Exosome populations comprising glyco-modified RNAS and methods of use thereof

Abstract

Extracellular vesicles or exosomes comprising glycoRNAs which direct an RNA of interest into said exosome and methods of use thereof are disclosed.

Description

[0001] EXOSOME POPULATIONS COMPRISING GLYCO-MODIFIED RNAS AND METHODS OF USE THEREOF

[0002] By

[0003] Megerditch Kiledjian

[0004] Sunny Shanna

[0005] Xinfu Jiao

[0006] CROSS REFERENCE TO RELATED APPLICATION

[0007] This application claims priority to US Provisional Application Number 63 / 678,359 filed August 1, 2024, the entire contents being incorporated herein by reference as though set forth in full.

[0008] STATEMENT OF GOVERNMENT SUPPORT

[0009] This invention was made with government support under grant number R35GM 149262 awarded by the U.S. National Institutes of Health. The government has certain rights in the invention.

[0010] FIELD

[0011] The present invention relates the fields of drug delivery and nucleic acid mediated cellular signaling, particularly that mediated by glyco-modified exosomal RNA. More specifically, compositions and methods related to glyco-modified RNAs, having distinct biochemical properties are provided which play crucial roles in metabolic processes, including immune responses and gene expression regulation. The exosomal glyco-modified RNAs described below can be used to advantage as valuable biomarkers for diagnosis and treatment of various pathological conditions. They can also be combined with RNA molecules of interest for delivery to target cells.

[0012] BACKGROUND

[0013] Several publications and patent documents are cited throughout the specification to more fully describe the state of the ail to which this invention pertains. Each of these citations is incorporated by reference herein as though set forth in full.

[0014] Chemical modifications play a pivotal role in the intricate management of biomolecular sorting within cells, ensuring the precise localization of diverse molecules1. Extensive research has characterized modifications such as phosphorylation, acetylation, and glycosylation, providing a comprehensive understanding of their roles in guiding proteins to specific cellular compartments1. In stark contrast, the sophisticated language of chemical modifications governing the sorting of nucleic acids remains largely unexplored. RNA modifications have long been known to influence the fate and function of coding and noncoding RNAs. The most well- characterized modification is the 7-methylguanosine (m7G) cap2added to the 5' end of virtually all mRNAs, which protects the mRNA and enables ribosomal translation. Another common modification is N6-methyladenosine (m6A)3, which affects -30% of mRNAs and impacts mRNA stability, localization, and translation4. More recently, a diverse family of non-canonical caps was discovered decorating the 5' ends of RNAs, including nucleotide metabolites like NAD, FAD, dpCoA, UDP-glucose, and UDP-N-acetylglucosamine (UDP-GlcNAc)5. The functions of most of these novel non-canonical caps remain unclear, except for NAD and FAD caps, which facilitate rapid RNA decay in eukaryotes6,7and arc employed by T4 bacteriophages in the RNAylation8of bacterial translational machinery. Notably, the 5’-FAD capping9of the Hepatitis C virus was demonstrated to shield the viral RNA from RIG-I-mediated innate immune recognition.

[0015] Recently the identification of glycoRNAs — small RNAs adorned with sugar molecules — on the surface of cells10has opened up new avenues for understanding RNA functionality beyond the intracellular environment. These glycoRNAs, with their distinct biochemical properties, have been proposed to play a crucial role in cellular processes, including immune response and gene expression regulation11,12. Our studies demonstrate the presence of glycoRNAs within extracellular exosome vesicles, suggesting a sophisticated sorting mechanism that leverages glycosylation as a determinant for exosomal packaging. This novel insight into exosomal cargo selection not only aligns with the emerging recognition of extracellular RNAs as potential biomarkers for disease but also underscores the intricate regulatory networks orchestrating RNA transport and function.

[0016] SUMMARY

[0017] Exosomes play a significant role in diverse cellular functions and responses, encompassing areas such as immune responses, viral pathogenicity, cardiovascular disease, neurological disorders, cancer and also serve as valuable biomarkers for various pathological conditions. Exosomcs exhibit approximately 40-160nm dimensions and a lipid bilaycr structure. These structures originate from endosomes and appear to furnish cells with an autonomous signaling mechanism facilitated by the molecular cargo enclosed within the exosome environment. This cargo encompasses a range of constituents such as DNA, RNA, lipids, metabolites, and cellular proteins. Surprisingly, the present inventors have identified RNA molecules within exosomes which exhibit a distinctive glycosylation modification pattern. Importantly, the glycosylation modification patterns identified occur on RNAs destined for incorporation into exosomes. Accordingly, the invention provides compositions comprising an exosomal population harboring internally modified glycoRNAs and an RNA encoding a molecule of interest for targeting said RNA molecule of interest into a target cell. Redirection of endogenous cellular machinery to target desired RNAs into exosomes provides an efficient and effective tool for the delivery of drugs as a target-specific therapy in different pathological and diagnostic conditions based on their safety, high biocompatibility, high bioavailability, and the ability to penetrate the blood-brain barrier. In addition, the exosomes of the invention are immune system friendly, have low adverse effects, and possess unique natural targeting capabilities to deliver their cargo to specific cell types or tissues. This ability to target specific cells greatly enhances the therapeutic potential of exosomes for RNA delivery.

[0018] In one embodiment, a composition comprising a population of exosomes harboring internally modified glycoRNAs and at least one RNA encoding a molecule of interest for endosome mediated delivery of said RNA molecule of interest into a target cell in a biological car ier is disclosed. In another embodiment, the glycoRNA comprises galactose or galactosamine and their derivatives. In yet another aspect, the glycoRNA is selected from at least one unique exosomal glycoRNA listed in Figure 6C. In certain embodiments, the glycoRNA is RNAase A resistant.

[0019] The therapeutic RNA can encode a useful therapeutic agent, including, but not limited to an inhibitory nucleic acid, a protein of interest, a vaccine antigen, an anti-tumor antigen, a single chain antibody, a cytokine, a hormone and an enzyme. In certain embodiments, the modified glycoRNA and said RNA encoding a molecule of interest are operably linked on a single RNA strand. The inhibitory nucleic acid can be selected from an siRNA, an antisense oligonucleotide, an shRNA, and a ribozyme having sufficient sequence homology to a nucleic acid encoding a protein targeted for inhibition, said inhibitory nucleic acid reducing expression of a target protein in said target cell.

[0020] In yet another aspect, the composition can further comprise a vector encoding UDP- galactose 4'-epimerase (GALE) and said internal RNA modification comprises at least one galactose, galactosamine or a derivative thereof.

[0021] In another embodiment, an exosome population is obtained from isolated cells from a subject, said exosomes comprising levels of unique internally modified glycoRNA biomarkers comparable to control subjects having a disease or metabolic disorder, indicating said subject has said disease or disorder.

[0022] The invention also provides a method for delivery of an exosome population harboring a therapeutic agent to a subject in need thereof, said agent comprising an RNA molecule of interest in combination with, or operably linked to an internally glycosylated RNA molecule (glycoRNA). An exemplary method comprises introducing said RNAs into a target cell, wherein RNA encoding said therapeutic agent can optionally be expressed and targeted to the exosome by said glycoRNA, followed by extrusion of said exosome from said cell. The extruded exosomes are then collected and delivered to a subject. Delivery can be systemic, topical or aerosol. In certain embodiments, the therapeutic agent is an inhibitory nucleic acid selected from an siRNA, an antisense oligonucleotide, an shRNA, and a ribozyme having sufficient sequence homology to a nucleic acid encoding a protein targeted for inhibition, said inhibitory nucleic acid reducing expression of a disease associated protein in said target cell. The RNA of interest can express at least one vaccine antigen, a protein of interest, an antibody or functional fragment thereof, and an anti-tumor antigen.

[0023] Also provided is a method wherein the RNA encodes an immunogen that elicits an immune response against one or more of Adenovirus, Bunyavirus, Caliciviridae, Coronavirus, Filovirus, Flavivirus, Hepadnavirus, Hepatitis viruses, Herpesvirus, Orthomyxovirus, Papovaviruses, Paramyxoviridae, Parvovirus. Pestivirus, Picornavirus, Poxviridae, Reovirus, Retrovirus, Rhabdovirus, and Togavirus.

[0024] In another embodiment, the RNA encodes an immunogen that elicits an immune response against a tumor antigen or oncoprotein. In this embodiment, the glycoRNA and RNA in said exosome or extracellular vesicle target a tumor antigen selected from NYESO- 1 , Her-2 / neu, MAGE-1, Tyrosinase, MUC1, CEA, Mam- A, hTERT, Sialyl-Tn, WT1, a- fetal protein, and CA- 125.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figures 1A - IB. Screening of UDP GlcNAc capped dccapping RNAs. Screening of UDP GlcNAc capped RNA decapping. (Fig. 1A) Schematic representation illustrating the structure of UDP-GlcNAc-capped RNA. (Fig. IB) Analysis of reaction products from in vitro decapping assays conducted with 25 nM recombinant mDXO and SpRai utilizing uniformly 32P-labeled triphosphate (pppUpG — ) or UDPGlcNAc-capped (GlcNAcppUpG) RNA (16 nucleotides) as substrates, denoted accordingly. The resultant products were separated via electrophoresis on 20% polyacrylamide gels containing 7 M urea.

