Engineered extracellular vesicles with individually regulated tropism

Abstract

Disclosed herein is a method for engineering extracellular vesicles (EVs) with desired cell tropism for target cells. The method includes reprogramming somatic cells toward the lineage of the target cells and harvesting EVs with desired cell tropism produced by these reprogrammed cells. In some embodiments, these EVs can be used to deliver diagnostic and / or therapeutic cargo to target cells in a subject in need thereof. Thus, also disclosed herein is a method for treating a disease or condition in a subject, comprising engineering reprogrammed cells to produce therapeutic EVs that deliver a therapeutic agent to a target cell.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 256,127, filed October 15, 2021, which is incorporated by reference herein in its entirety. [Background technology]

[0002] Exosomes are naturally secreted nanovesicles that have recently attracted great interest in the scientific and clinical communities for their role in cell-cell communication in almost all physiological and pathological processes. These 30-100 nm sized vesicles are released from cells into the extracellular space and eventually into biofluids in a tightly regulated manner. Their molecular composition reflects their cell of origin, may confer specific cell or tissue tropism and highlight their biological activity. Exosomes and other extracellular vesicles (EVs) carry a specific set of proteins, nucleic acids (DNA, mRNA, and regulatory RNA), lipids and metabolites that represent an attractive source of novel non-invasive markers via biofluid biopsy. Exosome shuttle molecules can maintain their biological activity and regulate and reprogram recipient cells. This multifaceted nature of exosomes holds great promise for improving cancer therapy characterizing them as novel diagnostic sensors as well as therapeutic effectors and drug delivery vectors. The natural biological activities of EVs, including their therapeutic payload and targeting behavior, can be tuned via genetic and chemical manipulation. However, identifying and testing suitable cell-targeting ligands is time consuming, and improved methods are needed to engineer EVs with desired cell tropism. Summary of the Invention [Means for solving the problem]

[0003] Disclosed herein is a method for engineering extracellular vesicles (EVs) with desired cell tropism for target cells, which includes reprogramming somatic cells toward the lineage of the target cells and harvesting the EVs produced by these "reprogrammed cells" with the desired cell tropism.

[0004] In some embodiments, reprogramming the somatic cell comprises intracellularly delivering a polynucleotide comprising a nucleic acid sequence encoding a reprogramming transcription factor (TF) into the somatic cell. Table 1 provides examples of reprogramming TFs.

[0005] In some embodiments, EVs are harvested from the reprogrammed cells 12 hours to 5 days after the reprogramming transcription factors are delivered to the somatic cells (including 12, 24, 36, or 48 hours and 1, 2, 3, 4, or 5 days after the reprogramming transcription factors are delivered to the somatic cells). Thus, in some embodiments, the "reprogrammed cells" are reprogrammed toward a target lineage, but are not fully differentiated. In some embodiments, the "reprogrammed cells" express cell markers indicative of progenitors of the target cell lineage.

[0006] In some embodiments, the reprogrammed cells express lineage-specific markers, but lack one or more markers of full lineage differentiation.

[0007] In some embodiments, these EVs can be used to deliver diagnostic and / or therapeutic cargo to target cells in a subject in need thereof. Accordingly, also disclosed herein are methods of treating a disease or condition in a subject, comprising engineering reprogrammed cells to produce therapeutic EVs that deliver a therapeutic agent to a target cell.

[0008] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0009] [Figure 1] We demonstrate that designer EVs with enhanced SC tropism can be generated ex vivo and deployed in vivo to treat neurofibromas. [Figure 2A] Figure 2C shows how SC-derived EVs are preferentially internalized by SCs compared to fibroblasts. On the other hand, fibroblast-derived EVs show enhanced tropism for fibroblasts. Figure 2C shows that SC-derived EVs can carry large plasmid copies (~10 kb) and transcripts. Figure 2D shows successful delivery to SCs using designer EVs. *p<0.05. [Figure 2B] We show how SC-derived EVs are preferentially internalized by SCs compared to fibroblasts, whereas fibroblast-derived EVs show enhanced tropism towards fibroblasts. [Figure 2C] This shows that SC-derived EVs can carry large plasmid copies (~10 kb) and transcripts. Figure 2D shows successful delivery to SCs using designer EVs. *p<0.05. [Figure 3A] Figure 3 shows that SC-derived EVs preferentially accumulated in the mouse sciatic nerve (after crush injury) after systemic delivery. Figure 3B shows a high magnification image of the dashed rectangle in Figure 3A, which shows the labeled EVs. Figure 3C shows that fibroblast-derived EVs showed no accumulation in the sciatic nerve after crush injury. [Figure 4A] We show how myoblast-derived EVs are preferentially internalized by myoblasts compared to fibroblasts, whereas fibroblast-derived EVs show enhanced tropism towards fibroblasts (*p<0.05). [Figure 4B]We show how myoblast-derived EVs are preferentially internalized by myoblasts compared to fibroblasts, whereas fibroblast-derived EVs show enhanced tropism towards fibroblasts (*p<0.05). [Figure 4C] Myoblast-derived EVs preferentially accumulated in muscle tissue (e.g., gastrocnemius) of mice after systemic delivery. White arrows point to colocalization of labeled EVs (purple) containing green fluorescent protein (GFP) cargo adjacent to multiple nuclei of myocytes and counterstained with DAPI (blue) in the tissue. [Figure 4D] We show that fibroblast-derived EVs did not show accumulation in muscle tissue (e.g., gastrocnemius) after systemic EV delivery. [Figure 5A] We show that N2A neurons efficiently internalize EVs derived from fibroblasts primed for neuronal reprogramming (approximately 100% uptake efficiency). [Figure 5B] Figure 1 shows that N2A neurons show a higher affinity for EVs derived from reprogramming fibroblasts compared to control EVs. *p<0.05. [Figure 6A] Shows how engineered EVs harvested 24 hours after transfection of fibroblasts with reprogramming factors SOX10 alone or the combination of SOX10+EGR2 are preferentially internalized by SCs compared to fibroblasts 6 hours after EV treatment (*p<0.05). [Figure 6B] Shows how engineered EVs harvested 24 hours after transfection of fibroblasts with reprogramming factors SOX10 alone or the combination of SOX10+EGR2 are preferentially internalized by SCs compared to fibroblasts 6 hours after EV treatment (*p<0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Before describing the present disclosure in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0011] Where a range of values ​​is provided, it is understood that, unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any expressly excluded limitations in the stated range. Where a stated range includes one or both of the upper and lower limits, ranges excluding one or both of those included upper and lower limits are also included in the disclosure.

[0012] 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are now described herein.

[0013] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference herein, and are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0014] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0015] The embodiments of the present disclosure employ, unless otherwise indicated, chemical, biological and other techniques within the skill of the art.

[0016] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to use the methods and probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric pressure. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0017] Before embodiments of the present disclosure are described in detail, it will be understood that, unless otherwise indicated, the disclosure is not limited to particular materials, reagents, reactants, manufacturing processes, etc., as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It is also possible that steps may be performed in differing order where this is logically possible.

[0018] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0019] definition The term "subject" refers to any individual who is the target of administration or treatment. A subject can be a vertebrate, e.g., a mammal. Thus, a subject can be a human or veterinary patient. The term "patient" refers to a subject under the care of a clinician, e.g., a physician.

[0020] The term "therapeutically effective" refers to the amount of the composition used being sufficient to alleviate one or more of the causes or symptoms of a disease or disorder. Such alleviation requires only a reduction or alteration, not elimination.

[0021] The term "pharmacologically acceptable" refers to compounds, substances, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0022] The term "carrier" means a compound, composition, substance, or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0023] The term "treatment" refers to the medical management of a patient with the intent of curing, alleviating, stabilizing, or preventing a disease, pathological condition, or disorder. The term includes active treatment, i.e., treatment specifically directed to ameliorating a disease, condition, or disorder, and also includes causal treatment, i.e., treatment directed to eliminating the cause of the associated disease, condition, or disorder. In addition, the term includes palliative treatment, i.e., treatment designed to relieve symptoms but not cure the disease, condition, or disorder; preventive treatment, i.e., treatment directed to minimize or partially or completely inhibit the onset of the associated disease, condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific treatment directed to ameliorating the associated disease, condition, or disorder.

[0024] The term "inhibit" refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% reduction, or any amount in between, compared to native or control levels.

[0025] The term "polypeptide" refers to amino acids linked together by peptide bonds or modified peptide bonds, such as peptide isosteres, and may contain modified amino acids other than the 20 genetically encoded amino acids. Polypeptides may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine ​​or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodation, methylation, myristoylation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to proteins, such as arginylation. (See Proteins - Structure and Molecular Properties 2nd Ed., TECreighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)).

[0026] As used herein, the term "amino acid sequence" refers to a list of abbreviations, letters, symbols, or words that represent amino acid residues. Amino acid abbreviations used herein are the conventional one-letter codes for amino acids and are represented as follows: A, alanine, B, asparagine or aspartic acid, C, cysteine, D aspartic acid, E, glutamate, glutamic acid, F, phenylalanine, G, glycine, H histidine, I isoleucine, K, lysine, L, leucine, M, methionine, N, asparagine, P, proline, Q, glutamine, R, arginine, S, serine, T, threonine, V, valine, W, tryptophan, Y, tyrosine, Z, glutamine or glutamic acid.

[0027] As used herein, the phrase "nucleic acid" refers to a naturally occurring or synthetic oligonucleotide or polynucleotide capable of hybridizing to a complementary nucleic acid by Watson-Crick base pairing, whether DNA or RNA, or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense. Nucleic acids can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0028] As used herein, a "nucleotide" is a molecule that contains a base moiety, a sugar moiety, and a phosphate moiety. Nucleotides can be linked together through their phosphate and sugar moieties to form an internucleoside linkage. The term "oligonucleotide" is sometimes used to refer to a molecule that contains two or more nucleotides linked together. The base moiety of a nucleotide can be adenine-9-yl (A), cytosine-1-yl (C), guanine-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is ribose or deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. A non-limiting example of a nucleotide would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).

