Extracellular vesicles and uses thereof for targeted delivery

Abstract

The present invention discloses extracellular vesicles or EVs designed for targeted delivery of bio- therapeutics and gene editing tools, to the cells and tissues. These engineered EVs comprise novel synthetic signal sequences, which facilitate efficient cargo loading. The EVs further comprise specific targeting moieties to locate and attach to target or recipient cells, components to evade immune system detection and potential rejection, inhibit phagocytosis by immune cells, minimize immunogenicity and reduce the likelihood of immune recognition as foreign entities.

Description

[0001] EXTRACELLULAR VESICLES AND USES THEREOF FOR TARGETED DELIVERY

[0002] PRIORITY CLAIM

[0003] The instant patent application is related to and claims priority from the India provisional application entitled, “EXTRACELLULAR VESICLES AND USES THEREOF FOR TARGETED DELIVERY”, Application No.: 202441056626, Filed: 25 July 2024, which is incorporated in its entirety herewith to the extent not inconsistent with the description herein.

[0004] TECHNICAL FIELD

[0005] The present invention relates to genetic engineering and gene therapy, particularly involving the use of extracellular vesicles (EVs) for targeted delivery of therapeutic agents and gene editing tools into specific cell types.

[0006] BACKGROUND

[0007] Extracellular vesicles (EVs) are small membrane-bound, naturally occurring nanoparticles secreted by cells that play a crucial role in intercellular communication by transporting proteins, lipids, and nucleic acids. These secreted by cells can be engineered for cargo delivery in vivo. Present in various body fluids such as blood, urine, and saliva, EVs are involved in numerous physiological and pathological processes, including immune responses, tissue repair, and cancer progression. Their ability to transfer bioactive molecules makes them valuable in research and clinical applications, particularly as biomarkers for disease diagnosis and as potential therapeutic delivery systems.

[0008] EVs possess several advantages over lipid nanoparticles (LNPs), viral vectors and virus-like particles (VLPs) due to their reduced immunogenicity, lower tumor risks, and minimal toxicity. They can cross cell and tissue barriers and inherently exhibit tissue tropism and repair characteristics. EVs are 30-1000 nm diameter, facilitate intracellular communication between synthesized donor cells and the internalized recipient cells that are nearby or at distance. This intracellular communication process is currently being explored for therapeutic cargo delivery of regenerative medicine.

[0009] Several methods exist for incorporating therapeutic proteins or gene editing molecules into EVs. One common technique is electroporation, used to introduce siRNA or drugs like doxycycline into EVs. However, electroporating Cas9 ribonucleoproteins (RNPs) into EVs have yielded variable results and also increased the quantity of damaged EVs. Recent studies explored the active encapsulation of Cas9 in EVs. These findings highlight the potential of EVs and underline the need for further improvement in generating engineered EVs.

[0010] EVs can be modified using both exogenous and endogenous methods. Exogenous modification involves physical techniques like electroporation to introduce specific cargo into already isolated EVs. However, this method faces several issues, such as EV precipitation and inconsistent results. Alternatively, endogenous modification involves genetically modifying cells to produce EVs that are already engineered. In this process, the desired protein or mRNA is attached to an EV membrane protein, allowing the cargo to be directed either inside the EV or onto its surface. Membrane proteins such as CD63, CD81, CD9, BASP1, and PTGFRN are commonly utilized for these genetic engineering techniques.

[0011] The delivery systems, like electroporation, engineered viral vectors and lipid nanoparticles have limitations for in vivo application and in specific HSPC targeting. The limitations include immunogenicity, poor specificity and targeting, short half-life upon entering systemic circulation and high toxicity. Thus, there is a need in the field to develop efficient systems that can reach bone marrow and release the payload into HSPCs, without any unwarranted off-target effects.

[0012] Hematopoietic stem and progenitor cells (HSPCs) are the fundamental source of all blood cell lineages throughout an individual’s lifetime. These multipotent cells possess the unique ability to self-renew and differentiate into all types of blood cells, including red blood cells, white blood cells, and platelets. Due to their regenerative capabilities, HSPCs are a focal point in research and clinical treatments for blood-related disorders, such as leukemia and lymphoma, and are utilized in stem cell transplantation therapies.

[0013] Hematopoietic stem cell transplantation (HSCT) has been a mainstay treatment for many of these conditions, involving the replacement of diseased HSCs with healthy or genetically modified cells (Genome engineered stem cell transplantation / HSPC gene therapy). However, current HSCT protocols are complex, costly, and associated with significant side effects, primarily due to the need for ex vivo manipulation of HSCs and the use of toxic conditioning regimens to enable engraftment.

[0014] Gene therapy protocols generally involve mobilization of HSPCs into the blood stream, collection of HSPCs by apheresis, ex vivo culture during which the HSPCs are electroporated with gene editing reagents, conditioning of patients with alkylating agent for myeloablation of bone marrow and the infusion of gene edited HSPCs. All these steps possess unique challenges and demand the requirement of a stem cell transplantation facility, onsite GMP facility, regular purchases of gene editing and stem cell culture reagents and highly trained human resources. Toxicity associated with bone marrow conditioning and the infections associated with delayed engraftment can risk the life of the patient. In vivo targeting of HSCs eliminates the need for cell harvest, ex vivo manipulation, and harsh conditioning regimens, thereby reducing procedural complexity, cost, and toxicity.

[0015] Whitley et al, is directed to the fusion of an octapeptide, derived from Src kinase, to the N-terminus of the Cas9 protein for encapsulation into EVs. (Whitley JA, Kim S, et.al., “Encapsulating Cas9 into extracellular vesicles by protein myristoylation.” Journal of Extracellular Vesicles 11, no. 4 (2022): el2196. doi:10.1002 / jev2.12196. PMID: 35384352; PMCID: PMC8982324.)

[0016] Zheng W et al., is directed to the screening and identification of scaffold proteins with high EV sorting ability for cargo loading. The study discloses TSPAN2 and TSPAN3 as efficient scaffold proteins for efficient transfer of luminal cargo proteins as well as surface display of different functional entities (Zheng W, Radler J, et al., “Identification of scaffold proteins for improved endogenous engineering of extracellular vesicles.” Nature Communication 14, no. 1 (2023): 4734. doi: 10.1038 / s41467-023-40453-0. PMID:37550290; PMCID: PMC10406850).

[0017] The published PCT application no. WO2021184022 is directed to engineering of EVs for targeted delivery of therapeutic cargo for treating Central Nervous System (CNS) disease, using tissuespecific ligands to increase tissue tropism to the cells, wherein the ligands and signal sequences are linked by scaffold proteins.

[0018] The published PCT application, WO2022147587 is directed to a targeted delivery of therapeutic cargo molecules using cell-penetrating peptide YARA as a scaffold protein to enhance cargo loading and targeting efficiency.

[0019] Gee et al., is directed to a CRISPR-Cas9 RNP delivery platform, for genome editing in vitro and in vivo using HIV Gag protein as scaffold (Gee, Peter, Mandy S.Y.Lung. et al., “Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping”. Nature Communications 11 (2020): 1334).

[0020] Liang, X. et al, is directed to loading of EV with cas9 for gene editing using CD63 protein as scaffold and VSV-G as fusogen (Liang, X, Gupta D, et al.; “Multimodal engineering of extracellular vesicles for efficient intracellular protein delivery”, bioRxiv, 2023).

[0021] The present invention addresses the challenges of in vivo HSC targeting and engineering EVs, by providing novel methods and compositions for producing engineered EVs, the in vivo targeting and modification of HSPCs, thereby advancing the field toward next-generation gene and cell therapies.

[0022] SUMMARY

[0023] Aspects of the present disclosure are directed to extracellular vesicles and uses thereof for targeted delivery. According to an aspect of the present disclosure, an extracellular vesicle (EV) is provided, wherein the EV encapsulates a biologically active cargo molecule functionally linked to a scaffold. The scaffold includes at least one extracellular vesicle targeting peptide sequence (ExTS) selected from SEQ ID NOs: 1-29. The ExTS possesses a net positive charge that facilitates the localization of the scaffold to the plasma membrane of a producer cell, enabling the biologically active cargo molecule to be encapsulated within the lumen of the EV during vesicle biogenesis.

[0024] In an embodiment, the biologically active cargo molecule is selected from a protein, RNA, DNA, or a small molecule. In another embodiment, the biologically active cargo molecule comprises at least one of a gene -editing component, a transcription factor, a cytotoxic protein, or an immunomodulatory factor.

[0025] In another embodiment, the gene -editing component includes zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), ribonucleoprotein (RNP) complexes containing CRISPR-Cas systems, base editors, prime editors, or epigenome editors. The cytotoxic protein includes diphtheria toxin A (DTA), DTA translocation domains, or PUMA.

[0026] In yet another embodiment, the extracellular vesicle includes a myristylation motif comprising at least one amino acid substitution selected from S3K, G3K, or L5K, and a polybasic region comprising at least one amino acid substitution selected from E21K, E21A, E27K, D17K, E23K, D16V, or D16K, or variants thereof.

[0027] In yet another embodiment, the scaffold further comprises at least one extracellular vesicle release enhancer peptide sequence (ExRE) selected from SEQ ID NOs: 30M-6, which enhances the budding and selective release of cargo-loaded vesicles from the plasma membrane of the producer cell. In yet another embodiment, the EV comprises one or more components selected from a cleavable cargo release linker, an endosomal escape sequence, a targeting ligand, and an anti-phagocytosis protein. The cleavable cargo release linker includes a pH-sensitive linker, a UV-cleavable PhoC12f linker, or a protease -cleavable linker selected from Cathepsin G, Cathepsin A, furin, or PRSS2. The endosomal escape sequence is selected from ZF5.3, a cell-penetrating peptide, a translocation domain, or a viral envelope protein. The targeting ligand includes membrane-anchored stem cell factor (maSCF), CXCR4, BaEV, CD117 antibody, or ScFV-CDl 17. The anti-phagocytosis protein is selected from CD47, CD24, or variants thereof. These components facilitate delivery of the cargo molecule to a recipient cell.

[0028] In yet another embodiment, the EV includes at least one surface display peptide sequence (ExDs) selected from SEQ ID NOs: 47-64, fused to at least one targeting ligand or binding domain. The targeting ligand or binding domain includes maSCF, a functional domain of SCF, CD117 singlechain variable fragment (scFv), or receptor-specific antibodies. These fusions enable selective binding of the EV to a receptor on the surface of the recipient cell.

[0029] In yet another embodiment, the recipient cell includes human hematopoietic stem and progenitor cells (HSPCs).

[0030] According to another aspect of the present disclosure, a genetically modified mammalian cell line is provided for the production of extracellular vesicles. The cell line includes a nucleic acid construct encoding a biologically active cargo molecule linked to a scaffold comprising at least one ExTS selected from SEQ ID NOs: 1-29. The ExTS exhibits a net positive charge that enables membrane localization and subsequent cargo encapsulation during vesicle formation.

[0031] In an embodiment, the biologically active cargo molecule is selected from gene-editing tools, transcription factors, cytotoxic proteins, immunomodulatory factors, or combinations thereof. In another embodiment, the cell line includes myristylation motifs and polybasic regions with specific amino acid substitutions.

[0032] In another embodiment, the scaffold includes an ExRE to promote vesicle budding and release.

[0033] In yet another embodiment, the producer cell line comprises additional nucleic acid constructs encoding a cleavable linker, an endosomal escape sequence, a targeting ligand, or an anti- phagocytosis protein. These components are incorporated into the EVs produced by the cell line to facilitate targeted delivery.

[0034] In yet another embodiment, the cell line expresses surface display peptides fused to targeting ligands or binding domains that enable selective binding to receptors on target cells.

[0035] In yet another embodiment, the producer cell is engineered to lack or silence the p2-microglobulin (B2M) gene to reduce immunogenicity.

[0036] In yet another embodiment, the producer cell is a derivative of HEK293, 293T, K562, HeLa, or U2OS, or a primary cell derived from mesenchymal stromal cells (MSCs), immortalized MSCs, induced pluripotent stem cells (iPSCs), embryonic stem cell-derived MSCs, HSPCs, hematopoietic cells, or organ-specific cells from the liver, spleen, pancreas, or kidney.

[0037] According to yet another aspect of the present disclosure, a method is provided for producing extracellular vesicles. The method includes introducing a nucleic acid construct encoding a biologically active cargo molecule and a scaffold comprising at least one positively charged ExTS sequence into a producer cell. The producer cell is cultured in serum-free medium or in the presence of vesicle -enhancing agents to promote secretion of EVs encapsulating the cargo molecule, followed by isolation of the secreted EVs from the culture medium.

[0038] In an embodiment, the method includes introducing gene-editing components, transcription factors, cytotoxic proteins, or immunomodulatory factors.

[0039] In another embodiment, the producer cell is engineered with myristylation motifs and polybasic regions to enhance membrane association.

[0040] In yet another embodiment, the scaffold includes an ExRE sequence to facilitate EV budding and release.