[0027] Figures 2A - 2E. Cellular RNAs undergo sugar modifications derived from N-acetyl- galactosamine. (Fig. 2A) Experimental design schematic illustrating the characterization of labeled RNA species using biorthogonal and cell-permeable Ac4GlcNaz, Ac4GalNaz, and Ac4ManNaz. (Fig 2B) Total RNAs from HeLa, isolated after 48 hours of incubation with media containing lOOpM of AC4G1CNAZ, Ac4GalNAz, or Ac4ManNAz following SPAAC reaction to conjugate biotin moieties. The resulting biotin-conjugated RNAs were resolved on an agarose gel and transferred onto a Nitrocellulose membrane. Visualization was achieved through nearinfrared (IR) IRDye® 800CW streptavidin-based detection for glyco -modified RNAs and with ethidium bromide for the total RNA using Odyssey Fc (Li-Cor Biosciences). Red asterisks indicate glyco RNAs derived from Ac4ManNAz, which are visible only upon extended exposure. (Fig. 2C) Ac4GalNaz-derived labeled total RNAs from both HeLa and HEK293T cells were fractionated into smaller (<200nts) and larger (>200nts) RNAs, followed by SPAAC reaction for biotin conjugation. The biotin-conjugated RNAs were then visualized as in b. (Fig. 2D) Gel analysis depicting the impact of the indicated nucleases on the glycoRNA. (Fig. 2E) To probe the presence of a 5' end cap associated with the detected azide, endogenous glycoRNA was treated with SpRai 1 to remove the GlcNAc cap. A total of 25 pg of RNA isolated from HEK23T cells, following incubation with lOOpM of Ac4GalNAz, underwent treatment with 100 nM of SpRail. The resultant reaction product was subjected to SPAAC and visualized as in b. The lack of significant reduction in the intensity of the TR-800 signal suggests the glyco-modification likely resides internally rather than at the 5' end.

[0028] Figures 3A - 3D. Analysis of Glyco RNAs in HEK293T and MCF7 cells and impact of RNase treatment. Total RNA from (Fig. 3 A) HEK293T or (Fig. 3B) MCF7 cells obtained following 48 hours of incubation with media containing lOOpM of Ac4GalNAz or Ac4ManNAz, underwent SPAAC reaction to attach biotin moieties. The resulting biotinylated RNAs were separated on an agarose gel and transferred onto a Nitrocellulose membrane. Detection was carried out using near-infrared (IR) IRDye® 800CW streptavidin-based labeling for glyco-modified RNAs and ethidium bromide staining for total RNA visualization, employing the Odyssey Fc imaging system (Li-Cor Biosciences) as in Figure 2. (Fig. 3C) Following 48 hours of incubation with media containing lOOpM of Ac4GalNAz, the enrichment of labeling for both total RNA and Poly A+ RNA was performed using the Poly(A)Purist™ MAG Kit (Invitrogen). The resulting fractions were assessed as described above. (Fig. 3D) Time course analysis at the indicated time of 15U micrococcal nuclease digestion of glycoRNAs at 37°C with or without the addition of the Ca2+, an essential cation for micrococcal nuclease activity.

[0029] Figures 4A - 4F. Analysis of Glyco-Modification in Exosomal RNAs. (Fig. 4A) Subcellular localization of glyco-modified RNAs was evaluated in Ac4GalNaz-labeled HeLa cells. Nuclear and cytoplasmic fractions were resolved, RNA isolated and biotin-conjugated RNAs were visualized as in Fig.2B. Western Blotting with GAPDH and histone H3 served as controls for fractionation. (Fig. 4B) Localization of glyco-modified RNAs in the soluble cytosolic compartment or membranous organelles was assessed using the ProteoExtract® Native Membrane Protein Extraction Kit (EMD Millipore). Membranous fractions (plasma membrane, ER, and Golgi) and cytosol were fractionated, and total RNAs from each fraction were subjected to SPAAC for biotin conjugation. The biotin-conjugated RNAs were then analyzed as described in (Fig. 4A). Western Blotting with GAPDH and Sec63 served as controls for fractionation. (Fig. 4C) Schematic illustration of the exosomal RNA isolation method employed. (Fig. 4D) Exosomal RNAs derived from HeLa cells were isolated and the resulting biotin-conjugated RNAs were analyzed after SPAAC as in (Fig. 4A). Western Blotting with GAPDH and CD9 served as controls for exosome purification. (Fig. 4E) Scatter plot analysis depicting Ac4GalNaz- derived labeled exosomal RNAs from HeLa cells. (Fig. 4F) Venn diagram illustrating the overlap between cellular RNA and exosomal RNAs gene sets (logFC>4), demonstrating shared and distinct transcripts.

[0030] Figures 5A - 5E. Affinity purification of the glyco RNAs and Oxford Nanopore sequencing. Fig. 5ASchematic representation illustrating the process of labeling and affinity purification of glyco-modificd RNAs. Fig. 5BSmall RNA (15 pg) underwent SPAAC reaction. Subsequently, the affinity-purified biotinylated RNA, representing glycoRNA, was eluted from streptavidin beads and visualized as in Fig. 2. Fig. 5C Scatter plot analysis depicting Ac4GalNAz-derived labeled RNAs from HEK293T cells and (Fig. 5D) HeLa cells. The graphs are color-coded based on RNA categories, and each dot's size corresponds to the respective RNA's length. (Fig. 5E) Northern blot analysis of RNA eluates obtained from affinity-purified glyco-RNA that was subjected to SPAAC from HeLa cells.

[0031] Figures 6A -6D. Exosomal glycoRNA. (Fig. 6A) Intact HeLa cells or (Fig. 6B) intact exosomes isolated from Hela cell cultures, were incubated for 48-hour with lOOpM Ac4GalNaz and subjected to micrococcal nuclease. GlycoRNAs subsequently isolated from the treated cells or exosomes were detected. (Fig. 6C) List of unique and common transcripts in the cellular and exosomal RNA of HeLa cells enriched over 8-fold (see Fig. 4F. (Fig. 6D) SPAAC reaction of the indicated amounts of HeLa small RNAs, and exosomal RNAs. The biotin-conjugated RNAs were visualized as in Fig. 2.

[0032] Figures 7A - 7C. Different patterns of glycoRNAs in iPSC and iN cells. (Fig. 7A) Overview of induced neuronal transdifferentiation. (Fig. 7B) Exosomal RNAs from human iPSCs and their derived induced neurons (iNs) collected after 48 hours of incubation with lOOpM Ac4GalNAz- containing media underwent SPAAC reaction for biotin conjugation. (Fig. 7Cc) Total RNA extracted from human (iPSCs) and their derived induced neurons (iNs) were harvested following a 48-hour incubation with media containing lOOpM Ac4GalNaz. Biotin-conjugated glycoRNAs were visualized as in Fig. 2.

[0033] Figures 8A - 8E. Biogenesis / Release and Impact on Glyco-Modified RNA Dynamics. (Fig. 8A) Schematic representation of exosomes, illustrating both ESCRT-dependent and independent pathways involved in exosomal release. (Fig. 8B) HGS (Vps27) knockdown using distinct DsiRNAs. HeLa cells were transfected with two different DsiRNA targeting HGS (Vps27), followed by a 72-hour incubation. The efficiency of gene knockdown was assessed through qRT- PCR analysis of total RNA extracted from these cells. (Fig. 8C) Examination of exosomal RNAs from HGS-dcplctcd or cells treated with Manumycin, GW4869, or a combination of Manumycin and GW4869. After 24-hour DsiRNA treatment or inhibitors, cells were exposed to lOOpM Ac4GalNaz, and RNA was isolated after 48 hours of incubation with media containing lOOpM AC4G1CNAZ. Subsequent SPAAC-mediated conjugation enabled the separation of resulting biotin-conjugated RNAs on an agarose gel and visualized as in Fig. 2B. (Fig. 8D) Representation of the average signal from glyco-modified RNA (n = 3) independent experiments, with error bars denoting ±SEM. (Fig. 8E) Analysis of cellular RNAs from the same set of HGS knockdown experiments (in panel c). After SPAAC-mediated conjugation, resulting biotin-conjugated RNAs were separated on an agarose gel and visualized as in Fig. 2B .

[0034] Figures 9A - 9C. Exosome directed intercellular transfer of glycoRNAs. (Fig. 9A) Experimental design overview demonstrating intercellular transfer of glycoRNAs from host cells to naive cells. (Fig. 9B) Total RNA from naive cells, with and without exposure to Ac4GalNaz-labeled exosomes isolated from HeLa cells, was incubated for 10 and 20 hours. After extensive washing to remove extracellular exosomes, total RNA was isolated from the naive cells and subjected to SPAAC-mediated conjugation to visualization the resulting biotin-conjugated RNAs as in Fig. 2. (Fig. 9C) RNA derived from naive cells subjected to incubation with and without Ac4GalNaz- labeled exosomes isolated from HeLa cells are shown following fractionation into small and large RNA fractions. GlycoRNAs were visualized as in Fig. 2.

[0035] Figures 10A -10C. Glycan addition is not mediated through amino-carboxypropyl-modified Uridine (acp3U). DTWD2 and TSR3 were knocked down in Hela cells with specific DsiRNAs. After 24 hours DsiRNA treatment, lOOpM Ac4GalNAz was added to the medium. Cells were harvested 48 hours after the addition of lOOpM Ac4GalNAz. (Fig. 10A) The efficiency of target gene knockdown was tested utilizing qRT-PCR analysis. (Fig. 10B) Azide-labeled GlycoRNAs from the control and corresponding knockdowns were subjected to biotin-conjugated SPAAC analysis and visualized as in Fig. lb. (Fig. 10C) Quantitation of the glycoRNAs in b using Image J. GlycoRNA levels in control knockdown were set as 1. Error bars show the + / -SD from three independent experiments (n = 3 replicates).

[0036] Figures HA -HD. Influence of Steady-State GalNAz Levels on GlycoRNAs. (Fig. 11 A) The N- acetyl galactosamine (GalNAc) salvage pathway generates UDP-GalNAc, with UDP-galactose 4'-epimerase (GALE) serving as the central enzyme mediating the interconversion between UDP-GlcNAc and UDP-GalNAc. GNE is a pivotal enzyme intcrconvcrting UDP-GalNAc and GlcNAc to ManNAc-6 phosphate, a step crucial for glycosylation events shaping cellular processes. (Fig. 11B) GALE and GNE depletion by DsiRNA. HEK293T cells were transfected with two distinct DsiRNA targeting GALE and GNE, followed by a 72-hour incubation. Total RNA extracted from these cells was subjected to qRT-PCR to assess gene knockdown efficiency. (Fig. 11C) Analysis of total RNAs from GALE and GNE depleted HEK293T cells. Post-24-hour DsiRNA treatment, lOOpM of Ac4GalNaz was introduced, and RNA isolation occurred after 48 hours of incubation with media containing lOOpM of Ac4GlcNAz. Following SPAAC-mediated conjugation, the resultant biotin-conjugated RNAs were separated on an agarose gel and transferred onto a Nitrocellulose membrane. Visualization was as in Fig. 1. (Fig. 1 ID) Inhibition of Oligosaccharyltransferase (OST) by NGL1. The effect of NGI-1, a specific and potent small molecule inhibitor of OST on glyco-modified RNA production was tested in cells and exosomes. NGI-1 treatment resulted in a dose-dependent loss of Ac4GalNaz labelled glycoRNA.

