Low immunogenic exosome-based RNA package and delivery system, composition and method of preparation thereof

Abstract

A low immunogenic exosome-based RNA package and delivery system capable of delivering a cargo RNA to a target site. In an embodiment, the low immunogenic exosome- based RNA package and delivery system comprises an exosome producing cell, one or more cargo RNA encoding plasmids and one or more fusion protein encoding plasmids, wherein the cargo RNA comprises a package RNA and a packaging domain and wherein the package fusion protein comprises an exosome associated transmembrane protein fused to a packaging protein. The present invention also provides an exosome composition comprising an exosome encapsulating a cargo RNA and a package fusion protein wherein the cargo RNA and the package fusion protein binds via the packaging domain and the packaging protein to form cargo RNA fusion protein complex. In an embodiment the packaging protein is endogenous to a subject. The present invention also provides a method of preparation of the exosome composition.

Description

[0001] Low Immunogenic Exosome-Based RNA Package and Delivery System, Composition and Method of Preparation Thereof

[0002]

[0003] Technical Field of the Invention

[0004] A low immunogenicity modular platform technology for packaging and delivering RNA in exosomes.

[0005] Background of the Invention

[0006] Small extracellular vesicles (EV) are ~50 to 150 nm sized particles which are constitutively shed by active cells and taken up by neighboring cells. EVs are an excellent delivery vehicle for various genetic therapeutics, as they are relatively inert, non-immunogenic, biodegradable, biocompatible and can transit across the blood brain barrier. While small interfering RNAs and mRNAs have been shown to be packaged into EVs and delivered to target cells, the ability to package and deliver nuclear functional non-coding RNAs via EVs to control epigenetic and transcriptional gene expression in target cells has remained unknown.

[0007] Extracellular vesicles (EV) are emerging as vehicles that can deliver therapeutic RNAs and proteins systemically as well as across the blood brain barrier. There is a need for a system, composition and method for packaging nuclear functional long non-coding RNAs into EVs that is capable of delivering epigenetic regulatory non-coding RNAs to recipient cells and transcriptionally control the RNA targeted genes promoter expression.

[0008] Summary of the Invention

[0009] An exosome-based RNA package and delivery system comprising an exosome producing cell, one or more cargo RNA encoding plasmids and one or more fusion protein encoding plasmids wherein the one or more cargo RNA encoding plasmids each encodes a cargo RNA wherein the cargo RNA comprises a package RNA and a packaging domain; wherein the one or more fusion protein encoding plasmids each encodes a fusion protein wherein the fusion protein comprises an exosome associated transmembrane protein fused to a packaging protein; wherein the exosome producing cell is capable of expressing the cargo RNA based on the one or more cargo RNA encoding plasmids when the exosome producing cell is transfected with the one or more cargo RNA encoding plasmids; wherein the exosome producing cell is capable of expressing the fusion protein based on the fusion protein encoding plasmid when the exosome producing cell is transfected with the one or more fusion protein encoding plasmids; wherein thepackaging protein and the packaging domain are capable of binding to one another to form a fusion protein cargo RNA complex; wherein the exosome producing cell is capable of producing one or more exosomes; wherein the exosome producing cell is capable of packaging the fusion protein cargo RNA complex into the one or more exosomes; and wherein the packaging protein is endogenous to a subject.

[0010] A method of packaging cargo RNA in exosomes using the system of claim 1 comprising the steps of: transfecting the exosome producing cell with the one or more fusion protein encoding plasmids and the one or more cargo RNA encoding plasmids, wherein the one or more fusion protein plasmid each encodes an exosome transmembrane protein fused to U1a packaging protein and wherein the one or more cargo RNA encoding plasmids each encodes a cargo RNA comprising a package RNA and a packaging domain; incubating the exosome producing cell to express the one or more fusion proteins and the cargo RNA; forming fusion protein-cargo RNA complex when U1a of the fusion protein binds to the UR domain of the cargo RNA; and packaging the fusion protein cargo RNA complex into exosomes when the exosome producing cell forms exosomes.

[0011] A RNA delivery exosome composition comprising an exosome, one or more cargo RNAs and one or more fusion proteins wherein each of the one or more fusion proteins comprises an exosome associated transmembrane protein fused to a packaging protein; wherein each of the one or more cargo RNAs comprises a package RNA and a packaging domain; wherein at least one of the one or more packaging domain binds to at least one of the one or more packaging proteins to form one or more fusion protein cargo RNA complexes; wherein the one or more fusion protein cargo RNA complexes is packaged into the exosome; and wherein the one or more packaging proteins is endogenous to a subject.

[0012] Brief Description of the Drawings

[0013] Figure 1 Exosome RNA packaging systems. (A) The EXOtic exosome packaging system is shown (1). The exosome surface expressed CD63 is fused to L7ae. The L7ae can bind to mRNAs containing the CD box domain, resulting in CD63-L7ae binding the mRNA of interest and shuttling it into the exosome. The exosomes bud from the surface of the transfected cell and can then be used to transfect neighboring cells where they deliver the mRNA payloads which are translated and expressed. (B) The U1a / UR RNA packaging system was developed and exosomesfrom dual-transfected, (CD63-fusion and L7ae-luciferase or U1a-luciferase) and the EXOtic booster, 293HEK producer cells were collected and exposed to non-transfected 293HEK cells. Luciferase expression was determined on day 2 post-treatment and contrasted with the producer cells. The results from a single experiment with the experimental mean and standard deviations are shown. (C) Fold change in normalized luciferase counts on day 2 of both L7ae + CD cells and U1a + L2 are shown.

[0014] Figure 2 The U1a / L2 system for packaging RNAs into exosomes. (A) A schematic is shown depicting the gene of interest (GOI) fused to the L2 (U1a stem loop), either alone or in a concatemeric configuration. (B) A schematic is shown depicting those L2 containing GFP transcripts assessed for packaging into exosomes. (C) Characterization of the various concatemeric L2 repeat domains embedded into the 3’ UTR of GFP. The relative expression of packaged GFP mRNA is shown in 293HEK cells treated with the different exosome packaged GFP constructs. The results of two independent experiments are shown with the standard deviations.

[0015] Figure 3 Safety of ULTRA-EVs in vivo. C57BL / 6 mice were injected with either PBS or 100 billion ULTRA-EV loaded with non-targeting asEGFP600-UR. (A) Blood urea nitrogen (BUN), creatinine (CRE), alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bilirubin (TBIL), glucose (GLU), total calcium (CA), total protein 30, albumin (ALB), globulin (GLOB), sodium (NA+), potassium (K+) chloride (CL-) and total carbon dioxide (tCO2) were measured from n = 4 PBS mice and n = 3 ULTRA-EV treated mice to assess signs of toxicity due to ULTRA-EV treatment after 24 hours using VetScan VS2 Chemistry Analyzer. Cytokine markers of inflammation such as interleukin (IL) 4; 5 and 6; interferon gamma (IFN Ɣ); Tumor Necrosis Factor (TNFα); IFNα and IFNβ were also measured (B) after 4 hours and (C) 72 hours of PBS or ULTRA-EV treatment in n = 4 mice to assess acute and delayed inflammatory response with Luminex xMAP multiplexing ELISA. For (A) and (C) the data are represented as mean ± standard deviation (SD) from triplicate treated samples. P- values by were calculated by student’s T tests between PBS and ULTRA-EV treated mice and were found to be non-significant.

[0016] Figure 4 Boosting ULTRA-EV expression. (A) HEK293T cells transfected with shRNA targeted to VPS4B or CHMP4C either alone or in combination enhances cellular production of EVs. EVs were collected and counted using nanoparticle tracking analysis. (B) Model of U1aLinked Transfer of RNA Across EV or ULTRA-EV. The EV protein CD63 is modified by fusing the high affinity RNA binding protein U1a which facilitates the recruitment of specific U1a bound RNAs (UR) into EVs. Production of EVs is increased by using shBooster (shVPS4B + shCHMP4C) and Connexin S368A and henceforth referred as ULTRA-EV. (C) Nanoluciferase activity was measured in HEK293T cells treated with EVs generated from cells expressing CD63-U1a and reporter nano-luciferase (nLuc) mRNA with UR and shBooster and ConnexinS368A or control. Protein concentration of lysate were used for normalization. For (A) and (C) data are represented as mean ± standard deviation (SD) from triplicate treated samples. P-values by were calculated by student’s T tests. ****p ≤ 0.0001.

[0017] Figure 5 Small hairpin RNA repression of VPS4B and CHMP4C in HEK293 cells. Triplicate transfected HEK293T cells were assessed 48 hours post-transfection for the expression of (A) VPS4B or (B) CHMP4C as determined by qRT-PCR relative to GAPDH housekeeping gene. HEK293T cells were transfected with shBooster and CD63-nLuc in varying ratios of 1:1; 1:4; and 1:9; only CD63-nLuc; and pcDNA as control. After 48 hours equal volume of 0.45 µm- filtered supernatant was subjected to (C) Nanosight Tracking Analysis (NTA) to count number of EVs and (D) nano-luciferase assay normalized by EVs count to evaluate nLuc cargo packaging efficiency. For A-D data are represented as mean ± standard deviation (SD) of triplicate treated samples and P-values by were calculated by student’s T tests. ***p ≤ 0.001; **p ≤ 0.01.

[0018] Figure 6 The modular shRNA exosome platform (shREX) technology. A three part transfection system (Table 2) is used to convert any cell into an shRNA exosome nanoparticle producing cell. The stable drug selected (Blastocidin, Puromycin, Hygromycin, and Neomycin) selected cells produce sustained extracellular vesicle shRNA (EV-shRNA) drugs that can be used for treating various human diseases.

[0019] Figure 7 Delivery of RNAi with EVs enriched with Ago2-dependent shRNAs. (A) An Ago2-dependent shRNA targeting GFP (Ago-shGFP) was transfected with a GFP-luciferase reporter and knockdown of >99% was obtained compared to a control (shControl). Error bars were generated by triplicate treatments and represent standard deviations. (B) EVs were harvested from HEK293 cells transfected with Ago-shGFP and the levels of mature RNAi effector was determined in the EVs and producer cells using RT-qPCR, showing an enrichment of 60-450 fold in the EVs. Each value represents a separate EV production (n=5). (C) The scaffold of the Ago2-shGFP was modified (V1-V5) and demonstrated that modification of theflanking sequence can further increase the mature RNAi effector in EVs. Errors were generated by duplicate treatments and represent the standard error of the mean. (D) The Ago-shRNA vector was transfected into HEK293 cells with vectors expected to enrich the catalytic Ago2 component in EVs: Ago2, EV-specific mutant Ago2-S387A, the EV-specific receptor CD63 known to bind endogenous Ago2, and a CD63 fused to Ago2. The vectors containing Ago2 resulted in a significant increase in the mature RNAi effector in EVs (up to ~300-fold) compared to Ago2- shRNA only, as determined by RT-qPCR. (E) EVs were purified from HEK293 cells transfected with the Ago-shGFP and Ago2 overexpression vectors and these EVs were added to the media of HEK293 cells stably expressing GFP (HEK293-GFP). At 48 hrs after the EV addition, the levels of GFP were significantly reduced by 50% in the treated samples and required both the Ago- shGFP and Ago2 to be effective. Error bars were generated by duplicate treatments and represent SEM.

[0020] Figure 8 ULTRA-EVs can deliver TGS-inducing lncRNAs. (A) In the HEK293T derived cell line whereby the PTEN promoter drives expression of luciferase, lncRNA PTENpg exon 1 asRNA α (Pg1asα) was over-expressed from either CMV or U1 promoter and luciferase activity was measured at days 2, 4 and 6 post-transfection. (B) Pg1asα was modified to Pg1asα-SU to express SIRLOIN and UR and sequence at 3’UTR to enable packaging into ULTRA-EV and for enabling nuclear localization of Pg1asα in recipient cells. Luciferase activity was measured from cells overexpressing Pg1asα and Pg1asα-SU. (C) Transwell assay assessment of ULTRA-EV producer cells with either Pg1asα or Pg1asα-SU as cargo were found to produce EVs that were taken up by PTEN-Luc expressing HEK293T cells as determined by luciferase activity measured from recipient cells after 5 days of co-culture. (D) EVs from HEK293T cells and ULTRA-EVs with either Pg1asα or Pg1asα-SU were added to HEK293T cells in the ratio of 10,000 EVs to 1 cell for 4 hours. RT-qPCR for Pg1asα was done for RNA isolated from whole cell or (E) from nuclear fraction of EV treated cell. Beta Actin was used to normalize in RT-qPCR from whole cell RNA fraction and U6 was used for nuclear RNA fraction. (F) Luciferase expression in PTEN-Luc HEK293T cells treated with EVs or ULTRA-EVs containing Pg1asα or Pg1asα-SU as cargo for 5 consecutive days. Luciferase analysis was done on day 7 after the first treatment. (G) Luciferase activity was measured from cells pretreated with EVs or ULTRA-EVs containing Pg1asα or Pg1asα-SU as cargo followed by DNA methyl transferase inhibitor (10µM Zebularine)treatment. For (B-G) data are represented as mean ± standard deviation (SD) from triplicate treated samples and P-values by were calculated by student’s T tests. ****p ≤ 0.0001.

[0021] Figure 9 Assessment of asEGFP600 delivery by EVs. (A) A schematic is shown depicting the antisense lncRNA, 600 bases in length and complimentary in sequence to EGFP (asEGFP600) with the UR domain towards the 3’UTR. The asEGFP600-UR can bind and block expression of nascent EGFP-Luciferase transcripts. (B) A luciferase assay from HEK293T cells transfected with varying ratios of EGFP-Luc (reporter) + pcDNA (control) plasmids to EGFP- Luc (reporter) + asEGFP600-UR (antisense GFP RNA expressing plasmid) to assess functionality of asEGFP-UR. (C) Luciferase assay from 5 days of transwell co-culture of cells producing ULTRA-EV with cargo with either asEGFP600 or asEGFP600-UR, with recipient cells which expressed EGFP-Luc cargo. (D) Enrichment of asEGFP600 lncRNA in EV or ULTRA-EV was calculated by RT-qPCR. (E) ULTRA-EVs were enriched from supernatant of producer cells expressing either asEGFP600 or asEGFP600-UR and added to EGFP-Luc expressing cells in the ratio of 10,000 to 1 every day for 3 days. EV from HEK293T were used as control. Luciferase assay from recipient cells was done after 4 days of EV treatment. For (B) to (E) data are represented as mean ± standard deviation (SD) from triplicate treated samples. P- values were calculated by student’s T tests. ***p ≤ 0.0001.

[0022] Figure 10 Characterization of ULTRA-EV with asEGFP600 lncRNA cargo. (A) Concentration and size distribution of ULTRA-EV packed with asEGFP600 was measured by Nanoparticle tracking analysis (NTA). NTA graph is representative of n = 3 replicates. (B) TEM image shows the morphology and size of ULTRA-EV packed with asEGFP600. Scale bar is 100 nm.

[0023] Figure 11 Illustrates an embodiment of the method of packaging RNA into exosomes of the present invention.

[0024] Figure 12 Illustrates various packaging proteins capable of binding to RNA.

[0025] Figure 13 illustrates the delivery of antisense RNA targeting influenza virus PB1 gene using various embodiments of the RNA delivery exosome of the present invention. Producer cells transfected in triplicate with plasmid combinations were co-cultured in a transwell assay with stable reporter recipient cells (12 well transwell, co-culture). After 72hrs the recipient cells are collected and assessed by qRT-PCR for GFP / beta-actin mRNA expression. All value represents fraction of reporter cell alone (±SEM, n=3). * p value < 0.05.

[0026] Figure 14 Illustrates various embodiments of the fusion protein encoding plasmids of the exosome-based RNA package and delivery system of the present invention for the delivery of GFP mRNA. CD63-L7ae+PTGFRN-U1a and CD81-U1a package and transfer GFP mRNA in EVs. Recipient cells were assessed for GFP mRNA compared to transient transfected producer HEK293 cells. The relative ratio of GFP mRNA in recipients was determined standardized to the producer cells at 48hrs post culture in transwell assays with untreated HEK293 recipient cells. The average of triplicate treated cells is shown with the standard errors of the mean. Significant differences are demarcated by (**) with P<0.01 from a paired T-test relative to no packaging GFP control cells. Dual fusion protein packing GFP mRNA into exosomes. CD63- L7ae+PTGFRN-U1a and CD63-U1a+PTGFRN-U1a package GFP mRNAs into EVs, but efficiencies are within the range of the GFP control background non-specific packaging, e.g. no fusion protein present.

[0027] Figure 15 Illustrates various embodiments of the exosome-based RNA package and delivery system of the present invention comprising two fusion protein encoding plasmids for the delivery of GFP mRNA.

[0028] Figure 16 illustrates the delivery of shRNA targeting influenza virus PB1 or PB2 gene using various embodiments of the RNA delivery exosome of the present invention. Recipient cells were collected at 72hrs post-co-culturing and qRTPCR carried out for GFP / beta-actin mRNA expression. All value represents fraction of untreated reporter cells alone (±SEM, n=3). P values from paired T-tests are shown.

[0029] Detailed Description of the Invention

[0030] As used in this specification and in claims which follow, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “an ingredient” includes mixtures of ingredients, reference to “an active pharmaceutical agent” includes more than one active pharmaceutical agent, and the like.

[0031] As used herein, the term “about” as a modifier to a quantity is intended to mean + or - 20% , + or - 15% , + or - 10% or + or - 5% inclusive of the quantity being modified.

[0032] As used herein, the term “subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.

[0033] As used herein, the term “effective amount” or “a therapeutically effective amount” of a drug or pharmacologically active agent comprises administering an amount necessary to achieve a desired result. The exact amount required will vary from subject to subject, depending on the species, age, general condition of the subject, the severity of the disease, the particular active agent, its mode of administration, the desired outcome, and the like. In certain embodiments of the present invention, a “therapeutically effective amount” of a compound or pharmaceutical composition is that amount effective for inhibiting progression or reversing of any disease disclosed herein in a subject or a biological sample (e.g., in cells). In certain embodiments, disease progression is inhibited by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%. In certain embodiments, the compound inhibits disease progression by at least about 25%, at least about 50%, at least about 75%, or at least about 90%. In certain embodiments of the present invention, a “therapeutically effective amount” refers to an amount of a compound or composition sufficient to effect reversal of disease. In certain embodiments, the disease is reversed by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or any numbers and number ranges falling within these values.