[0041] In yet another embodiment, additional constructs are introduced into the cell to express cleavable linkers, endosomal escape sequences, targeting ligands, and anti-phagocytosis proteins. In yet another embodiment, the cell line is engineered to produce surface display peptides fused to targeting ligands, which promote selective binding of EVs to specific recipient cells. In yet another embodiment, the producer cells are derived from immortalized or primary sources, including mesenchymal stromal cells, induced pluripotent stem cells, and organ-specific cells. In yet another embodiment, the recipient target includes hematopoietic cells, bone marrow, lung, liver, brain, heart, kidney, or tumor tissues.

[0042] Several aspects of the present disclosure are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the disclosure can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well known structures, materials, or operations are not shown in detail to avoid obscuring the features of the disclosure. Furthermore, the features / aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.

[0043] BRIEF DESCRIPTION OF DRAWINGS

[0044] Example embodiments of the present disclosure will be described with reference to the accompanying drawings briefly described below.

[0045] Figure 1A is a table depicting the five most positively charged N-terminal myristoylation sequences and their corresponding modified candidate sequences used as Extracellular vesicle targeting sequence for EV cargo targeting. Modified residue positions are highlighted.

[0046] Figure IB is a schematic depicting plasmid constructs for expression of ExTs-GFP fusion proteins in EV-producing cells.

[0047] Figure 1C is a schematic depicting the experimental workflow for GFP delivery using engineered EVs.

[0048] Figure ID is a set of flow cytometry plots depicting GFP signal in recipient cells treated with EVs produced from the cells transfected with GFP constructs bearing different N-terminal myristylation or engineered myristylation sequences.

[0049] Figure IE is a graph depicting the percentage of GFP-positive recipient cells following treatment with EVs produced from the cells transfected with GFP constructs bearing different N-terminal myristylation or engineered myristylation sequences.

[0050] Figure IF is a table depicting potential Extracellular vesicle targeting sequence (EXTS) candidates with engineered myristoylation and polybasic sequences and their net charge.

[0051] Figure 1G is a schematic depicting the experimental workflow for GFP delivery to recipient cells using EVs produced from ExTs-GFP-expressing cells.

[0052] Figure 1H is an image depicting GFP fluorescence in EV producer cells and GFP signal in recipient cells, with flow cytometry plots quantifying GFP-positive populations. Figure II is a graph depicting the percentage of GFP-positive recipient cells as measured by flow cytometry.

[0053] Figure 1J is a graph depicting the percentage of GFP-positive recipient cells following treatment with EVs produced from the cells transfected with GFP constructs bearing different N-terminal engineered myristylation and polybasic sequences.

[0054] Figure 2 A is a schematic depicting vector constructs incorporating ExRe motifs for EV release enhancement.

[0055] Figure 2B is a schematic depicting the experimental workflow for to identify potential Extracellular vesicle release enhancer (ExRe).

[0056] Figure 2C is a graph depicting GFP fluorescence intensity distribution in recipient cells following EV treatment, as measured by flow cytometry.

[0057] Figure 2D is a graph depicting the percentage of GFP-positive recipient cells treated with EVs produced from the cells transfected with GFP constructs bearing N-terminal EXTS and different L domains.

[0058] Figure 2E is a graph depicting the percentage of GFP-positive recipient cells treated with EVs produced from the cells transfected with GFP constructs bearing N-terminal EXTS and PPXY motif variants.

[0059] Figure 2F is a graph depicting GFP fluorescence intensity in recipient cells treated with EVs produced from the cells transfected with GFP constructs bearing N-terminal EXTS and duplicated PPXY motifs.

[0060] Figure 2G is a graph depicting the percentage of GFP-positive recipient cells treated with EVs produced from the cells transfected with GFP constructs bearing N-terminal EXTS and duplicated PPXY motifs.

[0061] Figure 2H is a graph depicting the number of GFP-positive Evs produced per cell, quantified by nanoparticle tracking analysis.

[0062] Figure 21 is a graph depicting the ratio of GFP-positive particles to total EVs as a measure of packaging efficiency.

[0063] Figure 2J is an image depicting confocal microscopy of producer cells showing GFP enrichment at the plasma membrane.

[0064] Figure 2K is a graph depicting the zeta potential of EVs engineered with ExTs or ExRe motifs.

[0065] Figure 2L is a pair of line graphs depicting intensity- weighted particle size distributions for ExTs- and ExRe-modified EVs based on dynamic light scattering.

[0066] Figure 2M is a graph depicting the mean size of fluorescently labeled EVs measured by nanoparticle tracking analysis.

[0067] Figure 2N is a set of graphs depicting particle concentration distributions of EV preparations based on size, measured by nanoparticle tracking analysis.

[0068] Figure 20 is a transmission electron microscopy (TEM) image of the engineered vesicles Figure 3A is a schematic depicting vector constructs expressing either Cas9 or Cre with ExTs and ExRe elements for editor EV generation.

[0069] Figure 3B is a schematic depicting the experimental workflow using a Cre-lox reporter system to evaluate gene editing activity.

[0070] Figure 3C is a set of flow cytometry contour plots showing RFP signal in recipient cells treated with EVs containing different Cre constructs.

[0071] Figure 3D is a graph depicting the percentage of RFP-negative recipient cells following treatment with EVs expressing different forms of Cre.

[0072] Figure 3E is an image depicting Sanger sequencing traces confirming editing at the B2M locus. Figure 3F is a graph depicting the percentage of indels at the B2M locus in recipient cells treated with increasing doses of editor EVs.

[0073] Figure 3G is a graph depicting the percentage of B2M-negative cells following editor EV treatment, based on flow cytometry.

[0074] Figure 3H is a set of flow cytometry plots depicting gene editing efficiency in the presence or absence of VSV-G.

[0075] Figure 31 is a graph depicting the percentage of B2M-negative recipient cells treated with VSV- G-containing editor EVs.

[0076] Figure 3J is a graph of biological replicate of figure 31.

[0077] Figure 3K is a schematic depicting EV constructs incorporating endosomal escape sequences.

[0078] Figure 3E is a graph depicting the percentage of indels at the BCE11A locus in cells treated with EVs engineered to express sgRNA from U6 or glutamine promoter and with endosomal escape mediated either by VSV-G or with ZF5.3.

[0079] Figure 4 A is a schematic outlining the approach of testing various surface modifications of the EVs for the HSPCs targeting.

[0080] Figure 4B shows a graph illustrating the bioluminescence in HSPCs (from donor- 1) following treatment with luciferase-loaded EVs displaying various HSPC-targeting motifs on their surfaces Figure 4C shows a graph illustrating the bioluminescence in HSPCs (from donor-2) following treatment with luciferase-loaded EVs displaying various HSPC-targeting motifs on their surfaces Figure 4D shows a FACS plot demonstrating GFP signal in HSPCs following treatment with GFP- loaded EVs displaying HSPC-targeting motifs (targeted) on their surfaces or without any targeting motifs (non-targeted).

[0081] Figure 4E presents a graph displaying GFP levels in HSPCs (donor- 1) after exposure to GFP- loaded EVs either presenting maSCF (mSCF) or lacking it (control) on their surface. HSPCs were cultured with varying concentrations of SCF to assess competition with maSCF displayed on the EVs.

[0082] Figure 4F presents a graph displaying GFP levels in HSPCs (donor-2) after exposure to GFP- loaded EVs either presenting maSCF (mSCF) or lacking it (control) on their surface. HSPCs were cultured with varying concentrations of SCF to assess competition with maSCF displayed on the EVs.

[0083] Figure 4G presents a graph displaying GFP levels in HEK-293T cells after exposure to GFP-loaded EVs either presenting maSCF (mSCF) or lacking it (control) on their surface.

[0084] Figure 4H presents a graph displaying the concentrations of maSCF on the EV particles, as quantified by ELISA.

[0085] Figure 41 presents a graph displaying the effect of maSCFs on the HSPC proliferation in vitro.

[0086] Figure 4J illustrates a schematic diagram of the experimental strategy used to assess the expression of different 'don’t eat me' signals — including CD47, CD24, and truncated CD47 — on the surface of EVs, in order to evaluate their effectiveness in protecting against phagocytosis by macrophages Figure 4K is a graph depicting relative luciferase signal in recipient macrophage cells following treatment with EVs engineered to express CD24, CD47, and both.

[0087] Figure 4L is a graph depicting relative luciferase signal in recipient macrophage cells following treatment with EVs engineered to express CD47 or truncated CD47.

[0088] Figure 5A is a schematic depicting the approach to demonstrate the bone marrow targeted delivery of EVs

[0089] Figure 5B is an image depicting organs of humanized mice under bioluminescence post infusion of non-targeted or target EVs containing luciferase

[0090] Figure 6A is a schematic depicting construct design and workflow for delivery of diphtheria toxin A (DTA) to HSPCs using engineered EVs.

[0091] Figure 6B is a graph depicting cell numbers following treatment with GFP- or DTA-loaded EVs, comparing targeted and non-targeted conditions.

[0092] Figure 6C is a graph depicting cell numbers following treatment with EVs containing DTA fused to either an intein linker or PhoC12f linker.

[0093] Figure 6D is a graph depicting cell numbers after treatment with EVs expressing DTA fused to different translocation domains (Dp-TL or Ch-TL).

[0094] Figure 6E is a graph depicting cell numbers following treatment with GFP- or DTA-loaded EVs produced with or without BaEV glycoprotein.

[0095] Figure 7A is a schematic depicting construct design and workflow for delivery of diphtheria toxin A (DTA) to AML cells (Kasumi) using engineered EVs.

[0096] Figure 7B is a graph depicting cell numbers following treatment with GFP- or DTA-loaded EVs, comparing targeted and non-targeted conditions.

[0097] Figure 7C is a graph depicting cell numbers following treatment with EVs containing DTA fused to either an intein linker or PhoC12f linker.

[0098] Figure 7D is a graph depicting cell numbers after treatment with EVs expressing DTA fused to different translocation domains (Dp-TL or Ch-TL).

[0099] Figure 7E is a graph depicting cell numbers following treatment with GFP- or DTA-loaded EVs produced with or without BaEV glycoprotein.

[0100] Figure 8A is a schematic depicting construct design for ExDs expression with surface display and mCherry co-expression.

[0101] Figure 8B is an image depicting mCherry and GFP fluorescence in producer cells expressing ExDs constructs.

[0102] Figure 8C is a graph depicting relative luciferase signal in recipient cells following treatment with EVs modified with different ExDs.

[0103] Figure 8D is a schematic depicting EVs engineered with targeting scaffolds on the surface and cargo loading sequences in the lumen.

[0104] Figure 8E is a schematic depicting the targeting of CD117-expressing recipient cells using mStrep- GFP EVs incubated with biotinylated anti-CD117 antibody.

[0105] Figure 8F is a schematic depicting the EXDS with different glycosylation mutations

[0106] Figure 8G presents a graph illustrating how glycosylation mutations in the EXDS of the Evs, influence the delivery of mCherry displayed on the cell surface.

[0107] Figure 8H is a graph depicting mean fluorescence intensity (MFI) of mCherry in recipient cells treated with EVs incorporating the same ExDs variants.

[0108] Figure 9 is a schematic depicting engineered EVs containing Cas9 ribonucleoprotein complexes in the lumen and surface modifications for HSPC targeting, immune and phagocytic evasion. In the drawings, like reference numbers generally indicate identical, functionally similar, and / or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number. In the drawings, like reference numbers generally indicate identical, functionally similar, and / or structurally similar elements.

[0109] DETAILED DESCRIPTION

[0110] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

[0111] Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0112] The terms “comprises”, “comprising”, or any other variations thereof, are intended to cover a nonexclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more compositions or elements or structures or components preceded by “comprises... a” does not, without more constraints, preclude the existence of other compositions or elements or other structures or other components or additional compositions or additional elements or additional structures or additional components.

[0113] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The compositions, methods, and examples provided herein are illustrative only and not intended to be limiting.

[0114] Embodiments of the present disclosure will be described below in detail with reference to the accompanying drawings.

[0115] The present invention provides novel compositions and methods for the targeted delivery of biologically active cargo molecules using engineered extracellular vesicles (EVs). These engineered EVs are designed for enhanced cargo encapsulation, efficient release from producer cells, precise targeting to recipient cells or tissues, and evasion of immune detection, thereby addressing significant challenges in gene therapy and therapeutic delivery. The invention further discloses genetically modified producer cell lines capable of continuous and optimized EV production, and methods for producing them. The invention further provides use of the EVs of the invention for various therapeutic and diagnostic applications.

[0116] “Extracellular Vesicles” or “EVs” as used herein, refer to small membrane -bound, naturally occurring nanoparticles secreted by cells, typically ranging from 30 to 1000 nm in diameter, that play a crucial role in intercellular communication by transporting proteins, lipids, and nucleic acids. EVs, particularly EVs (a sub -population of EVs whose size ranges between 100 and 1000 nm, formed by the direct outward budding of a mammalian cell membrane), are engineered for cargo delivery.For the purpose of this disclosure both terms “Extracellular Vesicles or EVs” may be used interchangeably.