[0037] Figures 12A-H. Glycosylated mRNAs identified in HeLa cells (Figs. 12A-12H and HeLa cell derived exosomes (Fig. 121). Ensembl gene identifier numbers for access to genetic information for each gene is listed. Ac4GalNAz-labeled glycoRNAs were generated in HeLa cells and exosomes isolated as described herein. Coding RNAs were isolated and biotinylated as described and strain-promoted alkyne azide cycloaddition (SPAAC) performed. GlycoRNAs bound to streptavidin beads were eluted with Trizol reagent and isolated RNA subjected to Illumina next generation sequencing.

[0038] DETAILED DESCRIPTION

[0039] Epitran scrip tomic modifications play pivotal roles in regulating RNA function, encompassing base alterations and the addition of both canonical m7G and noncanonical nucleotide metabolite caps. Recently, the spectrum of modifications has extended to include glyco modification at the 5’ cap or within the RNA. Despite this expansion, the functional implications of glyco modification on RNA remain elusive. Our study reveals that mammalian cells labeled with N-azidoacetylgalactosamine-tetraacylated (Ac4GalNAz) mainly produce small noncoding and nonpolyadenylated glyco-modified RNA (glycoRNA), primarily localized within exosome vesicles. The resistance of glycoRNA-containing exosomes to RNase treatment suggests that Ac4GalNAz-derived glycoRNA constitutes intraluminal cargo, distinct from recently reported cell surface glycoRNAs. Furthermore, wc demonstrate that exosome cargo can be transferred to naive cells, underscoring exosome-mediated intercellular communication of glycoRNAs. Inhibition of exosome biogenesis leads to the accumulation of intracellular glycoRNA while blocking glycan transfer to proteins concomitantly reduced the targeting of glycoRNA within exosomes. These findings highlight a correlation between protein and RNA glycosylation, suggesting that the accumulation of glycoRNA within exosomes is a regulated process. Our results support a functional role for glyco-modification in mediating RNA targeting into exosomes, offering new insights for enhancing recent advances in exosome-directed diagnostics and therapeutic applications.

[0040] DEFINITIONS

[0041] The term “glycoRNA” as used herein refers to RNAs with sialylated glycans or their derivatives including those derived from galactosamine. The sugars can be an N-linked monomer or polymer that may be branched. GlycoRNAs can include a single sugar or a single chain or branched polymer. The sugar (or glycan) moiety can be an N-linked monomer or polymer that may be branched.

[0042] GlycoRNAs in extracellular vesicles can be used as biomarkers, enabling rapid diagnosis and prognosis for specific diseases affecting different cell types. Furthermore, modifying RNA with glycans may be sensitive to immunotherapy medicines and thus provide new drug targets. In addition to immunotherapy, several other therapies, including radiotherapy and chemotherapy, have the potential to alter the glycan structure of glycoRNAs. The glyco modification may be used to target RNA of interest into the exosome for delivery into other cells, tissues or organisms.

[0043] “Exosomes” are small membrane extracellular vesicles (EV) of endocytic origin that are secreted by many cell types. For example, exosomes may have a diameter of about 40 to about 100 nm. They may be formed by inward budding of the late endosome leading to the formation of vesicle-containing multivcsicular bodies (MVB) which then fuse with the plasma membrane to release exosomes into the extracellular environment. Though their exact composition and content depends on cell type and disease state, exosomes all share certain characteristics. In certain aspects, the exosomes may be purified by ultracentrifugation in a sucrose gradient, then identified by the presence of marker proteins such as Alix and CD63 (Schorcy & Bhatnagar, 2008) or enrichment of tetraspanins and heat shock protein 70 (Lee, et al., 2011), all of which are specifically expressed on exosomes. Furthermore, exosomes can be isolated in vivo from malignant effusions and normal body fluids such as urine, blood, and cerebrospinal fluid, making them a promising source of diagnostic biomarkers. In some other aspects, exosomes can be isolated using isolation kits, such as those described below and exoEasy Maxi Kit from Qiagen, SmartSEC™.

[0044] As noted above, exosomes also have the potential for directional homing to specific target cells, depending on the physical properties of their membranes. Their effect can be local, regional or systemic. Exosomes do not contain a random sampling of their parent cell's cytoplasm, but are enriched in specific glycoRNAs, mRNAs, miRNAs and proteins. This cargo is protected from degradation by proteases and RNases while the vesicle is in the interstitial space, and retains bioactivity once taken up by a recipient cell. In this way, they facilitate the transfer of interactive signaling and enzymatic activities that would otherwise be restricted to individual cells based on gene expression. For example, it has been shown that mRNA for a reporter protein can be incorporated into exosomes, transferred to a recipient cell, and translated.

[0045] The exosomes produced or released by cells may be isolated and / or purified using several techniques. These include filtration, centrifugation, ion-chromatography, or concentration, either alone or in combinations. An exemplary purification method comprises a step of density gradient centrifugation. Another exemplary method comprises a step of ultrafiltration, either alone or coupled to a centrifugation step.

[0046] Selective purification or enrichment of physiologically active subpopulations of exosomes may be achieved via several procedures. In certain embodiments, effective exosomes may be concentrated to an enriched sample via use of specific surface protein markers and related separation techniques. In other embodiments, effective exosomes may be harvested from enriched ri ary cells cultures identified as capable of producing the effective exosomes. In further embodiments, based on screening procedures used to identify candidate effective exosome species, other exosomes may be fabricated using molecular engineering strategies designed to selectively produce exosomes containing the target (i.e., postulated) therapeutic molecular species. The latter may be confirmed by application of exosomes containing fabricated species to naive cultures, where the desired effect (c.g., increased myelination) may be verified.

[0047] In certain embodiments, the exosomes or vesicles may be loaded with therapeutic agents such as nucleic acid molecules, e.g., glycoRNAs alone or in combination with RNA molecules of interest encoding proteins useful for the treatment of disease or regulation of cellular' pathways. The methods may include, but are not limited to:

[0048] (a) Electroporation. By this method, a number of holes are made in cells / exosomes by briefly shocking them with an electric field of 100-200 V / cm. The DNA / RNA can enter the cells / exosomes through the holes made by the electric field.

[0049] (b) Lipofection. The method commonly called transfection and can be used to transform cells / exosomes with DNA / RNA via vesicles containing the desired genetic constructs. The vesicles fuse with the cell membrane (similar to how two oil spots at the top of a broth will fuse) and the contents of the vesicles and the cells are combined. There are a number of transfection kits in the market, ready for use, e.g. DeliverX siRNA Transfection Kit (cat. No. DX0002) from Panomics, FuGENE® HD Transfection Reagent (Cat. no. 04709691001) from Roche and LIPOFECTAMINE™ 2000 (Cat. No. 11668-027) from Invitrogen.

[0050] (c) Transformation using heat shock. Chilling cells / exosomes in the presence of divalent cations such as Ca2+ (in CaC12) makes their membranes become permeable to RNA or DNA plasmids or fragments. Cells or exosomes are incubated with the DNA and then briefly heat shocked (42° C. for 30-120 seconds), which causes the DNA to enter the cell. This method may work well for condensed circular plasmid DNAs and may work for exosomal or lipid nanovesicle constituents.

[0051] The above methods describe briefly how production and delivery of modified exosomes can be achieved to transfer RNA and DNA to recipient cells. Exosomes can be engineered to contain RNA / DNA or modified to contain the gene of interest and may be isolated and shifted to the recipient cells, to affect their biological function or survival. Consequently, the exosomes may dispose their content into the cytoplasm of the target cells, which in turn leads to translation of mRNA to specific proteins in the target cell. Further, exosomes are capable of carrying and transferring small coding and non-coding RNA such as microRNA and siRNA that may regulate translation of a specific gene. Modified or loaded exosomes being vesicles as earners of DNA or RNA as described herein can be used to treat inherited diseases in targeted cell types and organs. Modified or loaded exosome vesicles can also be used as carriers of DNA or RNA constructs for treatments of patients at risk for or having a disease such as cancer, heart disease, immunoinflammatory disease, a disease associated with in bom errors of metabolism, diabetes, etc. The RNA molecule of interest may also encode a vaccine antigen, a single chain antibody, or a therapeutic polypeptide, or for transfer through any biological membrane.

[0052] Changing or modifying the genetic material of exosomes by altering the conditions for the exosome-producing cells is achieved by changing pH, temperature, growing conditions, or using antibodies / chemicals toward exosome-producing cells. This results in alteration of the nucleic acid content.

[0053] To administer nucleic acids to recipient cells or tissues, DNA or RNA-containing exosomes can be administered to cells by addition of the exosomes to cell cultures in vitro, or injection of these exosomes intravenously, or by any other route, in vivo as is known in the art, such as nasally or intravenously. Exosomes can be targeted to any cell in the body, including cells in the cardiovascular system, skeletal muscle cells, joint cells, neural cells, gut cells, lung cells, liver cells or kidney cells, or cells in the immune system, or to any type of cell with any function or dysfunction in the body of humans or animals, including malignant cells. As disclosed in the invention herein, exosomes can be used to deliver genetic material to recipient cells to produce any drug or precursor of any drug, or to affect the function or metabolism of any drug, in any cell in humans or animals. In an alternative approach, cells from a cell population, (e.g., immune cells, cardiac cells, brain cells, liver cells, lung cells, muscle cells, mucosal cells, etc.) are contacted with a delivery vehicle comprising said glycoRNAs and or an RNA of interest, and optionally a nucleic acid encoding a glycosylating enzyme selected from (Galactose-4-Epimerase (GALE) for delivering the nucleic acids to the cell population of interest. The delivery vehicle can be a selected from a vector, a lipid nanoparticle or a combination thereof. Introduction of these components into the target cell generating glycoRNAs which home to the exosomes in the specific cell type which can then be harvested for production of exosome populations harboring the therapeutic molecule of interest.

