Designer extracellular vesicles for targeted delivery to Schwann cells

Abstract

Disclosed herein are designer extracellular vesicles (EVs) that target Schwann cells (SCs). For example, in some embodiments, the EVs are decorated with HRG1 / 2, NRG1, or a combination thereof. In some embodiments, these EVs can be used to deliver diagnostic and / or therapeutic cargo to Schwann cells of a subject in need thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 271,912, filed October 26, 2021, which is incorporated by reference herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing submitted in ST.26 format entitled "321501_2590_Sequence_Listing", created on October 25, 2022. The contents of the Sequence Listing are incorporated herein by reference in their entirety. [Background technology]

[0003] Neurofibromatosis type I (NF1) is an autosomal dominant genetic condition caused by mutations in the neurofibromin 1 (NF1) gene in Schwann cells (SCs). NF1 is characterized by peripheral nervous system tumors (PNSTs), including plexiform neurofibromas (pNFs), which cause neuronal dysfunction, deformity, painful damage to adjacent structures, and can undergo malignant transformation. There are currently no effective therapies to prevent or treat pNFs. Furthermore, there is a need for cell-specific delivery to SCs for diagnostic and therapeutic purposes. Summary of the Invention [Means for solving the problem]

[0004] Disclosed herein are designer extracellular vesicles (EVs) that target Schwann cells (SCs). For example, in some embodiments, the EVs are decorated with NRG1, NRG2, or a combination thereof. Other embodiments can incorporate Schwann cell molecules such as purinergic receptors (i.e., P2X4R) or RTKs (e.g., ErbB3). In some embodiments, these EVs can be used to deliver diagnostic and / or therapeutic cargo to Schwann cells of a subject in need thereof.

[0005] Thus, also disclosed herein are methods for treating any disease or condition associated with Schwann cells. Schwann cell-related diseases and potential therapeutic molecules for treatment include neurofibromatosis type 1 (NF1) and neurofibromatosis 1 gene, neurofibromatosis type 2 (NF2) and neurofibromatosis 2 gene, Charcot-Marie-Tooth disease (CMT) and targeting inhibiting PMP22 expression, Guillain-Barre syndrome (GBS, acute inflammatory demyelinating polyradiculopathy type), and schwannoma by targeting SMARCB1 or LZTR1 tumor suppressor genes. Chronic inflammatory demyelinating polyneuropathy (CIDP), leprosy, and Zika virus are all neuropathies involving Schwann cells. Thus, the disclosed EVs can be used to treat one or more of these neuropathies.

[0006] In some embodiments, these EVs carry functional neurofibromin 1 and can therefore be used to treat NF1 in a subject. EVs can also carry siRNA or RNAi molecules targeting Ras pathway genes to inhibit dysregulated cell proliferation. Small molecule inhibitors such as dabrafenib, selumetinib, or nilotinib can also be used in this case. Thus, also disclosed herein is a method of treating NF1 in a subject, comprising engineering cells of a subject to produce therapeutic EVs that target and deliver functional neurofibromin 1 to Schwann cells (SCs). Also disclosed is a method of harvesting ex vivo produced EVs and loading them with neurofibromin 1 for use in treating NF1 in a subject.

[0007] 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]