[0117] The present invention discloses extracellular vesicles, comprising a biologically active cargo molecule functionally linked to a scaffold protein.

[0118] “Biologically Active Cargo Molecule” as used herein may comprise gene -editing tools such as proteins, DNA, RNA, gene-editing complexes, peptides, or small molecules or therapeutic agents that are loaded into the EVs for targeted delivery. Examples include gene-editing components (e.g., ZFNs, TALENs, CRISPR-Cas systems, base editors, prime editors, epigenome editors), transcription factors, cytotoxic proteins (e.g., diphtheria toxin A (DTA), PUMA), or immunomodulatory factors.

[0119] The biologically active cargo molecule may be functionally linked to a protein or peptide sequence, referred to as a “Scaffold” in the present disclosure. The scaffold may facilitate its incorporation into or display on EVs., facilitating its incorporation into or display on EVs.

[0120] “Functionally linked” means that the biologically active cargo molecule is connected, either directly or indirectly, to another sequence such as a peptide, protein, or domain in such a way that the linkage does not abolish or substantially impair the functional activity of either component. For example, in some embodiments of the invention, a gene editor (e.g., Cas9) is functionally linked to an ExTS peptide sequence that allows the editor to be selectively localized to the plasma membrane without compromising its editing function — thereby promoting its encapsulation into EVs.

[0121] The present invention thus provides a platform for engineering extracellular vesicles to achieve highly efficient and targeted delivery of diverse biologically active cargo molecules. This platform overcomes limitations of existing delivery systems by integrating novel synthetic signal sequences for enhanced cargo loading, optimized EV release, precise cellular targeting, and robust immune evasion. In an aspect, a signaling peptide sequence called Extracellular Vesicle Targeting Sequence or ExTS were designed to drive desired payload or cargo into the EVs. The ExTs are synthetic peptide sequences (e.g., SEQ ID NOs: 1-29) that facilitate the efficient loading of therapeutic cargo into EVs by directing them to the plasma membrane of producer cells. ExTS typically include a lipidation motif (e.g., myristoylation, palmitoylation) and a polybasic region (PBR), engineered to possess an elevated net positive charge for enhanced membrane localization and protein enrichment of the scaffold at the plasma membrane of a producer cell. This localization is a key step that enables the efficient encapsulation of the biologically active cargo molecule into the EV lumen during EV biogenesis. The signaling peptide sequence, ExTs, when fused with an N-terminus of desired payload or cargo in a gene construct and transfected / transduced into mammalian cells, facilitates the trafficking of the payload / cargo to the plasma membrane of the cells upon expression of the gene construct. Subsequently, the payload / cargo is encapsulated into EVs, utilizing the natural sorting mechanisms of the ExTs sequence when the plasma membrane buds out.

[0122] “EV biogenesis” refers to the biological process by which extracellular vesicles (EVs) are generated within a cell and released into the extracellular environment. This process includes the sorting and packaging of biologically active molecules (e.g., proteins, RNAs, and lipids) into vesicular structures derived from either (i) inward budding of endosomal membranes to form multivesicular bodies (MVBs) that fuse with the plasma membrane to release exosomes', or (ii) direct outward budding and fission from the plasma membrane to form microvesicles. The biogenesis process enables selective encapsulation and secretion of molecular cargo for intercellular communication or therapeutic delivery.

[0123] A “Producer Cells” as used herein are mammalian cells that are genetically engineered to produce EVs encapsulating desired cargos. These cells are transduced with constructs of the present invention encoding sequences such as the Extracellular vesicle Targeting Signals (ExTs), Extracellular vesicle Display Signals (ExDs), and other relevant proteins. Producer cells can be established cell lines (e.g., HEK293T, K562, HeLa, U2OS, or derivatives thereof) or primary cells (e.g., mesenchymal stromal cells (MSCs), hematopoietic stem and progenitor cells (HSPCs), induced pluripotent stem cells (iPSCs), hematopoietic cells, liver cells, spleen cells, pancreatic cells, or kidney cells), and used to generate further cell lines with required genetic modifications.Figure 1 illustrates the efficacy of ExTS in facilitating plasma membrane targeting in producer cell lines and subsequent lumen loading of cargo.

[0124] The ExTS typically comprises a lipidation motif, such as a myristoylation motif, a palmitoylation motif, or other N-terminal lipidation sequences, or combinations thereof. The myristoylation motif can further comprise specific amino acid substitutions like S3K, G3K, and L5K, or variants thereof, which are shown to enhance cargo delivery. Figure 1A presents a table of N-terminal myristoylation sequences, including engineered variants with increased positive charges (e.g., S3K-CIB, L5K-CIB, S3K L5K-CIB), which serve as ExTS candidates. Figure IB schematically depicts plasmid constructs for expressing ExTs-GFP fusion proteins in EV-producing cells, while Figure 1C outlines the experimental workflow for GFP delivery using these engineered EVs. Microscopic analysis in Figure ID shows GFP localization in producer cells, demonstrating the concentration of GFP in the cytoplasm of Myr-GFP cell lines compared to diffuse expression in wild-type GFP cells. Additionally, the ExTS incorporates at least one polybasic region (PBR) or a variant thereof. The polybasic region can include amino acid substitutions such as E21K, E21A, E27K, D17K, E23K, D16V, and D16K, which are critical for enhancing membrane binding affinity in synergy with the lipidation signal. Figures IE and IF quantitatively demonstrate the fold increase in GFP-positive recipient cell populations and GFP content per cell, respectively, upon treatment with EVs produced from cells transfected with various ExTS-GFP constructs. Notably, the combination of G3K and E21A mutations in Myr+PBR sequences resulted in nearly a 400-fold increase in cargo delivery (Figure ID).

[0125] The biologically active cargo molecule encapsulated within the EV lumen can be diverse, including proteins, RNA, DNA, or small molecules. More specifically, these cargo molecules may comprise Gene-editing components such as zinc finger nucleases (ZFNs), transcription activatorlike effector nucleases (TALENs), CRISPR-Cas systems (which are ribonucleoprotein (RNP) complexes), base editors, prime editors, or epigenome editors; Transcription factors; Cytotoxic proteins including diphtheria toxin A (DTA), DTA translocation domains (e.g., Diphtheria translocase domain, Austwickia chelonae translocase domain), or PUMA (p53 upregulated modulator of apoptosis); Immunomodulatory factors or combinations thereof.

[0126] In an aspect of the present disclosure, a method is provided to facilitate active encapsulation of protein cargo into extracellular vesicles (EVs) by enriching the protein at the plasma membrane. As EVs are formed via plasma membrane budding, localization of a protein at the plasma membrane was evaluated as a strategy to increase its incorporation into EVs. N-terminal myristoylation was selected as the targeting mechanism. Myristoylation is a post-translational lipid modification involving the covalent attachment of a myristoyl group to the N-terminal glycine of a protein, promoting its membrane association.

[0127] To identify candidate myristoylation signals, 133 octapeptide sequences annotated as myristoylation motifs were obtained from the SVMyr dataset and ranked by net positive charge. Octapeptides derived from calcium and integrin-binding protein (CIB), transmembrane protein 106B, and brain acid soluble protein 1 (BASP1), each exhibiting a net positive charge of +3.553, were selected, along with sequences from LYN and SRC kinases. Each of the selected motifs was fused to the N-terminus of green fluorescent protein (GFP), and lentiviral vectors encoding these constructs were used to generate stable 293T producer cell lines.

[0128] Sequence alignment of the top five candidates indicated that residues at positions 2 and 5 tolerated substitution. To increase net positive charge, lysine residues were introduced at these positions. A leucine -to-lysine substitution at position 5 in the CIB-derived motif (L5K mutation) was generated. Wild-type GFP, which lacks a myristoylation signal, was used as a control. EVs were harvested from the stable cell lines expressing the various constructs and applied to recipient 293T cells.

[0129] Myristoylation motif tagging increased GFP delivery to recipient cells when compared to wildtype GFP. Among the tested sequences, the CIB-derived motif showed the highest delivery. The introduction of the E5K mutation into the CIB motif resulted in an increase in delivery of more than 50-fold relative to wild-type GFP. A combination of G3K and E5K mutations led to a decrease in delivery efficiency compared to E5K alone, indicating that motif composition influences encapsulation and delivery.

[0130] In an embodiment, a two-signal strategy was implemented by appending a polybasic region (PBR) to the Myr-CIB and Myr-CIB-E5K constructs, as it was observed that a majority of known myristoylated proteins found in EVs contain a PBR immediately following the myristoylation site. The PBR was modified by substituting acidic or neutral amino acids with basic amino acids to increase the overall net positive charge.

[0131] In an embodiment, incorporation of a PBR into Myr- or G3K-myr constructs increased GFP delivery to recipient cells. A construct containing the G3K mutation and a PBR with an E21A substitution (glutamic acid to alanine) resulted in a delivery increase of approximately 400-fold relative to wild- type GFP. In contrast, introduction of the D16V mutation (aspartic acid to valine) into the PBR reduced the amount of encapsulated GFP in EVs. These findings indicate that the composition and charge of residues in the targeting motif influence the efficiency of protein loading into EVs and subsequent delivery to recipient cells.Additional polybasic region modifications may include substitutions such as E27K, D17K, E23K, and D16K to increase the net positive charge and membrane affinity

[0132] In an aspect, in order to synthesize the ExTs, a lipidation signal such as Myristylation (Myr) signal was fused with the N-terminus of a GFP reporter (payload / cargo) and lentiviral-mediated Myr- GFP 293T stable cell lines were established for EV production). Various ExTs were fused with the N-terminus of GFP. The invention describes a lentiviral construct that integrates ExTs with a cargo / payload of interest, enhancing targeted delivery capabilities. Beginning with the EFl alpha promoter for robust expression, the construct incorporates ExTs to direct the payload / cargo into EVs. A self-cleaving P2A peptide separates the cargo from downstream elements, such as selection markers like puromycin, facilitating efficient expression and selection of transduced cells. These transduced cells that produce EVs are called producer cells.

[0133] In an embodiment, the efficacy of the above sequence in delivering the payload into a target cell was compared to the wild-type GFP. Wild-type GFP that has no Myr signal and solely relies on passive loading into EVs was used as a control. While WT-GFP cell lines showed GFP expression throughout the cells, the Myr-GFP cell lines had GFP expression concentrated in the cytoplasm (Fig 1H). The EVs were collected from both conditions and added to recipient cells, such as HEK293T cells. It was observed that Myr tagging facilitated around a 25-fold increase in the GFP cargo delivery compared to wild-type GFP.

[0134] In an aspect, a method to further enhance the payload / cargo loading into EVs was disclosed, wherein the ExTs comprises a myristoylation signal followed by a polybasic region (PBR) to enhance membrane binding affinity. The lipidation signal such as myristoylation, palmitoylation or prenylation serves as the primary membrane targeting signal. When this is combined with a secondary membrane affinity signal such as clusters of basic amino acids, PH domains, it further enhances the membrane association. Therefore, such a two-signal model (Myr-PBR) works synergistically to achieve optimal membrane binding. By tagging a payload / cargo to Myr+PBR, the cargo loading efficacy increased by 100-fold compared to no tagging. This was exemplified by using wild-type GFP as a payload / cargo. Fig. II and 1J show the fold increase in GFP-positive recipient cell population when treated with 20 ug of EVs and the fold increase in GFP content per cell when treated with 20 ug of EVs, respectively.

[0135] In an embodiment, the basic nature of the Myr+PBR sequence was increased to further improve membrane binding affinity. Acidic or neutral amino acids were replaced in the Myr+PBR region with basic amino acids. This increased the net charge of the 30-amino acid sequence (Fig IF).

[0136] For instance, in one amino acid sequence, a smaller amino acid, Glycine (G) was replaced with a larger, positively charged amino acid, Lysine (K) and in another a negatively charged amino acid Glutamic acid (E) was replaced with a neutral amino acid, Alanine (A). The G3K and E21A mutations further improved cargo delivery over the Myr+PBR sequence while a third mutation, such as D16V (replacing a negatively charged amino acid, Aspartic acid to hydrophobic amino acid, valine) drastically reduced the cargo encapsulated EVs suggesting the importance of signaling sequence in cargo loading.

[0137] In an aspect, ExTs tagging of payload / cargo facilitated over 400-fold improved delivery into the recipient cells when compared with EVs generated from cells that express the payload / cargo without ExTs. Myr+PBR with the combination of both G3K and E21 A mutations resulted in nearly 400-fold increase in cargo delivery (Fig 1H). In an embodiment, the amount of EVs added to the recipient cells ranged from 10 to 30 ug based on protein concentration or between IxlO7to IxlO8EV particles.

[0138] In an aspect, the ExTs can be used to deliver any one of the following payloads / cargoes: therapeutic proteins, DNA, RNA, gene editing protein complexes, or peptides to be expressed inside the lumen of the EV.

[0139] In an aspect, a method to encapsulate proteins of any gene editing system into EVs for gene editing in target cells such as HSPCs, MSCs, Macrophages, T-cells, hepatocytes but not limited to these is provided.

