TARGETING MICRO-RNAS FOR EXOSOMAL DELIVERY OR

CELLULAR RETENTION

DESCRIPTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims benefit of U.S. Provisional Application No.

62/703,566 filed July 26, 2018, the contents of which are incorporated herein by reference in their entirety.

STATEMENT OF FEDERAL FUNDING

[002] This invention was made with government support under DK082659 awarded by the National Institute of Health. The government has certain rights in the invention.

BACKGROUND

[003] Extracellular vesicles mediate cell to cell communication and are known to play a role in physiological and pathological processes. Extracellular vesicles may derive from the plasma membrane (e.g., microvesicles) or from the endosomal compartment (e.g., exosomes) and deliver their contents from origin to local or distant sites. microRNAs (miRNAs) are a class of non-coding RNAs that function as negative regulators of translation and are involved in many cellular processes. Exosomes carry mRNA, miRNA and other non-coding RNA that can be transferred to recipient cells. miRNAs were discovered in 1993 and are now known to mediate human disease. See Lee et al. (1993) Cell 75, 843-854. For example, it has been described that two human miRNA genes, mir-l5a and mir-l6-l, are downregulated or deleted in chronic lymphocytic leukemia (CLL). See Calin et al., Proc. Natl. Acad. Sci. USA (2002) 99,15524-15529. In addition, miRNAs are being explored clinically for the treatment of hepatitis C vims (HCV) infection. 5A? Lindow, M., and Kauppinen, S. (2012). J. Cell Biol. 199, 407-412.

[004] Exosomes are extracellular lipid vesicles released by every cell. They contain several classes of macromolecules including DNA, mRNA, proteins and micro- RNAs (miRNAs). Among all these molecules, exosomes seem to be particularly enriched in miRNAs. Exosomes have been demonstrated to be a very efficient delivery tool to transfer macromolecules to target cells where they can exert biological functions. For instance, exosomes can deliver miRNAs to repress gene expression in the target cell.

[005] Adipose tissue derived exosomes may have an especially potent effect in delivering miRNAs. Adipose tissue-derived miRNAs are released in vivo into the bloodstream and delivered to the liver, among many other potential tissue targets, where they can regulate hepatic expression of major metabolic genes such as fibroblast growth factor (FGF)-2l (see Thomouet al., Nature , 542:450-4555 (2017)).

[006] Despite extensive work in the last years, it remains unclear how miRNAs are selected and sorted into exosomes. Santangelo and colleagues described a GGCU motif that can enrich exosomal sorting in the mouse hepatocyte 3 A line ( see Santangelo et al. Cell Reports 17:799-808 (2016)), and Villarroya-Beltri and colleagues described a GGAG motif that can enrich exosomal sorting in in human lymphoblasts (see Villarroya- Beltri et al. Nature Communications 2980 (2013)). However, it is not yet clear whether exosomal sorting motifs can be broad to control sorting in a wide range of cells or alternatively if specific exosomal sorting can be cell-selective and limit sorting in only specific types of cells.

[007] The present application describes exosomal sorting and retention motifs that can be used therapeutically to direct miRNAs to desired cellular locations. Described herein are mechanisms that govern the selection of miRNAs into exosomes in a panel of different cell lines. Exosomal sorting motifs showed different levels of enrichment in individual cell lines, meaning that a sorting motif can be optimized to engineer artificial miRNAs by adding or removing a sequence specific to a particular cell type of interest. Similarly, motifs can be engineered to avoid exosomal sorting in particular cell types. SUMMARY

[008] In accordance with the description, this study analyzed miRNA motifs responsible for exosomal sorting or cellular retention in different cell lines. Five different mouse cell lines resembling major metabolic cells were cultured in vitro : 3T3-L1 (white adipocytes), BAT (brown adipocytes), C2C12 (muscle cells), AML12 (hepatocytes), and SVEC (vascular endothelial cells). Exosomes and cell lysates were collected, and miRNA profiling was performed to analyze miRNA expression. Motifs that regulate sorting of miRNAs into exosomes in cell-specific manner were determined. Conversely, motifs are described that limit exosomal sorting and enrich retention of miRNAs in the cell.

[009] In some embodiments, a method for producing miRNA-containing exosomes or exosome-like vesicles in vitro is provided comprising the steps of modifying a miRNA to include at least one exosomal sorting motif and/or removing any cellular retention motifs, and introducing the modified miRNA into a cell that produces exosomes or exosome-like vesicle under conditions that will result in expression of the modified miRNA. The exosomal sorting motif is selected from UGUG, GGAG, CAUG, GGCA/G, A/CGGG, CUGG, and CGGGAG. The cellular retention motif is selected from CAGU, ACAG, AUUG, UAGC, and CCCG. In some embodiments, the method further comprises collecting the produced exosomes or exosome-like vesicles that contain the modified miRNA.

[0010] In some embodiments, the exosomal sorting motif is UGUG.

[0011] In some embodiments, the exosomal sorting motif is GGAG.

[0012] In some embodiments, the exosomal sorting motif is CAUG.

[0013] In some embodiments, the exosomal sorting motif is GGCA/G.

[0014] In some embodiments, the exosomal sorting motif is A/CGGG.