[0034] As used herein, the term "nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof. The terms "polynucleotide," "oligonucleotide," "oligo" or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term "nucleotide" refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of nucleic acids contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acids contemplated herein include any types of RNA (e.g., long-non-coding RNA, antisense RNA, mRNA, siRNA, miRNA, shRNA, guide RNA, dicer substrate RNA, dicer substrate siRNAs (dsiRNAs) (dsiRNA are cleaved by the RNase I class endoribonuclease dicer into 21-23 base duplexes having 2-base 3'-overhangs siRNA), and any type of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term “long non-coding RNA (lncRNA)” refers to a transcript more than about 100nucleotides that are not translated into protein. In an embodiment, lncRNA comprises intergenic non-coding RNA (lincRNA), intronic non-coding RNA, sense lncRNA, or antisense lncRNA. In an embodiment, the term “antisense RNA” refers to a non-coding RNA comprising a nucleotide sequence at least in part reverse-complement to a target RNA. In an embodiment, the term “antisense RNA” and “long non-coding RNA” are used interchangeably to describe a non-coding RNA comprising a nucleotide sequence at least in part reverse-complement to a target RNA. The term "duplex" in the context of nucleic acids refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like. In an embodiment, the nucleotide sequence is provided using symbols ATCG (adenine (A), cytosine (C), guanine (G), and thymine (T)) for a DNA molecule, and provided using symbols AUCG (adenine (A), cytosine (C), guanine (G), and uracil (U)) for a RNA molecule. In an embodiment, the symbols T and U are used interchangeably in a nucleotide sequence to illustrate the DNA and RNA molecule made according to the nucleotide sequence respectively.

[0035] As used herein, the terms "polypeptide," "peptide" and "protein" generally refer to a polymer of amino acid residues. As used herein, the term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of corresponding naturally occurring amino acids or are unnatural amino acids. The term "protein", as generally used herein, refers to a polymer of amino acids linked to each other by peptide bonds to form a polypeptide for which the chain length is sufficient to produce tertiary and / or quaternary structure. In an embodiment, the "polypeptide," "peptide" or "protein" of the present invention is prepared from a plasmid encoding said "polypeptide," "peptide" or "protein". Therefore, the "polypeptide," "peptide" or "protein" of the present invention further comprises a nucleotide sequence encoding said "polypeptide," "peptide" or "protein" that could be converted using a genetic code such as but not limited to the standard genetic code.

[0036] As used herein, “sequence identity” and “% identity,” refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein a portion of the sequence in the comparison window may comprise additions or deletions as compared to the reference sequence for optimal alignment of the two sequences. The number of positions atwhich identical amino acid residues occur in both sequences is determined, yielding the number of matched positions, which is divided by the total number of positions in the window of comparison and the result multiplied by 100 to yield the percentage of sequence identity. The comparison window is the entire length of the sequence being referred to unless indicated otherwise.

[0037] As used herein, “% similarity” is calculated as described for “% identity,” with the exception that the hydrophobic residues Ala, Val, Phe, Pro, Leu, Ile, Trp, Met, and Cys are similar; the basic residues Lys, Arg, and His are similar; the acidic residues Glu and Asp are similar; and the hydrophilic, uncharged residues Gln, Asn, Ser, Thr, and Tyr are similar. The remaining natural amino acid Gly is not similar to any other amino acid in this context.

[0038] Extracellular vesicles (EV) are nanoparticles of cellular origin that are tolerated immunologically and that are believed to play a role in intercellular communicators and cargo delivery1-3. EV’s can range in size from 50 to 150nm in diameter and intrinsically package a variety of cellular cargo including protein, DNA and RNAs3. Packaging of cargo into EVs and macromolecules occurs during the late endosomal stage of the EV biogenesis pathway4. The difference between cellular and EV biomolecular cargo reveals that EV cargo packaging is tightly regulated and is not merely the collection of overproduced cellular materials5. Exosome transmembrane proteins such as CD63, CD9, and PTGFRN are specifically enriched in EVs and thus provide means to engineer EVs to package therapeutic biomolecules (Wang, Chen et al. 2018, Dooley, McConnell et al. 2021). In addition to the transmembrane proteins, EVs are also enriched with various types of RNAs, microRNAs, mRNAs and even circular non-coding RNAs (Zhang, Zhang et al. 2019), which are often recruited along with RNA binding proteins3. Moreover, several investigators have developed methods to package EVs with various cargo, including small interfering RNAs (siRNAs) (Liu, Li et al. 2015), microRNAs by embedding a putative EV specific miRNA scaffold (miR451 and mi155) into the RNA (Reshke, Taylor et al. 2020), mRNAs (Hung and Leonard 2016, Kojima, Bojar et al. 2018), including CRISPR (Li, Zhou et al. 2019), and recombinant proteins (Villamizar, Waters et al. 2021).

[0039] Kojima et al., 2018 (1) described an expression cassette consisting of mRNA CD63 exosome specific packaging modality and a separate “booster” consisting of proteins associated with increasing exosome production and enhancing exosome membrane fusion with host cells called the EXOtic system. In the EXOtic system, mRNA is generated to be expressed with a CD-box binding domain in the 3’ UTR. This CD-box domain interacts with the L7Ae protein, which is made in fusion with CD63, a main exosome membrane protein. In this manner the CD-box containing mRNA is packaged into those exosomes being generated from the plasmid transfected cells. A major drawback of the EXOtic system is that it is of bacterial origin, and therefore likely immunogenic for humans resulting in serious side effects.

[0040] The present invention provides an exosome-based RNA packaging and delivery system using a packaging mechanism of human origin rendering the system of the present invention low or non-immunogenic. Importantly, the low or non-immunogenicity of the present invention is critical for any application in or related to therapeutic use in humans. In an embodiment, the packaging mechanism of the present invention comprises the U1a / UR RNA packaging mechanism comprising U1 small nuclear ribonucleoprotein polypeptide A (U1a) and U1a-RBS domain (UR) capable of binding to one another. Therefore, in an embodiment, the exosome- based RNA packaging and delivery system is termed U1a Linked Transfer of RNA Across EV or ULTRA-EV. Example 1 in connection Figure 1A illustrates how an embodiment of the U1a / UR RNA packaging mechanism of the present invention comprising a CD63-U1a fusion protein and a U1 snRNA stem-loop 2 (L2) binding motif embedded in the 3’ UTR of a mRNA is able to package the mRNA into exosome. The U1a / UR system with only a single L2 motif in the 3’ UTR of luciferase works well for packaging and delivering various types of RNA as shown in the Example 1 in connection with Figures 1B-C. Various exosome transmembrane protein listed in Table 1 such as CD9 or PTGFRN may be used in placed of the CD63 protein in the fusion protein. In addition, other packaging mechanisms may also be used in place of U1a / UR such as shown in Figure 12.

[0041] Therefore, the present invention provides an exosome-based RNA package and delivery system comprising an exosome producing cell, one or more cargo RNA encoding plasmids and one or more fusion protein encoding plasmids. In an embodiment, the exosome producing cell comprises any cells capable of producing exosomes. In an embodiment, exosome producing cells comprise induced pluripotent stem cells (IPSCs), Mesenchymal stem cell (MSC), neural stem cells, and / or HEK293F cells1. In an embodiment, the one or more fusion protein encoding plasmids each encodes an exosome associated transmembrane protein fused to a packaging protein, and the one or more cargo RNA encoding plasmids each encodes a package RNA and a 1 h^ps: / / www.thermofisher.com / au / en / home / technical-resources / cell-lines / 2 / cell-lines-detail-29.htmlpackaging domain. In an embodiment, transfecting the exosome producing cell with the one or more fusion protein encoding plasmids causes the exosome producing cell to express a fusion protein comprising an exosome associated transmembrane protein fused to a packaging protein. In an embodiment, transfecting the exosome producing cell with the one or more cargo RNA encoding plasmids causes the exosome producing cell to express a cargo RNA comprising a package RNA and a packaging domain wherein the packaging domain is capable of binding to the packaging protein of the one or more fusion proteins. In an embodiment, the packaging protein is endogenous to a subject. In an embodiment, the expressed fusion protein binds to the expressed cargo RNA via the packaging protein and the packaging domain to form a fusion protein cargo RNA complex.

[0042] In an embodiment, the packaging protein comprises U1a protein. In an embodiment, the packaging domain comprises UR domain or L2 domain. In an embodiment, the package RNA comprises messenger RNAs (mRNA), non-coding RNA such as but not limited to long non- coding RNAs (lncRNA), circular RNAs, long-hairpin RNAs, antisense RNAs, siRNAs, shRNAs, miRNAs, guide RNAs (gRNAs) or a combination thereof. In an embodiment, the cargo RNA comprises a packaging domain in the 3’-end or in the 5’-end of the package RNA. In an embodiment, the exosome associated transmembrane protein comprises CD63, CD9, CD81, PTGFRN or a combination thereof. Other possible transmembrane proteins are listed in Y Yang et al.35which is hereby incorporated in its entirety as well as in Table 1A-C. Table 1A. Enriched proteins in ExosACate or Gene NameDProtein Descri tionDTable 1A. Enriched proteins in ExosAngTable 1B. Enriched proteins in sMVsBGeneTable 1C. Commonly identified proteins in Exos and sMVsCC t G N P ti D i ti B1Table 1C. Commonly identified proteins in Exos and sMVsCNotes: • A Exos enriched: protein selection category based on references: [1-8]. Raw mass spectrometry data is deposited in the PeptideAtlas and can be accessed at http: / / www.peptideatlas.org / PASS / PASS00749. • B sMV enriched: protein selection category based on references [1, 6]. Raw mass spectrometry data is deposited in the PeptideAtlas and can be accessed at http: / / www.peptideatlas.org / PASS / PASS00749. • C Co-Exos and sMV enriched: protein selection category based on references: [1-8]. • D Identified gene and protein information can be obtained from http: / / www.ncbi.nlm.nih.gov / gene and http: / / www.uniprot.org. • * Proteins that we consider specific markers for Exos and sMVs (based on unique predominant expression in purified EV types).

[0043] In an embodiment, the one or more fusion proteins comprise CD63-U1a fusion protein. In an embodiment, the nucleotide sequence of the CD63-U1a fusion protein encoding plasmid of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 1.

[0044] In an embodiment, the one or more fusion proteins comprise CD81-U1a fusion protein. In an embodiment, the nucleotide sequence of the CD81-U1a protein encoding plasmid of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 2.

[0045] In an embodiment, the one or more fusion proteins comprise PTGFRN-U1a fusion protein. In an embodiment, the nucleotide sequence of the PTGFRN-U1a protein encoding plasmid of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 3.

[0046] SEQ ID NO. 1: ATGGCGGTGGAAGGAGGAATGAAATGTGTGAAGTTCTTGCTCTACGTCCTCCTGCTG GCCTTTTGCGCCTGTGCAGTGGGACTGATTGCCGTGGGTGTCGGGGCACAGCTTGTC CTGAGTCAGACCATAATCCAGGGGGCTACCCCTGGCTCTCTGTTGCCAGTGGTCATCA TCGCAGTGGGTGTCTTCCTCTTCCTGGTGGCTTTTGTGGGCTGCTGCGGGGCCTGCAAGGAGAACTATTGTCTTATGATCACGTTTGCCATCTTTCTGTCTCTTATCATGTTGGTGG AGGTGGCCGCAGCCATTGCTGGCTATGTGTTTAGAGATAAGGTGATGTCAGAGTTTAA TAACAACTTCCGGCAGCAGATGGAGAATTACCCGAAAAACAACCACACTGCTTCGAT CCTGGACAGGATGCAGGCAGATTTTAAGTGCTGTGGGGCTGCTAACTACACAGATTG GGAGAAAATCCCTTCCATGTCGAAGAACCGAGTCCCCGACTCCTGCTGCATTAATGTT ACTGTGGGCTGTGGGATTAATTTCAACGAGAAGGCGATCCATAAGGAGGGCTGTGTG GAGAAGATTGGGGGCTGGCTGAGGAAAAATGTGCTGGTGGTAGCTGCAGCAGCCCT TGGAATTGCTTTTGTCGAGGTTTTGGGAATTGTCTTTGCCTGCTGCCTCGTGAAGAGT ATCAGAAGTGGCTACGAGGTGATGgaattcggcggaggcgggtccATGGCAGTTCCCGAGACC CGCCCTAACCACACTATTTATATCAACAACCTCAATGAGAAGATCAAGAAGGATG AGCTAAAAAAGTCCCTGTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGGATAT CCTGGTATCACGGAGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCAAGGA GGTCAGCAGCGCCACCAACGCCCTGCGCTCCATGCAGGGTTTCCCTTTCTATGA CAAACCTATGCGTATCCAGTATGCCAAGACCGACTCAGATATCATTGCCAAGATG AAA

[0047] Uppercase sequence denotes CD63, lower case is linker sequence, and bold sequence is the U1 small nuclear ribonucleoprotein polypeptide A (U1 snRNP A).

[0048] SEQ ID NO. 2:

[0049] ATGTCCGGACTCAGATCTCGAGCTCAAGCTTCCGGAGTGGAGGGCTGCACCA AGTGCATCAAGTACCTGCTCTTCGTCTTCAATTTCGTCTTCTGGCTGGCTGGAGGCGT GATCCTGGGTGTGGCCCTGTGGCTCCGCCATGACCCGCAGACCACCAACCTCCTGTAT CTGGAGCTGGGAGACAAGCCCGCGCCCAACACCTTCTATGTAGGCATCTACATCCTCA TCGCTGTGGGCGCTGTCATGATGTTCGTTGGCTTCCTGGGCTGCTACGGGGCCATCCA GGAATCCCAGTGCCTGCTGGGGACGTTCTTCACCTGCCTGGTCATCCTGTTTGCCTGT GAGGTGGCCGCCGGCATCTGGGGCTTTGTCAACAAGGACCAGATCGCCAAGGATGTG AAGCAGTTCTATGACCAGGCCCTACAGCAGGCCGTGGTGGATGATGACGCCAACAAC GCCAAGGCTGTGGTGAAGACCTTCCACGAGACGCTTGACTGCTGTGGCTCCAGCAC ACTGACTGCTTTGACCACCTCAGTGCTCAAGAACAATTTGTGTCCCTCGGGCAGCAA CATCATCAGCAACCTCTTCAAGGAGGACTGCCACCAGAAGATCGATGACCTCTTCTC CGGGAAGCTGTACCTCATCGGCATTGCTGCCATCGTGGTCGCTGTGATCATGATCTTC GAGATGATCCTGAGCATGGTGCTGTGCTGTGGCATCCGGAACAGCTCCGTGTACgaattcggcggaggcgggtccATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTATATCAAC AACCTCAATGAGAAGATCAAGAAGGATGAGCTAAAAAAGTCCCTGTACGCCATC TTCTCCCAGTTTGGCCAGATCCTGGATATCCTGGTATCACGGAGCCTGAAGATG AGGGGCCAGGCCTTTGTCATCTTCAAGGAGGTCAGCAGCGCCACCAACGCCCT GCGCTCCATGCAGGGTTTCCCTTTCTATGACAAACCTATGCGTATCCAGTATGCC AAGACCGACTCAGATATCATTGCCAAGATGAAATAA

[0050] SEQ ID NO. 3:

[0051] ATGGGGCGCCTGGCCTCCAGGCCGCTGCTGCTGGCGCTCCTGTCGTTGGCTCT TTGCCGAGGGCGTGTGGTGAGAGTCCCCACAGCGACCCTGGTTCGAGTGGTGGGCA CTGAGCTGGTCATCCCCTGCAACGTCAGTGACTATGATGGCCCCAGCGAGCAAAACT TTGACTGGAGCTTCTCATCTTTGGGGAGCAGCTTTGTGGAGCTTGCAAGCACCTGGG AGGTGGGGTTCCCAGCCCAGCTGTACCAGGAGCGGCTGCAGAGGGGCGAGATCCTG TTAAGGCGGACTGCCAACGACGCCGTGGAGCTCCACATAAAGAACGTCCAGCCTTCA GACCAAGGCCACTACAAATGTTCAACCCCCAGCACAGATGCCACTGTCCAGGGAAAC TATGAGGACACAGTGCAGGTTAAAGTGCTGGCCGACTCCCTGCACGTGGGCCCCAGC GCGCGGCCCCCGCCGAGCCTGAGCCTGCGGGAGGGGGAGCCCTTCGAGCTGCGCTG CACCGCCGCCTCCGCCTCGCCGCTGCACACGCACCTGGCGCTGCTGTGGGAGGTGCA CCGCGGCCCGGCCAGGCGGAGCGTCCTCGCCCTGACCCACGAGGGCAGGTTCCACC CGGGCCTGGGGTACGAGCAGCGCTACCACAGTGGGGACGTGCGCCTCGACACCGTG GGCAGCGACGCCTACCGCCTCTCAGTGTCCCGGGCTCTGTCTGCCGACCAGGGCTCC TACAGGTGTATCGTCAGCGAGTGGATCGCCGAGCAGGGCAACTGGCAGGAAATCCAA GAAAAGGCCGTGGAAGTTGCCACCGTGGTGATCCAGCCATCAGTTCTGCGAGCAGCT GTGCCCAAGAATGTGTCTGTGGCTGAAGGAAAGGAACTGGACCTGACCTGTAACATC ACAACAGACCGAGCCGATGACGTCCGGCCCGAGGTGACGTGGTCCTTCAGCAGGAT GCCTGACAGCACCCTACCTGGCTCCCGCGTGTTGGCGCGGCTTGACCGTGATTCCCT GGTGCACAGCTCGCCTCATGTTGCTTTGAGTCATGTGGATGCACGCTCCTACCATTTA CTGGTTCGGGATGTTAGCAAAGAAAACTCTGGCTACTATTACTGCCACGTGTCCCTGT GGGCACCCGGACACAACAGGAGCTGGCACAAAGTGGCAGAGGCCGTGTCTTCCCCA GCTGGTGTGGGTGTGACCTGGCTAGAACCAGACTACCAGGTGTACCTGAATGCTTCC AAGGTCCCCGGGTTTGCGGATGACCCCACAGAGCTGGCATGCCGGGTGGTGGACAC GAAGAGTGGGGAGGCGAATGTCCGATTCACGGTTTCGTGGTACTACAGGATGAACCGGCGCAGCGACAATGTGGTGACCAGCGAGCTGCTTGCAGTCATGGACGGGGACTGGA CGCTAAAATATGGAGAGAGGAGCAAGCAGCGGGCCCAGGATGGAGACTTTATTTTTT CTAAGGAACATACAGACACGTTCAATTTCCGGATCCAAAGGACTACAGAGGAAGACA GAGGCAATTATTACTGTGTTGTGTCTGCCTGGACCAAACAGCGGAACAACAGCTGGG TGAAAAGCAAGGATGTCTTCTCCAAGCCTGTTAACATATTTTGGGCATTAGAAGATTC CGTGCTTGTGGTGAAGGCGAGGCAGCCAAAGCCTTTCTTTGCTGCCGGAAATACATT TGAGATGACTTGCAAAGTATCTTCCAAGAATATTAAGTCGCCACGCTACTCTGTTCTC ATCATGGCTGAGAAGCCTGTCGGCGACCTCTCCAGTCCCAATGAAACGAAGTACATC ATCTCTCTGGACCAGGATTCTGTGGTGAAGCTGGAGAATTGGACAGATGCATCACGG GTGGATGGCGTTGTTTTAGAAAAAGTGCAGGAGGATGAGTTCCGCTATCGAATGTACC AGACTCAGGTCTCAGACGCAGGGCTGTACCGCTGCATGGTGACAGCCTGGTCTCCTG TCAGGGGCAGCCTTTGGCGAGAAGCAGCAACCAGTCTCTCCAATCCTATTGAGATAG ACTTCCAAACCTCAGGTCCTATATTTAATGCTTCTGTGCATTCAGACACACCATCAGTA ATTCGGGGAGATCTGATCAAATTGTTCTGTATCATCACTGTCGAGGGAGCAGCACTGG ATCCAGATGACATGGCCTTTGATGTGTCCTGGTTTGCGGTGCACTCTTTTGGCCTGGA CAAGGCTCCTGTGCTCCTGTCTTCCCTGGATCGGAAGGGCATCGTGACCACCTCCCG GAGGGACTGGAAGAGCGACCTCAGCCTGGAGCGCGTGAGTGTGCTGGAATTCTTGC TGCAAGTGCATGGCTCCGAGGACCAGGACTTTGGCAACTACTACTGTTCCGTGACTC CATGGGTGAAGTCACCAACAGGTTCCTGGCAGAAGGAGGCAGAGATCCACTCCAAG CCCGTTTTTATAACTGTGAAGATGGATGTGCTGAACGCCTTCAAGTATCCCTTGCTGAT CGGCGTCGGTCTGTCCACGGTCATCGGGCTCCTGTCCTGTCTCATCGGGTACTGCAGC TCCCACTGGTGTTGTAAGAAGGAGGTTCAGGAGACACGGCGCGAGCGCCGCAGGCT CATGTCGATGGAGATGGACgaattcggcggaggcgggtccATGGCAGTTCCCGAGACCCGCCC TAACCACACTATTTATATCAACAACCTCAATGAGAAGATCAAGAAGGATGAGCTA AAAAAGTCCCTGTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGGATATCCTG GTATCACGGAGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCAAGGAGGT CAGCAGCGCCACCAACGCCCTGCGCTCCATGCAGGGTTTCCCTTTCTATGACAA ACCTATGCGTATCCAGTATGCCAAGACCGACTCAGATATCATTGCCAAGATGAAA TAG

[0052] In an embodiment the amino acid sequence of the CD63-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 4.