[0140] To further optimize the production and yield of cargo-loaded EVs, the scaffold can further comprise at least one Extracellular Vesicle Release Enhancer (ExRE) peptide sequence, selected from SEQ ID NOs: 30-46. These sequences enhance the budding and selective release of cargo- loaded EVs from the plasma membrane of producer cells, often comprising viral late domain motifs (e.g., PSAP, PTAP, YPXL, PPXY, and their duplications / triplications) that hijack cellular machinery to facilitate particle budding.

[0141] In an embodiment, peptide sequences from three major viral late domain classes — PT / SAP, PPXY, and LYPXnL — were inserted between the engineered EXTS scaffold and a GFP reporter to assess their efficacy as extracellular vesicle (EV) release enhancers (ExREs) (FIG. 2A). Constructs containing PPXY (where X = S) or YPXL motifs significantly enhanced GFP cargo delivery to recipient cells (FIGS. 2B-2D), while PTAP or PSAP -containing constructs were less effective.

[0142] As depicted in Figure 2, ExRE elements significantly boost the release of cargo-encapsulated EVs. Figure 2A illustrates vector constructs incorporating various ExRe motifs for EV release enhancement, and Figure 2B shows the experimental workflow. Figures 2C-2G demonstrate that late domains featuring YPXL and PPXY motifs, particularly the PPEY variant and its duplication, significantly increase GFP delivery efficiency, suggesting their compatibility with ExTS and enhanced budding. Figure 2H and 21 quantify the increased number of GFP-positive EVs produced per cell and the improved relative packing efficiency when ExRE is utilized. Confocal microscopy in Figure 2 J confirms GFP enrichment at the plasma membrane, supporting the role of ExRE in EV biogenesis. The ExRE peptide sequence can comprise at least one motif selected from PSAP, PTAP, YPXL, PPSY, PPLY, PPPY, PPAY, and PPEY motifs, or combinations thereof. Furthermore, the ExRE peptide sequence can comprise at least two copies of a selected motif (e.g., PPEY -PPEY or PPEY -PPEY -PPEY), which has been shown to further amplify cargo delivery.

[0143] In other embodiments, the ExRE comprises a peptide selected from SEQ ID NOs: 30-46, each containing at least one of the following motifs: PSAP, PTAP, YPXL, PPSY, PPLY, PPPY, PPAY, or PPEY. Enhanced EV release and cargo encapsulation were observed when these motifs were fused downstream of the EXTS domain.

[0144] To optimize the PPXY motif, the central residue (X) was systematically substituted with amino acids commonly found in viral late domains. Among the variants, PPEY emerged as the most effective, showing improved delivery compared to the parental PPXY motif (FIG. 2E). Notably, tandem duplication of the motif (e.g., PPEY-PPEY) further enhanced EV budding and GFP cargo loading (FIGS. 2F and 2G), likely by improving host machinery recruitment.

[0145] EVs derived from EXTS-PPEY-PPEY constructs exhibited over a 10-fold increase in production (15 x 108particles / 48 hrs) compared to EXTS-only constructs (0.2 x 108particles) (FIG. 2H). These engineered EVs had consistent morphology (cuboidal), size (70-120 nm), and surface charge (zeta potentials of -26.32 mV and -27.58 mV) (FIGS. 2I-2N). Confocal microscopy confirmed that the vesicles originated from the plasma membrane and retained canonical EV markers without contamination from intracellular organelles (FIG. 2J).

[0146] Although EXTs improved cargo encapsulation and delivery, cargo-containing EVs constituted less than 5% of total EVs. Native EVs lacking exogenous cargo dominated production, limiting the yield of functional vesicles. The approach described herein selectively enhanced the release and delivery efficiency of cargo-encapsulated EVs.

[0147] For controlled release of the biologically active cargo molecule inside the recipient cell, the cargo molecule can be functionally linked to the ExTS via a cleavable cargo release linker (CRL) sequence, selected from SEQ ID NOs: 47-64. The CRL enables the release of the cargo molecule upon uptake by a recipient cell or exposure to a specific intracellular environment. The CRL may be selected from various types of inducible or cleavable linkers, including pH-sensitive linkers (e.g., mini-intein); UV-cleavable linkers (e.g., PhoC12f); Protease -cleavable linkers (e.g., sequences cleavable by Cathepsin G, Cathepsin A, furin, or PRSS2). Alternatively, the cleavable cargo release linker (CRL) can be replaced by a transient protein-protein interaction domain selected from FKBP-FRB or MCP-MS2, which enables cargo release through inducible dissociation.

[0148] In an embodiment, ExTs was tagged to the N-terminus of CRE through UV cleavable linker (PhOC12f) or a pH cleavable linker (intein) and a stable mammalian producer cell line that produces EVs loaded with Cre was established through puromycin selection. In addition to UV- and pH-sensitive linkers (e.g., PhoC12f and Intein), protease-cleavable linkers, such as those responsive to Cathepsin G, Cathepsin A, furin, or PRSS2, may be incorporated to facilitate release of the cargo within recipient cells

[0149] In an embodiment, ExTs was tagged to the N-terminus of Cas9-NLS (Nuclear Localisation Signal) and a stable mammalian producer cell line that produces EVs loaded with Cas9-NLS was established through puromycin selection. Another mammalian cell line such as HEK293T that stably expresses a sgRNA was established. The Cas9-NLS loaded EVs were collected and added to HEK293T cells that express sgRNA against CCR5 locus. When the Cas9-NLS was tested for gene editing at target locus CCR5, the efficacy of gene editing was much less compared to electroporating the target cells with cas9-NLS.

[0150] In yet another embodiment, a transient linker was added between ExTs and Cas9, to help Cas9 to detach from ExTs and release Cas9 for nuclear translocation. In an embodiment, Phocl2f was fused to ExTs and the purified EVs were exposed to UV for 30 sec to 2 minutes, cleaving Phocl2f, thereby releasing the cargo, Cas9.

[0151] In an aspect of the present disclosure, the gene editing efficacy may be improved by optimizing the linker efficiency, using alternative linkers. Furthermore, the yield and purity of the EVs may be increased by optimizing the culture conditions. In yet another embodiment, the loading efficacy of Cas9 into EVs may be improved by optimizing the ExTs-Cas9 fusion construct and using other means of incorporating this construct into the EVs. Furthermore, the design of sgRNA, modifications of Cas9, and use of other Cas proteins, may improve the nuclease activity or improve nuclear localisation signals and the efficiency of the EV loading. In an embodiment, the gene editing system is selected from Cas9-ribonucleoprotein (Cas9-RNP), Cre, TALENs, ZFN, Cpfl (Casl2a), AsCasl2f, Casl2b, Casl3a (C2c2), Casl3b, Casl4, base editors (ABE, CBE), prime editors, epigenome editors and meganucleases.

[0152] In an embodiment, cleavable or inducible cargo release linkers (CRLs) were identified to enable transient fusion of ExTs to the desired cargo, allowing controlled release upon delivery into recipient cells. These CRLs facilitate the separation of the cargo from the ExTs domain to permit cytoplasmic or nuclear translocation, depending on the intended intracellular destination. The CRL may be selected based on the desired intracellular trigger and includes, for example, UV-sensitive linkers such as PhoC12f, blue light-inducible systems such as CRY2-CIB1, pH-sensitive mini- inteins, protease -cleavable sequences responsive to Cathepsin G, Cathepsin A, furin, or PRSS2, and chemically inducible linkers such as the rapamycin-sensitive FRB-FKBP system.

[0153] In an embodiment, Cas9 was engineered for EV -based protein delivery by fusing the EXTS-EXRE membrane -targeting signal to its N-terminus, enabling its packaging into EVs. These EVs were produced using stable HEK293T cell lines expressing the fusion construct. When delivered in combination with a single-guide RNA (sgRNA).

[0154] In an embodiment, to enable intracellular release of Cas9 from the EV membrane, cleavable cargo release linkers (CRLs) were inserted between the EXTS-EXRE domain and the Cas9 protein (FIG. 3 A). A photo-sensitive linker (PhoC12f) and a pH-sensitive self-cleaving Intein were used. These linkers were designed to promote Cas9 release either prior to or upon cellular entry, thereby allowing nuclear translocation and functional genome editing (FIG. 3B). Alternatively, a UV- cleavable linker (e.g., PhoC12f), or a protease-cleavable linker responsive to enzymes such as Cathepsin G, Cathepsin A, furin, or PRSS2.

[0155] In an embodiment, Cre recombinase was used as a model cargo to evaluate CRL-mediated release and intracellular activity. Cre was fused to the EXTS-EXRE domain via either the PhoC12f or Intein linker, and the resulting constructs were stably expressed in producer cells. EVs collected from these cells were used to treat 293T reporter cells harboring a LoxP-flanked RFP cassette. Both EXTS-EXRE-PhoC12f-Cre and EXTS-EXRE-Intein-Cre constructs enabled efficient recombination at the LoxP sites, confirming the successful delivery and functional activity of Cre (FIGS. 3C and 3D). It was observed that CRL architecture supports intracellular release of protein cargo from EVs. In an embodiment, the CRL-enabled system was extended to Cas9 delivery. Stable producer cell lines expressing EXTS-EXRE-Intein-Cas9 were generated, and the corresponding EVs were applied to 293T cells expressing a sgRNA targeting the B2M locus. This resulted in efficient genome editing, as evidenced by loss of B2M expression. FIGS. 3E, 3F, and 3G confirming CRL insertion restores Cas9 functionality by enabling its nuclear localization post-delivery.

[0156] In an embodiment, co-packaging of Cas9 and sgRNA into the same EVs was achieved by generating stable producer cell lines co-expressing sgRNAs and either EXTS-EXRE-PhoC12f- Cas9 or EXTS-EXRE-Intein-Cas9. The resulting EVs enabled genome editing at two independent loci, B2M and HBB, in recipient cells (FIGS. 3H, 31, and 3J).

[0157] In an embodiment, an alternative approach to load Cas9 into EVs was developed using an MS2- based RNA-binding strategy. Specifically, Cas9 was engineered to carry MS2 stem loops, and stable producer lines expressing EXTS-EXRE-Intein-MS2 Coat Protein (MCP) were used to recruit Cas9 for EV packaging.

[0158] To eliminate reliance on viral components in the Editor EV system, the vesicular stomatitis virus glycoprotein (VSV-G), previously employed to promote endosomal escape, was removed. In its place, a synthetic endosomal escape domain, ZF5.3, was fused either downstream of the cargo release linker (CRL) or directly to the protein cargo (FIG. 3K). EVs generated using this configuration retained robust genome editing activity in recipient cells, even in the absence of VSV-G (FIG. 3H), thereby establishing a fully non-viral EV-based gene editing platform.

[0159] In an embodiment, engineered endosomal escape sequences were incorporated to enhance cytoplasmic release of EV cargo following endocytic uptake. These sequences include synthetic peptides such as ZF5.3 and viral envelope proteins such as VSV-G and BaEV, both of which possess fusogenic properties that disrupt endosomal membranes and facilitate intracellular delivery of the encapsulated payload.

[0160] For precise delivery, the engineered EV can further comprise at least one targeting moiety that directs the EV to a specific recipient cell. These moieties are selected from a range of ligands and antibodies, including Membrane-anchored stem cell factor (maSCF), CXCR4, BaEV, CD117 antibody, CD 117-single -chain variable fragment (ScFV-CD117) or combinations thereof. The specific recipient cell can be, for example, a hematopoietic stem and progenitor cell (HSPC), or other cells / tissues such as hematopoietic cells, bone marrow, lung, liver, brain, heart, kidney, and tumor cells (e.g., acute myeloid leukemia (AML) cells).

[0161] Figures 4 and 5 provide extensive support for the efficacy of these targeting moieties. Figure 4A outlines the approach for testing various surface modifications for HSPC targeting. Figures 4B and 4C show that luciferase-loaded EVs displaying maSCF, BaEV, or CD117-scFv significantly increase delivery to HSPCs. Figures 4D-4G demonstrate GFP signal in HSPCs and HEK293T cells, confirming cell-specific delivery. Figure 4H quantifies maSCF on EV particles, and Figure 41 shows its effect on HSPC proliferation. Figure 5 illustrates the in vivo bone marrow targeted delivery of EVs in humanized mice, showing preferential localization to bone marrow with reduced off-target accumulation when engineered with BaEV, CD117-ScFV, or maSCF.

[0162] To enhance the therapeutic efficacy of EVs by extending their circulation time and reducing premature clearance, the EV of the invention further comprises components involved in immune evasion strategies. The EV may comprise at least one anti-phagocytosis protein displayed on its surface, selected from CD47, CD24, or variants thereof. These proteins prevent phagocytosis of the EV by immune cells. Figure 4J illustrates the experimental strategy for assessing 'don’t eat me' signals like CD47 and CD24, while Figures 4K and 4L demonstrate their effectiveness in protecting against macrophage phagocytosis. The engineered EV may also be substantially devoid of P2 -microglobulin (B2M) protein on its surface. This is achieved by genetically modifying the producer cell line to knock out or silence the B2M gene, thereby eliminating MHC class I molecules from the EV surface and reducing immune recognition or clearance by recipient cells.