[0054] A "small nucleic acid inhibitor" refers to any sequence based nucleic acid molecule which, when introduced into a cell expressing the target nucleic acid, is capable of modulating expression of that target. siRNA, antisense, miRNA, shRNA and the like may be utilized in the methods of the invention.

[0055] The term "RNA interference" or "RNAi" refers generally to a process or system in which a RNA molecule changes the expression of a nucleic acid sequence with which RNA molecule shares substantial or total homology. The term "RNAi agent" refers to an RNA sequence that elicits RNAi.

[0056] The terms "miRNA" and "microRNA" refer to about 10-35 nt, preferably about 15-30 nt, and more preferably about 19-26 nt, non-coding RNAs derived from endogenous genes encoded in the genomes of plants and animals. They are processed from longer hairpin-like precursors termed pre-miRNAs that are often hundreds of nucleotides in length. MicroRNAs assemble in complexes termed miRNPs and recognize their targets by antisense complementarity. These highly conserved, endogenously expressed RNAs are believed to regulate the expression of genes by binding to the 3'-untranslated regions (3'-UTR) of specific mRNAs as well as other regions on targeted mRNAs. Without being bound by theory, a possible mechanism of action assumes that if the microRNAs match 100% their target, i.e. the complementarity is complete, the target mRNA is cleaved, and the miRNA acts like a siRNA. However, if the match is incomplete, i.e. the complementarity is partial, then the translation of the target mRNA is blocked. The manner by which a miRNA base-pairs with its mRNA target correlates with its function: if the complementarity between a mRNA and its target is extensive, the RNA target is cleaved; if the complementarity is partial, the stability of the target mRNA in not affected but its translation is repressed.

[0057] An "siRNA" refers to a molecule involved in the RNA interference process for a sequence-specific post-transcriptional gene silencing or gene knockdown by providing small interfering RNAs (siRNAs) that has homology with the sequence of the targeted gene. Small interfering RNAs (siRNAs) can be synthesized in vitro or generated by ribonuclease III cleavage from longer dsRNA and are the mediators of sequence- specific mRNA degradation. Preferably, the siRNA molecules of the invention are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA / RNA synthesizer. The siRNA can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions. Commercial suppliers of synthetic RNA molecules or synthesis reagents include Applied Biosystems (Foster City, Calif, USA), Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va., USA), ChcmGcncs (Ashland, Mass., USA) and Cruachem (Glasgow, UK).

[0058] The term "delivery" as used herein refers to the introduction of foreign molecule (i.e., and exosome comprising glycoRNA, a miRNA containing nanoparticle, etc.) into cells. The term "administration" as used herein means the introduction of a foreign molecule into a cell. The term is intended to be synonymous with the term "delivery".

[0059] The terms "construct", “cassette”, "expression cassette", “plasmid”, “vector”, “delivery vehicle”, or “expression vector” is understood to mean a recombinant nucleic acid, generally recombinant DNA, which has been generated for the purpose of the expression or propagation of a nucleotide sequence(s) of interest or is to be used in the construction of other recombinant nucleotide sequences.

[0060] The term “operably linked” can mean the positioning of components in a relationship which permits them to function in their intended manner. For example, a promoter can be linked to a polynucleotide sequence to induce transcription of the polynucleotide sequence.

[0061] The terms "complementarity" or “complement” refer to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 4, 5, and 6 out of 6 being 66.67%, 83.33%, and 100% complementary). "Perfectly complementary " means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. "Substantially complementary" as used herein refers to a degree of complementarity that is at least 40%, 50%, 60%, 62.5%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%, or percentages in between over a region of 4, 5, 6, 7, and 8 nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0062] Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0063] The term "earner" refers, for example to a diluent, adjuvant, excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

[0064] A pharmaceutical composition of the present invention can be administered by any suitable route, for example, by injection, by oral, pulmonary, nasal or other forms of administration. In general, pharmaceutical compositions contemplated to be within the scope of the invention, comprise, inter alia, pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions can include diluents of various buffer content (e.g., Tris HC1, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of components of a pharmaceutical composition of the present invention. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435 1712 which are herein incorporated by reference. A pharmaceutical composition of the present invention can be prepared, for example, in liquid form, or can be in dried powder, such as lyophilized form. Particular methods of administering such compositions are described infra.

[0065] In yet another embodiment, a pharmaceutical composition of the present invention can be delivered in a controlled release system, such as using an intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In a particular embodiment, a pump may be used [see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)]. In another embodiment, polymeric materials can be used [see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley: New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228: 190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard ct al., J. Ncurosurg. 71:105 (1989)]. In yet another embodiment, a controlled release system can be placed in proximity of the target tissues of the animal, thus requiring only a fraction of the systemic dose [see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115 138 (1984)]. In particular; a controlled release device can be introduced into an animal in proximity of the site of inappropriate immune activation or a tumor. Other controlled release systems are discussed in the review by Langer [Science 249:1527 1533 (1990)].

[0066] As used herein the term "biomarker" refers to a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.

[0067] As used herein, the phrase "effective amount" of a compound or pharmaceutical composition refers to an amount sufficient to modulate a biological activity associated with the disease to be treated. In the case of cancer, the activity can comprise tumor growth or metastasis in an animal, especially a human, including without limitation decreasing tumor growth or size or preventing formation of tumor growth in an animal lacking any tumor formation prior to administration, i.e., prophylactic administration.

[0068] As used herein, the terms "modulate", "modulating" or "modulation" refer to changing the rate at which a particular process occurs, inhibiting or promoting a particular process, reversing a particular process, and / or preventing the initiation of a particular process. Accordingly, if the particular process is tumor growth or metastasis, the term "modulation" includes, without limitation, decreasing the rate at which tumor growth and / or metastasis occurs; inhibiting tumor growth and / or metastasis; reversing tumor growth and / or metastasis (including tumor shrinkage and / or eradication) and / or preventing tumor growth and / or metastasis. A compound that increases a known activity, e.g., tumor growth or metastasis, is an “agonist”. One that decreases, or prevents, an undesirable malignant phenotype is an “antagonist” or “inhibitor”.

[0069] The following materials and methods are provided to facilitate the practice of the invention described herein. The methods are exemplary only as the skilled person is aware of that there are other methods and reagents known in the ait. Synthesis of 3’-(guanyl-5'-yl)-uridine 5 '-diphosphate N-acetylglucosamine, NAcGlcppUpG, capped RNAs via In Vitro Transcription

[0070] To generate RNAs capped with UDPGlcNAc we employed in vitro transcription using synthetic double-stranded DNA templates T7-GlcNAc-25 and T7-GlcNAc-40. Due to the challenge of incorporating uridinc-containing caps, we utilized a 5 ’-sugar- modified uridineguanosine (UpG) dinucleotide32- NAcGlcppUpG. This strategy was necessitated by the strong preference of T7 RNAP for transcription initiation with guanosine, which interacts with the N-7 and 0-6 of the purine ring33. While adenosine-containing cofactors can partially mimic these interactions and initiate transcription34, pyrimidine nucleotides like UDPGlcNAc do not facilitate efficient transcription initiation. Following in vitro transcription at 37°C overnight using the HiScribeTM T7 High Yield RNA Synthesis Kit (New England Biolabs (NEB)), RNA purification was performed employing MonarchORNA Cleanup Columns (NEB) according to the manufacturer’s protocol. For32P-labeled RNAs, the transcription reactions were performed in the presence of [a-32P] ATP.

[0071] In vitro Decapping Assays

[0072] Recombinant Nudix proteins, SpRail and mDXO with an N-terminal 6 x His-tag, were purified using His Bind Resin (Novagen).32P-labeled NAcGlcppUpG-cap or pppUpG capped RNAs were incubated with 50 nM of recombinant Nudix proteins and 25nM of SpRai 1 and mDXO in decapping buffer (10 mM Tris-HCl [pH 7.5], 100 mM KC1, 2 mM DTT, 2 mM MgCh, and 2 mM MnCh) at 37°C for 30 min. Reactions were terminated by heating at 90°C for 2 min. Decapped products were separated via electrophoresis on 7M urea 20% polyacrylamide gels, visualized using an Amersham Typhoon Biomolecular Imager (GE Healthcare).

[0073] Cell Culture

[0074] All cell lines were cultured at 37°C with 5% CO2. Specific cell types used in this study included HcLa, 293T, MCF7, and human-induced human pluripotent stem cells (iPSCs)35. HcLa, HEK293T, MCF7 cell lines were maintained in DMEM media supplemented with 10% exosome free-fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S). iPSCs were maintained in mTeSR™ hPSC medium (STEMCELL Technologies). Metabolic Labeling with Ac4GalNAz

[0075] Metabolic labeling of cells was achieved using N-azidoacetylgalactosamine-tetraacylated (Ac4GalNAz) or N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz) or N- azidoacetylglucosamine-tetraacylated (Ac4&cNAz.) Cells were cultured under standard conditions and treated with 100 pM Ac4GalNAz for specified durations. Following treatment, cells were harvested for subsequent RNA analysis.

[0076] RNA Isolation and treatment

[0077] Total RNA was extracted from treated and control cells using TRIzol reagent according to the manufacturer's instructions. The RNA was then treated with DNase I to remove any potential DNA contamination. To ensure the removal of proteins, samples were subjected to Proteinase K treatment and purified again using the MonarchORNA Cleanup Columns (NEB) according to the manufacturer’s protocol. For the following Figures, the RNA was directly precipitated without the use of a column: Figures 2D, 3D, 6A, 6B, 9B and 11D. To enrich for polyadenylated RNA, total RNA purified as above was used as the input for the Poly(A) Purist MAG Kit (Thermo Fisher Scientific) following the manufacturer’s protocol.