[0053] SEQ ID NO. 4: MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAV GVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFR QQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCG INFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM efggggsMAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQILDILVSRSLKMRG QAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAKMK

[0054] Uppercase sequence denotes CD63, lower case is linker sequence, and bold sequence is the U1 small nuclear ribonucleoprotein polypeptide A (U1 snRNP A).

[0055] In an embodiment, the amino acid sequence of the CD81-U1a fusion protein of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 5.

[0056] SEQ ID NO. 5:

[0057] MSGLRSRAQASGVEGCTKCIKYLLFVFNFVFWLAGGVILGVALWLRHDPQTTNL LYLELGDKPAPNTFYVGIYILIAVGAVMMFVGFLGCYGAIQESQCLLGTFFTCLVILFACE VAAGIWGFVNKDQIAKDVKQFYDQALQQAVVDDDANNAKAVVKTFHETLDCCGSSTL TALTTSVLKNNLCPSGSNIISNLFKEDCHQKIDDLFSGKLYLIGIAAIVVAVIMIFEMILSMV LCCGIRNSSVYEFGGGGSMAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQILDIL VSRSLKMRGQAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAKMK

[0058] In an embodiment, the amino acid sequence of the PTGFRN-U1a fusion protein of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 6.

[0059] SEQ ID NO. 6:

[0060] MGRLASRPLLLALLSLALCRGRVVRVPTATLVRVVGTELVIPCNVSDYDGPSEQNF DWSFSSLGSSFVELASTWEVGFPAQLYQERLQRGEILLRRTANDAVELHIKNVQPSDQGH YKCSTPSTDATVQGNYEDTVQVKVLADSLHVGPSARPPPSLSLREGEPFELRCTAASASP LHTHLALLWEVHRGPARRSVLALTHEGRFHPGLGYEQRYHSGDVRLDTVGSDAYRLSV SRALSADQGSYRCIVSEWIAEQGNWQEIQEKAVEVATVVIQPSVLRAAVPKNVSVAEGKELDLTCNITTDRADDVRPEVTWSFSRMPDSTLPGSRVLARLDRDSLVHSSPHVALSHVDAR SYHLLVRDVSKENSGYYYCHVSLWAPGHNRSWHKVAEAVSSPAGVGVTWLEPDYQVY LNASKVPGFADDPTELACRVVDTKSGEANVRFTVSWYYRMNRRSDNVVTSELLAVMD GDWTLKYGERSKQRAQDGDFIFSKEHTDTFNFRIQRTTEEDRGNYYCVVSAWTKQRNN SWVKSKDVFSKPVNIFWALEDSVLVVKARQPKPFFAAGNTFEMTCKVSSKNIKSPRYSV LIMAEKPVGDLSSPNETKYIISLDQDSVVKLENWTDASRVDGVVLEKVQEDEFRYRMYQ TQVSDAGLYRCMVTAWSPVRGSLWREAATSLSNPIEIDFQTSGPIFNASVHSDTPSVIRGD LIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSD LSLERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMD VLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMDEFGG GGSMAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQILDILVSRSLKMRGQAFVIF KEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAKMK

[0061] In an embodiment the nucleotide sequence of the UR domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 7 and the nucleotide sequence of the L2 domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID No. 8.

[0062] SEQ ID NO. 7: AATCCATTGCACTCCGGATT

[0063] SEQ ID NO. 8: AATCCATTGCACTCCGGATTT

[0064] In an embodiment, the exosome-based RNA package and delivery system of the present invention comprises an exosome producing cell, one or more cargo RNA encoding plasmids that encodes a shRNA and one or more UR or L2 domains, and a fusion protein encoding plasmid that encodes CD63-U1a fusion protein. In an embodiment, the exosome-based RNA package and delivery system of the present invention comprises an exosome producing cell, one or more cargo RNA encoding plasmids encoding a shRNA and one or more UR or L2 domains, and a CD81-U1a fusion protein encoding plasmid that encodes a CD81-U1a fusion protein. In an embodiment, the exosome-based RNA package and delivery system of the present invention comprises an exosome producing cell, one or more cargo RNA encoding plasmids encoding a shRNA and one or more UR or L2 domains, and a PTGFRN-U1a fusion protein encoding plasmid that encodes a PTGFRN-U1a fusion protein.

[0065] In an embodiment, the exosome-based RNA package and delivery system of the present invention comprises an exosome producing cell, one or more cargo RNA encoding plasmids thatencodes an antisense RNA and one or more UR or L2 domains, and a fusion protein encoding plasmid that encodes a CD63-U1a fusion protein. In an embodiment, the exosome-based RNA package and delivery system of the present invention comprises an exosome producing cell, one or more cargo RNA encoding plasmids that encodes an antisense RNA, and a CD81-U1a fusion protein encoding plasmid that encodes a CD81-U1a fusion protein. In an embodiment, the exosome-based RNA package and delivery system of the present invention comprises an exosome producing cell, one or more cargo RNA encoding plasmids that encodes an antisense RNA, and a PTGFRN-U1a fusion protein encoding plasmid that encodes a PTGFRN-U1a fusion protein.

[0066] In an embodiment, the exosome-based RNA package and delivery system of the present invention comprises an exosome producing cell, one or more cargo RNA encoding plasmids that encodes a mRNA, and a CD63-U1a fusion protein encoding plasmid that encodes a CD63-U1a fusion protein. In an embodiment, the exosome-based RNA package and delivery system of the present invention comprises an exosome producing cell, one or more cargo RNA encoding plasmids that encodes a mRNA and one or more UR or L2 domains, and a CD81-U1a fusion protein encoding plasmid that encodes a CD81-U1a fusion protein. In an embodiment, the exosome-based RNA package and delivery system of the present invention comprises an exosome producing cell, one or more cargo RNA encoding plasmids that encodes a mRNA, and a PTGFRN-U1a fusion protein encoding plasmid that encodes a PTGFRN-U1a fusion protein.

[0067] In an embodiment, any embodiment of the cargo RNA plasmid of the present invention further comprises stabilizing domains. In an embodiment, the stabilizing domain comprises OH domain and / or MorrisMotif domain wherein the nucleotide sequence of the OH domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 9 and the nucleotide sequence of the MorrisMotif domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 10.

[0068] SEQ ID NO. 9: CTGCAGATATCCAGCACAGTGGC

[0069] SEQ ID NO. 10: GCGCAGCGCGCGCAGCGC

[0070] Example 1 in connection with Figure 1C shows that a single L2 motif was only 50% as efficient at packaging luciferase mRNA into exosomes compared to the EXOtic system. Figure 2A shows that both 5’ and 3’ UTRs are able to accommodate the L2 domains and that packaging efficiency seems relatively concordant between the two conditions. In addition, Figures 2B-2Cillustrates that 4 to 6 repeats of the L2 domain appears sufficient to package GFP mRNAs at levels comparable to the EXOtic system (1). Collectively, these data indicate that the U1a / L2 RNA packaging system is comparable to the previously reported EXOtic system and could be a superior system with regards to the use of human-derived components that could be less or non- immunogenic than the EXOtic system when used in human. Therefore, in an embodiment, the packaging domain of the present invention comprises 1 to 10 repeats of the UR or L2 domains such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats including any numbers or number ranges falling within these values. In an embodiment, the packaging domain of the present invention comprises 1 to 10 repeats of the UR domain such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the UR domain including any numbers or number ranges falling within these values.

[0071] Furthermore, Example 4 in connection with Figures 3A, 3B and 3C show that exosome- based RNA delivery system of the present invention proved to be non-toxic and non- inflammatory when injected in C57BL / 6 WT mice. Therefore, the exosome-based RNA delivery system of the present invention is relatively safe in vivo and may have clinical potential to deliver RNA, especially non-coding RNA therapeutics capable of regulating gene transcription.

[0072] Notably, with two or more antibiotics, it is possible to package two or more package RNAs into one exosome using antibiotic selection of cargo RNA encoding plasmids. In an embodiment, this may be done using two or more distinct cargo RNA encoding plasmids, wherein each distinct plasmid comprises sequence coding for the one of the two or more distinct package RNAs and an antibiotic resistant gene corresponding to the one of the two or more antibiotics. The two or more distinct cargo RNA encoding plasmids are then transfected into the same exosome producing cells. Cells successfully transfected with the two or more distinct cargo RNA encoding plasmids are selected for by adding the two or more antibiotics to the cell culture medium. In an embodiment, the resulting exosome producing cells contain up to 2, 3, 4, 5, 6 or more distinct cargo RNA encoding plasmids, and can therefore produce up to 2, 3, 4, 5, 6 or more distinct cargo RNAs comprising up to 2, 3, 4, 5, 6 or more package RNAs and / or packaging domain. This may allow for simultaneous therapeutic effect on 2 or more targets of the distinct therapeutic package RNAs. For example, an exosome producing cell may be engineered to target the 4 genes (PRKN, PINK1, DJ1 and SNCA) whose expression is inhibited in Parkinson’s disease patients. More specifically, an exosome producing cell may be transfected with 4 distinct cargo RNA encoding plasmids to express the 4 distinct cargo RNAs tosimultaneously target and turn on expression of the 4 genes inhibited in patients with Parkinson’s disease for superior therapeutic effect. Delivery of multiple therapeutic RNAs at once is particularly useful for diseases involving multiple genes as well as diseases caused by viruses with multiple strains and variants. For example, delivery of multiple therapeutic RNAs at once is useful in treating illness associated with multiple genes such as Parkinson’s disease which involves several genes. Delivery of multiple therapeutic RNAs at once is also useful to target viruses with multiple variants and strains such as the influenza virus. In an embodiment, the plurality of distinct cargo RNAs provides synergistic therapeutic effect to a subject.

[0073] Moreover, by mixing and matching exosome transmembrane protein and packaging protein, it is possible to create multiple unique combinations of exosome transmembrane protein- packing protein. It is then possible to package a distinct package RNA with a particular unique combination of exosome transmembrane protein-packing protein. This may be beneficial in circumstances where certain combinations of package RNA, exosome transmembrane protein and packing protein work particular well such as exosome loading, exosome release, exosome targeting, toxicity, therapeutic effects etc….

[0074] In an embodiment, the packaging efficiency of the cargo RNA into the exosome using the exosome-based RNA package and delivery system of the present invention is related to the exosome associated transmembrane protein. In an embodiment where the cargo RNA comprises an antisense RNA, the exosome associated transmembrane protein comprises CD63, CD81, or PTGFRN. In an embodiment where the cargo RNA comprises a mRNA, the exosome associated transmembrane protein comprises CD63 or CD81. In an embodiment, the efficiency of packaging a mRNA into the exosome in the exosome-based RNA package and delivery system using a PTGFRN-U1a fusion protein plasmid is more than about 5%, about 10%, about 20%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, about 1200%, or about 1500% lower than the efficiency of packaging the same mRNA into the exosome in the exosome-based RNA package and delivery system using a CD63-U1a fusion protein plasmid or CD81-U1a fusion protein plasmid.

[0075] In an embodiment, the exosome-based RNA package and delivery system of the present invention comprises an exosome producing cell, one or more cargo RNA encoding plasmids and two or more fusion protein encoding plasmids. In an embodiment, the at least one of the two ormore fusion protein encoding plasmids encodes a packaging protein comprising an endogenous human protein component such as but not limited to CD63-U1a, CD81-U1a, and PTGFRN-U1a, and at least another one of the two or more fusion protein encoding plasmids encodes a packaging protein comprising a foreign protein component such as but not limited to CD63- L7ae, CD81-L7ae, and PTGFRN-L7ae. In an embodiment, the two or more fusion protein encoding plasmids each comprises a packaging protein comprising endogenous human protein components comprising CD63-U1a, CD81-U1a, or PTGFRN-U1a. In an embodiment, each of the two or more fusion protein encoding plasmids comprise a CD63-U1a fusion protein encoding plasmid or a CD81-U1a fusion protein encoding plasmid. In an embodiment, each of the two or more fusion protein encoding plasmids comprises a CD63-U1a fusion protein encoding plasmid or a PTGFRN-U1a fusion protein encoding plasmid. In an embodiment, each of the two or more fusion protein encoding plasmids comprises a CD81-U1a fusion protein encoding plasmid or a PTGFRN-U1a encoding fusion protein plasmid.

[0076] Example 1 in connection with Figure 4A show that repression of two genes from the ESCRT pathway, Charged Multivesicular Body Protein 4C (CHMP4C) and Vacuolar Protein Sorting 4B (VPS4B) using shRNA (shBooster) mediated RNA interference enhances release of EVs from EV producing cells. Furthermore, Example 1 in connection with Figures 5C and 5D show that 4:1 ratio of shBooster to CD63-nLuc provided the highest cargo packaging as compared to 1:1 and 9:1 ratios. Moreover, Figure 4C shows that transfecting mutant gap junction protein Connexin S368A in EV producing cells also increased overall delivery into the recipient cells.

[0077] Therefore, in an embodiment, the exosome-based RNA packaging and delivery system of the present invention further comprises a packaging boosting shRNA encoding plasmid that encodes packaging boosting shRNA capable of inhibiting the CHMP4C and VPS4B genes in the exosome producing cell when the exosome producing cell is transfected with the packaging boosting shRNA encoding plasmid. In an embodiment the packing boosting shRNA of the present invention comprising CHMP4C shRNA and VPS4B shRNA wherein the nucleotide sequence of CHMP4C shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 11 or SEQ ID NO. 12, and wherein the nucleotide sequence of VPS4B shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 13 or SEQ ID NO. 14.

[0078] SEQ ID NO. 11: GCAGAATAAGCGAGCTGCATT

[0079] SEQ ID NO. 12: GACAAATATCCGCCTTCCAAA

[0080] SEQ ID NO. 13: CGAAGATTTGAGAAACGAATT

[0081] SEQ ID NO. 14: GCTGATCCTAACCATCTTGTA

[0082] In an embodiment, the ratio of the number of the packaging boosting shRNA encoding plasmids of the present invention to the number of fusion protein encoding plasmids of the present invention transfecting the EV producing cell is from about 2:1 to about 8:1 such as about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1 or about 8:1 including any ratio and ratio ranges falling within these values. In an embodiment, the number of the packaging boosting shRNA encoding plasmid of the present invention to the fusion protein plasmid of the present invention transfecting the EV producing cell is from about 3:1 to about 5:1.

[0083] Moreover, Example 6 in connection with Figure 7B shows that Ago2 increased EV packaging of shRNA by up to several hundred fold. In addition, Ago2 with a S387A mutation further increased packaging of shRNA as illustrated in Figure 7D. Importantly, Figure 7E confirms that Ago-2 shRNAs packaged into EVs remain functional in the target cells. Therefore, in an embodiment, the exosome-based RNA packaging and delivery system of the present invention further comprises Ago2 plasmid that encodes Ago2 protein wherein the Ago2 protein is expressed by the exosome producing cells when transfected with the Ago2 plasmid and complexes with the cargo RNA of the present invention. In an embodiment, the Ago2 comprises S387A mutation.

[0084] Furthermore, Example 5 in connection with Figures 8A-G provides an example of an embodiment of the cargo RNA of the present invention PTEN pseudogene 1 antisense RNA 1 alpha (Pg1asα) that requires localization in the nucleus of the EV receiving cell. To enable nuclear localization of the EV delivered Pg1asα-UR RNA, a SINE-derived nuclear RNA LOcalizatIoN (SIRLOIN) sequences were added upstream of the UR sequences and expressed as Pg1asα-SU34,35. Figure 8B shows that the Pg1asα-SU RNA was found to functionally repress PTEN promoter driven luciferase activity comparable to Pg1asα. In the trans-well experimental system, whereby the EV producer cells are in the basolateral chamber and the PTEN-Luc HEK293T reporter cells were seeded in the apical chamber, Figure 8C shows that the EV containing Pg1asα-SU significantly repressed PTEN promoter driven luciferase activity compared to Pg1asα or control. To confirm this observation, the EVs were enriched fromsupernatants of the EV producer cells and added directly to HEK293T cells. In the HEK293T cells treated with EV of the present invention or EV of the present invention containing Pg1asα or the Pg1asα-SU, Pg1asα or Pg1asα-SU were detectable from whole cell RNA extracted after 4 hours of EV treatment confirming the delivery of lncRNA via EV of the present invention to recipient cells as illustrated in Figure 8D. However, significantly higher localization of Pg1asα- SU was observed in the nuclear fraction of ULTRA EV-treated cells compared to Pg1asα or control confirming the ability of SIRLOIN sequence in redirecting the lncRNA to nucleus in EV treated cells as shown in Figure 8E. To further confirm these observations enriched ULTRA-EVs were added daily to PTEN-Luc HEK293T cells in a ratio of 10,000 EVs to 1 cell for five consecutive days and a significant repression of PTEN promoter driven luciferase activity was observed as illustrated in Figure 8F.

[0085] Therefore, in an embodiment, the exosome-based RNA delivery system of the present invention wherein the cargo RNA plasmid further comprises a SIRLOIN nuclear localization sequence wherein the SIRLOIN is upstream of the UR sequence of the RNA. In an embodiment the nucleotide sequence of SIRLOIN is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 15.

[0086] SEQ ID NO. 15: CGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGA

[0087] In an embodiment, the exosome-based RNA package and delivery system of the present invention further comprises one or more modified myoferlin plasmids, wherein each of the one or more modified myoferlin plasmid of the present invention encodes a modified myoferlin protein of the present invention. In an embodiment, the modified myoferlin protein of the present invention comprises C2F, C2G, transmembrane domain, or a combination thereof. In an embodiment, the modified myoferlin protein of the present invention consists of C2F, C2G, transmembrane domain. In an embodiment, the modified myoferlin protein of the present invention consists of domains selected from the group consisting of C2F, C2G, transmembrane domain. In an embodiment, the amino acid sequence of the C2F domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 16. In an embodiment, the amino acid sequence of the C2G domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 17. In an embodiment, the amino acid sequence of the transmembrane domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 18.