[0015] In some embodiments, the exosomal sorting motif is CUGG.

[0016] In some embodiments, the exosomal sorting motif is CGGGAG.

[0017] In some embodiments, the miRNA comprises one exosomal sorting motif. In some embodiments, the miRNA comprises more than one exosomal sorting motif.

[0018] In some embodiments, subjects are intravenously injected or otherwise administered culture-derived exosomes containing a miRNA of interest and these exosomes deliver their miRNA cargo to a target cell, leading to the reduction of the expression of the gene of interest. In order to induce or increase the export of the desired miRNA to the exosomes, an exosomal sorting motif can be inserted in, or appended to, the miRNA sequence in the cultured cells and/or a cell retention motif removed. In fact, as different cell types seem to have different usage of the motifs (as shown in Figure 4), the exosomal enrichment may be further optimized by inserting the specific motif that this particular cell type preferentially uses to export its miRNAs. If the miRNA of interest contains a cellular retention motif, this sequence can be removed and replaced by an exosome-enrichment motif, without which exosomal enrichment and potential clinical use might be very limited.

[0019] Therefore, in some embodiments, the method described above further comprises administering the exosome or exosome-like vesicle to a subject.

[0020] Therapeutically, exosomes loaded with particular miRNAs may be used to treat diseases where decreasing the expression of a target gene is desired, such as oncogenes in cancer, or inflammatory, lipogenesis- or gluconeogenis-promoting genes in obesity and type 2 diabetes. Thus, in some embodiments, a method of treating a subject in need of gene silencing is provided comprising administering to the subject an exosome, wherein the exosome is produced in vitro by a) modifying a miRNA to include at least one exosomal sorting motif and/or removing any cellular retention motifs, and b) introducing the modified miRNA into a cell that produces exosomes or exosome-like vesicles under conditions that will result in expression of the modified miRNA, and collecting the produced exosome comprising the modified miRNA, wherein the exosomal sorting motif is selected from UGUG, GGAG, CAUG, GGCA/G, A/CGGG, CUGG, and CGGGAG and the cellular retention motif, if present, is selected from CAGU, ACAG, AUUG, UAGC, and CCCG.

[0021] In some embodiments, modifying the miRNA with an exosomal sorting motif results in more miRNA in the exosome as compared to an exosome produced with a miRNA not modified with an exosomal sorting motif. In some embodiments, the removal of the cellular retention motif results in more miRNA in the exosome as compared to an exosome produced with a miRNA comprising a cellular retention motif. [0022] In some embodiments, the miRNA contains a cellular retention motif and the cellular retention motif is removed.

[0023] Conversely, other applications might require miRNA production and retention into the cell. For instance, ex vivo cellular therapies imply the induction of the expression of genes in cells isolated from patients and later reintroduction of those back into the patient. If that gene is a miRNA, a cellular retention motif may be incorporated into its sequence in order to optimize the number of miRNAs that will be retained in the cells and reduce as much as possible its loss through exosomal secretion. In addition, this strategy may reduce the effect in other cells different from the transplanted by limiting the export and transfer of the inserted miRNA to other cells in the organism through exosomes when they are introduced back to the patient.

[0024] Thus, in some embodiments, a method for retaining miRNA inside a cell in vitro is provided comprising modifying a miRNA to include at least one cell retention motif and/or removing any exosomal sorting motifs, and introducing the modified miRNA into a cell that produces an exosome or exosome-like vesicle under conditions that will result in expression of the modified miRNA, wherein the cell retention motif is CAGU, ACAG, AUUG, UAGC, or CCCG, and the exosomal sorting motif, if present, is UGUG, GGAG, CAUG, GGCA/G, A/CGGG, CUGG, or CGGGAG.

[0025] In some embodiments, a method of treating a subject in need of gene silencing is provided comprising collecting the subject’s cells and manipulating them ex vivo to express an miRNA having at least one cellular retention motif and/or removing any exosomal sorting motifs, and b) administering the ex vivo manipulated cell comprising the modified miRNA to the same or different subject from which it was collected, wherein the cellular retention motif is selected from CAGU, ACAG, AUUG, UAGC, and CCCG, and the exosomal sorting motif, if present, is selected from UGUG, GGAG, CAUG, GGCA/G, A/CGGG, CUGG, and CGGGAG.

[0026] In some embodiments, the cellular retention motif is CAGU.

[0027] In some embodiments, the cellular retention motif is ACAG.

[0028] In some embodiments, the cellular retention motif is AUUG.

[0029] In some embodiments, the cellular retention motif is UAGC. [0030] In some embodiments, the cellular retention motif is CCCG.

[0031] In some embodiments, the miRNA comprises one cellular retention motif. In some embodiments, the miRNA comprises more than one cellular retention motif.

[0032] In some embodiments, the addition of the cellular retention motif reduces the export of the miRNA into an exosome or exosomal-like vesicle. In some

embodiments, the removal of the exosomal sorting motif reduces the export of the miRNA into an exosome or exosomal-like vesicle.

[0033] In some embodiments, the method further comprises administering the cell to a subject.

[0034] In some embodiments, the miRNA levels in non-implanted cell-types after administration to the subject are reduced as compared to levels in subject administered a non-modified miRNA containing cell.