[0163] Monocytes and macrophages can phagocytose EVs once they are injected in vivo, which reduces the number of EVs that reach the target organ. In an aspect, the ExDs can be used to extend the in vivo circulation time of EVs by cloning antiphagocytic factor (antigen binding sequence of CD47). The antigen binding sequence of CD47 was cloned downstream of ExDs to enable surface display. EVs generated from cell lines over expressing CD47-ExDs construct, were used to treat macrophages that stably express ExTs-nLuc. Macrophages treated with EVs from CD47- overexpressing cells exhibited lower levels of bioluminescence compared to those treated with control EVs, suggesting that CD47-O verexpressing EVs resist phagocytosis. Fig. 4K shows a bar graph of bioluminescence intensity in macrophages when treated with EVs displaying CD47. In other embodiments, the anti-phagocytosis protein displayed on the EV surface includes CD24 or engineered variants thereof, which similarly engage inhibitory receptors on phagocytic cells, thereby reducing clearance. In an embodiment, specialized EV producer cell lines were engineered to enhance immune evasion and modulate amino acid transport. To reduce recognition by host immune surveillance mechanisms, the Beta-2 Microglobulin (B2M) gene, encoding the light chain of MHC class I molecules, was knocked out. Loss of B2M expression eliminates MHC class I complexes from the cell surface, thereby diminishing immunogenicity, improving gene editing efficiency, and increasing extracellular vesicle (EV) yield. As a result, EVs derived from these B2M-deficient cells are substantially devoid of surface-expressed B2M protein, further reducing the risk of immune clearance in vivo.

[0164] Furthermore, in an embodiment, genes coding for amino acid transporters, namely, ASCT1 and ASCT2 were knocked out to produce B2M / _, ASCTl / _and ASCT2 / _producer cells. Knocking out of these genes reduced the formation of cell syncytia, a phenomenon wherein two or more individual cells fuse to form multinucleated cells. Cell syncytia hinders quality and yield of produced EVs.

[0165] Furthermore, these producer cells were modified to overexpress tetraspanins such as CD9 and envelope proteins such as BaEV, which serve as accessory proteins that further improve payload / cargo loading and sorting.

[0166] In an aspect, pseudotyping was used to modify the surface proteins of EVs to improve their endosomal escape, thereby improving the delivery efficiency. EVs encapsulating GFP through ExTs may be made to co-express any one of the following envelope proteins: VSV-G, HA protein, GALV, MLV, and BaEV, but not limited to these. In an aspect, BaEV pseudotype was used to improve the EV-mediated cargo / payload delivery into HSPCs by up to 2-fold. Other pseudotypes such as connexin, syntenin may also be used for the same purpose, although the efficacy of BaEV was significantly better compared to other pseudotypes when delivered to HSPCs.

[0167] Although EVs mediated the delivery into HSPCs, high doses were observed to reduce cell viability. In an embodiment, different methods to purify EVs to reduce toxicity were tested. By nanoparticle tracking analysis (NTA) of EVs, the GFP-contained EVs were estimated to be in the range of 200 to 900 nm, and the GFP-encapsulated EVs contributed only 0.04% of the total EVs, as most of the EVs were <100 nm in size. Selective purification of EVs larger than 100 nm enriched cargo-loaded EVs, reduced the number of EVs for cellular treatment and overcame the toxicity. A revised protocol reduced the EV count by 10-fold to saturate HSPCs with cargo. Fig 5D illustrates the EVs or EV subpopulation purified by different protocols and uptake of GFP in HSPCs.

[0168] The engineered EV may further comprise a surface display signal (ExDs), wherein the ExDs comprise transmembrane proteins, particularly modified PTGFRN protein that display ligands or targeting moieties on the surface of EVs, enhancing their ability to target specific cell types.The ExDs is functionally linked to at least one targeting ligand or binding domain, enabling selective binding of the extracellular vesicle to a recipient cell surface receptor. The ExDs can be fused to various targeting ligands or binding domains, including membrane-anchored stem cell factor (maSCF), a CD117-single -chain variable fragment (CD117-scFv), a receptor-specific antibody, or combinations thereof. These modifications enable high-density surface presentation of proteins on EVs.

[0169] In an aspect of the present disclosure, to facilitate surface display of proteins on EVs, ExDs were synthesized by modifying a transmembrane protein present abundantly on EVs, namely, PTGFRN, more precisely, a truncated version of PTGFRN del687. In an embodiment, the PTGFRN-del687 sequence is modified to include either a leucine zipper or a foldon sequence. These sequences are used for dimerization or trimerization, respectively, which enhances the display of PTGFRN- del687 on the surface of EVs. In an embodiment, in addition to these structural modifications, engineered N-glycosylation sites are introduced on the extracellular domain of PTGFRN-del687. Mutations such as V694N (Gl), S696N (G2), V734N (G3), or L745N (G4) were introduced to create new N-glycosylation sites on the extracellular surface of PTGFRN. These sites not only enhanced the stability of the displayed proteins by protecting them from protease activity during EV circulation but also improved specific cell (such as HSPCs) targeting when placed strategically. Fig. 8A provides a scheme of lentiviral vector construct comprising ExDs used for surface display of any protein. Efl a is the promoter that drives the expression of ExDs and display protein. A display protein such as mCherry is connected with the ExDs through a flexible linker.

[0170] Fig 8B is a fluorescence micrograph showing the EV producer cells displaying GFP and mCherry through ExTs and ExDs simultaneously. Fig. 8C shows a scheme of the workflow to produce EVs that display proteins on the surface and load other proteins inside the EV. It shows a cargo / payload (such as GFP) loaded into the lumen of the EV through ExTs and displaying a protein such as monomeric streptavidin on the surface of EV through ExDs. In order to analyse receptor-specific uptake, these EVs were purified and incubated with biotin-conjugated CD 117 antibody that binds to CD 117, a marker for HSPCs. These EVs are added to CD117 receptor-expressing HEK293T stable cell lines to analyse the receptor-specific uptake. The EVs that were pre-incubated with biotin-CD117 antibodies showed increased uptake of GFP compared to those EVs that were not conjugated to biotin-CD117 antibodies. The GFP uptake was analysed using Fluorescence Assisted Cell Sorter (FACS) (Fig. 8D and 8E). Fusion of ExTs-tagged cargo to the c-terminus of ExDs or co-expression of ExTs-cargo and ExDs-cell targeting signal resulted in simultaneous engineering of the surface and lumen of exosomes (e.g., cas9-encapsulated EVs displaying antibody against CD117 receptor of HSPCs).

[0171] In one embodiment, a trimeric form of PTGFRN was generated by operably linking PTGFRN to the Foldon oligomerization domain. To enhance the stability of the surface-displayed proteins and reduce susceptibility to ubiquitin-mediated degradation, one or more N-glycosylation motifs were introduced within the extramembrane region of the PTGFRN-based scaffold. In certain embodiments, site -directed mutagenesis was used to generate single or multiple substitutions introducing canonical N-glycosylation consensus sequences. Exemplary glycosylation site mutations include, but are not limited to: V694N (designated as Gl), S696N (G2), V734N (G3), and L745N (G4), as well as the insertion of a synthetic glycosylation sequence GNSTM.

[0172] In various implementations, stable producer cell lines were generated via single-copy genomic integration of the engineered constructs, enabling consistent production of engineered EVs. EVs were isolated and purified using standard methodologies and applied to recipient cells (e.g., HEK 293T cells) to evaluate delivery efficacy via reporter transfer assays.

[0173] In one embodiment, EVs displaying the trimeric PTGFRN scaffold with S696N and GNSTM modifications exhibited a statistically significant enhancement in reporter transfer to recipient cells, as compared to EVs displaying the wild-type PTGFRN-based scaffold (see, e.g., FIG. SAIB). These data demonstrate that strategic incorporation of N-glycosylation sites within the extramembrane domain of the ExDS scaffold increases both the surface stability and functional delivery efficiency of cargo proteins displayed on the EV membrane.

[0174] In an aspect, among the modified PTGFRN-del687 variants, the PTGFRN del687-foldon-S696N scaffold was selected as the ExDs (Extracellular Vesicle Display Signal) due to its superior efficacy in displaying cargo molecules onto EVs and delivering them to target cells. Furthermore, in an embodiment, any peptide / protein / cell targeting signal to be displayed on the EVs may be fused to the N-terminus of ExDs through a flexible linker such as G4S. In an embodiment, ExDs tagging enables any protein cargo to get enriched at the surface of the plasma membrane of the producer cells, which subsequently buds out as EVs. These purified EVs contain desired proteins such as streptavidin, ScFV, mCherry on their surface.

[0175] In an embodiment, the ExDs are used to purify the engineered EVs by cloning purification tags such as Flag, His, Myc, GST, biotin, and HA tag, but not limited to these.

[0176] In an aspect, a method for ex vivo gene editing is disclosed, wherein, the specialized producer cells (B2M / _, ASCTT / _and ASCT2 / _cells overexpressing CD9 and BaEV) are transfected with a plasmid or a lentiviral vector containing ExTs with a nucleotide sequence comprising SEQ ID NO. 9 fused (at its c-terminus) to a cleavable / inducible linker and a protein of gene editing system such as Cas9-NLS. In an embodiment, these producer cells are co-transfected with a plasmid expressing a sgRNA.

[0177] In yet another aspect, a method for in vivo gene editing is provided, wherein the specialized mammalian producer cells (B2M / _, ASCTT / _and ASCT2 / _cells overexpressing CD9 and BaEV) are transfected with one or more plasmids / lenti viral vector comprising the following genes: (i) ExTs with SEQ ID No.9 fused to a cleavable / inducible linker at the C-terminus and a protein of a gene editing system, Cas9-NLS fused at the N-terminus; (ii) sgRNA; (iii) ExDs; (iv) cell targeting signals such as Hematopoietic Stem and Progenitor Cell Targeting Signal (HSPCTs); and (v) antigen binding sequence of antiphagocytic factor. The transfected producer cells are cultured to obtain exosomes. The EVs thus produced are isolated and characterised to treat any recipient cells ex vivo. Alternatively, these purified exosomes may also be injected into an animal or patient for in vivo treatment.

[0178] In an embodiment, to achieve selective delivery of engineered extracellular vesicles (EVs) to hematopoietic stem and progenitor cells (HSPCs), the EV surface was modified to display one or more targeting moieties. In one aspect, four distinct surface ligands were utilized: (i) a membrane- anchored form of stem cell factor (maSCF), comprising a splice variant with enhanced affinity for the CD117 (c-Kit) receptor; (ii) a single-chain variable fragment (scFv) directed against CD117 (CD117-ScFv); (iii) the baboon endogenous retrovirus envelope (BaEV), which binds to ASCT1 and ASCT2 receptors on HSPCs; and (iv) the chemokine receptor CXCR4, which interacts with stromal cell-derived factor 1 (SDF1), a chemokine enriched in the bone marrow microenvironment.

[0179] In another embodiment, stable EXTS-nLUC 293T producer cell lines were generated to constitutively express a luminal reporter, and were subsequently transiently transfected with individual targeting constructs. The EVs were harvested 48 hours post-transfection, normalized by total protein or particle number, and incubated with primary human HSPCs. Luminescence-based quantification performed six hours post-treatment showed that EVs displaying one or more targeting ligands enhanced delivery to HSPCs relative to EVs lacking targeting moieties. In one aspect, EVs displaying maSCF or BaEV consistently exhibited the highest delivery efficiency across multiple donor samples.

[0180] In an embodiment, to evaluate the potential for soluble SCF to compete with maSCF-displaying EVs for CD117 binding, recombinant SCF was omitted or reduced in the culture medium. In one aspect, removal or reduction of soluble SCF increased delivery of a luminal GFP reporter from maSCF-EVs to HSPCs. Successful membrane display of SCF was confirmed using enzyme-linked immunosorbent assay (ELISA) and nanoparticle tracking analysis (NTA). In one aspect, dosedependent stimulation of HSPC proliferation was observed upon treatment with maSCF-decorated EVs.

[0181] In an aspect, EVs were co-engineered to display multiple targeting ligands including maSCF, BaEV, and CXCR4. This multiplexed targeting configuration resulted in enhanced delivery to HSPCs relative to EVs displaying any single ligand alone. In one embodiment, the co-display of CD117- and ASCT-binding ligands produced synergistic effects in targeting efficiency and was designated as the lead bone marrow-targeting configuration.

[0182] In an embodiment, EVs were further engineered to reduce clearance by macrophages and limit immunogenicity. In one aspect, this was achieved by displaying CD47 or a truncated CD47 variant (tCD47) on the EV surface. In one embodiment, p2-microglobulin (B2M) was knocked out in producer cells to reduce recognition by host immune surveillance pathways via MHC class I.