[0088] SEQ ID NO. 16 (C2F):

[0089] QFRELPDSVPQECTVRIYIVRGLELQPQDNNGLCDPYIKITLGKKVIEDRDHYIPNT LNPVFGRMYELSCYLPQEKDLKISVYDYDTFTRDEKVGETIIDLENRFLSRFGSHCGIPEE YCVSGV

[0090] SEQ ID NO. 17 (C2G):

[0091] PFNITPRKAKKYYLRVIIWNTKDVILDEKSITGEEMSDIYVKGWVPGNEENKQKT DVHYRSLDGEGNFNWRFVFPFDYLPAEQLCIVAKKEHFWSIDQTEFRIPPRLIIQIWDNDK FSLDDYLGFLELDLRH

[0092] SEQ ID NO. 18 (transmembrane domain):

[0093] PDLKAMNPLKAKTASLFEQKSMKGWWPCYAEKDGARVMAGKVEMTLEILNEK EADERPAGKGRDEPNMNPKLDLPNRPETSFLWFTNPCKTMKFIVWRRFKWVIIGLLFLLI LLLFVAV

[0094] In an embodiment, the modified myoferlin protein of the present invention comprises C2A, FerA, FerB, DysFN, transmembrane domain, or a combination thereof. In an embodiment, the modified myoferlin protein of the present invention consists of C2A, FerA, FerB, DysFN, transmembrane domain. In an embodiment, the modified myoferlin protein of the present invention consists of domains selected from the group consisting of C2A, FerA, FerB, DysFN, transmembrane domain. In an embodiment, the amino acid sequence of the C2A domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 19. In an embodiment, the amino acid sequence of the FerA domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 20. In an embodiment, the amino acid sequence of the FerB is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 21. In an embodiment, the DysFN domain comprises DysFN-1, DysFN-2, or a combination thereof. In an embodiment, the amino acid sequence of the DysFN-1 is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 22. In an embodiment, the amino acid sequence of the DysFN-2 is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 23. In an embodiment, the amino acid sequence of the transmembrane domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO 18.

[0095] SEQ ID NO. 19 (C2A):

[0096] MLRVIVESASNIPKTKFGKPDPIVSVIFKDEKKKTKKVDNELNPVWNEILEFDLRG IPLDFSSSLGIIVKDFETIGQNKLIGTATVALKDLTGDQSRSLPYKLISLLNERGQDTGATID LVIGYDPPSAPHPNDLS

[0097] SEQ ID NO. 20 (FerA):