[0035] In some embodiments, the cell is an adipocyte, muscle cell, hepatocyte, or vascular endothelial cell. In some embodiments, the adipocyte is a white adipocyte or brown adipocyte. In some embodiments, the white adipocyte is a 3T3-L1 cell. In some embodiments, the brown adipocyte is a BAT cell. In some embodiments, the muscle cell is a C2C12 cell. In some embodiments, the hepatocyte is an AML12 cell. In some embodiments, the vascular endothelial cell is a SVEC cell.

[0036] In some embodiments when the cell is a hepatocyte or endothelial cell, the sorting motifs are A/CGGG; CUGG; GGAG; and CGGGAG.

[0037] In some embodiments when the cell is a brown adipocyte, white adipocyte, or muscle cell, the exosomal sorting motifs are UGUG; CAUG; CUGG; and CGGGAG.

[0038] In some embodiments, the miRNA is any one of the miRNAs of SEQ ID Nos: 1-704.

[0039] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. [0040] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0041] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment s) and together with the description, serve to explain the principles described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figures 1 A-1D show comparisons of miRNAs among the different cell lines both in the exosomes and cell pellets. By comparing the expression of each miRNA in each cell line with respect to the other four, several cell-type enriched miRNAs for each cell type were identified (shown as outer circles labeled with cell type) in exosomes (Figure 1 A) and in the cell pellets (Figure 1C). Other miRNAs were not particularly enriched in any of the studied cell lines (shown as large inner circles). The top-lO enriched miRNAs for each type in the exosomes (Figure 1B) and cell pellets (Figure 1D) are shown.

[0043] Figures 2A-2E shows analysis of the exosome- and cell lysate-enriched miRNAs for each cell type. The cell lysate refers to the lysate generated from the cell pellet. Most of the miRNAs are specifically found only in the exosomes or in the cell lysate, suggesting the existence of a selective mechanism for exosomal sorting of miRNAs that varies for every cell type. Each cell type also had a smaller number of miRNAs that were present in both the exosome and in the cell lysate (shown in the overlapping region of the exosome and lysate fractions). In some cases, miRNAs detected in exosomes where not detected in the cell lysate and vice-versa; therefore, the total number of miRNAs for each cell type may not match the values in Figure 1.

[0044] Figure 3 shows a Venn diagram representing the number of miRNAs with a significant exosomal enrichment (top [“up”] numbers) or cellular enrichment (bottom [“down”] numbers) in each cell type. Overlapping regions indicate the number of miRNAs showing similar enrichment in more than one cell type.

[0045] Figures 4A and 4B show the main nucleotide motifs significantly enriched in miRNAs that are preferentially sorted into exosomes (Figure 4A) or retained in cells (Figure 4B). The numbers in the table indicate the percentage of miRNAs containing those motifs in each cell type. The presence of two nucleotides at a position means miRNAs were enriched when they contained a motif with either nucleotide at that position. The“total” value represents the percentage of miRNAs from each cell types that contained at least one of the motifs. Some miRNAs contained more than one motif. In this situation, the miRNA is only counted once for the total value, whereas it is counted in both columns referring to those two individual motifs.

[0046] Figures 5A-5E shows the main nucleotide motifs significantly enriched in miRNAs that preferentially are sorted to exosomes or retained in the cell and the number of miRNAs containing that sequence. The numbers in the left column indicate the order of abundance.

[0047] Figures 6A-6F show the effects of introducing or removing some sorting motifs. Figure 6 A shows the name, mature miRNA sequence, and introduced motif for the new miRNA constructs from miR-34c-5p and its wild-type version. Bold underlined text indicate nucleotides that were replaced. The miRNA constructs tested were miR-34c- 5p wt (SEQ ID NO: 648), miR-34c-5p - UGUG (SEQ ID NO: 701), miR-34c-5p-CAUG (SEQ ID NO: 702), and miR-34c-5p-CGGGAG (SEQ ID NO: 703). Figure 6B shows the predicted hairpin structure upon the nucleotide replacements (arrows) in the changes in the nucleotides in the guide strand (described in Figure 6A) and the passenger strand. High pairing possibility between nucleotides is shown in black, while low pairing probability in gray. Figure 6C shows exosomal enrichment measured as the difference between normalized expression of each miRNA version in exosomes divided by the normalized expression in the cell. In both exosomes and cells, expression was normalized in respect to miR-l38b-5p, which was shown to be equally abundant in both exosomes and cells. *P-value<0.05.

[0048] Figure 6D shows the name, mature miRNA sequence, and introduced motif for a new miRNA construct miR-693-3p - mut (SEQ ID NO: 704) and wild-type miR-693-3p (SEQ ID NO: 6). Bold underlined text indicates the nucleotide that was replaced. Figure 6E shows the predicted hairpin structure for miR-693-3p wild-type and the mutated version upon the nucleotide replacements (arrow) in the changes in the nucleotides in the guide strand (described in Figure 6D) and the passenger strand. High pairing possibility between nucleotides is shown in black, while low pairing probability in gray. Figure 6F shows exosomal enrichment measured as the difference between normalized expression of each miRNA version in exosomes divided by normalized expression in the cell. In both exosomes and cells, expression was normalized in respect to miR-l38b-5p. **P-value<0.0l.