[0008] [Figure 1]We show that designer EVs with enhanced SC tropism can be generated ex vivo and deployed in vivo to treat neurofibromas. TNTs transform the epidermis into a designer EV bioreactor to target SCs and be used to treat neurofibromas. [Figure 2A] The tunneling nanotube (TNT) platform (1: plasmid reservoir, 2: negative lead, and 3: positive lead) is shown. [Figure 2B] The tunneling nanotube (TNT) platform (1: plasmid reservoir, 2: negative lead, and 3: positive lead) is shown. [Figure 2C] This is an electron microscope photograph. [Figure 2D] We demonstrate pulsed field perforation and electrophoretically driving cargo into the skin. [Figure 2E] We demonstrate pulsed field perforation and electrophoretically driving cargo into the skin. [Figure 2F] We demonstrate pulsed field perforation and electrophoretically driving cargo into the skin. [Figure 2G] Simulation showing concentrated perforation (solid line) versus widespread perforation (dashed line) for TNT versus BEP. [Figure 2H] Simulation showing concentrated perforation (solid line) versus widespread perforation (dashed line) for TNT versus BEP. [Figure 2I] Gene expression vs. BEP is shown. *p<0.05. [Figure 3A] 1 shows the generation of EVs decorated with SC targeting ligands NRG1 and NRG2. [Figure 3B] 1 shows the generation of EVs decorated with SC targeting ligands NRG1 and NRG2. [Figure 4] The percentage of uptake of EVs decorated with the SC-targeting ligands NRG1 and NRG2 by PMEFs and SCs is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Before describing the present disclosure in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, 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.

[0010] 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.

[0011] 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.

[0012] 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 herein 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.

[0013] 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.

[0014] The embodiments of the present disclosure employ, unless otherwise indicated, chemical, biological and other techniques within the skill of the art.

[0015] 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.

[0016] 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.

[0017] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0018] 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.

[0019] 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.

[0020] The term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0021] 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, the carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0022] 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.

[0023] 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.

[0024] 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, PEGylation, 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)).

[0025] 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.

[0026] 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.

[0027] 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).

[0028] 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.

[0029] 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.

[0030] 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.

[0031] The term "operably linked" refers to a functional relationship between a nucleic acid and 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 to a 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] "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.

[0036] 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.

[0037] "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.

[0038] 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.

[0039] Schwann cell-targeted extracellular vehicles (EVs) Disclosed herein are Schwann cell (SC) targeted EVs capable of carrying therapeutic and / or diagnostic cargo. In some embodiments, the method comprises engineering cells of a subject to produce therapeutic EVs. In some embodiments, the method comprises harvesting ex vivo produced EVs and loading them with therapeutic cargo.

[0040] Engineering patient cells to produce therapeutic EVs Methods of reprogramming cells of a subject into EV-producing cells are disclosed, the method comprising intracellular delivery of a polynucleotide comprising a nucleic acid sequence encoding a SC targeting ligand and optionally a therapeutic cargo to the cells. In some embodiments, the cells can be any cells in the subject 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).

[0041] For example, disclosed herein are compositions and methods for reprogramming skin cells into EV-producing cells, both in vitro and in vivo, that can be used to treat NF1.

[0042] In some embodiments, the method comprises transfecting a cell of the subject with an expression vector encoding NRG1, NRG2, or any combination thereof, hi some embodiments, the method comprises transfecting a cell of the subject with an expression vector encoding neurofibromin.

[0043] The mRNA sequence of mouse NRG1 is provided in NCBI accession number NM_178591.3, which is incorporated by reference for this sequence. The mRNA sequence of mouse NRG2 is provided in NCBI accession number NM_001167891.3, which is incorporated by reference for this sequence.

[0044]

[0045] In some embodiments, human NRG1 mRNA encodes the following amino acid sequence: (SEQ ID NO:2, AAI50610.1).

[0046] In some embodiments, the human NRG2 cDNA has the following nucleic acid sequence:

[0047] In some embodiments, the human NRG2 mRNA encodes the following amino acid sequence: (Sequence number 4, AAI66615.1).

[0048] In some embodiments, EVs incorporate Schwann cell molecules, such as purinergic receptors (i.e., P2X4R) or RTKs (e.g., ErbB3).

[0049]

[0050] In some embodiments, the human P2RX4 mRNA encodes the following amino acid sequence: MAGCCAALAAFLFEYDTPRIVLIRSRKVGLMNRAVQLLILAYVIGPAFLKAAENFTLLVKNNIWYPKFNFSKRNILPNITTTYLKSCIYDAKTDPFCPIFRLGKIVENAGHSFQDMAVEGGIMGIQVNWDCNLDRAASLCLPRYSFRRLDTRDVEHNVSPGYNFRFAKYYRDLAGNEQRTLIKAYGIRFDIIVFGKAGKFDIIPTMINIGSGLALLGMATVLCDIIVLYCMKKRLYYREKKYKYVEDYEQGLASELDQ (SEQ ID NO: 6).