[0029] A nucleotide analog is a nucleotide that contains some type of modification in the base, sugar, and / or phosphate moieties. Modifications to nucleotides are well known in the art and may include, for example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, and 2-aminoadenine, as well as modifications in the sugar or phosphate moieties.

[0030] Nucleotide substitutes are molecules that have similar functional properties as nucleotides, but do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that recognize nucleic acids in a Watson-Crick or Hoogsteen fashion, but are bound together through moieties other than the phosphate moiety. Nucleotide substitutes can conform to a double helix type structure when interacting with an appropriate target nucleic acid.

[0031] The term "vector" or "construct" refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence is linked. The term "expression vector" includes any vector (e.g., a plasmid, cosmid, or phage chromosome) that contains a genetic construct in a form suitable for expression by a cell (e.g., linked to transcriptional control elements). "Plasmid" and "vector" are used interchangeably, as the plasmid is a commonly used form of vector. Furthermore, the invention is intended to include other vectors that serve equivalent functions.

[0032] The term "operably linked" refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcription and translation termination sites, and other signal sequences are examples of nucleic acid sequences that are operably linked to other sequences. For example, operably linked DNA to a transcriptional control element refers to a physical and functional relationship between the DNA and the promoter such that transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA.

[0033] For purposes herein, the % sequence identity of a given nucleotide or amino acid sequence C to, with, or against a given nucleic acid sequence D (which may alternatively be expressed as a given sequence C having or containing a particular % sequence identity to, with, or against a given sequence D) is calculated as follows: 100 x fraction W / Z, where W is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program in that program's alignment of C and D, and Z is the total number of nucleotides or amino acids in D. It will be understood that if the length of sequence C is not equal to the length of sequence D, then the % sequence identity of C to D will not be equal to the % sequence identity of D to C. Alignment for purposes of determining percent sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software.

[0034] By "specifically hybridize" it is meant that a probe, primer, or oligonucleotide recognizes and physically interacts (i.e., base pairs) with a substantially complementary nucleic acid (e.g., a c-met nucleic acid) under high stringency conditions and does not substantially base pair with other nucleic acids.

[0035] As used herein, the term "stringent hybridization conditions" means that hybridization generally occurs when there is at least 95%, preferably at least 97% sequence identity between the probe and the target sequence. An example of stringent hybridization conditions is overnight incubation in a solution containing 50% formamide, 5X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured, sheared carrier DNA (such as salmon sperm DNA), followed by washing the hybridization support in 0.1X SSC at about 65°C. Other hybridization and washing conditions are well known and are exemplified in Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY (1989), especially Chapter 11.

[0036] "Control elements" or "regulatory sequences" are vector enhancers, promoters, and untranslated regions of the 5' and 3' untranslated regions that interact with host cell proteins to effect transcription and translation. Such elements can vary in their strength and specificity.

[0037] A "promoter" is generally a sequence of DNA that functions when in a relatively fixed location in regard to the transcription start site. A "promoter" contains core elements required for basic interaction of RNA polymerase and transcription factors, and can contain upstream elements and response elements.

[0038] "Enhancer" generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' or 3' to the transcription unit. Additionally, enhancers can be within introns and within the coding sequence itself. They are usually 10-300 bp in length and function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers, like promoters, often also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression.

[0039] An "endogenous" enhancer / promoter is one that is naturally associated with a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that is juxtaposed to a gene by genetic manipulation (i.e., molecular biological techniques) so that transcription of that gene is directed by the associated enhancer / promoter.

[0040] Engineered extracellular vehicles (EVs) with designer tropism Disclosed herein is a method for engineering extracellular vesicles (EVs) with desired cell tropism for target cells, comprising reprogramming somatic cells toward the lineage of the target cells and harvesting EVs with desired cell tropism produced by these reprogrammed cells.

[0041] For example, methods for designer EVs that modulate pro-neuronal responses are described in Ortega-Pineda, et al. Adv. Healthcare Mater. 2022 11:210085 (incorporated by reference in its entirety for teachings on how to produce and use these EVs and their progeny).

[0042] Somatic cell reprogramming A method for reprogramming a somatic cell into a target cell is disclosed, the method comprising intracellularly delivering a polynucleotide comprising a nucleic acid sequence encoding a reprogramming transcription factor into the somatic cell. In some embodiments, the nucleic acid sequence is present in a non-viral vector. In some embodiments, the nucleic acid sequence is operably linked to an expression control sequence. In other embodiments, the nucleic acid is operably linked to two or more expression control sequences.

[0043] In some embodiments, the donor cells can be any donor cell capable of producing EVs, including, but not limited to, skin cells (e.g., fibroblasts, keratinocytes, skin stem cells), adipocytes, dendritic cells, peripheral blood mononuclear cells (PBMCs), pancreatic cells (e.g., ductal epithelial cells), liver cells (e.g., hepatocytes), immune cells (e.g., T cells, macrophages, myeloid-derived suppressor cells).

[0044] Transcription factors capable of reprogramming somatic cells into target cell types are known in the art. The diversity of cell types found in adult organisms is generated during development by lineage-specific transcription factors (TFs) that define and reinforce the specific gene expression patterns of each cell type. Fully differentiated cells can be stimulated to undergo significant cell fate changes by the induction of pluripotency in primary fibroblasts by a combination of four TFs (Yamanaka TFs) (Vierbuchen T, et al. Biotechnol. 2011 29(10):892-907). These transformations are understood as reversions from a mature state to a more primitive developmental state, with associated loss of developmentally important epigenetic information (Vierbuchen T, et al. Nature. 2010 463(7284):1035-41). Nevertheless, direct lineage reprogramming is considered an attractive approach in regenerative medicine as it offers an alternative with a relatively low risk of tumor development when compared to the use of pluripotent stem cells (Ruzittu S, et al. Direct Lineage Reprogramming: Harnessing Cell Plasticity between Liver and Pancreas. Cold Spring Harbor perspectives in biology. 2019, Xu J, et al. Cell Stem Cell. 2015 16(2):119-34).

[0045] Direct lineage reprogramming can induce cell fate conversion of functional cell types from one lineage to another without passing through an intermediate pluripotency stage. Several studies have shown that cell fate can be switched by introducing either single reprogramming TFs or their combinations, inspired by embryonic development or their in vivo ablation (Sancho-Martinez I, et al. Nature Cell Biology. 2012 14(9):892-9, Graf T, et al. Nature. 2009 462(7273):587-94). High expression of these TFs aims to overcome current epigenetic marks and promote the formation of target cells. This approach has been explored as an ideal alternative to reconstruct the traditional concept of epigenetic stability of differentiated cells to produce desired cell types and has been rapidly used to generate functional cells via direct conversion.

[0046] Although the generated cells usually lack the identity and functionality of mature cells, progenitor cells retain a plastic gene expression profile that correlates with global epigenetic remodeling. Progenitor cells with a fairly open chromatin structure become capable of responding to differentiation signals for redifferentiation into a functionally mature state, and proliferation of those cells allows the generation of abundant functional cells (Xie B, et al. Cell Research. 2019 29(9):696-710). Moreover, direct changes, including phenotypic changes, can be observed hours to days after TF overexpression and can proceed in the absence of cell proliferation.

[0047] There are "pioneer" TFs that function as master regulators of cell fate during normal development, interacting with chromatin and co-TFs (Morris SA. Development. 2016 143(15):2696-705). Often, reprogramming strategies use a combination of various TFs to overlay the program of the desired cell type by aiding in the transformation, with some TFs able to set fate specification and others able to set maturation, or alternatively functioning as repressors to delete the original cell identity (Ruzittu S, et al. Direct Lineage Reprogramming: Harnessing Cell Plasticity between Liver and Pancreas. Cold Spring Harbor perspectives in biology. 2019). Table 1 provides exemplary TFs for reprogramming cells. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0048] WO2018 / 119091 describes the use of ETV2, FOXC2, and FLU ("EFF factors") to reprogram somatic cells into angiogenic cells (incorporated by reference for the teaching of these reprogramming transcription factors). WO2020 / 028697 describes the use of Pdx1, Ng3, Mafa, and Tcf3 ("PMN-T factors") to reprogram somatic cells into insulin-producing cells (incorporated by reference for the teaching of these reprogramming transcription factors). In some embodiments, the transcription factor of the disclosed method is not an EFF or PMN-T factor.

[0049] A variety of methods are known in the art and are suitable for the introduction of nucleic acid into cells, including viral and non-viral mediated techniques.Exemplary non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated introduction, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated introduction, microinjection, microprojectile mediated introduction (nanoparticles), cationic polymer mediated introduction (DEAE-dextran, polyethyleneimine, polyethylene glycol (PEG), etc.) or cell fusion.

[0050] In some embodiments, after transfecting the target cells with transcription factors, the cells can then package the transfected genes (e.g., cDNA) into EVs, which can then induce other skin cells to form insulin-producing cells. Thus, a method of reprogramming skin cells into insulin-producing cells is also disclosed, comprising exposing somatic cells to extracellular vesicles produced from cells that contain Pdx1, Ng3, Mafa, and Tcf3.

[0051] The EVs secreted by donor cells can then be collected from the culture medium.These EVs can then be administered to skin cells to reprogram them into insulin-producing cells.In some embodiments, donor cells can be any cell from a subject that can produce EVs, including but not limited to skin cells (e.g., fibroblasts, keratinocytes, skin stem cells), adipocytes, dendritic cells, peripheral blood mononuclear cells (PBMCs), pancreatic cells (e.g., ductal epithelial cells), liver cells (e.g., hepatocytes), immune cells (e.g., T cells, macrophages, myeloid-derived suppressor cells).