[0098] LQTNIEALKSGIQGKIPANQLAELWLKLIDEVIEDTRYTLPLTEGKANVTVLDTQIR K

[0099] SEQ ID NO. 21 (FerB): [000100] WLDKLMQLTEEPQNSMPDIIIWMIRGEKRLAYARIPAHQVLYSTSGENASGKYCG KTQTIFLKYPQEKNNGP [000101] SEQ ID NO. 22 (DysFN-1): [000102] AVEKKFNSFAEGTFTVFAEMYENQALMFGKWGTSGLVGRHKFSDVTGKIKLKRE FFLP [000103] SEQ ID NO. 23 (DysFN-2): [000104] DPERSLLTEADAGHTEFTDEVYQNESRYPGGDWKPAEDTYTDANGDKAASPSEL TCP [000105] In an embodiment, any embodiment of the modified myoferlin protein of the present invention further comprises C2B, C2C, C2D, C2E domains, or a combination thereof. In an embodiment, the amino acid sequence of the C2B domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 24. In an embodiment, the amino acid sequence of the C2C domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 25. In an embodiment, the amino acid sequence of the C2D is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 26. In an embodiment, the amino acid sequence of the C2E is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 27. [000106] SEQ ID NO. 24 (C2B): [000107] PQDFQIRVRVIEGRQLSGNNIRPVVKVHVCGQTHRTRIKRGNNPFFDELFFYNVN MTPSELMDEIISIRVYNSHSLRADCLMGEFKIDVGFVYDEPGHAVMRKWLLLNDP [000108] SEQ ID NO. 25 (C2C):[000109] TFLLKIYRAEDIPQMDDAFSQTVKEIFGGNADKKNLVDPFVEVSFAGKKVCTNIIE KNANPEWNQVVNLQIKFPSVCEKIKLTIYDWDRLTKNDVVGTTYLHLSKIAASGGEVED FSSSGTGAASYTVNTGETEVGFVPTFGPCYLNLYGSPREYTGFPDPYDE [000110] SEQ ID NO. 26 (C2D): [000111] TPIVSCNFDRVYIYHLRCYVYQARNLLALDKDSFSDPYAHICFLHRSKTTEIIHSTL NPTWDQTIIFDEVEIYGEPQTVLQNPPKVIMELFDNDQVGKDEFLGRSIFSPVVKLNSEM DITPKLLWHPVMNGDKA [000112] SEQ ID NO. 27 (C2E): [000113] RNMKNFQMASITSPSLVVECGGERVESVVIKNLKKTPNFPSSVLFMKVFLPKEEL YMPPLVIKVIDHRQFGRKPVVG [000114] In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2A domain wherein the amino acid sequence of the C2A domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 19. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2B domain wherein the amino acid sequence of the C2B domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 24. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2C domain wherein the amino acid sequence of the C2C domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 25. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2D domain wherein the amino acid sequence of the C2D domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 26. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2E domain wherein the amino acid sequence of the C2E domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 27. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2F domain wherein the amino acid sequence of the C2F domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 16. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2G domain wherein the amino acid sequence of the C2G domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 17. In an embodiment, the modified myoferlin proteinof the present invention does not comprise the FerA domain wherein the amino acid sequence of the FerA domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 20. In an embodiment, the modified myoferlin protein of the present invention does not comprise the FerB domain wherein the amino acid sequence of the FerB domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 21. In an embodiment, the modified myoferlin protein of the present invention does not comprise the DysFN-1 domain wherein the amino acid sequence of the DysFN-1 domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 22. In an embodiment, the modified myoferlin protein of the present invention does not comprise the DysFN-2 domain wherein the amino acid sequence of the DysFN-2 domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to or similar to SEQ ID NO. 23. [000115] In an embodiment, the amino acid sequence of the modified myoferlin protein of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 28. [000116] SEQ ID NO. 28 (C2F-C2G): [000117] MVPAPPRQFRELPDSVPQECTVRIYIVRGLELQPQDNNGLCDPYIKITLGKKVIED RDHYIPNTLNPVFGRMYELSCYLPQEKDLKISVYDYDTFTRDEKVGETIIDLENRFLSRF GSHCGIPEEYCVSGVNTWRDQLRPTQLLQNVARFKGFPQPILSEDGSRIRYGGRDYSLDE FEANKILHQHLGAPEERLALHILRTQGLVPEHVETRTLHSTFQPNISQGKLQMWVDVFPK SLGPPGPPFNITPRKAKKYYLRVIIWNTKDVILDEKSITGEEMSDIYVKGWVPGNEENKQ KTDVHYRSLDGEGNFNWRFVFPFDYLPAEQLCIVAKKEHFWSIDQTEFRIPPRLIIQIWDN DKFSLDDYLGFLELDLRHTIIPAKSPEKCRLDMIPDLKAMNPLKAKTASLFEQKSMKGW WPCYAEKDGARVMAGKVEMTLEILNEKEADERPAGKGRDEPNMNPKLDLPNRPETSFL WFTNPCKTMKFIVWRRFKWVIIGLLFLLILLLFVAVLLYSLPNYLSMKIVKPNVYPYDVP DYA [000118] In an embodiment, the present invention also provides a polynucleotide encoding any embodiment of the modified myoferlin protein of the present invention. In an embodiment, the modified myoferlin protein encoding polynucleotide of the present invention encodes a C2A domain, a C2B domain, a C2C domain, a C2D domain, a C2E domain, a C2F domain, a C2G domain, a FerA domain, a FerB domain, a DysFN-1 domain, a DysFN-2 domain, atransmembrane domain, or a combination thereof. In an embodiment, the C2A domain is encoded by a C2A domain encoding polynucleotide wherein the nucleotide sequence of the C2A encoding polynucleotide at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 29. In an embodiment, the C2B domain is encoded by a C2B domain encoding polynucleotide wherein the nucleotide sequence of the C2B encoding polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 30. In an embodiment, the C2C domain is encoded by a C2C domain encoding polynucleotide wherein the nucleotide sequence of the C2C encoding polynucleotide at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 31. In an embodiment, the C2D domain is encoded by a C2D domain encoding polynucleotide wherein the nucleotide sequence of the C2D encoding polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 32. In an embodiment, the C2E domain is encoded by a C2E domain encoding polynucleotide wherein the nucleotide sequence of the C2E encoding polynucleotide at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 33. In an embodiment, the C2F domain is encoded by a C2F domain encoding polynucleotide wherein the nucleotide sequence of the C2F encoding polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 34. In an embodiment, the C2G domain is encoded by a C2G domain encoding polynucleotide wherein the nucleotide sequence of the C2G encoding polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 35. In an embodiment, the FerA domain is encoded by a FerA domain encoding polynucleotide wherein the nucleotide sequence of the FerA encoding polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 36. In an embodiment, the FerB domain is encoded by a FerB domain encoding polynucleotide wherein the nucleotide sequence of the FerB encoding polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 37. In an embodiment, the DysFN-1 domain is encoded by a DysFN-1 domain encoding polynucleotide wherein the nucleotide sequence of the DysFN-1 encoding polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 38. In an embodiment, the DysFN-2 domain is encoded by a DysFN-2 domain encoding polynucleotide wherein the nucleotide sequence of the DysFN-2 encoding polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identicalto SEQ ID NO. 39. In an embodiment, the transmembrane domain is encoded by a transmembrane domain encoding polynucleotide wherein the nucleotide sequence of the Transmembrane domain encoding polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 40. [000119] SEQ ID NO. 29 (nt C2A): [000120] ATGCTGCGAGTGATTGTGGAATCTGCCAGCAATATCCCTAAAACGAAATTTGG CAAGCCGGATCCTATTGTTTCTGTCATTTTTAAGGATGAGAAAAAGAAAACAAAGAA AGTTGATAATGAATTGAACCCTGTCTGGAATGAGATTTTGGAGTTTGACTTGAGGGGT ATACCACTGGACTTTTCATCTTCCCTTGGGATTATTGTGAAAGATTTTGAGACAATTGG ACAAAATAAATTAATTGGCACGGCGACTGTAGCCCTGAAGGACCTGACTGGTGACCA GAGCAGATCCCTGCCGTACAAGCTGATCTCCCTGCTAAATGAAAGAGGGCAAGATAC TGGGGCCACCATTGACTTGGTGATCGGCTATGATCCGCCTTCTGCTCCACATCCAAAT GACCTGAGC [000121] SEQ ID NO. 30 (nt C2B): [000122] CCACAGGACTTCCAGATCCGCGTCCGAGTGATTGAGGGCCGACAGTTAAGTG GCAACAACATAAGGCCTGTGGTCAAAGTTCACGTCTGTGGCCAGACACACCGAACA AGAATCAAGAGAGGAAACAACCCTTTTTTTGATGAGTTGTTTTTCTACAATGTCAACA TGACCCCTTCTGAATTGATGGATGAGATCATCAGCATCCGGGTTTATAATTCTCACTCT CTGCGGGCAGATTGTCTGATGGGGGAATTTAAGATTGATGTTGGATTTGTTTATGATGA ACCTGGCCATGCTGTCATGAGAAAGTGGCTTCTTCTCAATGACCCG [000123] SEQ ID NO. 31 (nt C2C): [000124] ACCTTCTTGCTGAAAATCTACCGAGCTGAGGACATCCCCCAGATGGATGATGC CTTCTCACAGACAGTAAAGGAAATATTTGGAGGCAATGCAGATAAGAAAAATCTCGT GGATCCTTTTGTAGAAGTTTCCTTTGCTGGAAAAAAGGTTTGTACAAACATAATTGAG AAGAATGCAAACCCAGAGTGGAATCAGGTCGTCAATCTTCAGATCAAGTTTCCTTCA GTGTGTGAAAAAATAAAACTAACAATATATGACTGGGACCGTCTTACTAAAAATGATG TAGTTGGAACAACATATCTACACCTCTCTAAAATTGCTGCCTCTGGTGGGGAAGTGGA AGATTTCTCATCTTCGGGAACTGGGGCTGCATCATATACAGTAAACACAGGAGAAACA GAGGTAGGCTTTGTTCCAACGTTTGGACCTTGTTACCTGAATCTTTATGGAAGCCCCA GAGAGTACACGGGATTCCCAGACCCCTATGATGAG [000125] SEQ ID NO. 32 (nt C2D):[000126] ACCCCCATTGTTTCCTGCAATTTTGACAGAGTCTACATCTACCATCTGCGCTGC TATGTCTATCAAGCCAGAAACCTCTTGGCTTTAGATAAGGATAGCTTTTCAGATCCATA TGCTCATATCTGTTTCCTCCATCGGAGCAAAACCACTGAGATCATCCATTCAACCCTG AATCCCACGTGGGACCAAACAATTATATTCGATGAAGTTGAAATCTATGGGGAACCCC AAACAGTTCTACAGAATCCACCCAAAGTTATCATGGAACTTTTTGACAATGACCAAGT GGGCAAAGATGAATTTTTAGGACGAAGCATTTTCTCTCCTGTGGTGAAACTGAACTC AGAAATGGACATCACACCCAAACTTCTCTGGCACCCAGTAATGAATGGAGACAAAGC C [000127] SEQ ID NO. 33 (nt C2E): [000128] AGAAATATGAAAAACTTCCAGATGGCTTCTATCACATCCCCCAGTCTTGTTGTG GAGTGTGGAGGAGAAAGGGTGGAATCGGTGGTGATCAAAAACCTTAAGAAGACACC CAACTTTCCAAGTTCTGTTCTCTTCATGAAAGTGTTCTTGCCCAAGGAGGAATTGTAC ATGCCCCCACTGGTGATCAAGGTCATCGACCACAGGCAGTTTGGGCGGAAGCCTGTC GTCGGC [000129] SEQ ID NO. 34 (nt C2F): [000130] CAGTTTCGGGAATTACCTGACAGCGTCCCACAGGAATGCACGGTTAGGATTTA CATTGTTCGAGGCTTAGAGCTCCAGCCCCAGGACAACAATGGCCTGTGTGACCCTTA CATAAAAATAACACTGGGCAAAAAAGTCATTGAAGACCGAGATCACTACATTCCCAA CACTCTCAACCCAGTCTTTGGCAGGATGTACGAACTGAGCTGCTACTTACCTCAAGA AAAAGACCTGAAAATTTCTGTCTATGATTATGACACCTTTACCCGGGATGAAAAAGTA GGAGAGACAATTATTGATCTGGAAAACCGATTCCTTTCCCGCTTTGGGTCCCACTGCG GCATACCAGAGGAGTACTGTGTTTCTGGAGTC [000131] SEQ ID NO. 35 (nt C2G): [000132] CCTTTCAACATCACACCCCGGAAAGCCAAGAAATACTACCTGCGTGTGATCAT CTGGAACACCAAGGATGTTATCTTGGATGAGAAAAGCATCACAGGAGAGGAAATGAG TGACATCTACGTCAAAGGCTGGGTTCCTGGCAATGAAGAAAACAAACAGAAAACAG ATGTCCATTACAGATCTTTGGATGGTGAAGGGAATTTTAACTGGCGATTTGTTTTCCCG TTTGACTACCTTCCAGCCGAACAACTCTGTATCGTTGCGAAAAAAGAGCATTTCTGGA GTATTGACCAAACGGAATTTCGAATCCCACCCAGGCTGATCATTCAGATATGGGACAA TGACAAGTTTTCTCTGGATGACTACTTGGGTTTCCTAGAACTTGACTTGCGTCAC [000133] SEQ ID NO. 36 (nt FerA):[000134] CTGCAAACAAATATAGAAGCTCTAAAATCAGGGATACAAGGTAAAATTCCTGC AAACCAGCTGGCTGAATTGTGGCTGAAGCTGATAGATGAAGTTATAGAAGACACGAG ATACACGTTGCCTCTCACAGAAGGAAAAGCCAACGTCACAGTTCTCGATACTCAGAT CCGAAAG [000135] SEQ ID NO. 37 (nt FerB): [000136] TGGCTTGATAAATTAATGCAGCTGACTGAAGAGCCACAGAACAGCATGCCTGA CATCATCATCTGGATGATCCGGGGAGAGAAGAGACTGGCCTATGCACGAATTCCCGCA CATCAGGTCTTGTACTCCACCAGTGGTGAGAATGCATCTGGAAAATACTGTGGGAAA ACCCAAACCATCTTTCTGAAGTATCCACAGGAGAAAAACAACGGGCCA [000137] SEQ ID NO. 38 (nt DysFN-1): [000138] GCTGTGGAGAAGAAGTTTAACAGCTTCGCAGAAGGAACTTTCACCGTCTTTG CTGAAATGTATGAAAATCAAGCTCTCATGTTTGGAAAATGGGGTACTTCTGGATTAGT AGGACGTCATAAGTTTTCTGATGTCACAGGAAAAATAAAACTCAAGAGGGAATTTTT TCTGCCT [000139] SEQ ID NO. 39 (nt DysFN-2): [000140] GATCCTGAAAGAAGCTTGCTGACTGAGGCAGATGCAGGTCACACGGAGTTCA CTGATGAAGTCTACCAGAACGAGAGCCGCTACCCCGGGGGCGACTGGAAGCCGGCC GAGGACACCTACACGGATGCGAACGGCGATAAAGCAGCATCACCCAGCGAGTTGAC TTGTCCT [000141] SEQ ID NO. 40 (nt TM): [000142] CCGGACCTCAAAGCCATGAACCCCCTTAAAGCCAAGACAGCCTCCCTCTTTGA GCAGAAGTCCATGAAAGGATGGTGGCCATGCTACGCAGAGAAAGATGGCGCCCGCG TAATGGCTGGGAAAGTGGAGATGACATTGGAAATCCTCAACGAGAAGGAGGCCGAC GAGAGGCCAGCCGGGAAGGGGCGGGACGAACCCAACATGAACCCCAAGCTGGACT TACCAAATCGACCAGAAACCTCCTTCCTCTGGTTCACCAACCCATGCAAGACCATGA AGTTCATCGTGTGGCGCCGCTTTAAGTGGGTCATCATCGGCTTGCTGTTCCTGCTTATC CTGCTGCTCTTCGTGGCCGTG [000143] In an embodiment, the modified myoferlin protein of the present invention further comprises a connexin 43 protein, wherein said dual protein comprising the modified myoferlin protein and the connexin 43 protein can be used to substantially enhance the efficacy and delivery of the RNA or nucleic acid payloads. In an embodiment, the connexin protein comprisesa S368A mutation. In an embodiment, the amino acid sequence of the connexin protein of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 41. [000144] SEQ ID NO. 41 (cnx43): [000145] MATTMGDWSALGKLLDKVQAYSTAGGKVWLSVLFIFRILLLGTAVESAWGDEQS AFRCNTQQPGCENVCYDKSFPISHVRFWVLQIIFVSVPTLLYLAHVFYVMRKEEKLNKK EEELKVAQTDGVNVDMHLKQIEIKKFKYGIEEHGKVKMRGGLLRTYIISILFKSIFEVAFL LIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTIFIIFMLVVSLVSLALNIIELFYVFF KGVKDRVKGKSDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSPPGYKLVTGD RNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNSKKLAA GHELQPLAIVDQRPSSRAASRASSRPRPDDLEI [000146] In an embodiment, the dual protein comprising a modified myoferlin protein fused with a connexin 43 protein is encoded by a dual protein encoding polynucleotide wherein the nucleotide sequence of the dual protein encoding polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 42. [000147] SEQ ID NO. 42: [000148] ATGGTGCCAGCCCCTCCCAGACAGTTTCGGGAATTACCTGACAGCGTCCCACA GGAATGCACGGTTAGGATTTACATTGTTCGAGGCTTAGAGCTCCAGCCCCAGGACAA CAATGGCCTGTGTGACCCTTACATAAAAATAACACTGGGCAAAAAAGTCATTGAAGA CCGAGATCACTACATTCCCAACACTCTCAACCCAGTCTTTGGCAGGATGTACGAACTG AGCTGCTACTTACCTCAAGAAAAAGACCTGAAAATTTCTGTCTATGATTATGACACCT TTACCCGGGATGAAAAAGTAGGAGAGACAATTATTGATCTGGAAAACCGATTCCTTTC CCGCTTTGGGTCCCACTGCGGCATACCAGAGGAGTACTGTGTTTCTGGAGTCAATACC TGGCGAGATCAACTGAGACCAACACAGCTGCTTCAAAATGTCGCCAGATTCAAAGGC TTCCCACAACCCATCCTTTCCGAAGATGGGAGTAGAATCAGATATGGAGGACGAGAC TACAGCTTGGATGAATTTGAAGCCAACAAAATCCTGCACCAGCACCTCGGGGCCCCT GAAGAGCGGCTTGCTCTTCACATCCTCAGGACTCAGGGGCTGGTCCCTGAGCACGTG GAAACAAGGACTTTGCACAGCACCTTCCAGCCCAACATTTCCCAGGGAAAACTTCAG ATGTGGGTGGATGTTTTCCCCAAGAGTTTGGGGCCACCAGGCCCTCCTTTCAACATCA CACCCCGGAAAGCCAAGAAATACTACCTGCGTGTGATCATCTGGAACACCAAGGATG TTATCTTGGATGAGAAAAGCATCACAGGAGAGGAAATGAGTGACATCTACGTCAAAGGCTGGGTTCCTGGCAATGAAGAAAACAAACAGAAAACAGATGTCCATTACAGATCTT TGGATGGTGAAGGGAATTTTAACTGGCGATTTGTTTTCCCGTTTGACTACCTTCCAGC CGAACAACTCTGTATCGTTGCGAAAAAAGAGCATTTCTGGAGTATTGACCAAACGGA ATTTCGAATCCCACCCAGGCTGATCATTCAGATATGGGACAATGACAAGTTTTCTCTG GATGACTACTTGGGTTTCCTAGAACTTGACTTGCGTCACACGATCATTCCTGCAAAAT CACCAGAGAAATGCAGGTTGGACATGATTCCGGACCTCAAAGCCATGAACCCCCTTA AAGCCAAGACAGCCTCCCTCTTTGAGCAGAAGTCCATGAAAGGATGGTGGCCATGCT ACGCAGAGAAAGATGGCGCCCGCGTAATGGCTGGGAAAGTGGAGATGACATTGGAA ATCCTCAACGAGAAGGAGGCCGACGAGAGGCCAGCCGGGAAGGGGCGGGACGAAC CCAACATGAACCCCAAGCTGGACTTACCAAATCGACCAGAAACCTCCTTCCTCTGGT TCACCAACCCATGCAAGACCATGAAGTTCATCGTGTGGCGCCGCTTTAAGTGGGTCAT CATCGGCTTGCTGTTCCTGCTTATCCTGCTGCTCTTCGTGGCCGTGCTCCTCTACTCTT TGCCGAACTATTTGTCAATGAAGATTGTAAAGCCAAATGTGTACCCATACGACGTCCC AGACTACGCTTAGGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCT TGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTG GCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTC TTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTC CTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGA ACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACAC CTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGA GTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTA CCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTC GAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAA AACACGATGATAATATGGCCACAACCATGGGTGACTGGAGCGCCTTAGGCAAACTCC TTGACAAGGTTCAAGCCTACTCAACTGCTGGAGGGAAGGTGTGGCTGTCAGTACTTT TCATTTTCCGAATCCTGCTGCTGGGGACAGCGGTTGAGTCAGCCTGGGGAGATGAGC AGTCTGCCTTTCGTTGTAACACTCAGCAACCTGGTTGTGAAAATGTCTGCTATGACAA GTCTTTCCCAATCTCTCATGTGCGCTTCTGGGTCCTGCAGATCATATTTGTGTCTGTAC CCACACTCTTGTACCTGGCTCATGTGTTCTATGTGATGCGAAAGGAAGAGAAACTGA ACAAGAAAGAGGAAGAACTCAAGGTTGCCCAAACTGATGGTGTCAATGTGGACATG CACTTGAAGCAGATTGAGATAAAGAAGTTCAAGTACGGTATTGAAGAGCATGGTAAGGTGAAAATGCGAGGGGGGTTGCTGCGAACCTACATCATCAGTATCCTCTTCAAGTCTA TCTTTGAGGTGGCCTTCTTGCTGATCCAGTGGTACATCTATGGATTCAGCTTGAGTGCT GTTTACACTTGCAAAAGAGATCCCTGCCCACATCAGGTGGACTGTTTCCTCTCTCGCC CCACGGAGAAAACCATCTTCATCATCTTCATGCTGGTGGTGTCCTTGGTGTCCCTGGC CTTGAATATCATTGAACTCTTCTATGTTTTCTTCAAGGGCGTTAAGGATCGGGTTAAGG GAAAGAGCGACCCTTACCATGCGACCAGTGGTGCGCTGAGCCCTGCCAAAGACTGT GGGTCTCAAAAATATGCTTATTTCAATGGCTGCTCCTCACCAACCGCTCCCCTCTCGC CTATGTCTCCTCCTGGGTACAAGCTGGTTACTGGCGACAGAAACAATTCTTCTTGCCG CAATTACAACAAGCAAGCAAGTGAGCAAAACTGGGCTAATTACAGTGCAGAACAAA ATCGAATGGGGCAGGCGGGAAGCACCATCTCTAACTCCCATGCACAGCCTTTTGATTT CCCCGATGATAACCAGAATTCAAAAAAACTAGCTGCTGGACATGAATTACAGCCACTA GCCATTGTGGACCAGCGACCTTCAAGCAGAGCCGCCAGTCGTGCCAGCAGCAGACC TCGGCCTGATGACCTGGAGATCTGA [000149] The present invention also provides a RNA delivery exosome composition comprising an exosome, one or more of any embodiment of the cargo RNA of the present invention and any embodiment of the one or more fusion proteins of the present invention wherein each of the one or more fusion proteins comprises an exosome associated transmembrane protein fused to a packaging protein, wherein the one or more cargo RNA comprises one or more package RNA and a packaging domain, wherein the packaging domain binds to the packaging protein to form a fusion protein and cargo RNA complex, and wherein the fusion protein and cargo RNA complex is packaged into the exosome. In an embodiment, the packaging protein is endogenous to a subject. In an embodiment, one of the one or more of the any embodiment of the cargo RNA comprises a package RNA distinct from the package RNA of another of the one or more of the any embodiment of the cargo RNA such that one exosome comprises two or more distinct package RNAs. In an embodiment, the package RNA comprises messenger RNAs, non-coding RNA such as but not limited to long non-coding RNAs, circular RNAs, long-hairpin RNA, antisense RNAs, siRNAs, shRNAs, miRNAs , guide RNAs (gRNAs) or a combination thereof. In an embodiment, the cargo RNA comprises the packaging domain in the 3’-end or in the 5’-end of the package RNA. In an embodiment, the exosome associated transmembrane protein comprises CD63, CD9, CD81, PTGFRN or a combination thereof. In an embodiment, thepackaging protein comprises U1a protein. In an embodiment, the packaging domain comprises a UR domain or a L2 domain. [000150] In an embodiment the one or more fusion proteins comprise CD63-U1a fusion protein. In an embodiment, the amino the acid sequence of the CD63-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 4. In an embodiment the one or more fusion proteins comprise CD81-U1a fusion protein. In an embodiment, the amino acid sequence of the CD81-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 5. In an embodiment the one or more fusion proteins comprise PTGFRN-U1a fusion protein. In an embodiment, the amino the acid sequence of the PTGFRN-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 6. [000151] In an embodiment, the RNA delivery exosome composition of the present invention comprises an exosome, one or more any embodiment of the cargo RNA of the present invention wherein the package RNA comprises a shRNA, and one or more any embodiment of the fusion proteins of the present invention wherein the at least one of the fusion protein comprises CD63- U1a of the present invention. In an embodiment, the RNA delivery exosome composition of the present invention comprises an exosome, one or more any embodiment of the cargo RNA of the present invention wherein the package RNA comprises a shRNA, and one or more of any embodiment of the fusion proteins of the present invention wherein at least one of the fusion protein comprises CD81-U1a of the present invention. In an embodiment, the RNA delivery exosome composition of the present invention comprises an exosome, one or more any embodiment of the cargo RNA of the present invention wherein the package RNA comprises a shRNA, and one or more any embodiment of the fusion proteins of the present invention wherein at least one of the fusion protein comprises PTGFRN-U1a of the present invention. [000152] In an embodiment, the RNA delivery exosome composition of the present invention comprises an exosome, one or more any embodiment of the cargo RNA of the present invention wherein the package RNA comprise an antisense RNA, and one or more any embodiment of the fusion proteins of the present invention wherein at least one of the fusion protein comprises CD63-U1a of the present invention. In an embodiment, the RNA delivery exosome composition of the present invention comprises an exosome, one or more any embodiment of the cargo RNA of the present invention wherein the package RNA comprises an antisense RNA, and one ormore any embodiment of the fusion proteins wherein at least one of the fusion proteins comprises CD81-U1a of the present invention. In an embodiment, the RNA delivery exosome composition of the present invention comprises an exosome, one or more any embodiment of the cargo RNA of the present invention wherein the package RNA comprises an antisense RNA, and one or more any embodiment of the fusion proteins wherein at least one of the fusion proteins comprises PTGFRN-U1a of the present invention. [000153] In an embodiment, the RNA delivery exosome composition of the present invention comprises an exosome, one or more cargo RNA of the present invention wherein any embodiment of the package RNA of the present invention comprises a mRNA, and one or more any embodiment of the fusion proteins wherein at least one of the fusion proteins comprises CD63-U1a of the present invention. In an embodiment, the RNA delivery exosome composition of the present invention comprises an exosome, one or more cargo RNA of the present invention wherein any embodiment of the package RNA comprises a mRNA, and any embodiment of the one or more fusion proteins wherein at least one of the fusion proteins comprises CD81-U1a of the present invention. In an embodiment, the RNA delivery exosome composition of the present invention comprises an exosome, one or more cargo RNA of the present invention wherein any embodiment of the package RNA comprises a mRNA, and one or more any embodiment of the fusion proteins wherein at least one of the fusion proteins comprises PTGFRN-U1a of the present invention. [000154] In an embodiment, the RNA delivery exosome composition of the present invention comprises an exosome, one or more cargo RNA of the present invention, and two or more distinct fusion proteins. In an embodiment, the two or more distinct fusion proteins comprise an endogenous human protein component such as but not limited to CD63-U1a, CD81-U1a, and PTGFRN-U1a, and a foreign protein component such as but not limited to CD63-L7ae, CD81- L7ae, and PTGFRN-L7ae. In an embodiment, the two or more distinct fusion proteins comprise at least two endogenous human protein components wherein each endogenous human protein component comprises CD63-U1a, CD81-U1a, or PTGFRN-U1a. In an embodiment, the two or more distinct fusion proteins comprise a CD63-U1a fusion protein and a CD81-U1a fusion protein. In an embodiment, the two or more distinct fusion proteins comprise a CD63-U1a fusion protein and a PTGFRN-U1a fusion protein. In an embodiment, the two or more distinct fusion proteins comprise a CD81-U1a fusion protein and a PTGFRN-U1a fusion protein. In anembodiment, the amino acid sequence of CD63-U1a protein is at least about 80%, 85%, 90%, 95%, or about 100% identical or similar to SEQ ID NO. 4. In an embodiment, the amino acid sequence of CD81-U1a protein is at least about 80%, 85%, 90%, 95%, or about 100% identical or similar to SEQ ID NO. 5. In an embodiment, the amino acid sequence of PTGFRN-U1a protein is at least about 80%, 85%, 90%, 95%, or about 100% identical or similar to SEQ ID NO. 6. [000155] In an embodiment, nucleotide sequence of the UR domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 7, and nucleotide sequence of the L2 domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 8. In an embodiment, the packaging domain of the present invention comprises 1 to 10 repeats of the UR or L2 domain. In an embodiment, the cargo RNA further comprises stabilizing domain comprising the OH domain and / or the MorrisMotif domain wherein the nucleotide sequence of the OH domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 9 and the nucleotide sequence of the MorrisMotif domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 10. [000156] In an embodiment, any embodiment of the cargo RNA of the exosome-based RNA delivery composition of the present invention further comprises a SINE-derived nuclear RNA LOcalizatIoN (SIRLOIN) sequences. In an embodiment, the SIRLOIN sequence is upstream of the packaging domain of the cargo RNA. In an embodiment, the cargo RNA of the exosome- based RNA delivery composition further comprises a SIRLOIN (SINE-derived nuclear RNA LOcalizatIoN) nuclear localization sequences wherein the SIRLOIN is upstream of the UR or L2 sequence of the cargo RNA. In an embodiment, nucleotide sequence of the SIRLOIN is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 15. [000157] In an embodiment, the RNA delivery exosome composition of the present invention further comprises one or more modified myoferlin protein. In an embodiment, the modified myoferlin protein is encapsulated within the exosome of any embodiment of the RNA delivery exosome composition of the present invention. In an embodiment, the modified myoferlin protein comprises C2F, C2G, transmembrane domain, or a combination thereof. In an embodiment, the modified myoferlin protein consists of C2F, C2G, and transmembrane domain. In an embodiment, the modified myoferlin protein consists of myoferlin domains selected fromthe group consisting of C2F, C2G, and transmembrane domain. In an embodiment, the amino acid sequence of the C2F domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 16. In an embodiment, the amino acid sequence of the C2G domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 17. In an embodiment, the C2G domain comprises an I-V mutation from the C2G domain of the wildtype human myoferlin protein to enhance the hydrophobicity of the C2G domain in the membrane and potentially offer enhanced stability. In an embodiment, the amino acid sequence of the transmembrane domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 18. In an embodiment, the modified myoferlin protein comprises C2A, FerA, FerB, DysFN, transmembrane domain, or a combination thereof. In an embodiment, the modified myoferlin protein consists of C2A, FerA, FerB, DysFN and transmembrane domain. In an embodiment, the modified myoferlin protein consists of myoferlin domains selected from a group consisting of C2A, FerA, FerB, DysFN and transmembrane domain. In an embodiment, any embodiment of the modified myoferlin protein of the present invention further comprises C2B, C2C, C2D, C2E, C2F, C2G domains, or a combination thereof. [000158] In an embodiment, the modified myoferlin protein does not comprise the C2A domain wherein the amino acid sequence of the C2A domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 19. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2B domain wherein the amino acid sequence of the C2B domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 24. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2C domain wherein the amino acid sequence of the C2C domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 25. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2D domain wherein the amino acid sequence of the C2D domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 26. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2E domain wherein the amino acid sequence of the C2E domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 27. In an embodiment, the modified myoferlin protein of thepresent invention does not comprise the C2F domain wherein the amino acid sequence of the C2F domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 16. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2G domain wherein the amino acid sequence of the C2G domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 17. In an embodiment, the modified myoferlin protein of the present invention does not comprise the FerA domain wherein the amino acid sequence of the FerA domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 20. In an embodiment, the modified myoferlin protein of the present invention does not comprise the FerB domain wherein the amino acid sequence of the FerB domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 21. In an embodiment, the modified myoferlin protein of the present invention does not comprise the DysFN-1 domain wherein the amino acid sequence of the DysFN-1 domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 22. In an embodiment, the modified myoferlin protein of the present invention does not comprise the DysFN-2 domain wherein the amino acid sequence of the DysFN-2 domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 23. [000159] In an embodiment, the modified myoferlin protein of the present invention is based upon a eukaryotic myoferlin protein. In some embodiments, the modified myoferlin protein of the present invention is based upon a human myoferlin protein. In an embodiment, any embodiment of the modified myoferlin protein of the present invention is in a form comprising a recombinant protein, a polypeptide, or a polynucleotide encoding said protein. In an embodiment, the modified myoferlin protein of the present invention comprises a truncated wild type human myoferlin protein. In an embodiment, the modified myoferlin protein of the present invention comprises at least a C2 domain. In an embodiment, the modified myoferlin protein of the present invention comprises at least two C2 domains. In an embodiment, the modified myoferlin protein of the present invention comprises a C2 domain, a FerA domain, a FerB domain, a DysF domain, a transmembrane domain, or a combination thereof. In an embodiment, the DysFN domain comprises DysFN-1, DysFN-2, or a combination thereof. In an embodiment, the modified myoferlin protein of the present invention comprises a myoferlin C2A domain, a myoferlin C2Fdomain, a myoferlin C2G domain, a myoferlin DysF domain, a myoferlin FerA domain, a myoferlin FerB domain, a myoferlin transmembrane domain, or a combination thereof. In an embodiment, the modified myoferlin protein of the present invention comprises a myoferlin C2A domain, a myoferlin DysF domain, a myoferlin FerA domain, and a myoferlin transmembrane domain. In an embodiment, the myoferlin DysFN domain comprises myoferlin DysFN-1, myoferlin DysFN-2, or a combination thereof. In an embodiment, the modified myoferlin protein of the present invention comprises a myoferlin C2F domain, a myoferlin C2G domain, and a myoferlin transmembrane domain. [000160] In an embodiment, any embodiment of the modified myoferlin plasmids of the present invention encodes a dual protein comprising a modified myoferlin protein and a connexin 43 protein, wherein the dual protein comprising the modified myoferlin protein and the connexin 43 protein can be used to substantially enhance the efficacy and delivery of RNA and nucleic acid payloads. In an embodiment, the connexin 43 protein comprises a S368A mutation. In an embodiment, the connexin 43 protein of the present invention comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 41. [000161] The present invention also provides a method 1000 of packaging any embodiment of the cargo RNA of the present invention into exosomes comprising the step 1010 of transfecting exosome producing cells with any embodiment of the fusion protein encoding plasmid of the present invention and one or more of any embodiment of the cargo RNA encoding plasmids of the present invention wherein the fusion protein encoding plasmid encodes any embodiment of the exosome transmembrane protein of the present invention fused to any embodiment of the U1a packaging protein of the present invention, wherein the one or more cargo RNA encoding plasmids encodes any embodiment of the one or more package RNA of the present invention and any embodiment the UR packaging domain of the present invention. Next, in step 1020, the exosome producing cells expresses the fusion protein comprising any embodiment of the exosome transmembrane protein of the present invention fused to any embodiment of the U1a packaging protein of the present invention. The exosome producing cells also expresses the one or more cargo RNA comprising any embodiment of the one or more package RNAs and one or more packaging domain. In step 1030, the fusion protein binds to the cargo RNA when the U1a packaging protein of the fusion protein binds to the UR packaging domain of the cargo RNA. Instep 1040, exosome producing cell then packages the fusion protein along with the bound cargo RNA into exosomes produced by the exosome producing cell. The packaging efficiency of packaging a cargo RNA of the present into an exosome using the method of packaging of the present invention can vary dependent on multiple factors known in the art such as but not limited to the type of fusion protein being transfected, the type of cargo RNA being transfected, the amount of the fusion protein plasmid being transfected, the amount of the cargo RNA plasmid being transfected, the ratio between the fusion protein plasmid and the cargo RNA plasmid being transfected, the confluency of the exosome producing cells, or the duration of the transfection process. In an embodiment, the method of packaging any embodiment of the cargo RNA of the present invention into exosomes further comprises the releasing of the exosomes packed using any embodiment of the method of packaging of the present invention from the exosome producing