[0183] In another embodiment, in vivo delivery of targeted EVs was assessed in a humanized mouse model. In one aspect, immunodeficient NSG mice were transplanted with human CD34+ HSPCs to establish bone marrow engraftment. Six weeks post-transplantation, mice were intravenously administered luciferase-labeled EVs either bearing or lacking targeting ligands. Bioluminescence imaging revealed that EVs lacking targeting signals exhibited no tissue-specific accumulation, whereas EVs displaying BaEV, CD117-ScFv, or maSCF preferentially localized to the bone marrow, with secondary signal observed in lung and spleen. In one embodiment, bone marrow accumulation of targeted EVs was reproducibly detected in all treated animals (n = 7). In an embodiment, to enable selective ablation of hematopoietic stem and progenitor cells (HSPCs) as an alternative to chemotherapy-based bone marrow conditioning, a cytotoxic extracellular vesicle (EV) payload system was developed. This system involved the expression of a biologically active toxin within the EVs, such as Diphtheria Toxin A (DTA), which induces apoptosis in target cells. Two fusion constructs were engineered: one in which DTA was fused to the C-terminus of the EXTS-EXRE-CRL scaffold along with its native translocation domain from Corynebacterium diphtheriae, and another in which the translocation domain was substituted with a homologous domain derived from Austwickia chelonae (FIG. 6A). More broadly, in various embodiments, the biologically active cargo delivered by EVs may include gene-editing components such as Cas9 ribonucleoprotein complexes, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), base or prime editors, or epigenome editors. Other possible cargos include transcription factors, cytotoxic proteins such as DTA, its translocation domains, or PUMA, and immunomodulatory agents including checkpoint inhibitors and CD47 -derived proteins.

[0184] In an aspect, transient expression of either fusion construct in HEK293T cells resulted in rapid cytotoxicity, with significant cell death within 72 hours and detectable inhibition of protein synthesis at 24 hours, thereby confirming functional DTA activity.

[0185] In an embodiment, to generate stable EV producer cells resistant to DTA toxicity, DPH1 was knocked out in 293T cells. In one aspect, B2M and ASCT2 knockouts were also introduced to reduce immune recognition and ligand competition. These modified producer cells were transduced with or without HSPC-targeting constructs (maSCF, CXCR4, tCD47, BaEV) and were engineered to express either DTA or a control GFP protein in the EV lumen.

[0186] In another embodiment, EVs produced from these cells were applied to primary HSPCs. In one aspect, targeted EVs containing DTA induced a >12-fold reduction in viable HSPCs by Day 4 post-treatment relative to targeted EVs carrying GFP. The non-targeted EVs, regardless of cargo, did not significantly affect HSPC viability. In one aspect, targeted EVs bearing maSCF and GFP promoted HSPC proliferation due to the known proliferative role of SCF.

[0187] In yet another embodiment, different linker sequences were tested for toxin loading into EVs. In one aspect, intein- and PhoC12f-based linkers were stably expressed in producer cells and resulted in comparable cytotoxicity against HSPCs (FIG. 6C). Substitution of the native translocation domain with the domain from A. chelonae maintained cytolytic potency, indicating that either domain supports endosomal escape (FIG. 6D). Targeted DTA-EVs lacking BaEV retained robust cytotoxicity, confirming that viral fusogens were not required when a competent translocation domain was present (FIG. 6E).

[0188] In an embodiment, the CD117-targeting platform was extended to applications in acute myeloid leukemia (AML), in which the CD117 receptor is overexpressed in more than 80% of patient cases. Engineered EVs were produced from a DPH1- / -, B2M- / -, and ASCT2- / - 293T cell line transduced with EXTS-EXRE-CRL-DTA-TL, maSCF, CXCR4, tCD47, and transiently transfected with BaEV (FIG. 7A).

[0189] In another embodiment, purified DTA-loaded EVs were applied to Kasumi cells, a CD117+ AML cell line. A dose -dependent increase in cell lysis was observed relative to non-targeted controls, as measured by trypan blue exclusion at five days post-treatment (FIG. 7B). BaEV surface display was found to be non-essential for AML cell lysis. Both the C. diphtheriae and A. chelonae translocation domains enabled endosomal escape and cytotoxic delivery of DTA in the absence of BaEV (FIGs. 7D-F).

[0190] In an aspect, at least one of Hematopoietic Stem and Progenitor Cell-Targeting Signals (HSPCTs) with nucleotide sequences comprising SED ID NOs: 47-64 may be combined with ExDs or expressed independently on the surface of EVs to target human hematopoietic stem cells in vivo as well as ex vivo. The HSPCTs includes CD117 antibody, ScFV-CD117, CXCR4, BaEV, and a modified membrane-bound SCF, all of which are known to bind to receptors expressed on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. In an embodiment, HSPCTS may be tagged to the N-terminus of ExDs to produce EVs that display HSPCTs on its surface. In yet another embodiment, any payload / cargo may be delivered into HSPCs in vivo through engineered EVs by ExDs displaying HSPCTs and ExTs.

[0191] In an aspect, an engineered ectosome comprising any one of HSPCTs with SEQ ID NOS. 47-64, is capable of delivering desired payload / cargo to the bone marrow with reduced off-target accumulation in other organs compared to EVs lacking targeting signals. In an embodiment, when these EVs co-display a cell envelope or pseudotype protein, BaEV and a HSPCTs such as membrane -bound SCF, their ability to target the bone marrow in vivo is further enhanced compared to EVs displaying either BaEV or membrane-bound SCF alone. Fig 6 depicts an engineered EV or ectosome that displays HSPC targeting mSCF through ExDs and encapsulates Cas9-RNP through ExTs. Furthermore, these engineered EVs co-display, BAEV, CD9 and CD47. The lower rates of cellular secretion of EVs limit gene editing applications, necessitating a high number of producer cells. Therefore, in an embodiment, the producer cells were pulsed with an electroporation system to induce membrane stimulation to increase ectosome shedding. When the producer cells were subjected to electroporation using the specific settings or parameters defined as "HSC 3" on a scalable electroporation system such as Maxcyte electroporation system, the number of EVs produced by these cells increased by 20 times compared to the number produced without this treatment. Furthermore, optimization of pulsing rate, number of cells pulsed, production confluency, and collection time led to an increase in the production of engineered EVs by over 100-fold (detailed in examples).

[0192] In an aspect, the EV producer cell lines are engineered to overexpress a desired payload or cargo fused to ExTs. Specifically, they are designed to (i) overexpress the desired cargo (e.g., Cas9) fused to ExTs, (ii) overexpress a desired cell targeting signal (e.g., HSPCTs) fused to ExDs, (iii) overexpress CD9 tetraspanin, (iv) overexpress BaEVRless, a modified envelope protein derived from Baboon endogenous retrovirus, (v) overexpress a truncated CD47 containing the SIRPa binding residues, (vi) knockout the B2M gene and (vii) knockout the ASCT1 and ASCT2 genes that serve as receptors for Baboon envelope pseudotyped EVs. The overexpression can be achieved through lentiviral vectors or by targeted insertion of the construct into any endogenous loci via gene editing. These producer cell lines may be derived from one of the following cells: HEK293T cells, immortalized bone marrow MSCs, iPSCs and iPSC-derived MSCs.

[0193] In an aspect, the EVs generated by the present disclosure may be used to target any of: Induced Pluripotent Stem Cells (iPSCs), Mesenchymal Stem Cells (MSCs), Hematopoietic Stem and Progenitor Cells (HSPCs), Hematopoietic cells and Adipose -Derived Stem Cells.

[0194] Once the producer cells are established, it can be turned into a cell factory for the continuous production of EVs. This allows off-site production and can be easily shipped to on-site for intravenous administration. Thus, the present disclosure introduces a novel method of utilizing engineered cells as a biofactory, which consistently generates engineered EVs. These vesicles are specialized to transport specific payloads / cargoes to targeted cells. A significant benefit of this system is the sustained production of vesicles loaded with therapeutic agents or gene editing proteins, primed for administration. The present disclosure offers multiple advantages over existing alternatives like lipid nanoparticles (LNPs), viral vectors, and virus-like particles (VLPs). These include reduced immunogenicity, minimized tumor risk, lower toxicity, and the ability to traverse cellular and tissue barriers. Moreover, these EVs naturally exhibit tissue targeting and regenerative properties.

[0195] In an aspect, the engineered EVs and the methods of their production enable a wide range of therapeutic and diagnostic applications. In an embodiment, the extracellular vesicles (EVs) described herein may be administered to a subject in an effective amount to modulate a biological process. The engineered EVs may deliver a biologically active cargo molecule to target cells in vivo to induce gene editing, cytotoxic activity, immune modulation, hematopoietic conditioning, or selective cell ablation.

[0196] The invention discloses a method for targeted delivery of a biologically active cargo molecule to a subject, comprising administering the engineered EV. The EV delivers the cargo to a specific cell or organ, such as hematopoietic cells, bone marrow, lung, liver, brain, heart, kidney, or tumor cells. The EVs of the invention may be used in targeted in vivo genome engineering of cells or tissues of various types, including base modifications and epigenome modifications. For instance, targeted cell ablation was achieved by selective in vivo elimination of cells or tissues, including tumor cells (e.g., targeted killing of CD 117-positive AML cells using DTA-loaded EVs). Figures 6 and 7 illustrate the efficient killing of HSPCs and AML cells, respectively, by DTA-loaded, targeted EVs. Other applications of the EVs of the invention may comrise targeted in vivo stimulation of proliferation of cells or tissues, or targeted overexpression of proteins in cells or tissues. Further, the EVs may be used for ex vivo genome engineering of cells or tissues, or ex vivo delivery of proteins, mRNA, DNA, small molecules, drugs, or radioactive materials and use as disease biomarkers and diagnostics. For example, Cas9- or base-editor-loaded EVs may be administered to correct disease -causing mutations in hematopoietic stem cells; Diphtheria toxin A-loaded EVs may ablate malignant or unwanted cell populations; CD47-engineered EVs may modulate immune clearance; and maSCF- or CXCR4-expressing EVs may promote hematopoietic progenitor proliferation or homing. Figure 9 provides a schematic depicting engineered EVs containing Cas9 ribonucleoprotein complexes in the lumen and surface modifications for HSPC targeting, immune and phagocytic evasion, illustrating the comprehensive capabilities of the invented platform for in vivo applications. The present invention thus provides a versatile, efficient, and safe platform for advanced therapeutic delivery, leveraging the natural properties of EVs combined with precise genetic engineering.

[0197] EXAMPLES

[0198] Example 1: Cell Culture

[0199] HEK293T cells were used for the production of functional extracellular vesicles (EVs) and were maintained in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; MP Biomedicals) supplemented with 10% fetal bovine serum (FBS) and 1% Antibiotic- Antimycotic (Gibco). Sublines derived from such a cell line were cultured under the same conditions. Lenti-X cells (Takara) and were also maintained using the same protocol as the HEK293T cells.

[0200] Mobilized peripheral blood (mPB) from leftover blood bags, following allogeneic transplantation, were obtained from Christian Medical College Hospital, Vellore, with prior approval from the Institutional Review Board (IRB). Mononuclear cells were isolated by density gradient centrifugation using Lymphoprep™ (#07801), and CD34+cells were enriched using the EasySep™ Human CD34 Positive Selection Kit II (Stemcell Technologies, Cat# 17856). CD34+hematopoietic stem and progenitor cells (HSPCs) were cultured at a density of 2 x 105cells / mL in StemSpan™ SFEM II medium supplemented with SCF (240 ng / mL), Flt3 (240 ng / mL), TPO (80 ng / mL), IL-6 (40 ng / mL), and lx Antibiotic- Antimycotic. All cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2.

[0201] Example 2: Transfection

[0202] For transiently expressing EV constructs, HEK293T cells and derived sublines were seeded in 10 cm dishes for EV packaging, cells were seeded at a density of 5 x 106cells per dish, 6-12 hours prior to transfection. Cells were transfected with 10 pg of the respective plasmid using FuGENE® (Promega, Cat #E2311) at a 1 :3 DNA-to-reagent ratio, following the manufacturer’s instructions.

[0203] Example 3: Stable Cell Line Generation

[0204] To generate a lentivirus, HEK293T or Lenti-X cells were seeded in 6-well plates at a density of 1 x 106cells per well (0.5 x 106cells / mL). After 6-12 hours, cells were transfected with 1 pg of the transgene along with 0.5 pg of second-generation envelope and packaging plasmids using FuGENE®. The medium was harvested 48 hours post-transfection, centrifuged at 3,000 x g for 10 minutes to remove cells and apoptotic bodies, and the supernatant was concentrated using a 4x lentiviral concentrator (in-house prepared). The supernatant was incubated at 4 °C for 12-16 hours. The next day, the virus was pelleted by centrifugation, resuspended in -100 pL of final volume, and used to transduce 0.5 x 106target cells plated at the time of transduction.