[0052] Exosomes and microvesicles are distinct EVs based on their biogenesis process and biophysical properties, including size and surface protein markers. Exosomes are homogeneous small particles with sizes ranging from 40 to 150 nm, and they usually originate from the endocytic recycling pathway. In endocytosis, endocytic vesicles form at the plasma membrane and fuse to form early endosomes. These mature to become late endosomes, where intraluminal vesicles become vesicle lumen. Instead of fusing with lysosomes, these multivesicular bodies fuse directly with the plasma membrane and release exosomes into the extracellular space. Exosome biogenesis, protein cargo sorting, and release involve endosomal sorting complexes required for transport (ESCRT complexes), as well as other associated proteins such as Alix and Tsg101. In contrast, microvesicles are directly produced through outward budding and fission of membrane vesicles from the plasma membrane, and therefore, their surface markers are highly dependent on the composition of the membrane of origin. Moreover, they tend to constitute a larger, more heterogeneous population of extracellular vesicles, ranging from 150 to 1000 nm in diameter. However, both types of vesicles have been shown to deliver functional mRNA, miRNA and proteins to recipient cells.

[0053] In some embodiments, the polynucleotide is delivered intracellularly to somatic cells or donor cells for EVs via genetic cancer, microparticles or nanoparticles suitable for such delivery, transfection by electroporation, three-dimensional nanochannel electroporation, tissue nanotransfection device, liposomes suitable for such delivery, or deep tumor tissue nanoelectroinjection device. In some embodiments, a viral vector can be used. However, in other embodiments, the polynucleotide is not delivered by a virus.

[0054] Electroporation is a technique in which an electric field is applied to a cell to increase the permeability of the cell membrane, allowing a cargo (e.g., a reprogramming factor) to be introduced into the cell. Electroporation is a common technique for introducing foreign DNA into cells.

[0055] Tissue nanotransfection allows for direct cytosolic delivery of cargo (e.g., reprogramming factors) into cells by applying very strong and focused electric fields through arrayed nanochannels that benignly nanoporate juxtaposed tissue cellular members and electrophoretically drive the cargo into the cells.

[0056] In one embodiment, the disclosed compositions are administered at a dose equivalent to a parenteral administration of about 0.1 ng to about 100 g per kg body weight, about 10 ng to about 50 g per kg body weight, about 100 ng to about 1 g per kg body weight, about 1 μg to about 100 mg per kg body weight, about 1 μg to about 50 mg per kg body weight, about 1 mg to about 500 mg per kg body weight, and about 1 mg to about 50 mg per kg body weight. Alternatively, the amount of the disclosed compositions administered to achieve a therapeutically effective dose is about 0.1 ng, 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 500 mg or more per kg of body weight.

[0057] To express the polypeptide or functional nucleic acid, the nucleotide coding sequence can be inserted into a suitable expression vector.Therefore, also disclosed is a non-viral vector comprising a polynucleotide comprising a nucleic acid sequence encoding two, three or four of the proteins selected from the group consisting of Pdx1, Ng3, Mafa and Tcf3, the nucleic acid sequence being operably linked to an expression control sequence.In some embodiments, the nucleic acid sequence is operably linked to a single expression control sequence.In other embodiments, the nucleic acid sequence is operably linked to two or more separate expression control sequences.

[0058] Methods for constructing expression vectors containing gene sequences and appropriate transcriptional and translational control elements are well known in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Press, Plainview, NY, 1989), and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York, NY, 1989).

[0059] Expression vectors generally contain regulatory sequences necessary elements for the translation and / or transcription of an inserted coding sequence. For example, the coding sequence is preferably operably linked to a promoter and / or enhancer to help control the expression of the desired gene product.

[0060] "Control elements" or "regulatory sequences" are vector enhancers, promoters, and untranslated regions of the 5' and 3' untranslated regions that interact with host cell proteins to effect transcription and translation. Such elements can vary in their strength and specificity.

[0061] A "promoter" is generally a sequence of DNA that functions when in a relatively fixed location in regard to the transcription start site. A "promoter" contains core elements required for basic interaction of RNA polymerase and transcription factors, and can contain upstream elements and response elements.

[0062] "Enhancer" generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' or 3' to the transcription unit. Additionally, enhancers can be within introns and within the coding sequence itself. They are usually 10-300 bp in length and function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers, like promoters, often also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression.

[0063] An "endogenous" enhancer / promoter is one that is naturally associated with a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that is juxtaposed to a gene by genetic manipulation (i.e., molecular biological techniques) so that transcription of that gene is directed by the associated enhancer / promoter.

[0064] Promoters used in biotechnology are of different types according to the intended type of control of gene expression. They can generally be divided into constitutive promoters, tissue- or developmental stage-specific promoters, inducible promoters, and synthetic promoters.

[0065] Constitutive promoters direct expression in virtually all tissues and are largely, if not completely, independent of environmental and developmental factors. Because their expression is usually not conditioned by endogenous factors, constitutive promoters are usually active across species and even kingdoms. Examples of constitutive promoters include CMV, EF1a, SV40, PGK1, Ubc, human beta actin, and CAG.

[0066] Tissue-specific or developmental stage-specific promoters induce the expression of a gene in a particular tissue(s) or in a particular developmental stage. In plants, promoter elements that express or affect the expression of genes in the vascular system, photosynthetic tissues, tubers, roots, and other vegetative organs, or seeds and other reproductive organs, can be found in heterologous systems (e.g., distantly related species, or even other kingdoms), but maximum specificity is generally achieved with homologous promoters (i.e., from the same species, genus, or family). This is probably because coordinate expression of transcription factors is required to regulate the activity of the promoter.

[0067] The performance of inducible promoters is conditioned not by endogenous factors, but by environmental conditions and external stimuli that can be artificially controlled. Within this group are promoters regulated by non-biological factors such as light, oxygen levels, high temperature, low temperature, and wounding. Some of these factors are difficult to control outside of the experimental environment, so promoters that respond to chemical compounds that are not naturally found in the organism of interest are of particular interest. In the same way, promoters that respond to antibiotics, copper, alcohols, steroids, and herbicides, among other compounds, have been adapted and refined to allow induction of gene activity at will and independently of other biological or non-biological factors.

[0068] The two most commonly used inducible expression systems for the study of eukaryotic cell biology are named Tet-Off and Tet-On. The Tet-Off system utilizes the tetracycline transactivator (tTA) protein, which is created by fusing one protein, TetR (tetracycline repressor), found in Escherichia coli bacteria, with the activation domain of another protein, VP16, found in herpes simplex viruses. The resulting tTA protein can bind to DNA at specific TetO operator sequences. In most Tet-Off systems, several repeats of such TetO sequences are placed upstream of a minimal promoter, such as the CMV promoter. The totality of several TetO sequences with a minimal promoter is called a tetracycline response element (TRE), because it responds to the binding of the tetracycline transactivator protein tTA by increasing the expression of the gene(s) downstream of that promoter. In the Tet-Off system, the expression of the TRE-controlled gene can be repressed by tetracycline and its derivatives. They bind to tTA and are unable to bind to the TRE sequence, thereby preventing transactivation of the TRE-controlled gene. The Tet-On system functions similarly, but in the opposite manner. In the Tet-Off system, tTA can only bind to the operator if it is not bound to tetracycline or one of its derivatives, such as doxycycline, in the Tet-On system, and the rtTA protein can only bind to the operator if it is bound to tetracycline. Thus, introduction of doxycycline into the system initiates transcription of the gene product. The Tet-On system may be preferred over the Tet-Off system due to its faster response.

[0069] In some embodiments, the nucleic acid sequences encoding the transcription factors disclosed herein are operably linked to the same expression control sequence. Alternatively, an internal ribosome entry site (IRES) element can be used to create a multi-gene or polycistronic message. The IRES element can bypass the ribosome scanning model of 5' methylated Cap-dependent translation and initiate translation at an internal site. The IRES element can be linked to a heterologous open reading frame. Multiple open reading frames can be transcribed together, each separated by an IRES, creating a polycistronic message. The IRES element allows each open reading frame to be accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message.

[0070] Disclosed is a non-viral vector that contains one or more polynucleotides disclosed herein operably linked to an expression control sequence.Examples of such non-viral vectors include oligonucleotides alone or in combination with suitable protein, polysaccharide, or lipid formulations.Non-viral methods present certain advantages over viral methods, simple large-scale production and low host immunogenicity being just two of them.Previously, low levels of transfection and gene expression held non-viral methods at a disadvantage, but recent advances in vector technology have led to molecules and techniques that have transfection efficiencies similar to those of viruses.

[0071] Examples of suitable non-viral vectors include, but are not limited to, pIRES-hrGFP-2a, pCMV6, pMAX, pCAG, pAd-IRES-GFP, and pCDNA3.0.

[0072] The disclosed compositions can be used in therapy in combination with a pharma- ceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., a material that can be administered to a subject together with a nucleic acid or vector, without causing any undesirable biological effects or interacting in a detrimental manner with any of the other components of the pharmaceutical composition with which it comes into contact. The carrier can necessarily be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as is well known to those skilled in the art.

[0073] The materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells), and they may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al. al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drug targeting to colon cancer), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-tropic tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989), and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand-induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through acidified endosomes where the receptors are sorted and then either recycled to the cell surface, stored intracellularly, or degraded in lysosomes.Internalization pathways perform a variety of functions, including nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligands, and regulation of receptor levels. Many receptors follow more than one intracellular pathway, depending on cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0074] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. AR Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharma- ceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharma- ceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to one skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and the concentration of the composition being administered.

[0075] Pharmaceutical carriers are known to those skilled in the art. These are most typically standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffer solutions at physiological pH. These compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0076] Pharmaceutical compositions may include, in addition to the molecule of choice, carriers, thickeners, diluents, buffers, preservatives, surface active agents, etc. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetic agents, etc.

[0077] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases and the like.

[0078] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0079] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets.Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable.

[0080] Some of the present compositions may potentially be administered as pharma- ceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-alkyl-, and arylamines, and substituted ethanolamines.

[0081] The compositions disclosed herein, including pharmaceutical compositions, can be administered in a number of ways, depending on whether local or systemic treatment is desired and the area to be treated. For example, the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. The compositions can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, intraperitoneal injection, transdermally, extracorporeally, ophthalmically, intravaginally, rectally, intranasally, topically, etc., including topical intranasal administration or administration by inhalation.