[0051] CTGTCTCTACAGGGGAAAAAAAAAAAAGAAACTGAGCCTTAAAGAGATGAAATAAATTAAGCAGTAGATCCAGGATGCAAAATCCTCCCAATTCCTGTGCATGTGCTCTTATTGTAAGGTGCCAAGAAAAACTGATTTAAGTTACAGCCCTTGTTTAAGGGGCACTGTTTCTTGTTTTTGCACTGAATCAAGTCTAACCCCAACAGCCACATCCTCCTATACCTAGACATCTCATCTCAGGAAGTGGTGGTGGGGGTAGTCAGAAGGAAAAATAACTGGACATCTTTGTGTAAACCATAATCCACATGTGCCGTAAATGATCTTCACTCCTTATCCGAGGGCAAATTCACAAGGATCCCCAAGATCCACTTTTAGAAGCCATTCTCATCCAGCAGTGAGAAGCTTCCAGGTAGGACAGAAAAAAGATCCAGCTTCAGCTGCACACCTCTGTCCCCTTGGATGGGGAACTAAGGGAAAACGTCTGTTGTATCACTGAAGTTTTTTGTTTTGTTTTTATACGTGTCTGAATAAAAATGCCAAAGTTTTTTTTCA (SEQ ID NO: 7, ERBB3 transcript variant 1).

[0052]

[0053] 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.

[0054] Various 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.

[0055] In some embodiments, EVs containing the disclosed nucleic acid sequences can be administered to cells of a subject, and then the cells in the subject can be induced to become EV-producing cells. Accordingly, a method of reprogramming a cell into an EV-producing cell is also disclosed, the method comprising exposing the cell to extracellular vesicles produced from a cell that contains or expresses the disclosed therapeutic gene.

[0056] 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.

[0057] In some embodiments, the polynucleotide is delivered intracellularly to the cell via a gene gun, a microparticle or nanoparticle suitable for such delivery, electroporation, transfection by three-dimensional nanochannel electroporation, tissue nanotransfection device, liposome suitable for such delivery, or deep local tissue nanoelectroinjection device. In some embodiments, a viral vector can be used. However, in other embodiments, the polynucleotide is not delivered by a virus.

[0058] 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.

[0059] 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.

[0060] To express a 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 disclosed herein, wherein the nucleic acid sequence is 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.

[0061] 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).

[0062] 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.

[0063] 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.

[0064] 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.

[0065] Tissue-specific or developmental stage-specific promoters induce the expression of genes in specific tissues or in specific developmental stages. 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.

[0066] 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.

[0067] 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 virus. 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 Tet-Off due to its faster response.

[0068] In some embodiments, the nucleic acid sequences 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.

[0069] 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.

[0070] Examples of suitable non-viral vectors include, but are not limited to, pIRES-hrGFP-2a, pCMV6, pMAX, pCAG, pAd-IRES-GFP, and pCDNA3.0.

[0071] 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.

[0072] Treatment EV Also disclosed are ex vivo produced EVs loaded with therapeutic cargo for use in the treatment of NF1. 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).

[0073] 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).

[0074] 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.

[0075] 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.

[0076] 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 buffers 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] Some of the compositions may potentially be administered as pharma- ceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric, hydrobromic, perchloric, nitric, thiocyanic, sulfuric, and phosphoric acid, and organic acids such as formic, acetic, propionic, glycolic, lactic, pyruvic, oxalic, malonic, succinic, maleic, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkylamines, and arylamines, and substituted ethanolamines.

[0082] 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.

[0083] Medical Cargo The disclosed extracellular vesicles may be loaded with therapeutic agents, and these extracellular vesicles deliver the agents to SCs. 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 or DNA). In some embodiments, the disclosed extracellular vesicles contain therapeutic RNA or DNA (also referred to herein as "cargo RNA" or "cargo DNA"). In certain embodiments, the cargo is neurofibromin 1 of the NF1 gene.