[0078] Strain-promoted alkyne azide cycloaddition (SPAAC)

[0079] To conjugate biotin to Ac4GalNAz-labeled sugars, a copper-free click chemistry approach was employed using dibenzocyclooctyne (DBCO)-PEG4-biotin. The SPAAC reactions were performed following the previously established protocol18. Here 25pg of Ac4GalNAz-labeled RNA-bearing azide RNA in pure water were mixed with lx volumes of MK-Gel Eoading Buffer (90% Formamide, 15mM EDTA, and 0.025% SDS) and 500 pM DBCO-biotin (Sigma). Typically, these reactions consisted of 20 pF MK-Gel Eoading Buffer, 18 pLRNA (from ADPRC reaction), and 2 pF of a lOmM DBCO reagent stock solution. The reactions were carried out at 55°C for 15 minutes to denature the RNA, halted by adding 310 pL water, followed by purification of the conjugated RNA using Monarch®RNA Cleanup Columns (NEB). The RNA underwent analysis either through gel electrophoresis or affinity purification as detailed below. To confirm that RNA molecules were labeled following the SPAAC reaction, the RNA was subjected to RNAse A (Ipg), RNase I (10U), micrococcal nuclease (15U) or DNase I (2U) at 37 °C for 1 hour before loading it onto the gel as described below. RNA gel electrophoresis, capillary blotting, and near-infrared fluorescent imaging

[0080] For blotting analysis of either enriched RNA following biotin-mediated affinity purification or directly after the SPAAC reaction, RNA was heat-incubated at 90°C for 2 minutes in formaldehyde loading buffer (FLB) (50% Formamide, 6% formaldehyde, 50mM HEPES, pH 7.8, 0.5pg / mL ethidium bromide, and 10% glycerol). Samples were loaded onto a 1.2% agarose gel and electrophoresed at 120V for 1 hour. Before transferring the gel onto a 0.45 pm nitrocellulose membrane (Cytiva), total RNA was visualized using a UV gel transilluminator. RNA transfer occurred overnight via passive; upward transfer facilitated by the capillary flow of the 10X SSC buffer. Following the transfer, RNA was cross-linked to the NC using UV-C light (0.2 J / cm2). Subsequently, the membrane was blocked with Odyssey Blocking Buffer, PBS (Li- Cor Biosciences) for 30 minutes at room temperature (RT). After blocking, IRDye® 800CW streptavidin (Li-Cor Biosciences) was diluted to 1:7,000 in Odyssey Blocking Buffer and used to stain the NC membrane for 30 minutes at RT followed by three washes with PBST (ResearchProductlntemational). Before scanning, the membranes were briefly rinsed in lx PBS and scanned by an Odyssey Fc (Li-Cor Biosciences) with the software set to auto-detect the signal intensity for both the 600 and 800 channels. The 600 channel was utilized for ethidium bromide, while the 800 channel was utilized for IRDye® 800CW streptavidin detection.

[0081] Subcellular Fractionation

[0082] To obtain highly purified nuclei and nuclei free cytoplasm, we employed a commercially available NE-PER® (Thermo Scientific) Nuclear and Cytoplasmic Extraction Kit for efficient cell lysis and extraction of separate cytoplasmic and nuclear protein fractions following the manufacturer’s instruction. Briefly, Ac4GalNAz labeled HeLa cells were harvested using trypsin- EDTA washed with PBS, and pelleted. After discarding the supernatant, the cell pellet was treated with ice-cold CER I. Cytoplasmic and nuclear proteins were extracted by vortexing the cell suspension, adding CER II, and centrifuging to collect the cytoplasmic fraction. The nuclear pellet was then suspended in NER, vortexed, and centrifuged to obtain the nuclear extract. One third of this fraction was used for the Western Blotting whereas the rest was used to extract RNA using a TRIzol reagent.

[0083] For cytosolic and crude membrane fraction isolation from the Ac4GalNAz -labeled HeLa cells, we utilized a commercial membrane protein extraction kit - The ProteoExtract® Native Membrane Protein Extraction Kit (EMD Millipore). This extraction method involves sequential lysis steps, initially releasing soluble cytosolic proteins and RNA, followed by the disruption of membranous organelles such as the plasma membrane, Golgi, and ER. In brief, cultured HeLa cells underwent media removal, followed by two washes with ice-cold Wash Buffer. Subsequently, Extraction Buffer I (containing protease inhibitor) was applied to the cells and incubated for 10 minutes at 4°C with gentle rocking, yielding the 'cytoplasm' fraction. This was followed by the addition of Extraction Buffer II (also supplemented with protease inhibitor) to the cells, which were further incubated for 30 minutes at 4°C with gentle rocking, resulting in the 'ER / membrane' fraction. Here again, l / 3ldof the fraction was used for Western Blotting whereas 2 / 3rdof it was used for RNA extraction using TRIzol extraction method.

[0084] Nanopore-Based RNA Sequencing

[0085] Five micrograms of small RNA were isolated from biological replicates of both HE29T, HeLa cells and HeLa exo somes and poly adenylated in a 25 pL reaction mix containing 2 pL of 10X E. coli poly(A) polymerase buffer, 2 pL of 10 mM ATP, 15-17 pL of nuclease-free water, and 1 pL of E. coli poly(A) polymerase (5 U / pL) (NEB), with incubation at 37°C for 2 minutes. To stop the reaction, 5 pL of 50 mM EDTA was added to reach a final concentration of 10 mM EDTA. Following incubation, 45 pL of RNase-free SPRI beads were added to the reaction and mixed on a mixer for 5 minutes at room temperature. The sample was then centrifuged, and the supernatant discarded after magnetic separation. The beads were washed twice with 200 pL of freshly-prepared 70% ethanol, and any residual ethanol was removed by air-drying the beads for 30 seconds. The beads were resuspended in 15 pL of nuclease-free water and incubated on ice for 5 minutes. The sample was cleared by magnetic separation, and the 15 pL eluate containing the polyadenylated RNA was retained.

[0086] Following the SPAAC reaction, enrichment was achieved through specific binding to streptavidin-coated magnetic beads, as detailed previously18. Briefly, RNA samples were incubated at 25°C for 30 minutes with 15 pL of pre-blocked magnetic Dynabeads™ MyOne™ Streptavidin Tl. Pre-blocking was carried out with 100 ng / pL of bacterial small RNAs in 100 pL of immobilization buffer, comprising 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 2 M NaCl. Following thorough washing with a wash buffer (10 mM Urea, 5 mM Tris-HCl [pH 7.5], 0.5 mM EDTA, and 1 M NaCl) for five cycles at 25°C for 5 minutes each, biotinylated RNAs were eluted by incubating the beads with 20 pL of MK-Gel Loading Buffer at 90°C for 2 minutes. Subsequently, the biotinylated RNAs underwent purification using Monarch® RNA Cleanup Columns (NEB) as outlined above and were eluted in 10 pL of nuclease-free water.

[0087] Nanopore RNA sequencing and subsequent bioinformatics analysis were carried out using a comprehensive pipeline. Nanopore sequencing libraries were prepared according to the manufacturer's instructions using the PCR-cDNA Barcoding Kit (SQK-PCB111.24) with R9.4.1 flow cells. Raw nanopore reads underwent quality control using NanoQC (https: / / github.com / wdecoster / nanoQC) to ensure data reliability. Subsequently, alignment to the reference genome (https: / / www.gencodegenes.org / human / release_43.html) was performed using Minimap2 (https: / / github.eom / lh3 / minimap2#uguide), resulting in the generation of a Binary Alignment Map (BAM) file.

[0088] Transcript quantification was performed using Salmon (version 1.10.1) with the aligned reads as input. The resulting quantification files (e.g., transcripts per million, TPM) were generated for downstream analysis. For differential expression analysis, edgeR (version 3.36.0) was employed. The quantification files from Salmon were converted to count matrices, which served as input for edgeR. The count data were normalized using the trimmed mean of M-values (TMM) method. Visualization of transcriptomic data was conducted using the ggplot2 package in R.

[0089] Exosome Isolation

[0090] Extracellular vesicles, including exosomes, were isolated using the Total Exosome Isolation Reagent (Invitrogen™). A total of 50 mL of cell culture media harvested from HeLa cell cultures underwent exosome isolation following a standardized protocol. Initially, the harvested media underwent triple centrifugation: first at 300 x g for 10 minutes, then at 2000 x g for another 10 minutes, and finally at 10,000 x g for 30 minutes at 4°C, aimed at effectively removing cells and debris. The resulting supernatant was carefully transferred to new tubes. Exosomes were isolated from the cell-free culture media using the Total Exosome Isolation (from cell culture media) reagent according to the manufacturer's instructions. Briefly, 50 mL of cell-free culture media was mixed with 0.5 volumes of the isolation reagent, vortexed thoroughly, and incubated overnight at 4°C. After incubation, samples were centrifuged at 10,000 x g for 1 hour at 2°C, and the supernatant was aspirated and discarded, leaving the exosome- containing pellet. Pellets were then resuspended in 400 pL of IX PBS. The isolated exosomes were subsequently subjected to RNA isolation using the TriZol extraction method and Western blot analysis using the cxosomc marker CD9 to confirm the presence of cxosomal markers.

[0091] Intercellular Transfer of GlycoRNAs via Exosomes

[0092] To investigate the transfer of glycoRNAs via exosomes, Ac4GalNaz-labeled exosomes were isolated from HeLa cells cultured in two 150 mm plates containing 30 mL of DMEM medium, as described above. The isolated exosomes were then incubated with naive cells for 10 and 20 hours. Following incubation, the cells underwent extensive washing to remove any extracellular exosomes. Total RNA was subsequently extracted from the naive cells and subjected to strain-promoted azide-alkyne cycloaddition (SPAAC)-mediated conjugation to enable visualization of biotin-conjugated RNAs.