[0082] Engineering reprogrammed cells to produce EVs In some embodiments, these EVs can be used to deliver diagnostic and / or therapeutic cargo to target cells in a subject in need thereof. Accordingly, also disclosed herein are methods of treating a disease or condition in a subject, comprising engineering reprogrammed cells to produce therapeutic EVs that deliver a therapeutic agent to a target cell.

[0083] In some embodiments, the disclosed EVs can be any vesicles that can be secreted by cells. Cells secrete extracellular vesicles (EVs) with a wide range of diameters and functions, including apoptotic bodies (1-5 μm), microvesicles (100-1000 nm in size), and vesicles of endosomal origin known as exosomes (50-150 nm).

[0084] The disclosed extracellular vesicles can be prepared by methods known in the art. For example, the disclosed extracellular vesicles can be prepared by expressing an mRNA encoding a cell-targeting ligand in a eukaryotic cell. In some embodiments, the cell also expresses an mRNA encoding a therapeutic cargo. The mRNA for the cell-targeting ligand and the therapeutic cargo can be expressed from a vector that is transfected into a suitable production cell for producing the disclosed EVs. The mRNA for the cell-targeting ligand and the therapeutic cargo can be expressed from the same vector (e.g., where the vector expresses the mRNA for the cell-targeting ligand and the therapeutic cargo from separate promoters), or the mRNA for the cell-targeting ligand and the therapeutic cargo can be expressed from separate vectors. The vector for expressing the mRNA for the cell-targeting ligand and the therapeutic cargo can be packaged in a kit designed to prepare the disclosed extracellular vesicles.

[0085] Medical Cargo The disclosed extracellular vesicles may be loaded with therapeutic agents, and these extracellular vesicles deliver the agents to target cells. Suitable therapeutic agents include, but are not limited to, therapeutic agents (e.g., small molecule drugs), therapeutic proteins, and therapeutic nucleic acids (e.g., therapeutic RNA). In some embodiments, the disclosed extracellular vesicles include therapeutic RNA (also referred to herein as "cargo RNA").

[0086] For example, in some embodiments, the cell targeting protein also includes an RNA domain (e.g., at the cytoplasmic C-terminus of the fusion protein) that binds to one or more RNA motifs present in the cargo RNA to package the cargo RNA into the extracellular vesicle before the extracellular vesicle is secreted from the cell. Thus, a protein can function as both a "cell targeting protein" and a "packaging protein." In some embodiments, the packaging protein can be referred to as an extracellular vesicle carrying protein or an "EV carrying protein."

[0087] Representative examples of nucleic acid therapeutic, prophylactic, and diagnostic agents include DNA plasmid vectors (e.g., expression vectors), RNA molecules (e.g., iRNA, siRNA, ribozymes, aptamers, repRNA, gRNA / sgRNA (guide RNA / single guide RNA for CRISPR-based gene editing)), and mRNA.

[0088] The cargo RNA of the disclosed extracellular vesicles can be of any suitable length. For example, in some embodiments, the cargo RNA can have a nucleotide length of at least about 10 nt, 20 nt, 30 nt, 40 nt, 50 nt, 100 nt, 200 nt, 500 nt, 1000 nt, 2000 nt, 5000 nt, or more. In other embodiments, the cargo RNA can have a nucleotide length of about 5000 nt, 2000 nt, 1000 nt, 500 nt, 200 nt, 100 nt, 50 nt, 40 nt, 30 nt, 20 nt, or 10 nt or less. In still further embodiments, the cargo RNA can have a nucleotide length within these anticipated nucleotide lengths, for example, a range of about 10 nt to 5000 nt or other ranges of nucleotide lengths. The cargo RNA of the disclosed extracellular vesicles can be relatively long, for example, the cargo RNA comprises an mRNA or another relatively long RNA.

[0089] In some embodiments, the therapeutic cargo is a membrane-permeable pharmacological compound that is secreted by cells and then loaded into EVs.

[0090] Transfection-based approaches have been proposed to achieve small RNA loading into EVs. Other reports have shown that vector-induced expression of small RNAs in cells can be used to achieve small RNA loading into EVs. Alternatively, EV donor cells may be directly transfected with small RNAs. Incubation of tumor cells with chemotherapeutic drugs is another method to package drugs into EVs. To stimulate the formation of drug-loaded EVs, cells are irradiated with ultraviolet light to induce apoptosis. Alternative approaches such as fusogenic liposomes also result in the loading of drugs into EVs.

[0091] In some embodiments, the therapeutic cargo is loaded onto the EVs by diffusion through a concentration gradient.

[0092] gRNA In some embodiments, the therapeutic RNA is a guide RNA (gRNA). The guide RNA may guide a Cas nuclease to a target sequence on a target nucleic acid molecule, where the guide RNA hybridizes and the Cas nuclease cleaves or modulates the target sequence. In some embodiments, the guide RNA binds to the nuclease and provides specificity for cleavage by the nuclease. In some embodiments, the guide RNA and the Cas protein may form a ribonucleoprotein (RNP) (e.g., a CRISPR / Cas complex). In some embodiments, the CRISPR complex may be a type II CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex may be a type V CRISPR / Cas complex, such as a Cpf1 / guide RNA complex. In some embodiments, the Cas nuclease may be a single protein Cas nuclease, e.g., a Cas9 protein or a Cpf1 protein. In some embodiments, the guide RNA targets cleavage by the Cas9 protein.

[0093] In some embodiments, guide RNAs for the CRISPR / Cas9 nuclease system include CRISPR RNA (crRNA) and tracrRNA (tracr). In some embodiments, the crRNA may include a targeting sequence that is complementary to and hybridizes with a target sequence on a target nucleic acid molecule. The crRNA may also include a flagpole that is complementary to and hybridizes with a portion of the tracrRNA. In some embodiments, the crRNA may parallel the structure of a naturally occurring crRNA transcribed from a bacterial CRISPR locus, where the target sequence serves as a spacer in the CRISPR / Cas9 system, and the flagpole corresponds to a portion of the repeat sequence adjacent to the spacer on the CRISPR locus.

[0094] The guide RNA may target any sequence of interest via the target sequence of the crRNA. In some embodiments, the degree of complementarity between the target sequence of the guide RNA and the target sequence on the target nucleic acid molecule may be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the target sequence of the guide RNA and the target sequence on the target nucleic acid molecule may be 100% complementary. In other embodiments, the target sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain at least one mismatch. For example, the target sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In some embodiments, the target sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1 to 6 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain five or six mismatches.

[0095] The length of the targeting sequence depends on the CRISPR / Cas system and components used. For example, different Cas proteins from different bacterial species have different optimal targeting sequence lengths. Thus, the targeting sequence may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the targeting sequence may comprise 18-24 nucleotides in length. In some embodiments, the targeting sequence may comprise 19-21 nucleotides in length. In some embodiments, the targeting sequence may comprise 20 nucleotides in length.

[0096] The flagpole may comprise any sequence that has sufficient complementarity with the tracrRNA to promote the formation of a functional CRISPR / Cas complex. In some embodiments, the flagpole may comprise all or a portion of the sequence of a naturally occurring crRNA that is complementary to the tracrRNA in the same CRISPR / Cas system (also called the "tag" or "handle"). In some embodiments, the flagpole may comprise all or a portion of a repeat sequence from a naturally occurring CRISPR / Cas system. In some embodiments, the flagpole may comprise a truncated or modified tag or handle sequence. In some embodiments, the degree of complementarity between the tracrRNA and the portion of the flagpole that hybridizes to the tracrRNA along the length of the shorter of the two sequences may be about 40%, 50%, 60%, 70%, 80%, or more, but less than 100%. In some embodiments, the tracrRNA and the portion of the flagpole that hybridizes to the tracrRNA are not 100% complementary along the length of the shorter of the two sequences due to the presence of one or more bulge structures on the tracr and / or wobble base pairs between the tracr and the flagpole. The length of the target flagpole may depend on the CRISPR / Cas system or tracrRNA used. For example, the flagpole may comprise 10-50 nucleotides in length, or more than 50 nucleotides in length. In some embodiments, the flagpole may comprise 15-40 nucleotides in length. In some embodiments, the flagpole may comprise 20-30 nucleotides in length. In some embodiments, the flagpole may comprise 22 nucleotides in length. When using dual guide RNAs, for example, there may be no upper limit to the length of the flagpole.

[0097] In some embodiments, the tracrRNA may comprise all or part of the wild-type tracr RNA sequence from a naturally occurring CRISPR / Cas system. In some embodiments, the tracrRNA may comprise a truncated or modified variant of the wild-type tracrRNA. The length of the tracr RNA may depend on the CRISPR / Cas system used. In some embodiments, the tracrRNA may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides in length. In certain embodiments, the tracr is at least 26 nucleotides in length. In additional embodiments, the tracr is at least 40 nucleotides in length. In some embodiments, the tracrRNA may comprise a specific secondary structure, such as, for example, one or more hairpin or stem-loop structures or one or more bulge structures.

[0098] In some embodiments, the guide RNA may comprise two RNA molecules, referred to herein as "dual guide RNA" or "dgRNA." In some embodiments, the dgRNA may comprise a first RNA molecule comprising a crRNA and a second RNA molecule comprising a tracrRNA. The first RNA molecule and the second RNA molecule may form an RNA duplex via base pairing between a flagpole on the crRNA and the tracrRNA.

[0099] In some embodiments, the guide RNA may comprise a single RNA molecule, referred to herein as a "single guide RNA" or "sgRNA." In some embodiments, the sgRNA may comprise a crRNA covalently linked to a tracrRNA. In some embodiments, the crRNA and the tracrRNA may be covalently linked via a linker. In some embodiments, the single guide RNA may comprise a stem-loop structure due to base pairing between a flagpole on the crRNA and the tracrRNA. In some embodiments, the sgRNA is a "Cas9 sgRNA" capable of mediating RNA-guided DNA cleavage by a Cas9 protein. In some embodiments, the sgRNA is a "Cpf1 sgRNA" capable of mediating RNA-guided DNA cleavage by a Cpf1 protein. In some embodiments, the guide RNA comprises sufficient crRNA and tracrRNA to form an active complex with a Cas9 protein and mediate RNA-guided DNA cleavage. In some embodiments, the guide RNA comprises sufficient crRNA to form an active complex with a Cpf1 protein and mediate RNA-guided DNA cleavage.