[0084] For example, in some embodiments, the cell targeting protein also includes an RNA domain (e.g., 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. Similarly, in some embodiments, the cell targeting protein also includes a DNA domain (e.g., the cytoplasmic C-terminus of the fusion protein) that binds to one or more DNA motifs present in the cargo DNA to package the cargo DNA 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."

[0085] The cargo RNA or cargo DNA of the disclosed extracellular vesicles may be of any suitable length. For example, in some embodiments, the cargo RNA or cargo DNA may 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 may 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 may have a nucleotide length within these anticipated nucleotide lengths, for example, a nucleotide length in the range of about 10 nt to 5000 nt or other ranges. The cargo RNA or cargo DNA of the disclosed extracellular vesicles may be relatively long.

[0086] In some embodiments, the therapeutic cargo is a membrane-permeable pharmacological compound that is secreted by cells and then loaded into EVs.

[0087] 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 way 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 drugs being loaded into EVs.

[0088] In some embodiments, the therapeutic cargo is loaded onto the EVs by diffusion through a concentration gradient.

[0089] method Disclosed herein are methods for delivering diagnostic or therapeutic cargo to Schwann cells using the disclosed EVs, and therefore, also disclosed herein are methods for treating any disease or condition associated with Schwann cells.

[0090] For example, schwannoma is a rare type of tumor that forms in the nervous system. Schwannoma tumors are often benign, meaning they are not cancerous. However, in rare cases, they can become cancerous.

[0091] Neurofibromatosis type I (NF1) is an autosomal dominant genetic condition caused by mutations in the neurofibromin 1 (NF1) gene in Schwann cells (SCs). NF1, also known as von Recklinghausen's disease, is characterized by the development of multiple noncancerous (benign) tumors (neurofibromas) of the nerves and skin as well as areas of abnormal skin color (pigmentation). Areas of abnormal skin pigmentation typically include light tan or light brown discolorations (café au lait spots), freckles in unusual locations such as under the arms (axillary region) or in the groin (inguinal region). Such skin pigmentation abnormalities are often evident by the age of 1 year and tend to increase in size and number over time.

[0092] At birth or early infancy, affected individuals may have relatively large, benign tumors consisting of bundles of nerves and other tissues (plexiform neurofibromas). Individuals with NF1 may also develop benign nodules in the colored area of ​​the eye (iris nodules), or tumors of the nerves of the visual pathway (optic gliomas). More rarely, affected individuals may develop certain malignant (cancerous) tumors.

[0093] NF1 may also be characterized by abnormally large head size (megacephaly) and relatively short stature. Additional abnormalities such as episodes of uncontrollable electrical activity in the brain (seizures), learning disabilities, and attention deficits, speech disorders, abnormally increased activity (hyperactivity), and skeletal deformities, including progressive curvature of the spine (scoliosis), inversion of the lower limbs (pseudoarthrosis), and improper development of certain bones, may also be present. Associated symptoms and findings may vary widely in scope and severity from person to person, even within the same family. Most people with NF1 have normal intelligence, but approximately 50% of children with NF1 develop learning disabilities.

[0094] According to a 1987 National Institutes of Health (NIH) Consensus Conference, a clinical diagnosis of NF1 may be made if a patient exhibits at least two of the following: (1) six or more cafe au lait spots measuring at least 5 millimeters [mm] (prepubertal) or 15 mm (postpubertal); (2) freckles in the armpit (axillary) or groin (inguinal) area; (3) abnormal pigmented masses in the colored part of the eye (iris nodule); (4) certain abnormalities in bone development in the head (sphenoid wing dysplasia) or abnormal deformations of the bones (pseudoarthrosis); (5) two or more neurofibromas of any type or one plexiform neurofibroma; and (6) an affected parent, sibling, or child with confirmed NF1.