[0205] Drug selection was initiated 48 hours post-transduction using 1 pg / mL puromycin and 200 pg / mL hygromycin. Cells were maintained under selection for at least three passages, with subculturing every 2-3 days. Example 4: Optimized Extracellular Vesicle (EV) Collection and Purification

[0206] EVs were collected from HEK293T cells either transfected with EV constructs or stably expressing the cargo of interest. Cells were cultured in media conditioned with 1% fetal bovine serum (FBS). Conditioned media was collected every 12 hours, with six collections performed per sample. Collected media samples were first centrifuged at 300 x g for 5 minutes to remove intact cells and large debris. The supernatant was then centrifuged at 2000 x g for 10 minutes to further eliminate residual cellular debris. To remove contaminating nucleic acids, the clarified media was treated with Benzonase (71206-3, Merck) at 25 U / ml in the presence of 2 mM MgCE and incubated on a shaker at room temperature for 2 hours. Following Benzonase treatment, samples were filtered through a 0.45 pm filter to remove remaining particulates. The filtered samples were then loaded onto an ExoFilter Maxi column (HBM-EXF-60050, Microgentas). After discarding the filtrate, an elution buffer was added to the filter to elute the EVs. The elution buffer was allowed to pass through the filter by gravity flow at room temperature. Once the elution buffer had passed through, the filter was centrifuged at 3000 x g for 30 seconds to recover any remaining EVs trapped in the filter. To concentrate the eluate and remove smaller contaminants, the eluate was loaded onto a centrifugal filter unit (Amicon® Ultra Centrifugal Filter, UFC905008 or UFC9100) with a molecular weight cut-off of either 50 kDa or 100 kDa, depending on the expected cargo size. This step was performed by centrifugation at 3500 x g for 15 minutes. The concentrated EV preparation was collected and stored at 4°C until further use.

[0207] Example 5: Optimization of Isolation and Purification of EVs

[0208] IHSPCs were treated with 20 pg of EVs, resulting in 30% GFP-positive cells with an MFI of 70. Increasing particle number increased delivery. High EV doses reduced viability.Benzonase treatment doubled viability. Filtration with 0.4 pm membranes and 35 nm SEC reduced GFP delivery.Fluorescence NTA showed GFP-containing EVs ranged from 100-900 nm. GFP-positive EVs constituted 0.04% of total; most EVs were <100 nm. Selection of EVs >100 nm enriched GFP-loaded particles, reduced particle count, and decreased toxicity. Four percent of EVs >100 nm contained GFP; EVs <100 nm contained none. Protocol was modified to include Benzonase digestion and 1 pm syringe filtration. GFP delivery to HSPCs was achieved with a tenfold lower EV count.

[0209] Example 6: Increasing EV production by Electroporation

[0210] The producer cells were pulsed with a scalable electroporation system, Maxcyte. An equal number of ectosome producer cells were pulsed with various codes of Maxcyte electroporator. A pulse code of HSC 3 yielded at least a 20-fold increase in engineered ectosonie production. By further optimizing the pulsing rate, the number of cells pulsed, production confluency, and collection time, we increased the production of engineered EVs by over 50-fold.

[0211] Example 7: Transmission electron microscopy

[0212] EVs were adsorbed to the carbon-coated formvar grid for 1-5 minutes at room temperature. The excess sample was gently blotted off with filter paper. The grid was washed once by adding a drop of deionized water for a few seconds and blotting again, to remove salts that might crystallize. A drop of (5-10 pL) of 1-2% (w / v) uranyl acetate was added to the grid for 30-60 seconds to stain the sample. The excess stain was removed with filter paper, and the grid was air-dried completely at room temperature. The grids were examined using Tecnai T12 transmission electron microscope at 60kV and imaged at various magnifications.

[0213] Example 8: EV size distribution and concentration measurement using NTA

[0214] Nanoparticle tracking analysis (NTA) was performed using the Particle Metrix, equipped with a 488 nm laser and a 500 nm emission filter. Calibration was performed using 100 nm polystyrene beads diluted 1:250,000 (v:v) in sterile water for both scatter and fluorescence modes. Instrument settings for calibration were sensitivity 85 and shutter 100 for scatter mode, and sensitivity 95 and shutter 100 for fluorescence mode. For sample measurements, extracellular vesicle (EV) samples were diluted in sterile water to a final volume of 1 mL and analyzed at 25°C and pH 7.0, with conductivity measured at <30 pS / cm. For each measurement, data were acquired at 30 frames per second in medium video resolution by scanning 11 positions per sample over 5 cycles, capturing 30 frames per position. After each sample, the system was thoroughly washed with distilled water to prevent cross-contamination.

[0215] Analysis was performed using ZetaView software with the following parameters: minimum area 10, maximum area 1000, minimum brightness 30, and a size classification of 5 nm per class. For scatter measurements, the system’s (standard operating procedure) SOP_EV_488 was used with instrument settings of sensitivity 85 and shutter 100. For fluorescence measurements, the SOP_EV_488_F500 was used with sensitivity 95 and shutter 100. Size distribution analysis was performed according to the relevant SOP for each measurement mode. Data quality was ensured by maintaining an average of 80-100 counted particles per frame and tracing over 1,000-5000 particles per sample. Concentrations were calculated using a dilution factor of 1,000 and a correction factor of 1 ,000,000, resulting in final particle concentrations expressed as particles per mL. Example 9: Western Blot Characterization of Extracellular vesicles

[0216] Cells were lysed in radioimmunoprecipitation assay (RIP A) buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and a 1% protease inhibitor cocktail. To facilitate lysis, cell suspensions were vortexed every 5 minutes over a 15-minute period, followed by sonication in a water bath sonicator for 2 minutes. Lysates were then centrifuged at 14,000 x g for 15 minutes at 4°C to remove cellular debris, and the supernatants were collected for further analysis.

[0217] Extracellular Vesicles (EVs) were collected based on particle count and were not subjected to centrifugation or sonication steps during the lysis process.

[0218] Protein concentrations were measured using the bicinchoninic acid (BCA) protein assay (Pierce), according to the manufacturer’s instructions. Cell lysate samples were normalized based on total protein content (30 pg), while extracellular vesicle (EV) samples were normalized by particle number, ranging from 1 x 1010to 1 x 1015particles, depending on the experimental requirements.

[0219] Equal amounts of protein and EV particles were mixed with Laemmli sample buffer (60 mM Tris- HCl pH 6.8, 10% glycerol, 2% SDS, 100 mM dithiothreitol, and 0.01% bromophenol blue) and denatured by heating at 95 °C for 5 minutes. Proteins were resolved on 10% SDS-PAGE gels at 80 V for 10 minutes followed by 100 V for 1 hour and 15 minutes in standard running buffer. Following electrophoresis, proteins were transferred to 0.45 pm PVDF membranes (Thermo Fisher Scientific) using a manual wet transfer method at 300 mA for 1 hour.

[0220] Following protein transfer, membranes were blocked with 4% bovine serum albumin (BSA) in TBS-T (Tris-buffered saline containing 0.05% Tween-20) for 1 hour at room temperature with gentle shaking. After blocking, membranes were incubated overnight at 4°C with primary antibodies diluted 1 : 1000 in the same blocking buffer. The following primary antibodies were used to characterize extracellular vesicles (EVs) in both cell lysates and isolated EV preparations: anti- CD9 (10626D), anti-CD81 (10630D), anti-CD63 (10628D), anti-syntenin (MA5-25984), and anti- TSG101 (MAI-23296) (all from Thermo Fisher Scientific); anti-Annexin V (8555, Cell Signaling Technology) as a specific marker for EVs; anti-calnexin (sc-23954, Santa Cruz Biotechnology) and anti-P-actin (sc-8432, Santa Cruz Biotechnology) as a loading control. In addition, the expression of target proteins was assessed using anti-SCF (703527) and anti-CXCR4 (358800), both from Thermo Fisher Scientific.

[0221] After primary antibody incubation, membranes were washed four times for 5 minutes each with TBS-T and incubated for 1 hour at room temperature with HRP-conjugated secondary antibodies: anti-mouse IgG (31437, Thermo Fisher Scientific) and anti-rabbit IgG (32260, Thermo Fisher Scientific), each diluted 1:10,000 in blocking buffer. The anti -mouse secondary antibody was used to detect CD9, CD63, CD81, calnexin, syntenin, TSG101, CXCR4, and P-actin, while the antirabbit secondary antibody was used to detect SCF.

[0222] After secondary antibody incubation, membranes were washed four times for 10 minutes each with TBS-T. Protein bands were detected using Western ECL Substrate and protein bands were visualized using a ChemiDoc Imaging System (Bio-Rad).

[0223] Example 10: Simultaneous loading of surface and lumen of EVs

[0224] To simultaneously engineer both the surface and lumen of EVs, stable HEK293T cell lines that express ExTs and ExDs were created by transducing with lentiviral constructs carrying Ex'Ts tagged with GFP and ExDs tagged with mCherry. The EVs produced from these stable cell lines were collected and used to treat recipient HEK293T cells. The recipient cells exhibited both GFP and mCherry, confirming the successful delivery of lumen and surface-engineered EVs. Additionally, HSPCs were treated with these engineered E Vs to confirm their delivery to HSPCs as well.

[0225] Example 11: Characterization of ExTs-tagged payload / cargo-loaded EVs

[0226] Among the various mutants prepared, the present disclosure presents the dual mutant, G3K+E21A containing Myr+PBR sequences, as the ExTs for payload / cargo encapsulation and delivery. The EVs produced from ExTs-GFP producer cells had a mean particle size of 176 nm ± 78 nm. EVs produced from ExTs tagged nanoLuciferase (nLUC) stable cell lines confirmed the presence of cargo (nLUC) in the EVs. NanoLuciferase is fused with two different ExTs (Myr+PBR and EXTS (Lumen-TS) and overexpressed in HEK293T cells. The produced EVs are directly measured for their bioluminescence. The relative light unit reading suggests that nanoLuciferase is encapsulated into EVs. ExTs facilitate cargo loading into EVs and can be used as a delivery platform. The cargo delivery effect of ExTs-GFP was further improved when the producer cells were co-expressed with a tetraspanin protein CD9 and envelope protein VSV-G. The surface proteins BaEV and CD9 enhance cargo delivery through engineered EVs. The GFP is fused with ExTs and overexpressed in the producer HEK293T cells in the presence of envelope proteins such as VSV-G, BaEV and tetraspanin CD9. The HSPCs were treated with purified EVs, and the GFP uptake was analyzed after 12 hours of treatment.

[0227] Example 12: In vitro gene editing of the cells using engineered Extracellular vesicles

[0228] Cells were seeded in 96-well plates at the densities of 5000 cells / well. Plating medium for Ev treatment consists of DMEM (high glucose, with L-glutamine) with 10% FBS and, optionally 1% penicillin-streptomycin. To modulate extracellular fluid viscosity, methyl cellulose (65 kDa) was incorporated into the culture medium at varying concentrations. Methyl cellulose was dissolved in pre -warmed cell culture medium to achieve final viscosities ranging from 1.0 to 5.0 centipoise (cP) at 37 °C. The concentrations used were as follows: 0.1% (1.0 cP), 0.2% (1.5 cP), 0.3% (2.0 cP), 0.4% (3.0 cP), 0.5% (4.0 cP) and 0.55% (5.0 cP), w / v methyl cellulose. The methyl cellulose was thoroughly mixed and allowed to hydrate at 4 °C overnight before use. 12 hrs post seeding engineered EVs were treated to the cells at a dose in the range of IxlO1- lxl020EVs / cell. Genomic DNA extraction was performed post 72-96 hrs of treatment using QuickExtract™ DNA Extraction solution. The cell pellet was then resuspended in approximately 20-50 pl of QuickExtract™ solution and subjected to sequential incubation at 68 °C and 98 °C. The DNA was directly processed for amplification of the target using the appropriate primers. PCR products were confirmed using gel electrophoresis and purified using NucleoSpin® Gel and PCR Clean-Up (Takara). For Sanger sequencing, the purified PCR amplicon was processed with BigDye Terminator™ v3.1 Cycle Sequencing, and the resulting chromatograms were analyzed by the EDITco ICE analysis for evaluating gene editing.

[0229] Example 13: In vitro Targeted uptake of engineered EVs

[0230] HSPCs were seeded in a 96 well plate at a density of 5000 cell / well cultured in SFEM medium supplemented with cytokines such as SCF, Flt3, TPO, IL6. Targeted EVs loaded with nLuc were treated to the cells at a dose in the range of IxlO1- IxlO20EVs / cell. 16 hrs post treatment. Cells were collected to analyse the uptake of nLUC into target recipient cells. Cells were incubated with 5ul of nLUC substrate (Purchased from Promega ( Cat.No. N4100) diluted in the ratio 1:200 placed in a black colour 96 well plate / Clear Bottom Plate and the luminescence was evaluated by measuring OD values acquired in an Elisa reader.

[0231] Example 14: In vitro targeted lysis of CD117+ HSPCs for Bone Marrow conditioning

[0232] HSPCs were seeded in a 96 well plate at a density of 5000 cell / well cultured in SFEM medium supplemented with cytokines such as SCF, Flt3, TPO, IL6. Engineered EVs loaded with either toxin or GFP were treated to the cells at a dose in the range of IxlO1- IxlO20EVs / cell. Four days post treatment cells were collected, and viability was assessed by tryphan blue dye exclusion assay and cell numbers were recorded.