cells. [000162] In an embodiment, at least one of the one or more package RNAs comprises a package RNA distinct from another of the one or more package RNAs so that more than one distinct package RNA is packaged into one exosome. [000163] In an embodiment, the method further comprises the step 1012 of inhibiting Charged Multivesicular Body Protein 4C (CHMP4C) and / or Vacuolar Protein Sorting 4B (VPS4B) gene expression in the exosome producing cells. In an embodiment, the step 1012 of inhibiting CHMP4C and / or VPS4B gene expression in the exosome producing cells is performed by transfecting the exosome producing cells with any embodiment of the packaging boosting shRNA encoding plasmid of the present invention. In an embodiment, the number of packaging boosting shRNA encoding plasmid of the present invention to the fusion protein plasmid transfecting the exosome producing cell is from about 2:1 to about 8:1 such as about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1 or about 8:1 including any ratio and ratio ranges falling within these values. In an embodiment, the number of the packaging boosting shRNA encoding plasmid of the present invention to the fusion protein plasmid of the present invention in the EV producing cell is from about 3:1 to about 5:1. [000164] In an embodiment, the method further comprises the step 1014 of transfecting the exosome producing cells with Connexin 43 S368A plasmid. In an embodiment, the step 1014 further comprises transfecting the exosome producing cells with one or more modified myoferlin protein plasmid, wherein each of the one or more modified myoferlin protein encoding plasmidencodes a modified myoferlin protein of the present invention. In an embodiment, the one or more modified myoferlin protein encoding plasmid further encodes a connexin 43 protein or a connexin S368A protein. In an embodiment, the method further comprises the step 1016 of transfecting the exosome producing cells with an Ago2 encoding plasmid so that the exosome producing cell expresses the Ago2 protein. In an embodiment, the Ago2 protein comprises a S387A mutation. In an embodiment, the Ago2 protein complexes with the cargo RNA. [000165] Examples [000166] Experimental Materials and Method [000167] CD63-U1a DNA sequence (5’-3’) [000168] ATGGCGGTGGAAGGAGGAATGAAATGTGTGAAGTTCTTGCTCTACGTCCTCCT GCTGGCCTTTTGCGCCTGTGCAGTGGGACTGATTGCCGTGGGTGTCGGGGCACAGCT TGTCCTGAGTCAGACCATAATCCAGGGGGCTACCCCTGGCTCTCTGTTGCCAGTGGTC ATCATCGCAGTGGGTGTCTTCCTCTTCCTGGTGGCTTTTGTGGGCTGCTGCGGGGCCT GCAAGGAGAACTATTGTCTTATGATCACGTTTGCCATCTTTCTGTCTCTTATCATGTTG GTGGAGGTGGCCGCAGCCATTGCTGGCTATGTGTTTAGAGATAAGGTGATGTCAGAGT TTAATAACAACTTCCGGCAGCAGATGGAGAATTACCCGAAAAACAACCACACTGCTT CGATCCTGGACAGGATGCAGGCAGATTTTAAGTGCTGTGGGGCTGCTAACTACACAG ATTGGGAGAAAATCCCTTCCATGTCGAAGAACCGAGTCCCCGACTCCTGCTGCATTAA TGTTACTGTGGGCTGTGGGATTAATTTCAACGAGAAGGCGATCCATAAGGAGGGCTGT GTGGAGAAGATTGGGGGCTGGCTGAGGAAAAATGTGCTGGTGGTAGCTGCAGCAGC CCTTGGAATTGCTTTTGTCGAGGTTTTGGGAATTGTCTTTGCCTGCTGCCTCGTGAAG AGTATCAGAAGTGGCTACGAGGTGATGgaattcggcggaggcgggtccATGGCAGTTCCCGAG ACCCGCCCTAACCACACTATTTATATCAACAACCTCAATGAGAAGATCAAGAAGG ATGAGCTAAAAAAGTCCCTGTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGG ATATCCTGGTATCACGGAGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCA AGGAGGTCAGCAGCGCCACCAACGCCCTGCGCTCCATGCAGGGTTTCCCTTTC TATGACAAACCTATGCGTATCCAGTATGCCAAGACCGACTCAGATATCATTGCCA AGATGAAA [000169] CD63-U1a amino acid sequence [000170] MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPV VIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVT VGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSG YEVMefggggsMAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQILDILVSRSLK MRGQAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAKMK [000171] Uppercase sequence denotes CD63, lower case is linker sequence, and bold sequence is the U1 small nuclear ribonucleoprotein polypeptide A (U1 snRNP A). [000172] Table 1 L2 domains assessed in Figure 2. Underlined sequence represents the U1 snRNA stem-loop II (SL2) sequence that was inserted into a non-coding region of a mRNA for packaging into exosomes by the CD63-U1a fusion protein. Concatemers of SL2 where inserted to improve CD63-U1a affinity and exosome packaging, and the repeats of the SL2 sequence are depicted (1x, 2x, 4x, and 6x). [000173] Table 2 Plasmid Drug Function t, s. g A g [00 [00Loop name number of Sequence (5’-3’) concatemers[000177] Plasmids Cell culture and transfection. DNA sequences coding for shRNA against VPS4B and CHM4C (Table 2) were cloned under H1 promoter in pCR2.1TOPO (Thermofisher) backbone to obtain shVPS4B and shCHM4C or shVPS4B+shCHMP4C (shBooster) vector. Plasmids expressing nLuc, CD63-nLuc, and Connexin43 S368A were a kind gift from Dr Martin Fusseneger (Kojima, Bojar et al.). Using oligonucleotide cloning strategy, DNA sequence for UR was cloned at the 3’end of a nLuc expression plasmid so that the nLuc mRNA will have a UR element at the 3’end. For creating antisenseEGFP600 expressing plasmids, a DNA fragment antisense to 600 bases of EGFP towards 3’ end was cloned in pcDNA3.1(+) mammalian expression vector using the NEBuilder® HiFi DNA Assembly Mix according to the manufacturer’s instructions (New England Biolabs). Sequences for U1 promoter specific primers were used to amplify the U1 promoter and inserted in pCR4 Topo T cloning vector. Sequence of Pg1asα 24,25 was inserted downstream of a U1 promoter or CMV promoter in pCDNA3.1 as gBlocks® (Integrated DNA Technology) using the NEBuilder® HiFi DNA Assembly Mix according to the manufacturer’s instructions (New England Biolabs). UR and SIRLOIN sequences were inserted in pCDNA-Pg1asα using an oligonucleotide cloning method. All the shRNA, oligonucleotide and DNA sequences are listed in Table 4. [000178] Table 4 Name Sequence GT g g ct g g t a c A A T C C G [00 [00(Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (GeminiBio) andincubated at 37°C and 5% CO2. To generate the PTEN-Luc cells, homology arms of the PTEN promoter were created with PCR products from the HEK293 genomic DNA and cloned into a vector with multiple cloning site (PTEN-MSC-PTEN), and then cloned in the Fluc-T2A-puro into the MCS using Gibson and a Gblock (PTEN-Fluc-T2A-Puro-Bgh-PTEN). The Fluc-T2A- Puro was designed to be inserted just after the translation start of the PTEN gene. For Cas9 mediated gene-editing, gRNAs were designed to cut the target site at the start of PTEN, ordered as gBlock (IDT) and inserted into px458 (Cas9-GFP). A single Cas9 expressing vector with sgRNA was transfected with a linearized PTEN promoter and transfected into HEK293 using Lipofectamine 3000 (Thermo Scientific). The cells were then selected with puromycin and cells expressing luciferase in frame with the PTEN promoter were generated. Insertion of the Fluc at the PTEN target was confirmed by PCR. For the remaining experiments HEK293T and PTEN- Luc+HEK293T were transfected using Lipofectamine 2000 (Thermo Scientific). [000181] Trans-well assay for functional evaluation of EVs. In this set up EV producer cells were seeded in baso-lateral chamber and EV recipient cells were seeded on the apical chamber on the top of insert with 0.4 µm pore size. Nearly 300,000 HEK293T cells were seeded in basolateral chamber per well of 24 well plate (Catalog number: 140620, Thermo Scientific). These cells were transfected with total 0.5 µg plasmid and 0.5 µl Lipofectamine 2000. Simultaneously trans well inserts were arranged in unused wells and seeded with 50,000 EV recipient cells in 100 µl complete DMEM and transfected if necessary. In the unused basolateral chamber with inserts, 500 µl complete DMEM was added. After 24 hours following transfection, media was changed to 500 µl of 10% exosome-depleted FBS+ DMEM. The trans-well containing recipient cells were placed on the top of producer cells and media was changed to 100 µl of 10% exo-depleted FBS+ DMEM carefully. Contact between media from baso-lateral chamber and apical chamber cells was ensured at all times. At the desired time point of readout, cells from trans well containing insert in a separate well were trypsinized and harvested for readout. [000182] Collection, purification and exposure of extra-cellular vesicles to cells. A total of 4 x 106 HEK293T cells were seeded in a 10 cm dish and 24 hours later transfected with 24.5 µg total of plasmids (14 µg of cargo RNA with or without UR, 3.5 µg each of CD63-U1a, Connexin43 S368A, shBooster plasmids). Six hours post-transfection the cell culture medium was replaced with exosome-depleted FBS (Thermo Scientific) containing DMEM. After each 24 hours, mediawas collected until 72 hours after transfection. The collected media was filtered through 0.22 µm Ultrafree® Centrifugal Filter Units (Millipore Sigma). Filtered supernatant was then subjected to sequential spins: a low-level spin at 800 x g for 5 mins to remove cells, followed by 2,000 x g for 20 mins to remove cell debris and then concentrated to 500 µl by ultrafiltration using Amicon Ultra-15100 kDa centrifugal filter device (Merck Millipore). After rinsing the columns with PBS, 500 µl of concentrated supernatant was applied on top of a qEV column (Izon Science) and three EV-rich fractions (2nd – 4th) were pooled and either analyzed directly or concentrated using an Amicon Ultra-4100 kDa centrifugal filter device (Merck Millipore) to the required concentration for administration in mice. The size distributions and absolute number of EVs were determined using the NanoSight NS300 (Malvern). For all in vitro experiments, EV to cell ratio of 104 EVs per cell was maintained. We have submitted all relevant data of our experiments to the EV-TRACK knowledgebase (EV-TRACK ID: EV210296) (Consortium, Van Deun et al.) [000183] RT-qPCR from cells and EVs. RNA was isolated from HEK293T cells (5 x105) or EVs (1x1010) using the Maxwell® RSC simply RNA Cells Kit or Maxwell® RSC miRNA Plasma and Serum Kit (Promega) according to manufacturer instructions. Equal amounts of RNA were used for Luna® Universal One-Step RT-qPCR Kit (NEB) according to the manufacturer’s instruction. Gene specific primers sequences are listed in Supplementary Table S1. Roche® LightCycler 96 was used to perform the RT-qPCR and results analyzed using the LightCycler 96 software (Roche). The PCR conditions were as follows: reverse transcription at 55 °C for 10 min; initial denaturation, 95 °C for 3 min; denaturation, 95 °C for 30 sec; annealing, 60 °C for 20 s for 35 cycles. [000184] Luciferase assay and Zebularine treatment. Nano-Glo® Luciferase assay system (Promega) was used to determine the luciferase activity in the cell lysates as per manufacturer’s instructions. Cells in 96 well plate were washed with PBS and harvested in Nano-Glo® luciferase assay buffer. Equal volume of 1:25 prediluted Nano-Glo® luciferase substrate was added to cell lysate and luciferase activity detected on the Promega GloMax Discover Microplate Reader Detection System with GM3000 Software (Promega). The firefly luciferase activity was measured from cell lysates using the Bright-Glo Luciferase Assay System (Promega). Cells were washed in PBS and suspended in Passive Lysis buffer (Promega) before adding equal amount of reconstituted Bright-Glo Luciferase substrate. A total of 50 µl of cell lysate and 50 µl luciferase substrate suspension was used for detecting luciferase activity. Total protein in 100 µl of celllysate was measured using BCA assay kit (Thermo Scientific). Luciferase activity was normalized by total protein for luciferase readout. For Zebularine treatment, fifty thousand PTEN-Luc+HEK293 cells pretreated with sEVs or ULTRA-EVs were seeded in 96well plate in triplicate. Final concentration of 10µM Zebularine was added for 12 hours followed by changing media to complete DMEM and allowed to grow for 12 more hours. No visible sign of cell death was seen and luciferase assay was done from these cells. [000185] Nanoparticle tracking analysis (NTA). Particle number and size distribution in the different EV subpopulations were measured by NTA using a Nanosight NS300 (Malvern Panalytical, Almelo, The Netherlands) with a camera level set at 13 and a detection threshold at 5. Samples were measured in triplicate. [000186] Transmission electron microscopy (TEM). The size and morphology of EV were analyzed by TEM with an FEI Tecnai™ transmission electron microscope. Samples were absorbed onto glow discharged carbon coated 200 mesh EM grids followed by conventional negative staining with 1% (w / v) uranyl acetate. Images were collected using an FEI Tecnai 12 transmission electron microscope (Thermo Fisher Scientific) equipped with a LaB6 filament and operated at an acceleration voltage of 120 kV. Images were recorded with a Gatan 2 k × 2 k CCD camera (Gatan, Inc.) using Gatan Microscopy Suite software GMS3 at a magnification of 30,000 × and a defocus value of ~1.5 μm. [000187] Animal experiments: Mice. Female C57BL / 6 mice (strain code 027) were purchased from Charles River Laboratories about 6 to 8 weeks in age. Mice were acclimatized and maintained in accordance with the Guide for the Care and Use of Laboratory Animals and were housed in Specific Pathogen Free conditions. Animal housing follows a 12 h light / 12 h dark cycle, wherein the temperature is maintained between 68–79°F and humidity was maintained between 30–70 percent. All experiments were performed according to the guidelines of the Institutional Animal Committee of the Beckman Research Institute of the City of Hope. Toxicity and cytokine analysis were done on 10-week-old mice. Hundred billion ULTRA-EV with non- targeting asEGFP suspended in 100 µl PBS were injected in four mice retro-orbitally while 100 µl PBS was injected in another four mice as control based on previous studies14. [000188] Toxicity analysis. Twenty-four hours after treatment, approximately ~200 µl of blood was collected in heparin tubes (Greiner Bio-One) for toxicity analysis using the VetScan Preventive Care Profile Plus kit (Abaxis). Immediately following collection, whole blood of eachanimal was applied to the reagent rotor containing the assay components and inserted into the VetScan Chemistry Analyzer device (Abaxis). [000189] Luminex cytokine analysis. Serum was collected for cytokine analysis at 4 and 72 hours post-IV injection using Essential Th1 / Th2 Cytokine 6-Plex Mouse ProcartaPlex and IFN- alpha / IFN-beta 2-Plex Mouse ProcartaPlex Panels (Invitrogen #EPX060-20831-901 and EPX02A-22187-901). Panels were analyzed by the Analytical Pharmacology Core (City of Hope, Duarte, CA) using the Flexmap 3D Luminex system (Luminexcorp). Cytokine concentrations were calculated using the Bio-Plex Manager 6.2 software (Bio-Rad) with afive parameter curve-fitting algorithm applied for standard curve calculations for duplicate samples. [000190] Statistical analysis. Statistical analysis was performed with GraphPad Prism 8.3 software. Data are shown as mean ± SEM of triplicates unless otherwise indicated. Statistical analysis was performed using a two-tailed Student’s t-test or one-way ANOVA with post hoc tests, as described. A P value less than 0.05 was designated as statistically significant. [000191] Example 1 U1a-RBS to Package RNA into Cell Derived Exosomes [000192] Figure 1A illustrates an embodiment of the U1a / L2 packing mechanism of the present invention. The U1a / L2 system utilizes endogenous systems in the human cell. We have taken advantage of the U1 small nuclear ribonucleoprotein polypeptide A (U1a) and its ability to bind the U1 snRNA stem-loop 2 (L2)(2) and generated a new U1a / L2 packaging system that contains a CD63-U1a fusion protein and the L2 binding motif embedded in the 3’ UTR of the mRNA to be packaged into the exosome as shown in Figure 1A. We find that this system, with only a single SL2 motif in the 3’ UTR of luciferase works (Figures 1B-C). However, a single L2 motif was only 50% as efficient at packaging luciferase mRNA into exosomes when contrasted with the previously reported EXOtic system as shown in Figure 1C. To determine if increasing the number of L2 motifs in the 3’ UTR affects the packaging efficiency we generated various concatemers of GFP mRNAs with L23’ UTR embedded motifs shown in Figure 2. To determine the penultimate number of L2 domains to embed in the 3’ UTR to enhance packaging of GFP mRNAs into exosomes we developed several different L23’ UTR domains shown in Figure 2B. First we interrogated whether or not the 5’ or 3’ end placement of a single L2 domain affects GFP mRNA packaging. We find that both 5’ and 3’ UTRs are able to accommodate the L2 domains and that packaging efficiency seems relatively concordant between the two conditions shown in Figure 3A. Next, we interrogated to what extent increasing the number of L2 domainsaffects packaging of the GFP mRNA and find that 4 to 6 repeats of the L2 domain appears sufficient to package GFP mRNAs at levels comparable to the EXOtic system (1) shown in Figure 2C. Collectively, these data indicate that the U1a / L2 RNA packaging system is comparable to the previously reported EXOtic system and could be a better system, with regards to avoiding immunological targeting, than the EXOtic system. [000193] Example 2 Development of ULTRA-EV system for RNA packaging and delivery [000194] To enhance the release of EVs from cells, two genes from the ESCRT pathway, Charged Multivesicular Body Protein 4C (CHMP4C) and Vacuolar Protein Sorting 4B (VPS4B), were repressed using shRNA mediated RNA interference (Colombo, Moita et al.). We observed an additive effect in increasing the cellular release of EV when both CHMP4C and VPS4B mRNA levels were reduced using RNAi (Figure 4A), possibly the result of inhibiting the formation of multivesicular bodies in the EV producer cells thereby limiting EV retention and ultimately enhancing EV release. Next, to determine the optimal ratio to facilitate CD63 coupled nanoluciferase (nluc) reporter cargo packaging and EV production boosting, a co-transfection of HEK293T cells with CD63-nLuc and shBooster (shCHMP4C and shVSPB) at varying ratios of 1:1; 4:1; and 9:1 was carried out and compared to only CD63-nLuc transfected cells (Figures 5C- D). While supernatant from transfected HEK cells demonstrated an shBooster dose dependent increase in EV production (Figure 5B) assessment of nluc activity from the supernatant of transfected cells revealed that the 4:1 ratio of shBooster to CD63-nLuc was the optimal ratio for cargo packaging (Figure 5C). It is unclear why this 4:1 ratio is the optimal ratio, but having determined the optimal ratio for protein cargo packaging in this system we next sought to determine if CD63 fused to U1a can recruit and package UR containing RNAs into EVs, and if delivery of this EV packaged UR linked RNA into recipient cells can be increased by the addition of a mutant gap junction protein Connexin S368A, as observed in a previous study from our group (Villamizar, Waters et al. 2021) (Figure 4B). To determine if CD63 fused to U1a can recruit and package UR containing RNAs into EVs, and if these EVs can deliver UR linked RNAs into recipient cells, we co-transfected reporter nLuc mRNA with UR at 3’end, CD63-U1a, shBooster and Connexin S368A into producer cells, harvested the resultant supernatants and passaged them into recipient cells. Notably, we observed an additive effect of each component in increasing the overall delivery and expression of nLuc into recipient cells (Figure 4C). These data suggest that RNA with UR element can be packaged into EVs with U1a-UR interaction andthat the delivery of these RNAs into recipient cells can be significantly enhanced by shBooster and mutant Connexin S368A. This modular system was then named U1a Linked Transfer of RNA Across EV or ULTRA-EV which can functionally deliver UR containing RNAs to recipient cells. [000195] Example 3 Antisense asEGFP600 delivered using ULTRA EVs [000196] We next tested whether ULTRA EV methodology can be applied for packaging and delivery of lncRNA via EVs. An artificial lncRNA, 600 bases in length complimentary and antisense to EGFP mRNA, which can block the translation of EGFP RNA and also contained the UR sequence at the 3’ end, was designed to be expressed under a CMV promoter (Figure 9A). Following co-transfection of plasmid expressing EGFP-Luc and asEGFP lncRNA, we observed a notable decrease in luciferase activity. Owing to the higher sensitivity of luciferase activity and 10 to 30 times shorter half-life of luciferase compared to EGFP (Ignowski and Schaffer 2004, Danhier, Krishnamachary et al. 2015), we found pronounced differences in luciferase activity after treating with asEGFP600 lncRNA (Figure 9B). To further validate that EVs were operative in the transfer of asEGFP600, a trans-well assay was set up whereby the EV secreting cells were seeded in basolateral chamber of trans-well containing cell culture dish and EGFP-Luc transfected cells were seeded in the apical chamber which received EVs from cells below through a 0.4 µm membrane. Following the exchange of EVs between ULTRA-EV producing cells and recipient cells, significant reduction of luciferase activity was seen in EGFP-Luc+ cells in apical chamber placed over cells producing ULTRA EVs with asEGFP600-UR compared to only asEGFP600 (Figure 9C). EV were harvested from supernatant of HEK293T cells which were previously transfected with either pcDNA; asEGFP600; asEGFP600 with ULTRA-EV components or asEGFP600-UR with ULTRA-EV components. Significantly higher incorporation of asEGFP600-UR lncRNA was seen in EVs enriched from HEK293T cells transfected with ULTRA-EV component compared to other parameters via RT-qPCR from total RNA extracted from EV (Figure 9D). Supporting the previous trans well experimental observation, ULTRA-EVs enriched from supernatants of HEK293T cells transfected to package asEGFP600-UR lncRNA demonstrated a similar effect in reducing luciferase activity in EGFP- Luc transfected cells when contrasted with controls (Figure 9E). These data confirm delivery of functional lncRNA via ULTRA-EVs, however to what extent these EV could be used in vivo and prove safe with regards to any RNA induced immune response remained largely unknown.[000197] Example 4 ULTRA-EV: U1a Linked Transfer of RNA Across EV does not increase acute toxicity or inflammation. [000198] To determine any off target immune effects of lncRNA packaged EVs a dose of 100 billion EVs was selected based on previous observations whereby delivery of reporter or therapeutic cargo appeared well tolerated in mice (Shrivastava, Ray et al. 2021). Prior to injection, enriched ULTRA-EV with non-targeting asEGFP600 lncRNA was confirmed for their size distribution using nanosight tracking analysis (Figure 10A) and morphology by transmission electron microscope (Figure 10B). Twenty-four hours following a single dose of 100 billion ULTRA-EVs containing the non-targeting asEGFP600 lncRNA cargo, serum biochemistry parameters including blood urea nitrogen (BUN), creatinine (CRE), alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bilirubin (TBIL), glucose (GLU), total calcium (CA), protein (Sonkoly, Bata-Csorgo et al.), albumin (ALB), globulin (GLOB), sodium (NA+), potassium (K+) chloride (CL-) and total carbon dioxide (tCO2) were measured and compared to PBS injected mice (Figure 3A). Notably, all readout values for blood clinical chemistry were found to be within reference intervals reported by others (Otto, Rathkolb et al. 2016). Several cytokines including interleukin (IL) 4; 5 and 6; interferon gamma (IFN Ɣ); Tumor Necrosis Factor (TNFα); IFNα and IFNβ were also measured from blood drawn from the ULTRA-EV or PBS treated control mice after 4 hours (Figure 3B) and 72 hours (Figure 3C) and no sign of acute or delayed onset of inflammation was observed. Collectively these data suggest that the ULTRA-EVs are well tolerated and do not appear to induce any notable toxicities or immune stimulatory properties in vivo. [000199] Example 5 Silencing of PTEN promoter by Pg1asα lncRNA-ULTRA-EV [000200] Previous studies have highlighted the role of PTEN pseudogene 1 antisense RNA 1 alpha (Pg1asα) in controlling PTEN transcription by directing epigenetic regulatory proteins to the PTEN promoter (Johnsson, Ackley et al. 2013, Lister, Shevchenko et al. 2017). An HEK293 cell line was created in which PTEN promoter drives expression of firefly luciferase. We validated this new cell line and found that when Pg1asα is expressed under a CMV promoter there is significant repression of PTEN promoter driven luciferase gene expression as measured on days 4 and 6 after transfection when compared to control (Figure 8A). Notably, Pg1asα expressed from the human U1 small nuclear RNA promoter did not show significant repression of PTEN promoter activity. Next, Pg1asα was expressed along with the UR sequence at the 3’UTR asPg1asα-UR to enhance Pg1asα packaging in the ULTRA-EV. The Pg1asα containing ULTRA- EV was also unable to repress PTEN promoter activity. As EV cargo is known to be delivered into the cytoplasm 32 or fuse with endo / lysosomal membranes to reach the cytoplasm (Heusermann, Hean et al.), we surmised that perhaps the Pg1asα packaged into the ULTRA-EVs was being limited by the biology of EV delivery and unable to localize to the nucleus to target the PTEN promoter. To assess this notion and enable nuclear localization of the ULTRA-EV delivered Pg1asα-UR, a SIRLOIN (SINE-derived nuclear RNA LOcalizatIoN) nuclear localization sequences was added upstream of the UR sequences and expressed as Pg1asα-SU 34,35. The Pg1asα-SU was found to functionally repress PTEN promoter driven luciferase activity comparable to Pg1asα (Figure 8B). Next, we evaluated the effect of the ULTRA-EVs in the trans-well experimental system, whereby the ULTRA-EV producer cells are in the basolateral chamber and the PTEN-Luc HEK293T reporter cells were seeded in the apical chamber (Figure 8C). In this system we observed that the ULTRA-EV containing Pg1asα-SU significantly repressed PTEN promoter driven luciferase activity compared to Pg1asα or control (Figure 8C). To confirm this observation, ULTRA-EV were enriched from supernatants of the ULTRA-EV producer cells and added directly to HEK293T cells. In the HEK293T cells treated with EV or ULTRA-EV containing Pg1asα or Pg1asα-SU, Pg1asα or Pg1asα-SU were detectable from whole cell RNA extracted after 4 hours of EV treatment confirming the delivery of lncRNA via ULTRA-EV to recipient cells (Figure 8D). However, significantly higher localization of Pg1asα- SU was observed in the nuclear fraction of ULTRA EV-treated cells compared to Pg1asα or control confirming the ability of SIRLOIN sequence in redirecting the lncRNA to nucleus in EV treated cells (Figure 8E). To further confirm these observations enriched ULTRA-EVs were added daily to PTEN-Luc HEK293T cells in a ratio of 10,000 EVs to 1 cell for five consecutive days and a significant repression of PTEN promoter driven luciferase activity was observed (Figure 8F). Lastly, since Pg1asα is known to repress PTEN promoter via DNA methylation, we assessed to what extent the DNA methylation via ULTRA-EV delivered Pg1asα-SU could be reversed by adding the DNA methylation inhibitor Zebularine. Notably, 10 µM Zebularine treatment was found to induce luciferase activity from cells wherein PTEN promoter was repressed by ULTRA-EV containing Pg1asα-SU as well as control cells, suggesting that the ULTRA-EV delivered Pg1asα-SU is operative in a manner concordant with RNA directed TGS (Weinberg and Morris 2016)(Figure 8G).[000201] We present observations here that cells can be engineered to package lncRNAs into EVs and that transcription modulating lncRNAs can be packaged and delivered by EVs to target cells. The ULTRA-EV approach is modular and is an advancement in the field in the terms of increased production of EVs as well as packaging of nuclear regulatory RNAs. Notably, the ULTRA EVs were capable of transferring cargo between cells and the EV cargo was primarily delivered to cytoplasm in the recipient cell. We hypothesized that as Pg1asα-SU is overexpressed in producer cells, it interacts with CD63-U1a and gets packaged in ULTRA-EVs rather than cycling into the nucleus. However, upon delivery to recipient cells the SIRLOIN sequence facilitates the nuclear localization of the EV packaged RNA and in the example presented here allowed for Pg1asα mediated PTEN promoter silencing. Applications of PTEN knockdown in neuro-regeneration has been shown previously. PTEN silencing via siRNA was shown to counteract the inflammatory response induced by oxygen and glucose deprivation during ischemic stroke36. Deletion of PTEN using AAV delivered Cre, in retinal ganglion cells of wildtype mice led to robust axon regeneration after optical nerve injury (Park, Liu et al. 2008). However, notably the promoter targeted endogenous lncRNA Pg1asα-SU is presumably more robust with regards to gene silencing than siRNA 36, as Pg1asα-SU is acting on promoter to direct epigenetic silencing, without introducing changes in the genome as has been done previously with Cre-recombinase (Park, Liu et al. 2008). [000202] The ULTRA-EV system is not without inherent limitations. Firstly, unlike mRNA, lncRNA delivered through ULTRA-EVs cannot make more copies of functional molecules in recipient cells. This might lead to a stoichiometrically insufficient number of lncRNAs delivered and little to no observable effect. For instance, we added purified ULTRA-EVs daily for 5 days to see repression of the targeted gene promoter. To compensate for this, repeated dosing of saturating amounts of EVs may be needed to see an effect. Secondly although packaging and delivery of lncRNAs was observed, the extent of promoter silencing exerted by ULTRA-EV delivered Pg1asα-SU varied highly between technical replicates and this could be the result of the SU system engaging endogenous U1a containing RNAs. As such we were able to observe biologically effective but statistically insignificant repression of PTEN promoter activity in some replicates intermittently. These observations can be attributed to variability in Pg1asα-SU packaging efficiency. However, this dichotomy can be assessed and the bona fide activity of those EVs containing the RNA determined by normalizing the effect of ULTRA-EV by totalnumber of Pg1asα-SU molecules packaged rather than total number of ULTRA-EVs, as many EVs were lacking in packaged RNA thus confounding the overall efficacy of the approach. Overall, a biologically repressive effect in PTEN promoter driven gene expression was observed via either overexpression of Pg1asα-SU or via ULTRA-EV mediated delivery. However, complete knockdown of PTEN may not be advantageous either, as complete repression of PTEN in transgenic mice led to an increased excitatory synapse formation on neurons, rendering them hyperexcitable and susceptible to seizures 39. Thus, subtle repressive effects of Pg1asα-SU may be desirable to realize its neuro-regenerative potential. Based on the observations presented here future research will require generation of a means to improve the EV packaging efficiency of the lncRNAs as well enhancing directed uptake of ULTRA-EVs into target cells. [000203] Another noteworthy experimental observation from this work is the development and utilization of transwell systems for screening the optimal combinations of ULTRA-EV components for functional cargo exchange. While transwells have been used previously for EV mediated reporter assays (de Jong, Murphy et al.), we find here that the transwell system provides a reproducible preliminary indication of the effects of engineered EVs on target cells and reduces nearly one week of efforts and resources of having to collect, purify, characterize and regular treatment of EVs in target cells before observing any effects. Exchange of EV through transwells also mimics the natural cellular dynamics better. While the effects seen due to transwell mediated EV exchange may also be confounded by other cellular secreted factors, the ULTRA-EV mediated RNA delivery was confirmed by adding cell derived manually enriched EVs to the target cells, which notably corroborated with effects seen with the transwell systems. Collectively, these observations strongly support the notion that transwell based systems are a useful tool in assessing EV dynamics and function in vitro. [000204] Example 6 EV packaging of shRNAs [000205] RNA interference [3] is a mechanism imbued in mammalian cells that can specifically turn off the production of proteins in cells in a sequence-specific and potent manner. In general, it works via the introduction of small-interfering RNAs (siRNA,15-30bp of double stranded RNA) that specifically target mRNAs via sequence complementarity, causing their subsequent degradation [4]. We and others have previously demonstrated that siRNAs targeted to the LTR of HIV, can result in transcriptional gene silencing, a form of RNA targeted silencing that can lock in silent state epigenetic marks to the LTR [5-7].[000206] To date multiple groups have presented methods to package siRNAs and shRNAs into EVs, ranging from direct transfection [8] to the embedding of a particular micro (mi)RNA scaffold (miR451 and mi155) into the siRNA [9]. We found that direct transfection of siRNAs into EVs greatly distorts the EVs and that the miRNA scaffolding approach was not reproducible in our hands. As such we turned to an approach to use Ago-dependent small-hairpin RNAs (shRNA) and overexpression of Ago2, which is naturally enriched into EVs