[0100] In some embodiments, the cargo comprises an expression cassette encoding a guide RNA. Thus, "guide RNA nucleic acid" can refer to a guide RNA (e.g., sgRNA or dgRNA) and a guide RNA expression cassette, which is a nucleic acid that encodes one or more guide RNAs.

[0101] In some embodiments, the nucleic acid may be a DNA molecule. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a crRNA. In some embodiments, the nucleotide sequence encoding the crRNA comprises a targeting sequence flanked by all or a portion of the repeat sequences from a naturally occurring CRISPR / Cas system. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a tracrRNA. In some embodiments, the crRNA and tracrRNA may be encoded by two separate nucleic acids. In other embodiments, the crRNA and tracrRNA may be encoded by a single nucleic acid. In some embodiments, the crRNA and tracrRNA may be encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and tracrRNA may be encoded by the same strand of a single nucleic acid. In some embodiments, the expression cassette encodes an sgRNA. In some embodiments, the expression cassette encodes a Cas9 nuclease sgRNA. In the following embodiments, the expression cassette encodes a Cpf1 nuclease sgRNA.

[0102] The nucleotide sequence encoding the guide RNA may be operably linked to at least one transcriptional or regulatory control sequence, such as a promoter, a 3'UTR, or a 5'UTR. In one example, the promoter may be a tRNA promoter, such as a tRNALys3, or a tRNA chimera. See Mefferd et al., RNA. 2015 21:1683-9; Scherer et al., Nucleic Acids Res. 2007 35:2620-2628. In certain embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters also include U6 and H1 promoters. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human U6 promoter. In some embodiments, the expression cassette is a modified nucleic acid. In certain embodiments, the expression cassette comprises modified nucleosides or nucleotides. In some embodiments, the expression cassette comprises a 5'-end modification (e.g., a modified nucleoside or nucleotide) that stabilizes the expression cassette and prevents its integration. In some embodiments, the expression cassette comprises double-stranded DNA with a 5'-end modification on each strand. In certain embodiments, the expression cassette comprises an inverted dideoxy-T or an inverted abasic nucleoside or nucleotide as the 5'-end modification. In some embodiments, the expression cassette comprises a label such as biotin, desthiobiotin-TEG, digoxigenin, and a fluorescent marker, including, for example, FAM, ROX, TAMRA, and AlexaFluor.

[0103] In certain embodiments, two or more guide RNAs can be used with the CRISPR / Cas nuclease system. Each guide RNA nucleic acid may contain a different targeting sequence, so that the CRISPR / Cas system cleaves two or more target sequences. In some embodiments, one or more guide RNAs can have the same or different properties, such as activity or stability within the CRISPR / Cas complex. When two or more guide RNAs are used, each guide RNA can be encoded on the same or different expression cassettes. The promoters used to drive the expression of two or more guide RNAs can be the same or different.

[0104] Template Nucleic Acid The formulations disclosed herein may include a template nucleic acid. The template may be used to modify or insert a nucleic acid sequence at or near a target site for a Cas nuclease.

[0105] In some embodiments, the template may be used in homologous recombination. In some embodiments, the template sequence or a portion of the template sequence may be incorporated into the target nucleic acid molecule by homologous recombination. In some embodiments, a single template may be provided. In other embodiments, two or more templates may be provided so that homologous recombination can occur at two or more target sites. For example, different templates may be provided to repair a single gene in a cell or two different genes in a cell. In some embodiments, multiple copies of at least one template are provided to the cell. In some embodiments, different templates may be provided in independent copy numbers or independent amounts.

[0106] In other embodiments, the template may be used for homology-directed repair, which involves the invasion of a DNA strand at the cleavage site of the nucleic acid. In some embodiments, homology-directed repair may result in the inclusion of the template sequence in the edited target nucleic acid molecule. In some embodiments, a single template may be provided. In other embodiments, two or more templates with different sequences may be used at two or more sites by homology-directed repair. For example, different templates may be provided to repair a single gene in a cell or two different genes in a cell. In some embodiments, multiple copies of at least one template are provided to the cell. In some embodiments, different templates may be provided in independent copy numbers or independent amounts.

[0107] In yet other embodiments, the template may be used in gene editing mediated by non-homologous end joining. In some embodiments, the template sequence is not similar to the nucleic acid sequence near the cleavage site. In some embodiments, the template or a portion of the template sequence is integrated. In some embodiments, a single template may be provided. In other embodiments, two or more templates with different sequences may be inserted into two or more sites by non-homologous end joining. For example, different templates may be provided to edit a single gene in a cell or two different genes in a cell. In some embodiments, different templates may be provided in independent copy numbers. In some embodiments, the template includes adjacent inverted terminal repeat (ITR) sequences.

[0108] In some embodiments, the template sequence may correspond to an endogenous sequence of the target cell. As used herein, the term "endogenous sequence" refers to a sequence that is native to the cell. The term "exogenous sequence" refers to a sequence that is not native to the cell or that is at a different location than its native location in the genome of the cell. In some embodiments, the endogenous sequence may be a genomic sequence of the cell. In some embodiments, the endogenous sequence may be a chromosomal or extrachromosomal sequence. In some embodiments, the endogenous sequence may be a plasmid sequence of the cell. In some embodiments, the template sequence may be substantially identical to a portion of the endogenous sequence in the cell at or near the cleavage site, but includes at least one nucleotide change. In some embodiments, repairing the cleaved target nucleic acid molecule with the template may result in a mutation that includes an insertion, deletion, or substitution of one or more nucleotides of the target nucleic acid molecule. In some embodiments, the mutation may result in one or more amino acid changes in a protein expressed from a gene that includes the target sequence. In some embodiments, the mutation may result in one or more nucleotide changes in an RNA expressed from the target gene. In some embodiments, the mutation may change the expression level of the target gene. In some embodiments, the mutation may result in an increase or decrease in expression of the target gene. In some embodiments, the mutation may result in a gene knockdown. In some embodiments, the mutation may result in a gene knockout. In some embodiments, the mutation may result in a restoration of gene function. In some embodiments, the mutation may result in a mutation in a target nucleic acid molecule cleaved with a template may result in a change in an exon sequence, an intron sequence, a regulatory sequence, a transcriptional control sequence, a translational control sequence, a splice site, or a non-coding sequence of the target gene.

[0109] In other embodiments, the template sequence may include an exogenous sequence. In some embodiments, the exogenous sequence may include a protein or RNA coding sequence operably linked to an exogenous promoter sequence such that when the exogenous sequence is integrated into the target nucleic acid molecule, the cell can express the protein or RNA encoded by the integrated sequence. In other embodiments, when the exogenous sequence is integrated into the target nucleic acid molecule, the expression of the integrated sequence may be regulated by an endogenous promoter sequence. In some embodiments, the exogenous sequence may be a chromosomal sequence or an extrachromosomal sequence. In some embodiments, the exogenous sequence may provide a cDNA sequence that codes for a protein or a portion of a protein. In still other embodiments, the exogenous sequence may include an exon sequence, an intron sequence, a regulatory sequence, a transcription control sequence, a translation control sequence, a splice site, or a non-coding sequence. In some embodiments, the integration of the exogenous sequence may result in the restoration of gene function. In some embodiments, the integration of the exogenous sequence may result in a gene knock-in. In some embodiments, the integration of the exogenous sequence may result in a gene knock-out.

[0110] The template can be of any suitable length. In some embodiments, the template can comprise 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, or more nucleotides in length. The template can be a single-stranded nucleic acid. The template can be a double-stranded or partially double-stranded nucleic acid. In certain embodiments, the single-stranded template is 20, 30, 40, 50, 75, 100, 125, 150, 175, or 200 nucleotides in length. In some embodiments, the template can comprise a nucleic acid sequence that is complementary to a portion of a target nucleic acid molecule that comprises the target sequence (i.e., a "homology arm"). In some embodiments, the template can comprise a homology arm that is complementary to a sequence located upstream or downstream of the cleavage site on the target nucleic acid molecule. In some embodiments, the template may include a first homology arm and a second homology arm (also referred to as a first and a second nucleotide sequence) that are complementary to sequences located upstream and downstream of the cleavage site, respectively. When the template includes two homology arms, each arm may be of the same length or different lengths, and the sequence between the homology arms may be substantially similar or identical to the target sequence between the homology arms, or may be entirely unrelated. In some embodiments, the degree of complementarity between the first nucleotide sequence on the template and the sequence upstream of the cleavage site, and between the second nucleotide sequence on the template and the sequence downstream of the cleavage site, may allow for homologous recombination, e.g., high fidelity homologous recombination, between the template and the target nucleic acid molecule. In some embodiments, the degree of complementarity may be about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100%. In some embodiments, the degree of complementarity may be about 95%, about 97%, about 98%, about 99%, or about 100%. In some embodiments, the degree of complementarity may be at least 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be 100%.

[0111] In some embodiments, the template comprises ssDNA or dsDNA that includes flanking inverted terminal repeat (ITR) sequences. In some embodiments, the template is provided as a vector, a plasmid, a minicircle, a nanocircle, or a PCR product.

[0112] Chemically modified RNA Modified nucleosides or nucleotides may be present in therapeutic RNA. The term "modified" RNA refers to the presence of one or more non-naturally occurring and / or naturally occurring components or structures that are used in place of or in addition to the standard A, G, C, and U residues. In some embodiments, modified RNA is synthesized with non-standard nucleosides or nucleotides and is referred to herein as "modified." Modified nucleosides and nucleotides can include one or more of: (i) modifications in the phosphodiester backbone linkages, e.g., replacement of one or both of the non-bridging phosphate oxygens and / or one or more of the bridging phosphate oxygens (exemplary backbone modifications); (ii) modifications, e.g., replacement of a component of the ribose sugar, e.g., the 2' hydroxyl of the ribose sugar (exemplary sugar modifications); (iii) wholesale replacement of a phosphate moiety with a "dephosphorylated" linker (exemplary backbone modifications); (iv) modifications or replacement of naturally occurring nucleobases, including with non-standard nucleobases (exemplary base modifications); (v) replacement or modification of the ribose phosphate backbone (exemplary backbone modifications); (vi) modifications of the 3' or 5' terminus of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group, or conjugation of a moiety, cap, or linker (such 3' or 5' cap modifications can include sugar and / or backbone modifications); and (vii) modifications or replacement of the sugar (exemplary sugar modifications).