[0233] Example 15: In vitro targeted lysis of CD117+ Acute Myeloid Leukemia cells

[0234] Kasumi cells were seeded in a 96 well plate at a density of 5000 cell / well cultured in RPMI 1640 with 10% FBS. Engineered EVs loaded with either toxin or GFP were treated to the cells at a dose in the range of IxlO1- IxlO20EVs / cell. 5 days post treatment cells were collected, and viability was assessed by tryphan blue dye exclusion assay and cell numbers were recorded.

[0235] Example 16: In vivo gene editing of HSPCs using engineered EVs with HSPC targeting signals

[0236] To test in vivo gene editing of HSPCs, EVs were engineered to display targeting signals such as CD117 antibody, ScFV-CD117, CXCR4, BaEV, and membrane -bound SCF, all binding to receptors on HSPCs in the bone marrow. To test the ability of these engineered EVs to deliver cargo to HSPCs in vivo, humanized mice were generated by transplanting human HSPCs into immunodeficient NBSGW mice. After allowing six weeks for the human HSPCs to engraft, the engineered EVs carrying a luciferase reporter (nLUC) were injected into the mice via the tail vein and the localization of the EVs was tracked using bioluminescence imaging. While EVs without targeting signals (control) accumulated primarily in the liver and spleen, EVs displaying BaEV, scFV-CD117, or membrane-bound SCF were able to efficiently reach the bone marrow with reduced off-target accumulation in other organs. Furthermore, co-displaying both BaEV and membrane -bound SCF on the same EVs enhanced their ability to target the bone marrow in vivo compared to EVs displaying either signal alone.

[0237] Example 17: In vivo targeting

[0238] Female NSG mice (6-8 weeks old) were humanized by intravenous injection of 5 x 105purified day 0 human CD34+cells. Before transplantation, mice were conditioned with two doses of busulfan (25 mg / kg body weight) administered 48 hours apart. Ten weeks post-humanization, freshly purified 2E10 engineered EVs encapsulating NanoLuc (nLuc) quantified using NTA were administered via tail vein injection. Biodistribution of the engineered EVs was assessed 6 hours after injection using an IVIS Spectrum imaging system (PerkinElmer). Major organs — including bone marrow, lungs, liver, kidneys, spleen, heart, skin, and intestine — were harvested and imaged following administration of the NanoLuc substrate FFz (Promega N4100).

Claims

CLAIMS:

1. An extracellular vesicle (EV), comprising a biologically active cargo molecule functionally linked to a scaffold, wherein the scaffold comprises at least one extracellular vesicle targeting peptide sequence (ExTS) selected from SEQ ID NOs: 1-29, and wherein the ExTS comprises an elevated net positive charge for localization of the scaffold to the plasma membrane of the producer cell enabling the encapsulation of the biologically active cargo molecule into the EV lumen during EV biogenesis.

2. The extracellular vesicle of claim 1, wherein the biologically active cargo molecule is selected from a protein, RNA, DNA, and a small molecule.

3. The extracellular vesicle of claim 2, wherein the biologically active cargo molecule is selected from at least one: a. gene-editing component, selected from zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), ribonucleoprotein (RNP) complexes comprising CRISPR-Cas, base editors, prime editors, or epigenome editors; b. transcription factor; c. cytotoxic protein, selected from diphtheria toxin A (DTA), DTA translocation domains, or p53 upregulated modulator of apoptosis (PUMA); d. immunomodulatory factor; or e. combinations thereof.

4. The extracellular vesicle of claim 1, further comprising atleast one myristylation motif comprising at least one amino acid substitutions selected S3K, G3K, L5K or variant thereof; and at least one polybasic region comprising at least one amino acid substitutions selected from E21K, E21A, E27K, D17K, E23K, D16V and D16K or a variant thereof.

5. The extracellular vesicle of claim 1, wherein the scaffold further comprises at least one extracellular vesicle release enhancer peptide sequences (ExRE) selected from SEQ ID NOs: SO- 46, wherein the ExRE enhances budding and selective release of the cargo-loaded extracellular vesicles from the plasma membrane of the producer cell.

6. The extracellular vesicle of claim 1, further comprising at least one component selected from:i. a cleavable cargo release linker (CRL) selected from at least one: pH-sensitive linker, UV-cleavable PhoC12f linker, and a protease -cleavable linker selected from Cathepsin G, Cathepsin A, furin, and PRSS2; ii. an endosomal escape sequence selected from: ZF5.3, a cell -penetrating peptide, a translocation domain, or a viral envelope protein; iii. a targeting ligand selected from: membrane-anchored stem cell factor (maSCF), CXCR4, BaEV, CD117 antibody, ScFV-CD117, or a combination thereof; and iv. an anti-phagocytosis protein selected from: CD47, CD24, or variants thereof; wherein the EV comprises at least one selected component, and enables the delivery of the biologically active cargo molecule to a recipient cell.

7. The extracellular vesicle of claim 1, further comprising a surface display peptide sequence (ExDs) selected from SEQ ID NOs: 47-64, and wherein the at least one sequence is fused to at least one targeting ligand or binding domain selected from membrane-anchored stem cell factor (maSCF), functional domain of SCF, CD 117- single-chain variable fragment (CD117-scFv), receptor specific antibodies or combinations thereof wherein the at least one targeting ligand or binding domain enables selective binding of the extracellular vesicle to the recipient cell surface receptor.

8. The EV of claim 7, wherein the recipient cell is human hematopoietic stem cells (HSPCs).

9. A genetically modified mammalian producer cell line for production of extracellular vesicles (EVs), wherein the cell comprises a nucleic acid construct encoding a biologically active cargo molecule functionally linked to a scaffold; wherein the scaffold comprises at least one extracellular targeting peptide sequence (ExTS) selected from SEQ ID NOs: 1-29; and wherein the ExTS comprises an elevated net positive charge for localization of the scaffold to the plasma membrane of the producer cell enabling the encapsulation of the biologically active cargo molecule into the EV lumen during EV biogenesis.

10. The producer cell line of claim 9, wherein the biologically active cargo molecule is selected from at least one:a. gene-editing component, selected from zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), ribonucleoprotein (RNP) complexes comprising CRISPR-Cas, base editors, prime editors, or epigenome editors; b. transcription factor; c. cytotoxic protein, selected from diphtheria toxin A (DTA), DTA translocation domains, or p53 upregulated modulator of apoptosis (PUMA) ; d. immunomodulatory factor; or e. combinations thereof.

11. The producer cell line of claim 9, further comprising: at least one myristylation motif comprising at least one amino acid substitutions selected S3K, G3K, L5K or variant thereof; and at least one polybasic region comprising at least one amino acid substitutions selected from E21K, E21A, E27K, D17K, E23K, D16V and D16K or a variant thereof.

12. The producer cell line of claim 9, wherein the scaffold further comprises at least one extracellular vesicle release enhancer peptide sequences (ExRE) selected from SEQ ID NOs: 30- 46, wherein the ExRE enhances budding and selective release of the cargo-loaded extracellular vesicles from the plasma membrane of the producer cell.

13. The producer cell line of claim 9, further comprising at least one nucleic acid construct encoding at least one component selected from: i. a cleavable cargo release linker (CRL) selected from at least one pH-sensitive linker or a UV-cleavable PhoC12f linker, or a protease-cleavable linker selected from Cathepsin G, Cathepsin A, furin, or PRSS2; ii. an endosomal escape sequence selected from ZF5.3, a cell-penetrating peptide, a translocation domain, or a viral envelope protein; iii. a targeting ligand selected from membrane-anchored stem cell factor (maSCF), CXCR4, BaEV, CD117 antibody, ScFV-CD117, or a combination thereof; and iv. an anti-phagocytosis protein selected from CD47, CD24, or variants thereof; wherein the producer cell line expresses at least one additional component, and wherein the at least one additional component is configured to be incorporated into an extracellular vesicle produced by the cell line, enhancing the delivery of the biologically active cargo molecule from the extracellular vesicle to a recipient cell.

14. The producer cell line of claim 9, further comprising at least one nucleic acid construct encoding at least one surface display peptide sequence (ExDs) selected from SEQ ID NOs: 47-64, wherein the at least one ExD sequence is fused to at least one targeting ligand or binding domain selected from: membrane-anchored stem cell factor (maSCF), functional domain of SCF, CD117-single-chain variable fragment (CD117-scFv), receptor specific antibodies or combinations thereof; and wherein the at least one targeting ligand or binding domain enables selective binding of an extracellular vesicle produced by the cell line to a recipient cell surface receptor.

15. The producer cell line of claim 9, wherein the P2 -microglobulin (B2M) gene is knocked out or silenced to prevent the host immune response.

16. The producer cell of claim 9, wherein the producer cell is a genetically modified derivative of a cell line selected from: HEK293, 293T, K562, HeLa, or U2OS, ESC-derived MSCs, hematopoietic stem and progenitor cells (HSPCs), induced pluripotent stem cells (iPSCs), hematopoietic cells, liver cells, spleen cells, pancreatic cells, or kidney cells and primary cell selected from mesenchymal stromal cells (MSCs), immortalized MSCs, iPSC.

17. A method for producing extracellular vesicles (EVs), the method comprising: i. providing a producer cell; ii. introducing into a producer cell line a nucleic acid construct encoding a biologically active cargo molecule functionally linked to a scaffold, wherein the scaffold comprises at least one extracellular targeting peptide sequence (ExTS) selected from SEQ ID NOs: 1-29; wherein the scaffold comprises an elevated net positive charge for localization of the scaffold to the plasma membrane of the producer cell enabling the encapsulation of the biologically active cargo molecule within into the EV lumen during EV biogenesis; iii. culturing the producer cell in serum free medium or in the presence of EV specific enhancers, for the secretion of EVs encapsulating the cargo molecule within the EV lumen; and iv. isolating the secreted EVs from the culture medium.

18. The method of claim 17, wherein the method further comprises introducing into the producer cell line at least: a. gene -editing component, selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-Cas systems, baseeditors, prime editors, and epigenome editors, wherein the CRISPR-Cas systems are ribonucleoprotein (RNP) complexes; b. transcription factor; c. cytotoxic protein, selected from the group consisting of diphtheria toxin A (DTA), DTA translocation domains, and p53 upregulated modulator of apoptosis (PUMA) ; d. immunomodulatory factor; or e. combinations thereof.

19. The method of claim 17, further comprising introducing into the producer cell at least myristylation motif comprising at least one amino acid substitutions selected S3K, G3K, L5K or variant thereof; and at least one polybasic region comprising at least one amino acid substitutions selected from E21K, E21A, E27K, D17K, E23K, D16V and D16K or a variant thereof.

20. The method of claim 17, further comprising introducing into the producer cell at least one construct encoding extracellular vesicle release enhancer peptide sequences (ExRE) selected from SEQ ID NOs: 30M-6, wherein the ExRE enhances budding and selective release of the cargo- loaded extracellular vesicles from the plasma membrane of the producer cell.

21. The method of claim 17, further comprising introducing into the producer cell at least one nucleic acid construct encoding at least one component selected from: i. a cleavable cargo release linker (CRL) selected from at least one pH-sensitive linker comprising a UV-cleavable PhoC12f linker, or a protease-cleavable linker selected from Cathepsin G, Cathepsin A, furin, or PRSS2; ii. an endosomal escape sequence selected from ZF5.3, a cell-penetrating peptide, a translocation domain, or a viral envelope protein; iii. a targeting ligand selected from membrane-anchored stem cell factor (maSCF), CXCR4, BaEV, CD117 antibody, ScFV-CD117, or a combination thereof; and iv. an anti-phagocytosis protein selected from CD47, CD24, or variants thereof; wherein the at least one component is incorporated into an extracellular vesicle produced by the cell line, enhancing the delivery of the biologically active cargo molecule from the extracellular vesicle to a recipient cell.

22. The method of claim 17, further comprising introducing into the producer cell at least one nucleic acid construct encoding at least one surface display peptide sequence (ExDs) selected from SEQ ID NOs: 47-64; wherein the at least one ExD sequence is fused to at least one targeting ligand or binding domain selected from membrane-anchored stem cell factor (maSCF), functional domain of SCF, CD117-single-chain variable fragment (CD117-scFv), receptor specific antibodies or combinations thereof; and wherein the at least one targeting ligand or binding domain enables selective binding of an extracellular vesicle produced by the cell line to a recipient cell surface receptor.

23. The method of claim 17, wherein the producer cell comprises a genetically modified cell line derived from HEK293, 293T, K562, HeLa, or U2OS cells, or from genetically modified primary cells selected from mesenchymal stromal cells (MSCs), immortalized MSCs, iPSC / ESC- derived MSCs, hematopoietic stem and progenitor cells (HSPCs), induced pluripotent stem cells (iPSCs), hematopoietic cells, liver cells, spleen cells, pancreatic cells, or kidney cells.

24. The method of claim 22, wherein the recipient cell or tissue is selected from the group consisting of hematopoietic cells, bone marrow, lung, liver, brain, heart, kidney, and tumor cells.

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