DESCRIPTION OF THE EMBODIMENTS

[0049]“Exosomes” as used herein are membrane-surrounded, endosomal-derived vesicles that are present in many biological fluids, including blood, urine, and cultured medium of cell cultures. Exosomes may also be referred to as secreted vesicles. It will be understood that exosomes as described herein may, in certain non-limiting embodiments, also encompass exosome-like vesicles that may vary somewhat from typical exosomes but are still functionally and/or structurally similar or related. Reference to exosome- producing cells herein may include other suitable exosome-like vesicle-producing cells which produce exosome-like vesicles which may vary somewhat from typical exosomes but are still functionally and/or structurally similar or related. For instance, exosomes as described herein may include, in certain non-limiting embodiments, other suitable exosome-like vesicles between 50- l50nm (which contain exosomal markers), and/or larger exosome-like vesicles of l00-600nm.

[0050]“microRNA” or“miRNA” as used herein refers to small non-coding RNA molecules that are evolutionary conserved. miRNAs are naturally occurring in an organism. Alternatively, a miRNA may be designed artificially and not be present in any organism. A miRNA may be chemically modified, for example, to improve stability. A miRNA may affect RNA silencing and post-transcriptional regulation of gene expression.

[0051]“Protein” as used herein, is a protein, polypeptide, or peptide. As such, a “protein” as used in this application may refer to only a portion of a full-length protein that is the product of a gene.

[0052]“Cell (or cellular) retention motif,” as used herein, refers to a sequence of nucleotides that when naturally or artificially present or appended to a miRNA cause the miRNA to be substantially retained in the endosome. [0053]“Exosome (or exosomal) sorting motif,” as used herein, refers to a sequence of nucleotides that when naturally or artificially present or appended to a miRNA cause the miRNA to be substantially present or exported to or into an exosome.

[0054] miRNA constructs (also sometimes referred to herein as“miRNA”) as described herein may be chemically synthesized using, for example, solid phase synthesis, or other methods known in the art. miRNA may also be prepared by cellular or in vitro expression from a suitable expression vector as will be known in the art. Variants, chemically modified analogues, and structural mimics of miRNA as described herein may also be possible.

[0055] miRNA constructs may be introduced into a cell, expressed in a cell, or caused to be produced by a cell, using any of a number of well-known methods.

Introduction of a miRNA into a cell may include expression of the nucleic acid construct within a cell using a method as described herein, or using a suitable method known in the art, and/or may include direct introduction of the miRNA construct into the cell via, for example, transfection. Expression vectors (either viral, plasmid, or other) may be transfected, electroporated, or otherwise introduced into cells, which may then express the miRNA construct s). Alternatively, nucleic acid constructs themselves may be directly introduced into cells, for example via transfection or electroporation (i.e. using a transfection reagent such as but not limited to Lipofectamine™, Oligofectamine, or any other suitable delivery agent known in the art), or via targeted gene or nucleic acid delivery vehicles known in the art. Many delivery vehicles and/or agents are well-known in the art, several of which are commercially available. Delivery strategies for nucleic acids are described in, for example, Yuan et ah, Expert Opin. Drug Deliv. (2011) 8:521- 536; Juliano et al, (2012) Acc. Chem. Res. 45: 1067-1076; and Rettig et al. Mol. Ther. (2012) 20: 483-512. Examples of transfection methods are described in, for example, Ausubel et al. (1994) Current Protocols in Molecular Biology, John Wiley & Sons, New York. Expression vector examples are described in, for example, Cloning Vectors: A Laboratory Manual (Pouwels et al., 1985, Supp. 1987). It will be understood that introduction of a nucleic acid construct into a cell may refer to the production of a nucleic acid within a cell from a gene (i.e. transcription), such an exogenous gene which has been introduced into the cell.

[0056] In some embodiments, a cell already expresses a miRNA and that miRNA is modified in vitro to contain an exosomal sorting or cellular retention motif.

[0057] In some embodiments, the miRNA comprises a native sequence that is present in the subject organism. In some embodiments, the miRNA does not comprise a native sequence. In some embodiments, the miRNA is non-natural.

[0058] In some embodiments, the miRNA is non-naturally prepared ex vivo. In some embodiments, the miRNA alters gene function.

[0059] Autologous or heterologous exosomes may be prepared.

[0060] In some embodiments, autologous exosomes are prepared.“Autologous exosomes” refers to exosomes that are prepared from the same subject who would receive the exosomes after ex vivo manipulation.

[0061] In some embodiments, heterologous exosomes are prepared.

“Heterologous exosomes” refer to exosomes that are prepared from a different individual than the subject who receives the exosomes after ex vivo manipulation.

[0062] In some embodiments, the exosomes are produced by cells in vitro. In some embodiments, the isolated exosomes are formed inside the cell in compartments known as multi vesicular endosomes (MVE) or multivesicular body (MVB). In some embodiments, exosomes are released from a cell without a trigger or signal. In some embodiments, exosomes are released from a cell based on a signal, such as binding of a cell-surface receptor.