[0095] NF1 symptoms usually begin in childhood, and depending on the circumstances, a definite diagnosis may be made by age 4. The disorder is progressive throughout life. In some cases, NF1 symptoms are reported to worsen during puberty, pregnancy, or when hormonal changes occur, but this correlation is not yet fully understood. The extent and severity of NF1 symptoms vary widely among affected individuals, and the rate of progression of the disorder is unpredictable. However, the majority of patients (approximately 60%) are reported to have a "mild" form of the condition.

[0096] Often the first sign of NF1 is the appearance of multiple brown spots on the skin (café au lait spots) or freckles in the armpits (axillae) or groin (inguinal), which may occur at birth or early infancy. Iris nodules may also be present early in life and occur in approximately 97% of affected individuals, strongly suggesting an NF1 diagnosis.

[0097] Multiple noncancerous (benign) tumors (neurofibromas) develop in NF1 along the superficial layers (sheaths) of nerves under the skin or in deeper regions of the body. Neurofibromas can form in any organ in the body. Skin (cutaneous) neurofibromas, or less distinct neurofibromas (plexiform neurofibromas), can be disfiguring. Sometimes tumors can develop in the brain, on nerves exiting the brain, and / or in the spinal cord. The total number of neurofibromas in an adult can range from a few to hundreds or thousands, and the number of these tumors tends to increase with age. Pain can result from the affected peripheral nerve or as a result of a localized mass effect on adjacent structures. In 8-15% of affected individuals, neurofibromas can become cancerous (malignant peripheral nerve sheath tumors). This is accompanied by pain, weight loss, night sweats, and requires urgent evaluation and treatment.

[0098] Approximately 15% of people with NF1 develop brain tumors (gliomas), almost always occurring during childhood. These frequently occur in the nerves of the eye (optic gliomas) and can affect vision or lead to blindness. Additionally, a variety of other tumors can develop in patients with NF1, including gastrointestinal stromal tumors (GISTs). Women with NF1 have a 3.5-fold increased risk of developing breast cancer and a 5-fold increased risk of developing breast cancer before age 50.

[0099] Orthopedic problems may develop with NF1, including curvature of the spine (scoliosis), abnormal growth of the skull (sphenoid wing dysplasia), or conditions characterized by loss of bone tissue, fracture, and abnormal healing, and deformation of weight-bearing long bones (pseudoarthrosis). Additionally, disorders of bone density (osteopenia and osteoporosis) are more common in people with NF1 than in the general population. The process by which these conditions develop is not fully understood, but they have been associated with decreased levels of activated vitamin D, increased levels of parathyroid hormone, and increased markers of bone destruction. NF1 patients tend to be below average height for their age, below average muscle strength, and above average head size.

[0100] High blood pressure (hypertension) is seen more frequently in the NF1 population than in the general population. The cause of this is unknown, but it may not be directly related to NF1, but may be due to associated changes in the blood vessels leading to the kidneys (renal artery stenosis). More rarely, patients with NF1 are at risk of developing tumors in the adrenal glands (pheochromocytoma), which can cause severely elevated blood pressure if not treated.

[0101] Sexual development may be delayed or occur early (precocious puberty) in individuals with NF1. (For more information on this disease, select "precocious puberty" as a search term in the Rare Diseases Database.) In addition, more than 50% of people with NF1 experience learning disabilities, such as attention deficit hyperactivity disorder (ADHD). Seizures may occur. Other symptoms include headaches, numbness, and / or weakness.

[0102] In a localized form of NF1 known as segmental neurofibromatosis, the abnormal pigmentation and / or tumors may be limited to one area of ​​the body.

[0103] Neurofibromatosis 2 (NF2) is a rare disorder that is genetically distinct from NF1. NF2 is characterized by benign tumors in both the auditory nerve (vestibular schwannoma) and other areas of the body. Other tumors of the central nervous system, including meningiomas and / or ependymomas, may also develop. Individuals with NF2 typically do not have cafe au lait spots or abnormal skin freckles. Other symptoms of NF2 include balance problems, buzzing or ringing in the ears (tinnitus), progressive hearing loss, and facial weakness. In some embodiments, the disclosed compositions and methods can be used to treat NF2.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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 than once. 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.