[0093] DsiRNA-mediated knockdown and NGI-1 treatment

[0094] HeLa cells were seeded 18 hours prior to transfection to achieve an optimal -50% confluency in a 6-well plate. Before transfection, each well was supplemented with 1.0 mL of complete medium containing serum and antibiotics, followed by an incubation period of 30 to 60 minutes. DsiRNA, at a final concentration of 5.0 nM per well, was diluted into 100 pL of PepMute™ Transfection Buffer working solution and thoroughly mixed. Subsequently, 2.4 p L or 3.6 pL of PepMute™ reagent was added and thoroughly mixed by pipetting. The mixture was then incubated at room temperature for approximately 15 minutes to facilitate the formation of the transfection complex. The transfection mix was carefully added drop-wise to the cells, with gentle rocking of the plate, before being returned to the CO2 incubator. After 24 hours, lOOpM of Ac4GalNAz was added to each well and the cells were incubated for another 48 hours. Gene silencing was assessed 72 hours post-transfection, and the labeled RNA was assessed by blotting as described above. In the case of NGI-1, Manumycin, or GW4869 treatment, cells were treated with the respective compounds for 24 hours. Following this treatment, Ac4GalNAz was added, and the cells were incubated for an additional 48 hours before harvesting either the cells or the media to assess their impact on intracellular or extracellular RNA labeling, respectively.

[0095] Inducing Neuronal Transdifferentiation from Induced Pluripotent Stem Cells

[0096] Human iPSCs were cultured on Matrigel-coated plates in mTeSR™ medium (STEM CELL Technologies), and directly differentiated into induced neurons (iNs) using an inducible lentiviral system as previously described35. For neuronal transdifferentiation, iPSCs were dissociated into single cells using Accutasc and seeded onto Matrigcl-coatcd supplemented with Y-compound (5 pM) and transduced with lentiviruses encoding rtTA (FUW-M2rtTA) and NGN2 (Tet-O-Ngn2-puro). Transfected cells were grown in neurobasal culture medium containing B27 supplement (Invitrogen). Doxycycline (2 pg / ml) was added 24 hrs posttransfection to induce NGN2 and infected cells were selected with the addition of puromycin (2 g. ml) for 48 hrs. Cells were cultured for 7 days in neurobasal culture medium supplemented with B27 and doxycycline.

[0097] During the transdifferentiation process, cells were treated with 100 pM Ac4GalNAz for 48 hours on day 7 of induction. After this labeling period, the cells were washed with PBS and harvested for RNA extraction. Total RNA was extracted using TriZol. The labeled RNA was subjected to biotin-azide click chemistry to attach a biotin moiety to the labeled RNA molecules and the biotinylated RNA was visualized using IRDye® 800CW streptavidin detection.

[0098] The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.

[0099] EXAMPLE I

[0100] GlycoRNAs, with their distinct biochemical properties, have been proposed to play a crucial role in cellular processes, including immune response and gene expression regulation11,12. Our studies demonstrate the presence of glycoRNAs within extracellular exosome vesicles, suggesting a sophisticated sorting mechanism that leverages glycosylation as a determinant for exosomal packaging. This novel insight into exosomal cargo selection not only aligns with the emerging recognition of extracellular RNAs as potential biomarkers for disease but also underscores the intricate regulatory networks orchestrating RNA transport and function. The data show that the glycosylation pattern on glycoRNA serves as a selective signal for their inclusion into exosomes, thereby influencing the repertoire of messages conveyed between cells and modulating recipient cell behavior.

[0101] Identification of a distinct class of human glyco-modified small RNAs via metabolic labeling with N-Azidoacetylgalactosamine-Tetraacylated (Ac4GalNAz)

[0102] Among the recently discovered noncanonical RNA cap structures5, one of the most unique modifications emerges from a structurally diverse array of nucleotide sugars. Remarkably, eukaryotic cellular RNA molecules have been identified to contain 5' uridine diphosphate N-acetyl glucosamine (UDP-GlcNAc) and UDP-glucose (UDP-Glc) moieties. Quantification via mass spectrometry has revealed a strikingly higher abundance of UDP GlcNAc compared to the extensively studied NAD-capped RNAs5. To elucidate the functional role of these novel 5'-end noncanonical modifications, the identification of the decapping enzyme stands as a crucial milestone, as recently demonstrated for NAD-capped RNAs7 13. Considering the remarkable diversity of the substrates targeted by the DXO family of proteins14, we determined whether DXO and Rail proteins possess the capacity to hydrolyze a GlcNAc cap. In vitro, transcribed UDP-GlcNAc capped RNAs (Fig. 1A) were efficiently decapped by His- tagged recombinant Dxo or Rail from E. coli. (Fig. IB). Consistent with their respective activities15, Dxo degrades the RNA following removal of the GlcNAc cap while Rail does not have exonuclease activity and leaves the RNA intact following UDP-GlcNAc removal (Fig. IB). These findings demonstrate the Dxo family of proteins can hydrolyze a GlcNAc cap.

[0103] To determine whether the UDP-GlcNAc caps are detected on endogenous RNA, we leveraged and implemented the N-azidoacetylgalactosamine-tetraacylated (Ac4GalNAz) metabolic labeling approach16(Fig. 2A). This approach takes advantage of metabolic cross-talk between the N-acetylgalactosamine salvage and O-GlcNAcylation pathways for the tagging and identification of O-GlcNAcylated proteins16and an approach analogous to a recent report for the identification of internal N-glycan modifications on small RNA with N- azidoacetylmannosamine-tetraacylated (Ac4ManNAz)10(Fig. 2A). Three different glyco derivatives, Ac4GalNAz, Ac4ManNAz, and N-azidoacetylglucosamine-tetraacylated (AC4G1CNAZ) were used as potential labeling agents in HeLa cells, human embryonic kidney (HEK) 293 and breast cancer MCF7 cell lines (Fig. 2 A and Fig. 3 A, Fig. 3B), for the identification of 5' UDP-GlcNAc caps on cellular RNA.

[0104] To visualize the azide-labeled RNA, we employed strain-promoted azide-alkyne click chemistry (SPAAC)1748. Interestingly, a distinct temporal and selective Ac4GalN Az-dependent pattern in the biotinylated RNA species was evident (Fig. 2B). Moreover, the migration of the glycoRNA on agarose gel electrophoresis indicated the modification occurred exclusively within smaller RNAs (Fig. 2B). Although no detectable biotinylated species were discerned when employing Ac4ManNAz or AC4G1CNAZ for labeling, a faint larger migrating species was detected with the Ac4ManNAz upon overexposure (Fig. 2B). The presence of slower migrating glyco RNA detected with Ac4ManNAz. Considering the prominent signal with the Ac4GalNAz labeled RNA, wc focused the subsequent analysis on Ac4GalNAz glycolatcd RNAs.

[0105] To further confirm the size distribution of the faster migrating GalNAz RNA, the size was validated through a size-dependent RNA precipitation and silica column-based fractionation method, segregating transcripts into "large" (>200 nt) and "small" (<200 nt) categories. Consistently, glycoRNAs extensively co-fractionated with the small RNA population (Fig. 2C) and were primarily devoid of polyadenylated RNA (Fig. 3C). Interestingly, the detected glycoRNA was not sensitive to treatment with the sequence restricted nuclease, RNase A, yet sensitive to the more sequence independent RNase I nuclease where RNA degradation was evident (Fig. 2D). Moreover, the signal was eliminated upon exposure to the more pleotropic nuclease, micrococcal nuclease prior to resolution on the gel with the expected Ca2+dependance (Fig. 4D and Fig. 3D). Consistent with the signal being derived from RNA, it was resistant to DNase treatment (Fig. 2D). These findings suggest that cells labeled with Ac4GalNAz predominantly incorporate the azide label into relatively small structured cellular RNA species.

[0106] To determine whether the azide detected on the endogenous RNA was constituted by a 5' end cap, the RNA was treated with SpRail to remove the GlcNAc cap. Surprisingly, a decrease in the intensity of the IR-800 signal was not evident implying that the glyco-modification is not on the 5' end but likely an internal modification (Fig. 2E). Collectively, our findings indicate that the prevailing glycoRNA detected in HEK293T, and HeLa cells predominantly represent an internal GalNAc modification rather than a UDP-GlcNAc cap.

[0107] Transcriptome-wide mapping of Glyco-modified RNAs identified a diverse group of small noncoding RNAs.

[0108] To identify glyco-modified transcripts, small RNA fractions were first isolated from Ac4- GalNAz-labeled HeLa and HEK293T cells (Fig. 5A). Following SPAAC and affinity purification with streptavidin beads, the captured biotinylated RNAs (Fig. 5B)), were next subjected to Oxford nanopore sequencing. Given that Oxford nanopore sequencing primarily targets poly(A)- containing RNAs, an additional polyadenylation step was introduced using E. coli poly(A) polymerase. This step was necessary since the Ac4GalNAz-labeled RNAs did not yield detectable poly(A) RNAs (Fig. 4C). The sequencing outcomes revealed a diverse collection of small noncoding RNAs, including various isoforms of snoRNAs, snRNAs, vault RNAs, and Y RNAs in both HeLa and HEK293T cells (Figs. 5C and 5D). A parallel experiment without Ac4GalNAz-labclcd RNA served as a negative control, ensuring the specificity of the approach. Enrichment results were further validated by Northern blot analysis of two representative RNAs, RNY3 and 5S rRNA, using32P labeled transcript-specific probes (Fig. 5E). Analysis of the identified RNAs revealed a remarkable congruence with previously identified exosomal RNAs19,20, including Y RNAs, vault RNAs, and rRNAs suggesting a close association between glycoRNAs and membrane organelles.

[0109] Exosomal RNAs are modified with glyco-derived modifications.

[0110] To begin unraveling the role of the glyco modifications of RNA in cellular physiology, we evaluated their intracellular localization. Two distinct biochemical approaches were utilized to determine the distribution of glycoRNAs within cellular compartments. The first involved fractionation of nuclei away from total cytosol while the second further refined the cytoplasmic fraction into the soluble cytosolic compartment and membranous organelles. IR-800 signal was almost exclusively detected in RNAs derived from Ac4GalNAz-labeled total cytoplasmic fraction but not the nuclear’ fraction (Fig. 4A). Moreover, the cytoplasmic glycoRNAs were predominantly detected within the membrane fraction relative to the soluble fraction (Fig. 4B). These observations suggest the glycoRNAs are primarily associated with membranes. However, although Ac4MalNAz-labeled RNAs were recently shown to localize to the extracellular surface of the HeEa cell membrane10, the glycoRNAs described here are likely distinct from the previous report. First, micrococcal nuclease treatment of intact cells did not appreciably diminish glycoRNA detection (Fig. 6A) indicating the Ac4GalNAz-derived RNAs are not localized on the cell surface. Second, distinct categories of small RNAs (sRNAs) were recently delineated within the endoplasmic reticulum (ER)21suggesting internal membranes may house the glycoRNAs. Third, the correspondence of the identified glycoRNAs (Figs. 5C, 5D) to exosome RNAs strongly support exosomal association19,20.