[0063] In some embodiments, exosomes are approximately 30 to 100 nm, 20 to 90 nm, 30 to 80 nm, 40 to 70 nm, or 50 to 60 nm. In some embodiments, exosomes are approximately 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 200 nm in size.

[0064] In some embodiments, the exosomes are derived from adipose tissue. In some embodiments, exosomes secreted from fat or adipose tissue may be termed fat- derived exosomes. In some embodiments, this adipose tissue can be inguinal, epididymal, or brown adipose tissue (BAT). In some embodiments, this adipose tissue can be brown fat, beige fat, or white fat. [0065] In some embodiments, an exosome is derived from BAT tissue. In some embodiments, BAT is characterized by numerous small lipid droplets and a higher concentration of mitochondria compared with white fat. In some embodiments, BAT occurs in high concentrations in certain anatomical locations, such as between the shoulder blades, surrounding the kidneys, the neck and supraclavicular area, and along the spinal cord. In some embodiments, BAT occurs in the upper chest and neck, especially paravertebrally.

[0066] This description and exemplary embodiments should not be taken as limiting. For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term“about,” to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0067] It is noted that, as used in this specification and the appended claims, the singular forms“a,”“an,” and“the,” and any singular use of any word, include plural referents unless expressly and unequivocally limited to one referent. As used herein, the term“include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

EXAMPLES

[0068] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way. Example 1. Cell lines

[0069] This study analyzed 5 different mouse cell lines resembling major metabolic cells: 3T3-L1 (white adipocytes), BAT (brown adipocytes), C2C12 (muscle cells), AML12 (hepatocytes) and SVEC (vascular endothelial cells).

[0070] 3T3-L1 cells (ATCC, catalog nr CL-173) were grown in growth medium (DMEM-high glucose supplemented with 10% fetal bovine serum, 1% penicillin /streptomycin, and 0.2 % normocin). For the experiments, cells were grown to reach full confluence and differentiated to mature adipocytes. Upon addition of differentiation cocktail containing 0.5 mM IBMX, insulin 5 pg/mL and dexamethasone 0.25 pM in growth medium for 72 hours, cells were maintained in growth medium only

supplemented with insulin 5 pg/ml for 8 days to obtain fully differentiated adipocytes.

[0071] Brown pre-adipocytes (BAT) were generated as described previously (Fasshauer M et al., J Biol Chem 275(33):25494-50l (2000)) and grown in DMEM-high glucose, 20 % fetal bovine serum, 1 % penicillin/streptomycin and 0.2% normocin. For the experiments, cells were grown to full confluence and differentiated to mature brown adipocytes. To induce differentiation, cells were incubated for 24 hours in growth medium supplemented with 0.5 mM IBMX, 0.125 mM indomethacin, 2 pg/ml dexamethasone, 20 nM insulin, and 1 nM T3 hormone. After that, cells were grown in culture medium only supplemented with 20 nM insulin and 1 nM T3 for 6 days. All reagents for 3T3-L1 and BAT differentiation were purchased from Millipore-Sigma.

[0072] AML12 hepatocytes were purchased from ATCC (catalog nr CRL-2254) and grown in DMEM/F12 high glucose, 10% fetal bovine serum, 1%

penicillin/streptomycin and 0.2% normocin supplemented with insulin-transferrin- selenium-sodium pyruvate mixture (ITS-A, Thermofisher), 2.5 mM L-Glutamine (Thermofisher), 15 mM HEPES (Millipore-Sigma) and dexamethasone 40 ng/ml.

[0073] SVEC endothelial cells were purchased from ATCC (catalog nr CRL- 2181) and cultured in growth medium.

[0074] C2C12 myoblasts (ATCC, catalog nr CRL-1772) were grown in growth medium. Upon confluence, cells were differentiated by growing the cells in DMEM-high glucose supplemented with 2% horse serum, 1% penicillin/streptomycin and 0.2% normicin for 6 days and used for the experiments.

Example 2. Exosome isolation and analysis

[0075] For exosome isolation, all cell lines cells were grown to full confluence. When cells required differentiation (3T3-L1, BAT, and C2C12), they were differentiated as described in Example 1. To collect exosomes, cells were washed with PBS and incubated for 72 hours in exosome-free medium consisting of DMEM-high glucose, 10% exosome-depleted fetal bovine serum (SBI), and 1% penicillin/streptomycin. Medium was collected and exosomes were isolated by differential centrifugation protocol (Thery C et al., Curr Protoc Cell Biol Chapter 3: Unit 3.22 (2006)). Briefly, medium was successively centrifuged at 500 g, 2,000 g and 10,000 g. Supernatant was later ultracentrifuged at 100,000 g for 70 min using a SW-28 rotor. Pellets were washed with PBS and centrifuged again at 100,000 g for 70 min. Pellets were resuspended in 1 mL TRIzol reagent (Thermofisher) to generate a cell lysate for further RNA isolation.

[0076] Similarly, cells that had produced the exosomes were washed with PBS after the incubation in exosome-free medium and 1 mL TRIzol reagent was added for further RNA isolation. This sample represents the cell lysate.