[0113] Exemplary embodiments Embodiment 1. A composition comprising extracellular vesicles (EVs) produced from donor cells engineered to express NRG1, NRG2, or a combination thereof.

[0114] Embodiment 2. The composition of embodiment 1, wherein the donor cells are autologous.

[0115] Embodiment 3. The composition of embodiment 1 or 2, wherein the donor cells are skin cells.

[0116] Embodiment 4. A composition according to any one of embodiments 1 to 3, wherein the EV encapsulates a therapeutic cargo.

[0117] Embodiment 5. The composition of embodiment 4, wherein the therapeutic cargo comprises a nucleic acid encoding NF1 or neurofibromin.

[0118] Embodiment 6 The composition of embodiment 1, wherein the EVs selectively target Schwann cells.

[0119] Embodiment 7. A method for selectively delivering a therapeutic cargo to Schwann cells of a subject, comprising administering to the subject an effective amount of embodiment 4.

[0120] Embodiment 8. A method of treating neurofibromatosis type 1 (NF1) in a subject, comprising administering to the subject an effective amount of a composition described in any one of embodiments 1-6.

[0121] Embodiment 9. A method for treating neurofibromatosis type 1 (NF1) in a subject, comprising intracellular delivery of a polynucleotide comprising a nucleic acid sequence encoding NRG1, NRG2, or a combination thereof, and a nucleic acid sequence encoding neurofibromin to skin cells of the subject, wherein the skin cells produce EVs that are decorated with NRG1, NRG2, or a combination thereof and encapsulate neurofibromin as a therapeutic cargo.

[0122] 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

[0123] Example 1 Designer EVs for targeted delivery Nanotransfection is used to engineer fibroblasts to produce EVs with tropism for SCs and deliver therapeutic payloads to SCs systemically (Figure 1). Tunneled nanotubes (TNTs) are also used to engineer skin patches to release (in situ) designer EVs with tropism for SCs and deliver therapeutic payloads to SCs systemically (Figure 1).

[0124] 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). Therefore, 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.

[0125] 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.

[0126] TNTs can be used for non-viral (i.e., no capsid size limit) gene delivery in vivo. TNTs use silicon nanochannels and electric fields to deliver cargo to solid tissue tissues in a fast (~100 ms), efficient, and gentle manner (Figure 2). This is achieved by a combination of nanoscale electroporation and electrophoresis. In silico and in vivo studies have validated the superiority of TNT compared to standard bulk electroporation (BEP). TNTs will be used to drive gene therapy of cutaneous neurofibromas (cNFs) and plexiform neurofibromas (pNFs) in mice.

[0127] In addition to enabling direct gene delivery to solid tissues, TNT can also drive the release of designer EVs from the epidermis, with gene copies and transcripts loaded onto the TNT. Studies suggest that TNT-treated skin can produce EVs that may be used to amplify transfection beyond the skin. Here, TNT is used to "force" SC-targeted designer EVs (in vivo) into the epidermis that target cNFs and pNFs throughout the body via specific receptor-ligand interactions, in addition to systemic delivery of ex vivo-generated designer EVs.

[0128] An EV-based approach to deploying gene therapy for NF1 is produced in some embodiments by dispatching SC-targeted EVs from the skin. For example, in addition to delivering therapeutic genes directly to cNFs, TNTs are used to program the epidermis to release designer EVs with SC tropism throughout the body.

[0129] Full-length NF1 is used as a model cargo, but the proposed EV technology can be used to deliver different types of therapeutic cargo (e.g., CRISPR / Cas9).