[0113] The modifications listed above can be combined to provide modified RNAs that include nucleosides and nucleotides (collectively "residues") that can have two, three, four, or more modifications. For example, modified residues can have modified sugars and modified nucleobases. In some embodiments, each base of the RNA is modified, e.g., all bases have modified phosphate groups, such as phosphorothioates. In certain embodiments, all or substantially all of the phosphate groups of the RNA molecule are replaced with phosphorothioate groups. In some embodiments, the modified RNA includes at least one modified residue at or near the 5' end of the RNA. In some embodiments, the modified RNA includes at least one modified residue at or near the 3' end of the RNA.

[0114] In some embodiments, the RNA comprises one, two, three, or more modified residues. In some embodiments, the RNA comprises one, two, three, or more modified residues at each of the 5' and 3' ends of the RNA. In some embodiments, the RNA comprises 5, 10, 15, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more modified residues. In some embodiments, at least 5% (e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%) of the positions in the modified RNA are modified nucleosides or nucleotides.

[0115] Unmodified nucleic acids may be susceptible to degradation, for example, by cellular nucleases. For example, nucleases may hydrolyze the phosphodiester bonds of nucleic acids. Thus, in one aspect, the RNAs described herein may contain one or more modified nucleosides or nucleotides, for example, to introduce stability against nucleases. In certain embodiments, the cargo RNAs described herein may contain one or more modified nucleosides or nucleotides, for example, to introduce stability against nucleases. In some embodiments, the modified RNA molecules described herein may exhibit reduced innate immune responses when introduced into a cell population, both in vivo and ex vivo. The term "innate immune response" includes cellular responses to exogenous nucleic acids, including single-stranded nucleic acids, including induction of cytokine (particularly interferon) expression and release and cell death.

[0116] In some embodiments of backbone modifications, the phosphate group of the modified residue may be modified by replacing one or more oxygens with different substituents. Furthermore, modified residues, such as those present in modified nucleic acids, may include large-scale replacement of unmodified phosphate moieties with modified phosphate groups, as described herein. In some embodiments, backbone modifications of the phosphate backbone may include modifications that result in either uncharged linkers or charged linkers with asymmetric charge distribution.

[0117] Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. The phosphate atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the atoms or groups of atoms described above can make the phosphorus atom chiral. The stereogenic phosphorus atom can have either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). The backbone can also be modified by replacement of the bridging oxygen (i.e., the oxygen that connects the phosphate to the nucleoside) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). Replacement can occur at either or both bridging oxygens.

[0118] The phosphate group can be replaced by a non-phosphorus-containing linking group in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties that can replace the phosphate group can include, but are not limited to, for example, methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino.

[0119] Scaffolds can also be constructed that can mimic nucleic acids in which the phosphate linker and ribose sugar are replaced by nuclease-resistant nucleoside or nucleotide surrogates. Such modifications can include backbone and sugar modifications. In some embodiments, the nucleobases can be tethered by alternative backbones. Examples can include, but are not limited to, morpholino, cyclobutyl, pyrrolidine, and peptide nucleic acid (PNA) nucleobase surrogates.

[0120] Modified nucleosides and modified nucleotides may include one or more modifications to the sugar, i.e., sugar modifications. For example, the 2' hydroxyl group (OH) may be modified, e.g., replaced by a number of different "oxy" or "deoxy" substituents. In some embodiments, modifications to the 2' hydroxyl group may enhance the stability of the nucleic acid, since the hydroxyl can no longer be further deprotonated to form a 2'-alkoxide ion.

[0121] Examples of 2' hydroxyl group modifications include alkoxy or aryloxy (OR, where "R" can be alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), polyethylene glycol (PEG), O(CH2CHO) n In some embodiments, the 2' hydroxyl group modification may include CH2CH2OR, where R is, for example, H or an optionally substituted alkyl, and n may be an integer from 0 to 20 (e.g., 0-4, 0-8, 0-10, 0-16, 1-4, 1-8, 1-10, 1-16, 1-20, 2-4, 2-8, 2-10, 2-16, 2-20, 4-8, 4-10, 4-16, and 4-20). In some embodiments, the 2' hydroxyl group modification may be 2'-O-Me. In some embodiments, the 2' hydroxyl group modification may be a 2'-fluoro modification, which replaces the 2'-hydroxyl group with fluorine. In some embodiments, the 2' hydroxyl group modification may be a 2' hydroxyl group modification, which replaces the 2'-hydroxyl group with fluorine, for example, C 1-6 Alkylene or C 1-6 They may also include "locked" nucleic acids (LNAs) that may be linked to the 4' carbon of the same ribose sugar via a heteroalkylene bridge, exemplary bridges being methylene, propylene, ether, or amino bridges, O-amino (where amino can be, for example, NH, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino), and aminoalkoxy, O(CH). n-amino (wherein amino can be, for example, NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the 2' hydroxyl group modification can include an "unlocked" nucleic acid (UNA) in which the ribose ring lacks a C2'-C3' bond. In some embodiments, the 2' hydroxyl group modification can include a methoxyethyl group (MOE), (OCH2CH2OCH3, e.g., a PEG derivative).

[0122] A "deoxy" 2' modification can be any of the following: hydrogen (i.e., a deoxyribose sugar (e.g., partially in the overhanging portion of a dsRNA)), halo (e.g., bromo, chloro, fluoro, or iodo), amino (wherein amino can be, for example, -NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid), NH(CH2CH2NH) n These can include CH2CH2-amino (wherein amino can be, for example, as described herein), -NHC(O)R (wherein R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), cyano, mercapto, alkyl-thio-alkyl, thioalkoxy, as well as alkyl, cycloalkyl, aryl, alkenyl, and alkynyl, which can be optionally substituted, for example, with amino, as described herein.

[0123] Sugar modifications can include sugar groups that contain one or more carbons and have the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, modified nucleic acids can include nucleotides that contain, for example, arabinose as the sugar. Modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. Modified nucleic acids can also include one or more sugars that are in the L-form (e.g., L-nucleosides).

[0124] The modified nucleosides and modified nucleotides described herein that can be incorporated into modified nucleic acids can include modified bases, also referred to as nucleobases. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or completely replaced to provide modified residues that can be incorporated into modified nucleic acids. The nucleobases of the nucleotides can be independently selected from purines, pyrimidines, purine analogs, or pyrimidine analogs. In some embodiments, the nucleobases can include, for example, naturally occurring bases and synthetic derivatives of bases.

[0125] Pharmaceutical Compositions and Administration Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. AR Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharma- ceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharma- ceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to one skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and the concentration of the composition being administered.

[0126] Pharmaceutical carriers are known to those skilled in the art. These are most typically standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffer solutions at physiological pH. These compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0127] Pharmaceutical compositions may include, in addition to the molecule of choice, carriers, thickeners, diluents, buffers, preservatives, surface active agents, etc. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetic agents, etc.

[0128] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases and the like.

[0129] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0130] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets.Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable.

[0131] Some of the present compositions may potentially be administered as pharma- ceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-alkyl-, and arylamines, and substituted ethanolamines.

[0132] The compositions disclosed herein, including pharmaceutical compositions, can be administered in a number of ways, depending on whether local or systemic treatment is desired and the area to be treated. For example, the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. The compositions can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, intraperitoneal injection, transdermally, extracorporeally, ophthalmically, intravaginally, rectally, intranasally, topically, etc., including topical intranasal administration or administration by inhalation.

[0133] method Disclosed herein are methods for delivering diagnostic or therapeutic cargo to target cells using the disclosed EVs, and therefore for treating any disease or condition associated with the target cells.

[0134] disease In some embodiments, the disclosed EVs can be used to treat neurofibromatosis type 1 (NF1) by delivering NF1. NF1-loaded EVs selectively target diseased Schwann cells (SCs) in peripheral nerves via functionalization with SC-targeting ligands or by reprogramming fibroblasts into SCs to produce EVs with tropism for SCs and efficiently deliver therapeutic cargo against NF1.

[0135] In some embodiments, the disclosed EVs can be used to treat acute respiratory distress syndrome (ARDS) by delivering anti-inflammatory cargos such as IL-4, IL-10, FGF7, mIR-146a, etc. Engineered EVs carrying anti-inflammatory cargos preferentially target specific cellular components in the alveolar microenvironment of injured lungs, suppressing inflammation and promoting recovery in patients with ARDS.

[0136] In some embodiments, the disclosed EVs can be used to treat type II diabetes by delivering transcription factors such as Pdx1, Ngn3, or MafA. Engineered EVs as targeted delivery vehicles of transcription factors to induce cellular reprogramming of pancreatic ductal cells towards an insulin-producing phenotype.

[0137] In some embodiments, the disclosed EVs can be used to treat neurological conditions (neurodegenerative diseases, brain, and nerve damage) by delivering transcription factors such as Acsl1, Brn2, Myt1l, etc. EVs carrying reprogramming transcription factors that regulate electrophysiological activity in neural cells and drive pro-neurogenic reprogramming in non-neuronal cells and potential deployment of functionalized EV-based therapies for the brain. In some embodiments, the disclosed EVs can be used to treat neurological conditions (neurodegenerative diseases, brain, and nerve damage) by delivering dopamine or dopamine-enhancing precursors (e.g., dopamine genes such as DRD1, DRD2, DRD3, DRD4, DRD5, and transporter DAT1).

[0138] In some embodiments, the disclosed EVs can be used to treat cancer by delivering anti-metastatic and anti-tumor genes, such as Timp3 and Rarres2. Non-viral transfection of myeloid-derived suppressor cells (MDSCs) can release engineered EVs to regulate the translocation and overexpression of anti-metastatic and anti-tumor genes in tumor cells / tissues in a targeted manner.