[0077] For RNA isolation and miRNA profiling, samples in TRIzol were added to 200 pL chloroform (Millipore-Sigma). After mixing, samples were centrifuged at 12,000 g for 15 min. Upper liquid phase was collected and RNA was precipitated by adding 2- propanol (Millipore-Sigma) and ammonium acetate (Millipore-Sigma) and incubating at - 20° C overnight. Samples were centrifuged at 12,000 g for 30 min and RNA pellets were washed twice with 70% ethanol and resuspended in nuclease-free water (Qiagen). Both exosomal and cell pellet miRNA profilings were performed using a mouse a QuantiMir for cDNA synthesis (SBI) and miRNome miRNA profiling kit (SBI) following manufacturer’s protocol. RNA amount used for the experiment was 275 ng per sample.

[0078] For bioinformatic analysis, an arbitrary threshold (80) was used to extract ct value from the qPCRs. Samples that did not have ct value <= 35 in at least 2 of the replicates for a given miRNA were considered non-detected and therefore filtered out. Ct values were normalized using mean ct of all detected miRNAs of each sample. Package Limma for R software (Ritchie ME et al., Nucleic Acids Research 43(7), e47 (2015)) was used for the analysis. Bioinformatics was used to compare the normalized expression in the cell and in the exosome of each given miRNAs with a false discovery rate(FDR)of <

0.1. When the FDR for a given miRNA was < 0.1, it was considered significant.

Example 3. Identification of cell type-enriched miRNAs in exosomal and cellular fractions

[0079] Cells described in Example 1 were cultured in exosome-free medium, and exosomes were collected from the medium after 72 hours. RNA was isolated from the collected exosomes as well as from the cell pellets, converted to cDNA and subjected to a qPCR-based miRNA profiling to detect miRNA expression.

[0080] Among the 709 mouse miRNAs analyzed included in the miRNA profiling kit, 697 were detected in at least one of the cell types. By comparing the expression of each miRNA in each cell type to the expression in the other four, several miRNAs were identified that were particularly enriched in the exosomes derived from one of the cell types. Figures 1A (exosomes) and 1C (cell pellets) shows overall counts of miRNAs that had enrichment in a particular cell type. The outer circles in Figures 1 A and 1C show the number of miRNAs with enrichment in a particular cell type. Similarly, some other miRNAs were found selectively in the cell pellets from one particular cell type.

[0081] The top- 10 enriched miRNAs for each cell type in exosomes (Figure 1B) and cell pellets (Figure 1D). Different cell types had different miRNAs with higher enrichment, and miRNAs that were enriched in one particular cell type were not necessarily highly expressed in other cell types.

[0082] As expected, most measured miRNAs were not uniquely representative of one of the cell types either in the exosomal (453 miRNAs) or in the cellular fraction (467 miRNAs) (Figures 1A and 1C). Some of the cell-enriched miRNAs were previously reported to mediate important functions in the tissues that these cells resemble. For instance, miR-l9a and miR-l22 that were significantly higher in AML 12 hepatocytes (Figures 1B and 1D) are known to be expressed in mouse liver where they regulate glycogen synthesis and lipid metabolism, respectively (see Dou L et al., Sci Rep

26(5): l l602 (2015) and Esau C et al., CellMetab 3(2):87-98 (2006)). Other examples are miR-l and miR-l33a/b that were found to be enriched in C2C12 myotubes and previously reported to mediate crucial functions in skeletal muscle and heart (see Zhao Y et al., Cell 129(2):303-317 (2007) and Chen JF et al., Nat Genet 38(2):228-33 (2006)). Similarly, miR-l 46b seems to activate adipogenesis and was enriched in 3T3-L1 adipocytes (see Ahn J et al., EMBOMolMed 10: 1602-12 (2013)). All these data suggest that this study efficiently identified cell-type enriched miRNAs and that the cell lines used here resemble metabolic distinct tissues.

[0083] After identifying exosome and cell pellet-specific miRNAs, the miRNA population contained in the exosomes was compared to the cellular content of each cell type. If exosomes simply represent a sample of the miRNAs that are found in the cell, there should be a perfect match between the specific miRNAs found in exosomes and the cell pellets for each cell type. If, in contrast, there is selectivity in the loading of exosomes, populations of exosomal and cellular miRNAs would be at least partially different.

[0084] As shown in Figures 2A-2E, there is an incomplete match in the miRNA population between exosome and cell lysate samples from each cell type. Some miRNAs are cell type-specific regarding both compartments, as shown by the number of miRNAs contained in the region of overlap between the exosome and lysate samples for each cell type. However, many other miRNAs were selectively found in the cellular or in the exosome fraction of a given cell type, and they were not specifically found in the other fraction. These data suggest the presence of a sorting mechanism of miRNAs into exosomes that is specific for one cell type versus another.

