mode. 1 ml of sample diluted in DPBS was loaded into the cell, and the instrument measured each sample at 11 different positions throughout the cell. After automated analysis of all positions and removal of any outlier positions, the mean, median, and mode (expressed as diameter) sizes were calculated by the optimized instrument software.

[0133] FACS analysis. After treatment, cells were detached from the cell culture plate with 0.05% trypsin-EDTA and washed once with DPBS. Finally, cells were resuspended in DPBS and kept on ice (less than 1 h) until analysis on an Attune NxT flow cytometer (Thermo Scientific, Illinois, USA). Each sample was incubated with 10 μg / mL DAPI (Merck Millipore, Massachusetts, USA) immediately before analysis. The gating strategy is shown in Figure S1C. Data were analyzed using FlowJo software (BD Bioscience, New Jersey, USA).

[0134] Live cells were visualized under an EVOS M5000 microscope at 20x magnification. Image analysis was performed using ImageJ software (NIH, Maryland, USA).

[0135] Example 3: Results We developed a novel method to control cargo loading into EVs on demand: these EVs are equipped with nonviral fusogenic factors, thereby facilitating delivery of EV-cargo to recipient cells.

[0136] To sensitively measure this process, we tracked the fate of luciferase-tagged cargo. Cargo loading was enabled by a drug-reversible, inducible dimerization system. Briefly, donor cells were transfected with a plasmid encoding FKBP-tagged CD63, a classical membrane EV marker, and FRB-NanoLuciferase (NLuc), a protein normally present in the cytoplasm. Upon addition of a dimerization agent, FRB-Nluc interacts with FKBP-CD63 and is recruited to secreted EVs, which promotes their delivery to acceptor cells. This phenomenon can be further enhanced if EVs are equipped with syncytin 1, a mammalian fusogenic protein that induces fusion between the EV membrane and the plasma membrane of acceptor cells.

[0137] The first application is expected to be the development of "edited EVs" that will deliver the cas9 editing apparatus to target cells / tissues. Indeed, edited EVs containing cas9 and guide RNA for the plasma membrane-localized receptor CxCR4 and decorated with syncytin 1 have improved delivery capabilities. Therefore, Syn1+ edited EVs can efficiently knock out CxCr4 within approximately 30% of acceptor cells (Figure 11). Another application would be the delivery of toxins by "killer EVs" to specifically eliminate tumor-containing cells / tissues.

[0138] Using this novel method, we demonstrate herein that the catalytic domain of diphtheria toxin (DTA), which is involved in protein synthesis inhibition and ultimately cell death, can be delivered to acceptor cells via functionalized EVs, resulting in protein synthesis inhibition and cell death in the acceptor cells.

[0139] This novel method and the applications resulting from it are expected to open new doors in precision medicine, especially if EVs will be equipped with antibodies raised against cell-specific antigens.

[0140] References Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are incorporated by reference into this disclosure.

Claims

1. functionalized with ERV syncytin, loaded with one or more cargoes of interest, and optionally functionalized with a targeting moiety; Isolated extracellular vesicles (EVs).

2. The isolated EV of claim 1, wherein the ERV syncytin is selected from the group consisting of human syncytin (e.g., HERV-W and HERV-FRD), mouse syncytin, syncytin-Ory1, syncytin-Car1, syncytin-Rum1, or functional orthologs thereof.

3. 2. The isolated EV according to claim 1, wherein the ERV syncytin is a syncytin-1 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 (SDGGGX2DX2R) and capable of binding to the ASCT1 receptor, preferably the ASCT2 receptor.

4. The isolated EV of claim 3, wherein the syncytin-1 polypeptide comprises the amino acid sequence (SDGGGVQDQAR) shown in SEQ ID NO:

3.

5. The isolated EV of claim 4, wherein the syncytin-1 polypeptide comprises an amino acid sequence having 70% identity with the amino acid sequence ranging from the amino acid residue at position 21 to the amino acid residue at position 538 in SEQ ID NO:

1.

6. The isolated EV of claim 1, wherein the cargo is selected from the group consisting of organic molecules, polymers, polypeptides, polynucleotides and small organic compounds having a molecular weight of more than 50 daltons and less than about 2,500 daltons.

7. The isolated EV of claim 6, wherein the cargo is a polynucleotide, more particularly an RNA or DNA molecule.

8. The isolated EV of claim 6, wherein the cargo is a DNA targeting endonuclease, such as a polypeptide selected from the group consisting of a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a CRISPR-associated endonuclease, a base editing enzyme, and a prime editor.

9. The isolated EV of claim 6, wherein the cargo is a toxin.

10. The isolated EV of claim 6, wherein the toxin is diphtheria toxin or a toxic fragment thereof.

11. 7. The isolated EV of claim 6, wherein the diphtheria toxin comprises residues 1 to 389 of SEQ ID NO:

4.

12. The isolated EV of claim 1, comprising a structural polypeptide capable of forming a dimer with a cargo polypeptide.

13. 13. The isolated EV of claim 12, wherein the structural polypeptide and cargo polypeptide are fused, either directly or via a linker, to their respective domains capable of dimerizing in the presence of a compound.

14. 14. The isolated EV of claim 13, wherein a structural polypeptide is fused to an FKBP domain and a cargo polypeptide (e.g., a toxin) is fused to an FRB domain (or vice versa), which allows the FKBP and FRB domains to dimerize in the presence of rapamycin during production of the EV.

15. The isolated EV of claim 14, comprising a loading system in which a transmembrane protein is fused to an FKBP2 domain.

16. The isolated EV of claim 15, wherein the transmembrane protein is a tetraspanin.

17. The isolated EV of claim 16, wherein the tetraspanin is CD63.

18. The isolated EV of claim 15, wherein the loading system consists of the amino acid sequence shown in SEQ ID NO:

7.

19. 1. A method of treatment in a subject in need thereof, comprising: Administering a therapeutic amount of an isolated EV according to any one of claims 1 to 18 to a subject. method.

20. 20. The method of claim 19 for treating cancer.

21. A quantity of isolated EV according to any one of claims 1 to 18. Pharmaceutical compositions.

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