[0139] In some embodiments, the disclosed EVs are used to promote wound healing by delivering pro-angiogenic transcription factors such as Etv2, Fli1, and Foxc2. EVs loaded with pro-angiogenic transcription factors may induce direct reprogramming of human fibroblasts into endothelial cells, promote healing after injury, and develop vascularized skin grafts for regenerative medicine applications.

[0140] In some embodiments, the disclosed EVs can be used to treat low back pain by delivering transcription factors such as FOXF1 and / or Brachyury. Engineered EVs can deliver transcription factors that can reprogram degenerated cells in the intervertebral disc (IVD), such as human nucleus pulposus (NP) cells, to a healthy anabolic phenotype to increase proteoglycans and reduce factors associated with inflammation, catabolism, and pain that are important for maintaining healthy IVD structure and function.

[0141] In some embodiments, the disclosed EVs can be used to treat calcific aortic stenosis by delivering transcription factors such as CEBPα and PU.1. EV-based therapeutics selectively deliver reprogramming transcription factors to diseased valves, inducing cellular reprogramming of endothelial cells towards a pro-healing (M2) macrophage-like phenotype, promoting resorption of calcified tissue and reducing inflammation.

[0142] In some embodiments, the disclosed EVs can be used to treat Alzheimer's disease (AD) by delivering amyloid beta antagonists such as shRNA, miR, siRNA, etc. Skin-derived EVs have been explored using a model of AD (3xTgad) in disease onset and progression. These skin-derived EVs have been found to be preferentially taken up by neurons and can introduce mRNA into these cells. Increased amyloid beta as well as increased gliosis in the hippocampus have been found in AD models, which may be mediated by EVs. Skin-derived EVs have also been found to target brain tissue / cells for delivery of cDNA, mRNA, and proteins, which may be of therapeutic nature. AD progression in the hippocampus has also been found to be partially mediated by EVs, and thus EVs may be exploited to deliver therapeutic cargo to AD-affected cells.

[0143] In some embodiments, the disclosed EVs can be used to treat stroke by delivering pro-angiogenic transcription factors such as Etv2, Fli1, and Foxc2. Transfection of fibroblasts with transcription factors tends to produce exosomes carrying pro-angiogenic / angiogenic transcripts. This suggests that increased intracranial perfusion may be regulated, in part, by exosome-driven autocrine and / or paracrine vasculogenesis and angiogenesis.

[0144] In some embodiments, the disclosed EVs can be used to treat peripheral nerve injury by delivering pro-angiogenic transcription factors such as Etv2, Fli1, and Foxc2. Using reprogramming factor genes via tissue nanotransfection (TNT), delivery of angiogenic cell therapy to the contused nerve resulting in increased vascularity, reduced macrophage infiltration, and improved recovery of electrophysiological parameters.

[0145] Production method Administration The disclosed EVs can be administered to a subject by any suitable means. Administration to a human or animal subject can be selected from parenteral, intramuscular, intracerebral, intravascular, subcutaneous, or transdermal administration. Typically, the delivery method is by injection. Preferably, the injection is intramuscular or intravascular (e.g., intravenous). A physician will be able to determine the administration route required for each particular patient.

[0146] The EVs are preferably delivered as a composition. The composition may be formulated for parenteral, intramuscular, intracerebral, intravascular (including intravenous), subcutaneous, or transdermal administration. Compositions for parenteral administration may include sterile aqueous solutions that may also contain buffers, diluents, and other suitable additives. The EVs may be formulated in pharmaceutical compositions that may include, in addition to the EVs, pharma- ceutically acceptable carriers, thickeners, diluents, buffers, preservatives, and other pharma-ceutically acceptable carriers or excipients, and the like.

[0147] Parenteral administration is generally characterized by injection, such as subcutaneous, intramuscular, or intravenous. Formulations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent immediately prior to use, including subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products, ready to be combined with a vehicle immediately prior to use, and sterile emulsions. Solutions can be either aqueous or non-aqueous.

[0148] For intravenous administration, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof. Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharma- ceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations, including phenol or cresol, mercuric, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride, must be added to parenteral formulations packaged in multi-dose containers. Isotonicity agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone.

[0149] Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents for metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment. The concentration of the pharmacologic active compound is adjusted to provide an effective amount to produce the desired pharmacological effect upon injection. The exact dose depends on the age, weight, and condition of the patient or animal, as is known in the art.

[0150] Unit dose parenteral preparations can be packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration must be sterile, as is known and practiced in the art.

[0151] A therapeutically effective amount of the composition is administered. The dose can be determined according to various parameters, in particular the severity of the condition, age, and weight of the patient being treated, the route of administration, and the required regimen. A physician will be able to determine the route of administration and dosage required for any particular patient. The optimal dosage can vary depending on the relative potency of the individual constructs, and can generally be estimated based on the EC50 found to be effective in in vitro and in vivo animal models. Generally, dosages are 0.01 mg to 100 mg / kg of body weight. A typical daily dose is about 0.1 to 50 mg per kg of body weight, preferably about 0.1 mg / kg to 10 mg / kg, depending on the potency of the particular construct, the age, weight, and condition of the subject being treated, the severity of the disease, and the frequency and route of administration. Different dosages of the constructs can be administered depending on whether administration is by intramuscular or systemic (intravenous or subcutaneous) injection.

[0152] Preferably, the dose for a single intramuscular injection is in the range of about 5-20 μg. Preferably, the dose for a single or multiple systemic injection is in the range of 10-100 mg / kg of body weight.

[0153] Due to clearance of the construct (and degradation of any targeted molecules), the patient may have to be repeatedly treated, for example, daily, weekly, monthly, or yearly, or more. One of skill in the art can easily estimate the repetition rate of dosing based on the measured residence time and concentration of the construct in bodily fluids or tissues. After successful treatment, it may be desirable to have the patient undergo maintenance therapy, with the construct administered at a maintenance dose ranging from 0.01 mg to 100 mg / kg of body weight, once or more per day to once every 20 years.

[0154] Although several embodiments of the invention have been described, it should be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. EXAMPLES

[0155] Example 1: Designer EVs for targeted delivery Nanotransfection is used to engineer fibroblasts to produce EVs with tropism for SCs and to systemically deliver therapeutic payloads to SCs (Figure 1).

[0156] Neurofibromatosis type 1 (NF1) is driven by mutations / deletions of the NF1 gene in SCs. Such mutations / deletions lead to neurofibromas, as NF1 has many important functions (e.g., regulating Ras-GTPase). Thus, gene therapy to restore NF1 function has been investigated. Retrovirus and adeno-associated virus vectors have been used to transfect the GAP-associated domain of NF1 to restore function. However, these studies often have low efficiency. Moreover, viral vectors cannot carry full-length NF1 due to capsid size limitations.

[0157] Viral vectors have become the gold standard for gene therapy. However, although promising, viruses have several limitations beyond the capsid size constraint. Viral vector-induced immunity may, for example, hinder remedying or raise biosafety concerns. Thus, EVs have emerged as promising therapeutic carriers for gene therapy. Compared to most carrier systems (viral or synthetic), EVs can package large cargoes and exhibit improved biocompatibility, reduced immunogenicity, enhanced stability, and an innate ability to cross biological barriers. Thus, a significant amount of research has been devoted to engineering therapeutic EVs for various diseases. However, there is currently a lack of research on designer EVs for delivering therapeutic payloads for NF1.

[0158] An EV-based approach to deploying gene therapy for NF1 is produced, in some embodiments, by reprogramming fibroblasts into SCs to produce EVs with tropism for SCs.

[0159] Full-length NF1 is used as an exemplary therapeutic cargo, however, the proposed EV technology can be used to deliver different types of therapeutic cargo (e.g., CRISPR / Cas9).

[0160] To assess whether SC-derived EVs exhibit tropism for SCs, EVs were isolated from nanotransfected SCs (ATCC). Myt1l plasmid (~10.4 kb) was used as a large model cargo. In vitro coculture of SCs and fibroblasts exposed to EVs derived from nanotransfected SCs and fibroblasts shows that SC-derived EVs were preferentially captured by SCs and showed higher affinity for fibroblast-derived EVs compared to fibroblasts (Figure 2A-B). qRTPCR analysis shows that EVs were able to package MYT1L and mediated gene transfer to SCs (Figure 2C-D). To test tropism in vivo, a crush injury in the sciatic nerve of mice was performed to induce local recruitment of SCs. SC-derived EVs were labeled and delivered via the tail vein, and then the nerve was harvested 6 h later. Imaging of the nerve revealed that SC-derived EVs clearly accumulated at the site of crush injury (Figure 3A-B). Taken together, these data demonstrate the ability to obtain EVs with higher tropism for SCs. Because SCs are not an abundant cell source, methods are being developed to confer SC tropism to EVs derived from more readily available cell sources (e.g., dermal fibroblasts).

[0161] EVs were isolated from myoblasts nanotransfected with a plasmid encoding Pmax-GFP to assess whether myoblast-derived EVs exhibit tropism for myoblasts. In vitro co-culture of myoblasts and fibroblasts exposed to myoblast- and fibroblast-derived EVs showed that myoblast-derived EVs were preferentially captured by myoblasts, similar to fibroblasts, further revealing an affinity for fibroblast-derived EVs (Figure 4A-B).

[0162] In vivo studies were performed to test the tropism of myoblast-derived EVs. In these experiments, myoblast-derived EVs were labeled and delivered via the tail vein, and 6 h later, different muscle tissues (e.g., triceps, quadriceps, abdominal, and gastrocnemius) were harvested. Imaging of muscle tissues showed accumulation of myoblast-derived EVs, where the cargo of labeled EVs (GFP) colocalized with multiple nuclei of muscle cells counterstained with DAPI (blue) (Figure 4C), compared to fibroblast-derived EVs, where the signal was absent (Figure 4D). Taken together, these data demonstrate the ability to obtain EVs with higher tropism for muscle tissue when the EVs are derived from myoblasts.