[0010] , to enhance shRNA packaging into EVs. The shRNAs are processed by Ago2 making this approach programmable and a distinct advantage over miRNA scaffolds. To confirm this system, we expressed an Ago-shRNA targeted to green fluorescent protein (GFP) (Ago-shGFP) (Fig. 7A) and Ago2-shGFP is enriched ~60-450 fold in the EVs above the levels observed in the cells (Fig. 7B). Next, we modified the shRNA scaffold and observed that modifications of the flanking sequence further increased the mature RNAi effector in EVs (Fig. 7C) and observed that Ago2 containing the S387A mutation facilitates increased EV packaging of the shGFP (Fig. 7D). To determine if the EV packaged shRNAs were functional upon uptake in target cells, EVs were purified from shRNA / Ago2 transfected HEK293 cells and added to HEK293 cells stably expressing GFP (HEK293-GFP) and a ~50% reduction in GFP was observed (Fig. 7E), suggesting that the Ago-2 shRNAs packaged into these EVs remain functional in the target cells. Collectively, these data demonstrate that we can package shRNAs into EVs and these EVs can deliver functional shRNAs to target cells. In this application we will develop the previously validated LTR targeted (sh362), package these shRNAs into EVs and determine the ability of these EVs to epigenetically repress virus expression in vitro and in vivo. [000207] Example 7 EV transfer of antisense RNAs targeting influenza virus PB1 protein [000208] To determine the efficacy of EV transfer of RNAs the repression of the PB1-GFP reporter gene was assessed. Repression of this transgene indicates the fidelity of asRNA transfer as the asRNA represses the transgene and therefore GFP expression in the recipient reporter cells as determined by qRT-PCR at various times post co-culture in transwell plates. Various combinations of packaging constructs, e.g. CD63-U1a, PTGFRN-U1a, CD81-U1a, CD63-L7ae were assessed (Table 5) for delivery of antisense RNAs targeted to Flu (as1 and as2) or delivery of GFP mRNA. All fusion constructs were found to be functional, and the ability to package particular RNAs, e.g. as2 vs. as1 (Figure 13) vs. GFP mRNAs (Figure 14). Notably all the U1acontaining constructs demonstrated effective RNA-EV repression of reporter gene expression (Figure 13), suggesting that CD63, CD81 and PTGRFN U1a fusions are functional standouts in the RNex platform that can be used to package therapeutic RNAs, antisense non-coding RNAs and mRNAs or various combinations or RNAs. Interestingly, we find that there are notable differences between the various packaged RNAs, e.g. asRNA (as1 flu and as2 flu)(Figure 13) vs. mRNA (GFP)(Figure 14), whereby only the CD63-and CD81 U1a fusions worked for both asRNAs and GFP mRNA. This observation demonstrates that the 3’ UTR tag, presents differently to the fusion proteins, and that these differences may likely be due to the upstream 5’ RNA sequence appended to the 3’ UTR tag (Table 6), as the 3’ UTR is consistent across the various EV packaged RNAs, whereas the upstream 5’ UTRs are different sequences. We also observed that dual packaging was inefficient with the GFP mRNA (Figure 15) but found to be effective with non-coding RNAs, specifically the PTGFRN-U1a and CD63-L7ae dual packaging observed in Figure 13. Collectively, these data suggest that our RNex packaging system is functional relative to the control L7ae system, but superior as the U1a / UR system uses endogenous Human protein components (e.g. U1a), whereas the L7ae is a foreign RNA binding protein. We also note that the RNeX packaging system is varied and that each RNA packaged needs to be assessed with the various fusion proteins to determine the optimal packer. [000209] Table 5 Plasmid combinations transfected into HEK293 producer cells for knockdown of the GFP reporter gene by the asRNA (as2). Sample (IG-002) Plasmid to co-transfect into HEK293 cells[00ATCC ATAGCGGTAGAAAAAGCTCGTGAATTCAAATGTCCCTGTCCGATTTATGTAAGACTTC TTCTTGCTCATATGGATCCGGGTACCGTGATCCGAAAGGTGAGTACCCTTCGAATCCAT TGCACTCCGGATTTTTGA [000211] as1-PB1: TCATTAATTCCAGACACTCCAAAACTGGGCAGCTCCATCTGAAATTGGCTACAAATCC ATAGCGGTAGAAAAAGCTCGTGAATTCAAATGTCCCTGTCCGATTTATGTAAGACTTC TTCTTGCTCATAT [000212] Table 6 Domains required for RNex packaging. The bold underlined text represents the bridge sequence between the U1a and CD domains and the entire U1a / CD motif depicted in boldthat can be amended to the 3’Tail domain to engineer the packaging of any RNA, including mRNA and / or non-coding RNAs into exosomes by CD63, CD81 or PTGFRN fusions. ’ ’ T[000213] Example 8 EV transfer of shRNAs targeting influenza virus PB1 / PB2 protein [000214] The following plasmid combinations (Table 7 and Table 8) were used to generate shEV producer HEK293 cells, which were grown in transwell plate with IG-002 or IG-008 reporter cell lines. Recipient cells were collected at 72hrs post-co-culturing and qRTPCR carried out for GFP / beta-actin mRNA expression (Figure 16). All value represents fraction of untreated reporter cells alone (±SEM, n=3). P values from paired T-tests are shown. [000215] Table 7 Those plasmids used to generate the stable shRNA-EV cell system. Plasmids 1-3 are transfected into desired producer cells and the cultures then drug selected. The cells are then triple drug selected and populations or single cells selected to result in the stable shRNA-EV producer lines. Plasmid Drug Function selection resistance anng A og[000216] Table 8 Plasmid combinations used to generate shRNA packaged EVs. All shRNAs require p88 and p87 in all formulations to enhance shRNA packaging and endosomal release. Sample Plasmid to co-transfect into HEK293 cells and drug selec3on DNA total l [00 GGCT [00 CCCG[000219] It can be appreciated by those skilled in the art that changes could be made to the examples described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular examples disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims. 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Mol Neuropsychiatry 5(Suppl 1): 60-71. Sonkoly, E., Z. Bata-Csorgo, A. Pivarcsi, H. Polyanka, A. Kenderessy-Szabo, G. Molnar, K. Szentpali, L. Bari, K. Megyeri, Y. Mandi, A. Dobozy, L. Kemeny and M. Szell (2005). "Identification and characterization of a novel, psoriasis susceptibility-related noncoding RNA gene, PRINS." J Biol Chem 280(25): 24159-24167. Villamizar, O., S. A. Waters, T. Scott, N. Grepo, A. Jaffe and K. V. Morris (2021). "Mesenchymal Stem Cell exosome delivered Zinc Finger Protein activation of cystic fibrosis transmembrane conductance regulator." J Extracell Vesicles 10(3): e12053. Wang, X., Y. Chen, Z. Zhao, Q. Meng, Y. Yu, J. Sun, Z. Yang, Y. Chen, J. Li, T. Ma, H. Liu, Z. Li, J. Yang and Z. Shen (2018). "Engineered Exosomes With Ischemic Myocardium-Targeting Peptide for Targeted Therapy in Myocardial Infarction." J Am Heart Assoc 7(15): e008737. Weinberg, M. S. and K. V. Morris (2016). "Transcriptional gene silencing in humans." Nucleic Acids Res 44(14): 6505-6517. Zhang, J., X. Zhang, C. Li, L. Yue, N. Ding, T. Riordan, L. Yang, Y. Li, C. Jen, S. Lin, D. Zhou and F. Chen (2019). "Circular RNA profiling provides insights into their subcellular distribution and molecular characteristics in HepG2 cells." RNA Biol 16(2): 220-232. Zheng, Y., R. He, P. Wang, Y. Shi, L. Zhao and J. Liang (2019). "Exosomes from LPS-stimulated macrophages induce neuroprotection and functional improvement after ischemic stroke by modulating microglial polarization." Biomater Sci 7(5): 2037-2049.Zhu, X., M. Badawi, S. Pomeroy, D. S. Sutaria, Z. Xie, A. Baek, J. Jiang, O. A. Elgamal, X. Mo, K. Perle, J. Chalmers, T. D. Schmittgen and M. A. Phelps (2017). "Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells." J Extracell Vesicles 6(1): 1324730. Zhuang, L., W. Xia, D. Chen, Y. Ye, T. Hu, S. Li and M. Hou (2020). "Exosomal LncRNA- NEAT1 derived from MIF-treated mesenchymal stem cells protected against doxorubicin- induced cardiac senescence through sponging miR-221-3p." J Nanobiotechnology 18(1): 157. Yang, Y., Hong Y., Cho E., Kim G., and Kim I. (2018). “Extracellular vesicles as a platform for membrane-associated therapeutic protein delivery.” J Extracell Vesicles. 2018; 7(1): 1440131.