[0111] To directly determine whether glycoRNAs localized within extracellular’ membrane vesicles, the schematic outlined in Fig. 4C was employed for exosomal RNA isolation. Fractionation of the exosome was validated utilizing Western blot analysis of GAPDH and exosome marker CD922(Fig. 4D). RNA extracted from Ac4GalNAz-labeled HeEa cell exosomes were subjected to SPAAC analysis, and the resulting biotin-conjugated RNAs were evaluated. SPAAC analysis demonstrated that the Ac4GalNAz-derived glycoRNA were almost exclusively contained within HcLa cell exosomes (Fig. 4D). Importantly, the glycoRNAs were not detected on the extracellular surface. Subjecting the Ac4GalNAz-labeled exosomes to micrococcal nuclease had no effect on the labeled glycoRNA content (Fig. 6B) indicating that glycoRNAs are encapsulated within the exosomal lumen, rather than being surface-associated.

[0112] The composition of exosomal glycoRNAs was next addressed. Glyco-modified exosome RNAs were affinity purified using streptavidin beads, and the eluted biotinylated RNAs were subjected to Oxford nanopore sequencing (Fig. 4E). A striking correlation in the RNA species was evident between the glycoRNAs from cellular and exosomal sources (Fig. 4F, Fig. 6C). This finding underscores the consistency and similarity in the glyco-modification profiles between cellular and exosomal RNA populations.

[0113] To assess the relative abundance of glyco-modified RNAs in both exosomes and cells, we compared the labeling of RNA used for the SPAAC. Given that exosomes predominantly contained small RNAs (sRNAs), our focus narrowed to the small RNA fraction within cellular RNAs. Intriguingly, HeLa exosomes harbored a disproportionate percentage of labeled glycoRNAs compared to cellular sRNAs (Fig. 7B) indicting a potential functional significance for the glycosylation to target RNA to the exosome.

[0114] ESCRT-dependent and independent pathways govern the release of glyco-modified RNA-enriched exosomes.

[0115] The formation of exosomes involves an active contribution of the endosomal- sorting complex required for transport (ESCRT) machinery and a key component of this activity is the protein Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate (HGS) also known as Vps2723,24(herein referred to as HGS). A notable mechanistic dichotomy driving ESCRT- dependent and ESCRT-independent pathways exists25(Fig. 8A). Manumycin-A (MA)22, a natural microbial metabolite is a potent inhibitor of exosome biogenesis and secretion which is ESCRT- dependent with no discernible impact on cell growth. Similarly, ceramide, whose biosynthesis is regulated by neutral sphingomyelinase 2 (nSMase2) is critical for ESCRT-independent secretion of exosomes26, and inhibition of nSMase2 by GW486926leads to a block of exosome release. To substantiate the presence of glycoRNAs in exosomes and examine the influence of ESCRT-dcpcndcnt and independent pathways, wc employed HGS knockdown using two distinct DsiRNA as well as the MA and GW4869 inhibitors targeting exosome biogenesis. The efficiency of HGS knockdown in HeLa cells was monitored by quantitative real-time polymerase chain reaction (qRT-PCR) 72 hours post DsiRNA transfection (Fig. 8B). As depicted in Figs. 8C and 8D, the depletion of HGS and the administration of MA and GW4869 resulted in a significant reduction in glyco-modified exosomal RNAs, evidenced by a decrease in the IR-800 signal. To reinforce the assertion that this decrease in signal stemmed from the inhibition of exosomal release, we also examined cellular RNAs from the same set of HGS knockdown experiments from Fig. 8C. As anticipated, a corresponding increase in cellular glycoRNAs (Fig. 8E) was observed, indicating a block in exosome release and corresponding accumulation of exosome and its glycoRNA cargo within the cytoplasm. In summary, our findings underscore the intricate involvement of both ESCRT-dependent and independent pathways in the biosynthesis of glycoRNAs within exosomes.

[0116] Distinctive Signatures of Glyco-Modified Exosomal RNAs Across Human iPSCs, and Induced Neurons

[0117] To elucidate the presence of glyco-modified exosomal RNAs across diverse cell types and explore their distinctive signatures we investigated their fate during induced neuronal transdifferentiation (Fig. 7A). Total Ac4GalNAz-labeled cellular and exosomal RNAs were extracted from human induced pluripotent stem cells (iPSCs) and their derived induced neuron cells (iNs) were subjected to SPAAC reaction after 48 hours of incubation with lOOpM Ac4GalNAz-containing media. Importantly, exosomal RNAs from iPSCs and iNs exhibit a remarkable distinction in their size profile (Fig. 7A), which was also evident in the cellular RNAs (Fig. 7B). iPSC-derived exosomes are characterized by the prevalence of slower migrating RNAs, while iNs showcase faster migrating RNAs (sRNAs), aligning with the profiles observed in the more differentiated HeLa and HEK293T cell lines suggesting differentiation dependent distribution of exosomal glycoRNAs.

[0118] Intercellular Communication of Glyco-Modified Exosomal RNA Exosome-mediated transfer of RNAs has been shown as a novel mechanism of genetic exchange between cells27. To assess the capacity of cells to communicate through the cxosomc- directed transfer of glycoRNA, naive HeLa cells were incubated with and without Ac4GalNAz- labeled exosomes isolated from HeLa cells for 10 and 20 hours (Fig. 9A). Following extensive washing to eliminate extracellular exosomes, total RNA was isolated and visualized through SPAAC-mediated biotin conjugation. Remarkably, glyco-modified exosomal RNAs were detected in the naive HeLa cells (Figs. 9B and 9C), demonstrating exosome-mediated cellular communication of glycoRNA.

[0119] Inhibition of Protein Glycosylation curtails Exosomal glycoRNA

[0120] A recent report28has unveiled the amino-carboxypropyl-modified Uridine (acp3U) as a crucial site for attaching N-glycans to RNA. Building on this finding and supported by genetic knockout data targeting the DTWD2 enzyme responsible for cellular acp3U in mammalian cells, we determined whether acp3U was a primary RNA modification essential for N-glycosylation of RNA in our system. DsiRNA-mediated knockdowns of DTWD2 and another relevant amino- carboxy-propyl transferase, TSR3 were carried out in HeLa cell lines to assess the impact of acp3U on Ac4GalNAz-derived glyco-modification. The efficiency of knockdown was evaluated through qRT-PCR (Fig. 10A). Interestingly, knockdown of either DTWD2 or Tsr3 did not manifest a discernible impact on the levels of Ac4GalNaz-derived glyco-modification (Figs. 10B and 10C) suggesting the observed RNA glycosylation is not mediated through acp3U conjugation.

[0121] We next set out to identify the specific sugar precursor responsible for the glyco modification and targeted Galactose-4-Epimerase (GALE) and UDP-Mannose-4-Epimerase (GNE)29. GALE and GNE fulfill crucial roles in converting UDP-Glucose to UDP-GalNAc, a process integral to the production of CMP-sialic acid utilized by the Golgi for sialylated glycoconjugation in proteins (Fig. 11 A). The GalNAc salvage pathway involves GALE as a central enzyme mediating the interconversion between UDP-GlcNAc and UDP-GalNAc. GALE's enzymatic activity generates key nucleotide sugars essential for glycosylation in the endoplasmic reticulum (ER) and Golgi29. Simultaneously, GNE converts UDP-GalNAc and GlcNAc to ManNAc-6 phosphate, a crucial step in glycosylation events shaping cellular processes. HEK293T cells were transfected with two distinct DsiRNAs targeting GALE and GNE, followed by a 72-hour incubation. Total RNA extracted from these cells was subjected to qRT- PCR to assess the efficiency of gene knockdown (Fig. 1 IB). After 24 hours of DsiRNA treatment, Ac4GalNAz was added to the media and RNA was isolated after 48 hours of incubation, resolved by agarose gel and visualized (Fig. 11C). Significantly, the substantial increase in the IR-800 signals following the knockdown of both GALE and GNE strongly suggested an elevated relative steady state of Ac4GalNAz. These observations implicate Ac4GalNAz as the source of the observed modifications. Furthermore, our findings underscore that the steady-state levels of Ac4GalNAz play a pivotal role in determining the abundance of glycoRNAs.

[0122] Next, we investigated the impact of NGI-1, a specific and potent small molecule inhibitor of oligosaccharyltransferase (OST)30, which transfers branched sugars onto protein in the endoplasmic reticulum, on glycoRNA production. Interestingly, NGI-1 treatment resulted in a dose-dependent loss of glycoRNA labeling for both cellular and exosomal RNAs with Ac4GalNaz (Fig. 1 ID). These data indicate that a block of protein glycosylation also leads to a decrease in RNA glycosylation and a potential coordination between protein and RNA glycosylation. Overall, our results underscore the shared regulatory pathways involving GalNAc and its associated enzymes in orchestrating protein and RNA glyco modifications.

[0123] Using a slightly different modification and isolation protocol, additional coding glycated RNAs were identified in Hela cells (Figs. 12A-12H) and in exosomes (Fig. 121).

[0124] Discussion

[0125] Our investigation into glycoRNAs within exosomes has provided novel insights into the intricate mechanisms of RNA sorting and the potential functional implications of RNA glycosylation. The findings presented in this manuscript align with the emerging understanding of extracellular RNAs (exRNAs) as multifaceted players in intercellular communication11 12. Particularly, the identification of glycoRNAs on the cell surface, as well as the development of membrane-coated nanoparticles for RNA analysis, underscores the complexity and regulatory potential of RNA outside the canonical intracellular environment.