[0130] Example 2 Development of designer EVs for SC targeted delivery We develop designer EV formulations with enhanced tropism for SCs. This is done by nanotransfecting mouse dermal fibroblasts (MDFs) with plasmids to overexpress SC-targeting ligands (HRG1 / 2, NRG1) or promote their conversion to SCs (SOX10, EGR2). The working hypothesis is that nanotransfecting MDFs with SC-targeting ligand plasmids will lead to the release of functionalized EVs that will preferentially bind to SCs via ligand-receptor interactions (HRG1 / 2-ErbB2 / 3, NRG1-EGF / ErbB3). Thus, a method is needed to confer SC tropism to EVs derived from more readily abundant cells.

[0131] EVs decorated with targeting ligands: A nanotransfection platform is fabricated and used to deliver plasmids for SC targeting ligands to MDFs as previously described. Briefly, MDFs (ScienCell) are seeded in direct contact with the nanochannels and a pulsed electric field (~250 V, 10 ms pulses, 10 pulses) is used to deliver the plasmids. A sham plasmid with the same backbone is used as a control. Plasmids are transfected individually or mixed equimolarly in the two permutations. EVs are isolated from the supernatant at 6-72 h using the ExoQuick kit. Functionalization of EVs with targeting ligands is assessed by Western blot (WB). EV concentration and size are quantified using a NanoSight.

[0132] Selective uptake: Selective SC uptake of designer EV formulations is assessed in co-cultures of SC and MDF. SC (ATCC) and MDF 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 Upon exposure to EVs / ml, selective uptake by SCs and MDFs is assessed by confocal microscopy. SC-derived EVs are used as a positive control.

[0133] 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. Briefly, homozygous Nf1 fl / fl Mate 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 / flApproximately 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 the experimental strain. 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.

[0134] Delivery of NF1 to cNF / pNF: Once the optimal set of ligands is identified, EVs carrying full-length NF1 as a model cargo are generated. MDFs are co-nanotransfected with NF1 (~13.4 kb, Origene) and optimized ligand plasmids (1:1 ratio). Positive control EVs are prepared by delivering NF1 to SCs. Negative control EVs are prepared by co-delivering sham + ligand plasmids. At 6-72 h, designer EVs are harvested from the supernatant and EV functionalization and loading of NF1 are assessed by WB and qRT-PCR. Selective uptake of NF1-loaded EVs by SCs is assessed. qRT-PCR is used to evaluate NF1 expression in SCs. EVs are transfected daily for approximately 10 min. 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.

[0135] Example 3 Development of a TNT protocol for in situ expansion of SC-targeted EVs An alternative approach will be tested to deploy EV-based therapy against NF1 by using TNT to produce SC-targeted EVs to treat neurofibromas in the epidermis. Using skin as an in situ source of therapeutic EVs may eliminate the need for separation / purification and thus facilitate scale-up and application. The working hypothesis is that TNT-based co-delivery of NF1+optimized formulation of SC-targeting ligands may (1) result in direct delivery of NF1 to SCs in cNFs, and (2) result in the release of epithelial EVs carrying NF1 and "decorated" with SC-targeting ligands. Thus, continuous drainage of such EVs to peripheral lymph nodes or systemic circulation is likely to result in systemic spread beyond the skin and homing to SCs in cNFs and pNFs.

[0136] Fabrication of TNT device: The TNT platform is fabricated using wafer-scale cleanroom procedures from 4 inch Si wafers as shown previously. Briefly, nanochannels (~300 nmφ, ~20 μm deep) are etched into the Si using a combination of projection and contact lithography and deep reactive ion etching. Characterization at each step of the process by electron microscopy ensures the quality of the devices. The processed wafer is mounted in a plastic case to produce 1 cm sized wafers in which the plasmid reservoirs will be formed. 2 The device is diced.