Claims

What is claimed is:

1. An exosome-based RNA package and delivery system comprising an exosome producing cell, one or more cargo RNA encoding plasmids and one or more fusion protein encoding plasmids wherein the one or more cargo RNA encoding plasmids each encodes a cargo RNA wherein the cargo RNA comprises a package RNA and a packaging domain; wherein the one or more fusion protein encoding plasmids each encodes a fusion protein wherein the fusion protein comprises an exosome associated transmembrane protein fused to a packaging protein; wherein the exosome producing cell is capable of expressing the cargo RNA based on the one or more cargo RNA encoding plasmids when the exosome producing cell is transfected with the one or more cargo RNA encoding plasmids; wherein the exosome producing cell is capable of expressing the fusion protein based on the fusion protein encoding plasmid when the exosome producing cell is transfected with the one or more fusion protein encoding plasmids; wherein the packaging protein and the packaging domain are capable of binding to one another to form a fusion protein cargo RNA complex; wherein the exosome producing cell is capable of producing one or more exosomes; wherein the exosome producing cell is capable of packaging the fusion protein cargo RNA complex into the one or more exosomes; and wherein the packaging protein is endogenous to a subject.

2. The system of claim 1, wherein the packaging protein comprises U1a protein.

3. The system of claim 1, wherein the packaging domain comprises UR domain or L2 domain wherein nucleotide sequence of the UR domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 7 and wherein nucleotide sequence of the L2 domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO.

8.

4. The system of claim 3, wherein the packaging domain of the present invention comprises 1 to 10 repeats of the UR domain or the L2 domain.

5. The system of claim 1, wherein the package RNA comprises messenger RNAs (mRNA), non-coding RNAs, or a combination thereof, wherein the non-coding RNAs comprises long non-coding RNAs (lncRNA), circular RNAs, long-hairpin RNA, antisense RNAs, small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), guide RNAs (gRNAs) or a combination thereof.

6. The system of claim 1, wherein the exosome associated transmembrane protein comprises CD63, CD9, CD81, PTGFRN or a combination thereof.

7. The system of claim 1, wherein at least one of the one or more cargo RNA encoding plasmids encodes a package RNA distinct from package RNA encoded by another of the one or more cargo RNA encoding plasmids such that the exosome producing cell is capable of packaging two or more distinct package RNAs into one of the one or more exosomes produced by the exosome producing cell.

8. The system of claim 1, wherein the one or more fusion protein encoding plasmids comprise a CD63-U1a fusion protein encoding plasmid that encodes a CD63-U1a fusion protein, a CD81-U1a fusion protein encoding plasmid that encodes a CD81-U1a fusion protein, or a PTGFRN-U1a protein encoding plasmid that encodes a PTGFRN-U1a fusion protein.

9. The system of claim 8, wherein nucleotide sequence of the CD63-U1a fusion protein encoding plasmid is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 1, wherein nucleotide sequence of the CD81-U1a fusion protein encoding plasmid is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 2, and wherein nucleotide sequence of the PTGFRN-U1a fusion protein encoding plasmid is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO.

3.

10. The system of claim 8, wherein the amino acid sequence of the CD63-U1a fusion protein is about at least 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 4, wherein the amino acid sequence of the CD81-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 5 and wherein the amino acid sequence of the PTGFRN-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 6.

11. The system of claim 1, wherein at least one of the one or more fusion protein encoding plasmids comprises a CD63-U1a fusion protein encoding plasmid that encodes a CD63- U1a fusion protein and another one of the one or more fusion protein encoding plasmids comprises a CD81-U1a protein encoding plasmid that encodes a fusion CD81-U1a protein, wherein the amino acid sequence of the CD63-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 4, and wherein amino acid sequence of the CD81-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO.

5.

12. The system of claim 1, wherein at least one of the one or more fusion protein encoding plasmids comprises a CD63-U1a fusion protein encoding plasmid that encodes a CD63- U1a fusion protein and another one of the one or more fusion protein encoding plasmids comprises a PTGFRN-U1a fusion protein encoding plasmid that encodes a PTGFRN-U1a fusion protein, wherein the amino acid sequence of the CD63-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 4, and wherein amino acid sequence of the PTGFRN-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO.

6.

13. The system of claim 1, wherein at least one of the one or more fusion protein encoding plasmids comprises a CD81-U1a fusion protein encoding plasmid that encodes a CD81- U1a fusion protein and another one of the one or more fusion protein encoding plasmids comprises a PTGFRN-U1a fusion protein encoding plasmid that encodes a PTGFRN-U1a fusion protein, wherein the amino acid sequence of the CD81-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 5, and wherein amino acid sequence of the PTGFRN-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO.

6.

14. The system of claim 1, wherein the one or more of the cargo RNA encoding plasmids further comprise one or more stabilizing domain comprising OH domain and / or MorrisMotif domain wherein the nucleotide sequence of the OH domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 9 andthe nucleotide sequence of the MorrisMotif domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO.

10.

15. The system of claim 1, further comprising boosting shRNA encoding plasmids wherein the packaging boosting shRNA encoding plasmids encode a packaging boosting shRNA capable of inhibiting CHMP4Cgene, VPS4B gene, or a combination thereof of the exosome producing cell when the exosome producing cell is transfected with the one or more packaging boosting shRNA encoding plasmids.

16. The system of claim 15, wherein the nucleotide sequence of the packaging boosting shRNAs is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, or a combination thereof.

17. The system of claim 15, wherein the ratio of the number of the packaging boosting shRNA encoding plasmids to the number of fusion protein encoding plasmids used in transfecting the exosome producing cell is from about 2:1 to about 8:

1.

18. The system of claim 1, further comprising one or more modified myoferlin protein encoding plasmids that each encodes a modified myoferlin protein.

19. The system of claim 18, wherein the modified myoferlin protein comprises C2F, C2G, transmembrane domain, or a combination thereof.

20. The system of claim 19, wherein the modified myoferlin protein comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO.

28.

21. The system of claim 18, wherein the modified myoferlin protein comprises C2A, FerA, FerB, DysFN, transmembrane domain, or a combination thereof, wherein the DysFN domain comprises a DysFN-1 domain, a DysFN-2 domain, or a combination thereof.

22. The system of claim 18, wherein the modified myoferlin further comprises a connexin 43 protein.

23. The system of claim 22, wherein the connexin 43 protein comprises a S368A mutation.

24. The system of claim 1, further comprising Ago2 protein encoding plasmids wherein the exosome producing cells express Ago 2 protein when transfected with the Ago2 protein encoding plasmids and wherein the Ago2 proteins expressed by the exosome producing cell is capable of complexing with the cargo RNA.

25. The system of claim 24, wherein the Ago2 protein comprises a S387A mutation.

26. The system of claim 1, wherein the one or more cargo RNA encoding plasmids further comprises a SINE-derived nuclear RNA LOcalizatIoN (SIRLOIN) sequence wherein the SIRLOIN sequence is upstream of the packaging domain of the one or more cargo RNA, and wherein the nucleotide sequence of SIRLOIN is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO.

15.

27. A method of packaging cargo RNA in exosomes using the system of claim 1 comprising the steps of: a. transfecting the exosome producing cell with the one or more fusion protein encoding plasmids and the one or more cargo RNA encoding plasmids, wherein the one or more fusion protein encoding plasmids each encodes an exosome transmembrane protein fused to U1a packaging protein and wherein the one or more cargo RNA encoding plasmids each encodes a cargo RNA comprising a package RNA and a packaging domain; b. incubating the exosome producing cell to express the one or more fusion proteins and the one or more cargo RNA; c. forming fusion protein-cargo RNA complex when the U1a packaging protein of the expressed fusion protein binds to the UR domain of the expressed cargo RNA; and d. packaging the fusion protein cargo RNA complex into exosomes produced by the exosome producing cell.

28. The method of claim 27, wherein at least one of the one or more cargo RNA encoding plasmids encodes a package RNA distinct from package RNA encoded by another of the one or more cargo RNA encoding plasmids such that the exosome producing cell is capable of packaging two or more distinct package RNAs in a single exosome.

29. The method of claim 27, wherein the one or more cargo RNA further comprise one or more stabilizing domains comprising a OH domain and / or a MorrisMotif domain wherein the nucleotide sequence of the OH domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 9 and the nucleotide sequence of the MorrisMotif domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 10.

30. The method of claim 27, wherein the one or more cargo RNA further comprises a SINE- derived nuclear RNA LOcalizatIoN (SIRLOIN) sequence wherein the SIRLOIN sequence is upstream of the packaging domain of the one or more cargo RNA, and wherein the nucleotide sequence of SIRLOIN is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO.

15.

31. The method of claim 27, wherein the transfecting step further comprises transfecting the exosome producing cells with packaging boosting shRNA encoding plasmids that causes the exosome producing cells to express packaging boosting shRNA capable of inhibiting Charged Multivesicular Body Protein 4C (CHMP4C) gene, Vacuolar Protein Sorting 4B (VPS4B) gene expression, or a combination thereof in the exosome producing cells to increase production of exosomes by the exosome producing cells.

32. The method of claim 31, wherein the nucleotide sequence of the packaging boosting shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, or a combination thereof.

33. The method of claim 31, wherein the ratio of the number of the packaging boosting shRNA encoding plasmids to the number of fusion protein encoding plasmids used in the step of transfecting the exosome producing cell is from about 2:1 to about 8:

1.

34. The method of claim 27, wherein the transfecting step further comprises transfecting the exosome producing cells with one or more modified myoferlin protein encoding plasmids, wherein each of the one or more modified myoferlin protein encoding plasmids each encodes a modified myoferlin protein.

35. The method of claim 34, wherein the modified myoferlin protein comprises C2F, C2G, transmembrane domain, or a combination thereof.

36. The method of claim 35, wherein the modified myoferlin protein comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO.

28.

37. The method of claim 34, wherein the modified myoferlin protein comprises C2A, FerA, FerB, DysFN, transmembrane domain, or a combination thereof, wherein the DysFN domain comprises a DysFN-1 domain, a DysFN-2 domain, or a combination thereof.

38. The method of claim 34, wherein the one or more modified myoferlin protein further comprises a connexin 43 protein.

39. The method of claim 38, wherein the connexin 43 protein comprises a S368A mutation.

40. The method of claim 27, wherein the transfecting step further comprises transfecting exosome producing cells with Ago2 encoding plasmid that encodes the Ago2 protein.

41. The method of claim 40, wherein the Ago2 protein comprise S387A mutation.

42. A RNA delivery exosome composition comprising an exosome, one or more cargo RNAs and one or more fusion proteins wherein each of the one or more fusion proteins comprises an exosome associated transmembrane protein fused to a packaging protein; wherein each of the one or more cargo RNAs comprises a package RNA and a packaging domain; wherein at least one of the one or more packaging domain binds to at least one of the one or more packaging proteins to form one or more fusion protein cargo RNA complexes; wherein the one or more fusion protein cargo RNA complexes are packaged into the exosome; and wherein the one or more packaging proteins is endogenous to a subject.

43. The exosome composition of claim 42, wherein at least one of the package RNA of the one or more cargo RNA is distinct from package RNA of another of the one or more cargo RNA such that the exosome comprises two or more distinct package RNAs.

44. The composition of claim 42, wherein the one or more cargo RNA further comprises one or more stabilizing domain comprising the OH domain and / or the MorrisMotif domain wherein the nucleotide sequence of the OH domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 9 and the nucleotide sequence of the MorrisMotif domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO.

10.

45. The composition of claim 42, wherein each of the one or more cargo RNA further comprises a SINE-derived nuclear RNA LOcalizatIoN (SIRLOIN) sequence wherein the SIRLOIN sequence is upstream of the packaging domain of the one or more cargo RNA,wherein the nucleotide sequence of SIRLOIN is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO.

15.

46. The composition of claim 42, wherein the packaging protein comprises a U1a protein.

47. The composition of claim 42, wherein the packaging domain comprises a UR domain or a L2 domain.

48. The composition of claim 47, wherein nucleotide sequence of the UR domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 7 and nucleotide sequence of the L2 domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO.

8.

49. The composition of claim 47, wherein the packaging domain comprises 1 to 10 repeats of the UR domain or the L2 domain.

50. The composition of claim 42, wherein the package RNA comprises messenger RNAs (mRNA), non-coding RNAs, or a combination thereof, wherein the non-coding RNAs comprises long non-coding RNAs (lncRNA), circular RNAs, long-hairpin RNA, antisense RNAs, small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), guide RNAs (gRNAs) or a combination thereof.

51. The composition of claim 42, wherein the exosome associated transmembrane protein comprises CD63, CD9, CD81, PTGFRN or a combination thereof.

52. The composition of claim 42, wherein the one or more fusion proteins comprise CD63- U1a fusion protein, CD81-U1a fusion protein or PTGFRN-U1a fusion protein.

53. The composition of claim 52, wherein amino the acid sequence of the CD63-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 4, wherein amino the acid sequence of the CD81-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 5 and wherein amino the acid sequence of the PTGFRN-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO.

6.

54. The composition of claim 52, wherein one of the one or more fusion protein comprises CD63-U1a protein and another of the one or more fusion protein comprises CD81-U1a protein, wherein the amino acid sequence of the CD63-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQID NO. 4, and wherein amino acid sequence of the CD81-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO.

5.

55. The composition of claim 52, wherein one of the one or more fusion protein comprises CD63-U1a protein and another of the one or more fusion protein comprises PTGFRN- U1a protein, wherein the amino acid sequence of the CD63-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 4, and wherein amino acid sequence of the PTGFRN-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO.

6.

56. The composition of claim 52, wherein one of the one or more fusion protein comprises CD81-U1a protein and another of the one or more fusion protein comprises PTGFRN- U1a protein, wherein the amino acid sequence of the CD81-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 5, and wherein amino acid sequence of the PTGFRN-U1a fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO.

6.

57. The composition of claim 42, further comprising Ago2 protein complexed with the cargo RNA.

58. The composition of claim 57, wherein the Ago2 protein comprises S387A mutation.

59. The composition of claim 42, further comprising one or more modified myoferlin protein.

60. The composition of claim 59, wherein the modified myoferlin protein comprises C2F, C2G, transmembrane domain, or a combination thereof.

61. The composition of claim 60, wherein the modified myoferlin protein comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO.

28.

62. The composition of claim 59, wherein the modified myoferlin protein comprises C2A, FerA, FerB, DysFN, transmembrane domain, or a combination thereof, wherein the DysFN domain comprises a DysFN-1 domain, a DysFN-2 domain, or a combination thereof.

63. The composition of claim 59, wherein the modified myoferlin further comprises a connexin 43 protein.

64. The composition of claim 63, wherein the connexin 43 protein comprises a S368A mutation.

Images