[0163] Studies have shown that nanotransfection of fibroblasts with ASCL1, BRN2, and MYT1L (ABM) leads to their reprogramming into neurons. We assessed whether EVs released during the reprogramming process exhibit tropism towards neurons (Figure 5). Uptake studies show that EVs obtained from ABM-nanotransfected fibroblasts were preferentially internalized by neurons compared to control EVs. These results highlight the ability to use reprogramming to generate EVs from fibroblasts with enhanced tropism towards other cells.

[0164] Example 2: Development of designer EVs for SC targeted delivery Develop designer EV formulations with enhanced tropism for SCs. This is done by nanotransfecting mouse dermal fibroblasts with plasmids to promote their conversion to SCs (SOX10, EGR2). The working hypothesis is that EVs from fibroblasts "primed" to convert to SCs will exhibit tropism for SCs. SCs are not an abundant source of cells to readily generate EVs. Thus, a method is needed to confer SC tropism to EVs derived from more readily abundant cells.

[0165] EVs derived from fibroblasts that transform into SCs: EVs exhibit tropism towards the cells / tissues from which they originate. Data show that SC-derived EVs are preferentially internalized by SCs and can be exploited to confer cell-specific tropism towards EVs in a reprogramming approach. Here, fibroblasts are converted into SCs and EVs are harvested at different stages of the reprogramming process. Briefly, fibroblasts are nanotransfected with expression plasmids for SOX10 and EGR219, and EVs are harvested from the supernatant on days 1-21. EVs isolated from fibroblasts nanotransfected with a sham plasmid serve as controls. SC-directed reprogramming of fibroblasts is assessed by qRT-PCR and immunostaining for SC-specific markers (S100, O4, and MPZ) on days 7-21. EV concentration and size are quantified using a NanoSight.

[0166] Selective uptake: Selective SC uptake of designer EV formulations is assessed in co-cultures of SC and fibroblasts. SC (ATCC) and fibroblasts are mixed in a 1:1 ratio. Cells and EVs are labeled with fluorophores of different wavelengths (approximately 490, 560, and 650 nm). Co-cultures are incubated for approximately 10 min. 9 ~10 10 EVs / ml were exposed to SCs and selective uptake by fibroblasts was assessed by confocal microscopy. EVs derived from SCs are used as positive controls. Results show that EVs collected 24 h after transfection of fibroblasts with reprogramming factors SOX10 alone or SOX10+EGR2 are preferentially internalized by SCs compared to fibroblasts 6 h after EV treatment *p<0.05 (Figure 6A-B).

[0167] Biodistribution: To identify EV preparations with enhanced tropism for SCs in CNF / pNF, we use a mouse model of NF1 in which the Nf1 allele is inactivated in SOX10+ cells, leading to the formation of cNF and pNF. fl / flMate Nf1 mice (stock number: 017640, JAX) with tamoxifen-inducible SOX10-CreERT2 mice (stock number: 027651, JAX). fl / - :SOX10-CreERT2 + / 0 Mice were transfected with Nf1 fl / fl Approximately 25% of the offspring have a homozygous genotype for the Nf1flox allele and a hemizygous genotype for the SOX10-CreERT2 allele (Nf1 fl / fl :SOX10-CreERT2 + / 0 ), used as experimental lines. Progeny homozygous for the Nf1flox allele, null for the SOX10-CreERT2 allele (Nf1 fl / fl :SOX10-CreERT2 0 / 0 ) are used as controls. Mice are treated with tamoxifen at approximately 1 month of age. EV preparations are injected via the tail vein approximately 6 months after tamoxifen induction, when cNF / pNF lesions / symptoms (dirty fur, hunched back, lameness, quadriplegia) are identified. EVs derived from the SC are used as positive controls. EVs are fluorescently tagged with MemGlow. Mice are injected daily for approximately 10 12 A bolus of EVs / gram body weight is injected and mice injected 1-5 times are compared. Mice are euthanized 24 h after the last injection and cNF lesions, spinal cord / sciatic nerve (to examine pNF), liver, lungs, spleen, and kidneys are harvested and imaged by IVIS to evaluate EV distribution. Tissues are then processed for histological examination. Neurofibromas are immunostained for S100β, GAP43, SOX10, Iba1, and mast cells. The presence of EVs in tissue sections is quantified by confocal imaging.

[0168] Delivery of NF1 to cNF / pNF: Once the optimal time point for EV recovery from fibroblasts primed to transform into SCs is identified, the NF1 plasmid is nanotransfected into fibroblasts being transformed (or have been transformed), and 6-72 hours later, EVs are isolated from the supernatant. Positive and negative control EVs are obtained. NF1 loading is assessed by qRT-PCR. EVs are harvested daily, approximately 10 12 Tamoxifen-treated Nf1fl / fl:SOX10-CreRT2 cells via the tail vein with a bolus of EVs / gram body weight + / 0 NF1 was delivered to mice and compared between mice injected 1-5 times. Biodistribution was assessed. NF1 delivery cNF / pNF and function was assessed by qRT-PCR, in the case of NF1, immunostaining for neurofibromin, p-ERK, and quantification of SOX10+ and mast cells. Additional assessments included quantification of neurofibroma number and volume, as well as TUNEL and BrdU staining. To verify whether EVs are carrying NF1, laser capture microdissection (LCM) was used to isolate fluorescently tagged portions of tissue sections (indicating accumulation of tagged EVs) and PCR / qRT-PCR was used to quantify NF1 plasmid / mRNA at their location.

[0169] Equivalents and Incorporation by Reference All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent was specifically and individually indicated and incorporated by reference in its entirety for all purposes. This incorporation by reference statement is intended by the applicant to relate to all individual publications, database entries (e.g., Genbank sequence or GeneID entry), patent application, or patent, each of which is clearly identified in accordance with 37 CFR §1.57(b)(2), even if such citation is not immediately adjacent to the specific statement incorporated by reference. The inclusion within this specification of a specific statement incorporated by reference (if any) does not in any way weaken this general statement incorporated by reference. The citation of a reference herein is not intended as an admission that the reference is relevant prior art, nor does it constitute an admission as to the contents or date of these publications or documents.

[0170] While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention.

[0171] 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 a disclosed invention belongs.

Claims

1. 1. A method for producing engineered extracellular vesicles (EVs) with desired cell tropism for a target cell, comprising: (a) intracellularly delivering to a population of somatic cells at least one polynucleotide comprising a nucleic acid sequence encoding at least one reprogramming transcription factor, the polynucleotide being configured to reprogram the somatic cells toward a target cell lineage and produce reprogrammed cells; (b) recovering the EVs from the reprogrammed cells.

2. The method of claim 1 , wherein the somatic cells are fibroblasts, keratinocytes, adipocytes, macrophages, or myoblasts. (i) the target cell lineage is a glutamatergic neuron, and the reprogramming transcription factor is selected from the group consisting of Ascl1, Brn2, Myt1l, and NeuroD1; (ii) the target cell lineage is a dopaminergic neuron and the reprogramming transcription factor is selected from the group consisting of Ascl1, Lmxla, and Nurr1; (iii) the target cell lineage is a cardiomyocyte and the reprogramming transcription factor is selected from the group consisting of GATA4, HAND2, Myocd, Tbx5, mIR-1, and mIR-133; or (iv) The method of claim 2, wherein the target cell lineage is a motor neuron and the reprogramming transcription factor is selected from the group consisting of Ascl1, Brn2, Myt1l, Lhx3, Hb9, IsI1, and Ngn2.

4. The method of claim 1 , wherein the somatic cells are myoblasts. (i) the target cell lineage is a hepatocyte, and the reprogramming transcription factor is selected from the group consisting of Hnf4α, Hnf1α, Hnf6, CEBPA, ATf5, Prox1, p-53-siRNA, and c-Myc; (ii) the target cell lineage is a melanocyte, and the reprogramming transcription factor is selected from the group consisting of MITF, SOX10, and PAX3; (iii) the target cell lineage is an endothelial cell and the reprogramming transcription factor is selected from the group consisting of Etv2, Foxc2, and fli1; (iv) the target cell lineage is hematopoietic progenitor and mature cells and the reprogramming transcription factor is Oct4; (v) the target cell lineage is a monocyte-like progenitor cell and the reprogramming transcription factor is selected from the group consisting of Sox2 and mIR-125b; (vi) the target cell lineage is a Schwann cell and the reprogramming transcription factor is selected from the group consisting of Sox10 and EGR2; (vii) the target cell lineage is a cardiomyocyte progenitor cell and the reprogramming transcription factor is selected from the group consisting of ETS2 and MESP1; or (viii) The method of claim 2, wherein the target cell lineage is an adipocyte and the reprogramming transcription factor is selected from the group consisting of Prdm16 and C / EBPβ.

6. The method of claim 1 , wherein the somatic cells are endothelial cells.

7. 7. The method of claim 6, wherein the target cell lineage is a hematopoietic progenitor cell and the reprogramming transcription factor is selected from the group consisting of Fosb, Gfi1, Runx1, and Spi1.

8. The method of claim 1 , wherein the somatic cells are pancreatic exocrine cells.

9. 9. The method of claim 8, wherein the target cell lineage is a beta cell and the reprogramming transcription factor is selected from the group consisting of activated MAPK and STAT3.

10. 10. The method of claim 1, wherein the EVs are harvested from the reprogrammed cells before the cells are fully differentiated.

11. 2. The method of claim 1, wherein the EVs are recovered from the reprogrammed cells 12 to 48 hours after the reprogramming transcription factors are delivered to the somatic cells.

12. The method of claim 1, wherein the reprogrammed cells express lineage-specific markers but lack one or more markers of full lineage differentiation.

13. The method of claim 1, wherein the EV is decorated with at least one targeting ligand.

14. 14. The method of claim 13, wherein the targeting ligand is a glutamate targeting ligand.

15. 10. The method of claim 1, further comprising manipulating the reprogrammed cells to produce a therapeutic cargo that is encapsulated within the EV.

16. 10. The method of claim 1, further comprising loading the EV with a therapeutic cargo.

17. Engineered extracellular vesicles (EVs) obtained by the method described in any one of claims 1 to 16.

18. A composition for delivering a therapeutic cargo to a target cell, comprising the engineered EV of claim 17.

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