[0137] Transfection: The optimized formulation for plasmid and targeting ligand for NF1 is co-TNT (1:1 ratio). TNT inhibits tamoxifen-induced Nf1 fl / fl :SOX10-CreERT2 + / 0TNT is performed directly on mouse cNF or normal skin. Skin is depilated prior to TNT. Sham plasmid only and Sham + TNT with targeting ligand plasmid serve as controls. Plasmid is delivered by applying a pulsed electric field (250 V, 10 ms pulse, 10 pulses) across a pair of electrodes located between the plasmid reservoir and the skin. As the TNT procedure is only approximately 100 ms in duration, 1–4 spots are TNT per mouse. To evaluate re-dosing effects, TNT is performed weekly for 1–5 weeks. Mice are euthanized and TNT-treated skin, cNF lesions, spinal cord / nerve, liver, spleen, kidneys, and lungs are harvested. To track epidermal EVs, skin is pre-TNT treated with EV tracker plasmid (pCT-CD63-GFP, Systems Bio) 24 h prior to TNT of NF1 and targeting ligand plasmid.

[0138] TNT Results: Successful delivery / expression of NF1 in cNFs or normal skin is assessed by LCM of epidermis and dermis followed by PCR / qRT-PCR and immunostaining to evaluate plasmid delivery and expression (mRNA and protein levels). EVs are isolated from skin biopsies and decoration and loading are characterized by WB and qRT-PCR. Biodistribution of CD63-GFP tagged EVs is evaluated. EV-based delivery / expression of NF1 in non-direct TNT treated cNFs and pNFs is characterized by LCM / qRT-PCR to analyze GFP+ areas where epidermal EVs have accumulated. Functional assessment in cNFs and pNFs is also performed.

[0139] Example 4 Protocol for generating EVs EVs were isolated from the culture medium 24 hours after transfection of donor cells. The medium was centrifuged at 2,000 g for 30 minutes at 4°C to remove dead cells and debris. After centrifugation, the cell-free culture medium was filtered and concentrated using a Vivaspin filter (Sartorius, 76408-886) with a molecular weight cut-off of 300 kDa. EVs were subsequently isolated using one of two methods: 1) the concentrated solution containing EVs was then subjected to size-exclusion chromatography on a qEV automated fraction collector (Izon Science Ltd.) using a qEV Original SEC column, and three EV-enriched fractions were collected and stored for subsequent analysis, or 2) Total Exosome Isolation Reagent (Thermo Fisher Scientific, 44-783-59) was added to the supernatant containing the cell-free culture medium according to the manufacturer's instructions. All EVs were characterized in solution by measuring their concentration and size distribution by nanoparticle tracking analysis (NTA) technique, and prior to any experiments, quantitative qRT-PCR was used to verify the loading of molecular cargo within the EVs.

[0140] 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 the disclosed invention belongs. Publications cited herein and the material for which they are cited are specifically incorporated herein by reference.

[0141] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A composition comprising extracellular vesicles (EVs) produced from donor cells engineered to express NRG1, NRG2, HRG1, HRG2, or a combination thereof.

2. The composition of claim 1 , wherein the donor cells are autologous.

3. The composition of claim 1 , wherein the donor cells are skin cells.

4. The composition described in claim 3, wherein the skin cells are fibroblasts, keratinocytes, or skin stem cells.

5. 10. The composition of claim 1, wherein the EV encapsulates a therapeutic cargo.

6. The composition of claim 5 , wherein the therapeutic cargo comprises neurofibromin 1 or a nucleic acid encoding neurofibromin.

7. The composition of claim 1 , wherein the EVs selectively target Schwann cells.

8. The composition of claim 5 for selectively delivering a therapeutic cargo to Schwann cells of a subject.

9. 7. The composition of claim 6 for treating neurofibromatosis type 1 (NF1) in a subject.

10. 1. A composition for treating neurofibromatosis type 1 (NF1) in a subject, comprising: the composition comprises a polynucleotide comprising a nucleic acid sequence encoding NRG1, NRG2, HRG1, HRG2, or a combination thereof, and a nucleic acid sequence encoding neurofibromin; the composition is delivered intracellularly to skin cells of the subject, thereby reprogramming the skin cells into EV-producing cells; The composition, wherein the EV-producing cells produce EVs decorated with NRG1, NRG2, HRG1, HRG2, or a combination thereof, and encapsulating neurofibromin as a therapeutic cargo.

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