Example 4. Enrichment and depletion of miRNAs from exosomes

[0085] In order to understand how some miRNAs are preferentially loaded into the exosomes whereas others preferentially remain in the cell, the expression of each particular miRNA was compared between the exosomal and the cellular fraction. This approach allowed separation of miRNAs that are particularly enriched in the exosomes compared to the cells where they were produced (expression in exosomes would be significantly higher than in the cell), or in contrast miRNAs that are enriched in the cell pellets but rarely go to the exosomes (expression significantly lower in the exosomes than in the cell). As shown in Figure 3, miRNAs were identified with a significant enrichment in the exosomes (top [“up”] values) or in the cell pellet (bottom [“down”] values) in each cell type. The sorting into up and down groups was done by comparing the normalized expression of a miRNA in the exosome (measured by comparing to the mean of all detected miRNAs) to the normalized expression in the cell pellet. For a miRNA to be placed“up”, it needed to have a significant higher expression in the exosome compared to cell lysate as determined by a cut-off <0.1 in the false discovery rate. Similarly for a miRNA to be placed“down,” it needed to have a significant higher expression in the cell pellet compared to cell lysate as determined by a cut-off <0.1 in the false discovery rate.

[0086] A total of 19 miRNAs were identified that were significantly enriched in the exosomes from every cell type, and 49 miRNAs were identified that were

significantly depleted from the exosomes of all cell lines analyzed in this study; these data are shown in the center values that overlapped between all cell types. Another interesting finding is the high degree of similarity between brown adipocytes (BAT) and muscle cells (C2C12) regarding which miRNAs are enriched or depleted in the exosomes (Figure 3), as represented by the high number of miRNAs (35) enriched in exosomes shared by both cell types. Similarly, the number of miRNAs (27) in the intersection of BAT, C2C12, and 3T3-L1 cells is also high, which further contributes to the finding of similarity in enrichment data between BAT and C2C12 cells.

Example 5. Identification of RNA motifs associated with exosomal or cellular miRNA enrichment in different cell types

[0087] The potential mechanism that made some miRNAs preferentially sorted to exosomes or be retained in the cell was explored. In particular, nucleotide sequences of the miRNAs were investigated to determine if they could determine the fate of the sorting.

[0088] Table 1 shows all detected miRNAs and their mature sequences. The code 1 indicates significant enrichment of that miRNA expression in exosomes from the cell type referred in the column, code -1 indicates significant cell enrichment, and code 0 indicates no difference between exosomal and cellular expression. The identified exosomal enrichment motifs are highlighted in bold whereas the cellular enrichment motifs are underlined. Some miRNAs did not comprise either an exosomal sorting motif or a cellular retention motif. Some miRNAs comprised both an exosomal sorting motif and in a cellular retention motif. Nucleotides that were within both an exosomal sorting and within a cellular retention motif are noted with bold underlined font.

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Table 1 : Detected miRNAs and their sorting in different cells

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[0089] As shown in Figure 4A, 6 main nucleotide motifs in the mature miRNAs were identified that were significantly more abundant in the miRNAs that had

preferential sorting to exosomes. All values for individual cell types indicates the percentage of miRNAs in the exosome fraction of that cell type that contained a given motif. The“total” value represents the percentage of miRNAs from each cell types that contained at least one miRNA containing one of the motifs. These motifs could be found anywhere in the miRNA sequence.

[0090] The presence of these specific sequences was able to explain between 62- 70% of the miRNA enrichment in the exosomes of the different cell lines. One of these sequences (GGAG) was previously reported to mediate exosome sorting in a cell type not studied here (human lymphoblasts, see Ritchie 2015), which suggests that these motifs might be evolutionarily conserved.

[0091] Interestingly, BAT (Figure 5A), C2C12 (Figure 5B) and 3T3-L1 (Figure 5C) show a very similar pattern in the abundance of these exosomal sorting motifs, predominantly using UGUG; CAUG; or CUGG. In contrast, AML12 (Figure 5D) and SVEC (Figure 5E) predominantly use A/CGGG; CUGG; or GGAG exosomal sorting motifs.

[0092] Likewise, nucleotide motifs that might be associated with cellular enrichment and guide their retention were investigated. As shown in Figure 4B, five nucleotides sequences in the mature miRNA were significantly more abundant in those miRNAs that were retained in the cell. In this case, these five motifs were able to explain 34-56% of the miRNAs significantly enriched in the cells. As shown in Figure 5, there is a clear hierarchy in the abundance of these cellular enrichment motifs, being CAGU > ACAG > AUUG > UAGC > CCCG in almost all the cases.

[0093] Thus, these data describe sorting motifs for enrichment of miRNAs in exosomal and cellular fractions. Some of these miRNA motifs were unique to particular types of cells, while other motifs were found across a range of cell types. Example 6. Introduction or removal of motifs

[0094] In order to analyze whether the discovered motifs play a role in exosome sorting, experiments were performed to introduce or remove some of these motifs. Wild- type sequences for pre-miR-34c and pre-miR-693 and their flanking genomic 100 base pairs upstream and downstream were obtained from Ensembl database, flanked by restriction enzyme sites and ordered through Integrated DNA Technologies. For the mutations of the sequences in order to introduce or remove exosomal motifs, indicated nucleotides were changed in the guide strand sequence as well as complementary nucleotides in the passenger strand to maintain the same pre-miRNA structure, as predicted by RNAfold Webserver (University of Wien). These sequences were equally flanked by genomic 100 bp upstream and downstream and restriction enzymes. For both wild-type and mutated version, the sequences were cloned into the backbone lentivirus vector upon removal of the scramble miRNA cassette (MMIR000, System Biosciences). Plasmids were used to transfect BAT pre-adipocytes and positively incorporated cells were selected 6 days later by Flow Cytometry (MoFlo Cell Sorter, Beckman Coulter) for GFP signal.