[0126] The biogenesis and specific sorting of glycoRNAs into exosomes, as revealed by our study, indicate a targeted mechanism that facilitates cell-cell communication and could have implications for the regulation of gene expression. The higher degree of glyco-labeled RNA within cxosomcs relative to cellular RNA suggests glycoRNA arc preferentially encapsulated within exosomes. Considering that glyco-modification of proteins is an important prerequisite for protein sorting into exosomes, it appears that glyco-modification fulfills a similar function for RNA and serves as the conduit for RNA trafficking into exosomes. Moreover, the disruption of oligosaccharyltransferase which transfer branched glycol moieties onto a protein in the ER with the use of NGI-1 inhibitor, altered the level of targeted glycoRNAs within exosomes (Fig. 11D) indicating that accumulation of glycoRNAs to exosomes is a regulated process. This finding indicates that the same pool of glycol moieties are utilized to modify both protein and RNA and implicates a common regulatory pathway.

[0127] Although we initially set out to identify UDP-GlcNAc capped RNA, two lines of evidence suggest the glycoRNA species detected are likely not glyco-capped RNA but rather consist of internally glyco modified RNA. First, the pleotropic decapping enzyme family of DXO and Rail which can decap GlcNAc-capped RNA, do not remove the glyco moiety from the glycoRNA (Fig. 2E). Second, the remarkable resistance to RNase A (Fig. 2D) and susceptibility to other nucleases (RNase I and micrococcal nuclease) strongly suggests internal modification of the RNA that impedes nucleases either indirectly by affecting the structure or directly by virtue of its modification of the RNA. Its enhanced resistance to nucleases is also indicative of a potential function for glycosylation in the stability of the modified RNA. A detectable resistance to RNAse A treatment was also recently reported for cell surface RNAs detected following Ac4ManNAz treatment of cells31indicating a common feature of the glyco modification in providing enhanced nuclease resistant and stability onto an RNA.

[0128] The finding in this report complement the recent findings that identified glycoRNA derived from Ac4ManNAz that appear as slow migrating glycoRNAs on an agarose gel and can reside on the cell surface10 11. Importantly, the Ac4GalNAz glycoRNA reported here appears to be distinct and considerably more abundant with the assay parameters used (Fig. 2, Fig. 3A, Figure 12). Moreover, the Ac4ManNAz-derived glycoRNAs are predominantly on the cell surface28while the Ac4GalNAz glycoRNA we detect in the exosome are resistant to nuclease treatment of the intact cell as well as the intact exosome and are intraluminal. These findings indicate there are at least two classes of glycoRNAs. In conclusion, our findings contribute to a growing body of evidence that positions extracellular RNAs, and specifically glycoRNAs, as significant entities in the extracellular milieu. The role of RNA glycosylation in the sorting and stabilization of exRNAs within exosomes opens new avenues for research and potential clinical applications. Moreover, the use of glycoRNAs as either biomarkers for disease diagnosis, or for linkage in dicistronic RNAs for delivery into the exosomes of target cells for treatment or prevention of disease is now achievable.

[0129] EXAMPLE II

[0130] As mentioned above, the exosomes described herein can be used to advantage to deliver therapeutic RNAs to patients in need thereof. Target viral antigens are specific molecules on a virus that are recognized by the host's immune system, particularly by antibodies and T cells. Target oncoprotein antigens are specific molecules present on cancer cells. These antigens are crucial for the development of vaccines and antiviral therapies or anticancer therapies as they represent the targets for immune responses aimed at neutralizing or eliminating the vims or cancer.

[0131] Target viral antigens are specific molecules on a vims that are recognized by the host's immune system, particularly by antibodies and T cells. Target oncoprotein antigens are specific molecules present on cancer cells. These antigens are cmcial for the development of vaccines and antiviral therapies or anticancer therapies as they represent the targets for immune responses aimed at neutralizing or eliminating the vims or cancer.

[0132] Targeting strategies include without limitation, antibody-mediated neutralization where antibodies can bind to viral antigens, neutralizing the virus and preventing it from infecting cells. It may also be desirable to induce T cell-mediated immunity. T cells can recognize viral antigens or oncoprotein antigens presented on infected cells or tumor cells, respectively, and destroy them.

[0133] The exosomes or extracellular vesicles described herein can also be used to advantage to develop vaccines. These can be designed to elicit immune responses against specific viral antigens, leading to long-term protection. Antiviral drugs can also be generated which target viral proteins or nucleic acids involved in different stages of the viral life cycle, such as entry, replication, or assembly.

[0134] In some embodiments, the glycoRNA can encode an immunogen that can elicit an immune response against one of the following virus family members: Adenovirus, Bunyavirus, Caliciviridae, Coronavirus, Filovirus, Flavivirus, Hepadnavirus, Hepatitis viruses, Herpesvirus, Orthomyxovirus, Papovaviruses, Paramyxoviridae, Parvovirus, Pestivirus, Picomavirus, Poxviridae, Reovirus, Retrovirus, Rhabdovirus, and Togavirus. For example, if Covid is to be treated, a nucleic acid encoding a spike protein or an immunogenic variant or fragment thereof could be loaded into exosomes. For influenza, targeting HA or NA would be suitable. For RSV, F-protein could be targeted. For Herpes simplex, glycoproteins C (gC) and G (gG) could be targeted. For Herpes zoster, glycoprotein E could be targeted.

[0135] RNAs can be generated in cells either from a transiently transfect construct or by stably integration. Constructs would encode an RNA that contains the glycosylation element and the target gene of interest transcribed by a eukaryotic promoter and containing pre-mRNA processing elements to express the target mRNA. The RNA could also be generated from a genomic modification. The glycosylation element can be incorporated into an endogenous gene of interest (e.g., CRISPR technology) to express the target RNA of interest containing the glycosylation element to enable localization of the said RNA into exosomes.

[0136] Isolated exosomes containing the glycosylated target RNA of interest can be used to transfer the said RNA into recipient cells.

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[0171] While certain features of the invention have been described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the ait. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising a population of exosomes or extracellular vesicles harboring internally modified glycoRNAs and at least one RNA encoding a molecule of interest for exosome mediated delivery of said RNA molecule of interest into a target cell in a biological carrier.

2. The composition of claim 1, wherein said glycoRNA comprises galactose or galactosamine and their derivatives.

3. The composition of claim 1, wherein said glycoRNA is selected from at least one unique exosomal glycoRNA listed in Figure 6C or Figure 121.

4. The composition of claim 1, wherein said glycoRNA is RNAase A resistant.

5. The composition of claim 1, wherein said RNA of interest is an inhibitory nucleic acid.

6. The composition of claim 1 or claim 3, wherein said modified glycoRNA and said RNA encoding a molecule of interest are operably linked on a single RNA strand.

7. The composition of claim 5, wherein said inhibitory nucleic acid is an RNA selected from an siRNA, an antisense oligonucleotide, an shRNA, and a ribozyme having sufficient sequence homology to a nucleic acid encoding a protein targeted for inhibition, said inhibitory nucleic acid reducing expression of said protein in said target cell.

8. The composition of any one of claims 1-7, wherein said RNA of interest encodes at least one vaccine antigen, a protein of interest, an antibody or functional fragment thereof, and an antitumor antigen.

9. The composition of claim 1, wherein said RNA of interest encodes a single chain antibody.

10. The composition of any one of the preceding claims, wherein composition further comprises a vector encoding UDP-galactose 4'-epimerase (GALE) and said internal RNA modification comprises at least one galactose.

11. A composition comprising an isolated exosome or extracellular vesicle population comprising unique internally modified glycoRNA biomarkers, said biomarkers being indicative of the presence of a disease or metabolic disorder in said subject.

12. The composition of claim 1 , wherein said glycoRNA is shown in Figure 6C.

13. The composition of claim 1, wherein said glycoRNA is shown in Figure 121.

14. The composition of any of claims 11-13, wherein said disease or disorder is selected from viral infection, cancer, diabetes, and neurological disorders.

15. The composition of claim 11, wherein said modified glycoRNA biomarker levels are comparable to those determined in a control subject having said disease or disorder.

16. The composition of claim 14, wherein said disease is cancer and said RNA in said exosome or extracellular’ vesicle encodes a tumor antigen selected from N YES 0-1, Her-2 / neu, MAGE-1, Tyrosinase, MUC1, CEA, Mam-A, hTERT, Sialyl-Tn, WT1, a- fetal protein, and CA-125.

17. A method for delivery of an exosome population harboring a therapeutic agent to a subject in need thereof, said agent comprising an RNA molecule of interest in combination with, or operably linked to an internally glycosylated RNA molecule (glycoRNA), said method comprising a) introducing said RNAs into a target cell, wherein RNA encoding said therapeutic agent is expressed and targeted to the exosome by said glycoRNA, followed by extrusion of said exosome from said cell; b) collecting said extruded exosome comprising said RNAs, and c) delivering said exosome population to a subject.

18. The method of claim 17, wherein said inhibitory nucleic acid is an RNA selected from an siRNA, an antisense oligonucleotide, an shRNA, and a ribozyme having sufficient sequence homology to a nucleic acid encoding a protein targeted for inhibition, said inhibitory nucleic acid reducing expression of said protein in said target cell.

19. The method of any one of claims 17 or 18, wherein said RNA of interest encodes at least one vaccine antigen, a protein of interest, an antibody or functional fragment thereof, and an antitumor antigen.

20. The method of claim 17 or claim 18, wherein said RNA of interest encodes a single chain antibody.

21. The method of of claim 17, wherein the RNA encodes an immunogen that elicits an immune response against one or more of Adenovirus, Bunyavirus, Caliciviridae, Coronavirus, Filovirus, Flavivirus, Hcpadnavirus, Hepatitis viruses, Herpesvirus, Orthomyxovirus, Papovaviruscs, Paramyxoviridae, Parvovirus, Pestivirus, Picornavirus, Poxviridae, Reovirus, Retrovirus, Rhabdovirus, and Togavirus.

22. The method of claim 19, wherein the RNA encodes an immunogen that elicits an immune response against a tumor antigen or oncoprotein.

23. The method of claim 17, wherein said RNA in said exosome or extracellular vesicle targets a tumor antigen selected from NYESO-1, Her-2 / neu, MAGE-1, Tyrosinase, MUC1, CEA, Mam- A, hTERT, Sialyl-Tn, WT1, a- fetal protein, and CA-125.

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