[0095] Exosome isolation was performed again by ultracentrifugation method. RNA was isolated from the exosomes and cell bodies by TRIzol method. In order to measure the presence of the wild-type or mutated versions of miR-34c-5p and miR-693- 3p, RNA was retrotranscribed by miRCURY LNA RT Kit (Qiagen 339320) following manufacturer’s instructions. Specific LNA primers (Qiagen) were used in quantitative real-time PCR (qPCR) to distinguish wild-type and mutated versions of these two miRNAs. Expression for each miRNA was normalized respect to miR-l38-5p, which has expression essentially identical between exosomes and cells.

[0096] miR-34c-5p (SEQ ID NO: 648) is a miRNA that was significantly enriched in the cell bodies of all cell types except hepatocytes (Table 1). The motifs UGUG or CAUG were introduced towards the 3’ end of the miR-34c-5p sequence with minimal changes in the miRNA sequence (Figure 6A) in order to maintain the normal pre-miRNA structure (Figure 6B), which is essential to display a regular processing by Dicer and potential recognition by RNA-binding proteins (RBPs) (see Bartel DP. Cell 173(1):20-51 (2018) and Dominguez D et al., Mol Cell 70(5):854-867.e9 (2018)).

Expectedly, when the wild-type version of miR-34c-5p was introduced, there was a higher presence in the cell body compared to the exosome fraction. However, when the mutant versions were introduced there was a shift in the mutated miR-34c-5p

distribution, with expression being much more abundant in the exosomal fraction for both motifs and leading to a nearly statistically significant 4-fold enhancement of exosomal enrichment by UGUG introduction (SEQ ID NO: 701) and a significant lO-fold increase by CAETG introduction (SEQ ID NO: 702) (Figure 6C).

[0097] Additionally, a novel 6-mer motif CGGGAG combining two shorter identified motifs, CGGG & GGAG, was introduced in a CGGGAG mutant (SEQ ID NO: 703). The novel motif displays a 24- to 80-fold enrichment in exosome-enriched miRNAs from endothelial cells and hepatocytes. Due to this huge enrichment, the CGGGAG was termed a SuperEXOmotif. In this case, exosomal abundance was increased to a larger extent, leading to a final enrichment of 20-fold in abundance in exosomes versus cell bodies, which is much higher than the other motifs (Figure 6C). Very interestingly, the introductions of the EXOmotif CAETG and SuperEXOmotif CGGGAG implied a complete shift in miRNA distribution from a very cellular-prone miRNA to a highly exosomal enriched miRNA (Figure 6C). These data clearly suggest that these motifs play a role in miRNA sorting to exosomes.

[0098] In addition, the impact of removal of the identified exosome motif ETGUG on exosome sorting was assessed. Wild-type miR-693-3p (SEQ ID NO: 6) is normally enriched in the exosomal fraction of all cell types. A mutated version of miR-693-3p lacking a ETGUG motif (SEQ ID NO: 704) was studied. Again, these minor changes in the sequence (Figure 6D) did not alter the expected secondary structure of this miRNA (Figure 6E). When the wild-type version was overexpressed, miR-693-3p was found enriched in the exosome fraction. However, when the mutated version of miR-693-3p lacking a UGUG motif showed partially impaired sorting (Figure 6F), again suggesting that this motif could be important for exporting miRNA into exosomes. Example 7. Embodiments

[0099] The following numbered items provide embodiments as described herein, though the embodiments recited here are not limiting.

[00100] Item 1. A method for producing exosomes or exosome-like vesicles comprising miRNA in vitro comprising:

modifying a miRNA to include at least one exosomal sorting motif and/or removing any cellular retention motifs;

introducing the modified miRNA into a cell capable of producing an exosome or exosome-like vesicle under conditions that will result in expression of the modified miRNA; and

optionally, collecting the produced exosomes or exosome-like vesicles, wherein the exosomal sorting motif is UGUG, GGAG, CAUG, GGCA/G, A/CGGG, CUGG, or CGGGAG, and the cell retention motif, if present, is CAGU, ACAG, AUUG, UAGC, or CCCG.

[00101] Item 2. A method of treating a subject in need of gene silencing comprising administering to the subject an exosome, wherein the exosome is produced in vitro by a) modifying a miRNA to include at least one exosomal sorting motif and/or removing any cellular retention motifs, and b) introducing the modified miRNA into an exosome- or exosome-like vesicle producing cell under conditions that will result in expression of the modified miRNA, and collecting the produced exosome comprising the modified miRNA, wherein the exosomal sorting motifs is selected from UGUG, GGAG, CAUG, GGCA/G, A/CGGG, CUGG, and CGGGAG and the cellular retention motif, if present, is selected from CAGU, ACAG, AUUG, UAGC, and CCCG.

[00102] Item 3. The method of item 1 or 2, wherein the miRNA comprises one exosomal sorting motif.

[00103] Item 4. The method of item 1 or 2, wherein the miRNA comprises more than one exosomal sorting motif.

[00104] Item 5. The method of item 1, further comprising administering the exosome or exosome-like vesicle to a subject. [00105] Item 6. The method of item 1 or 2, wherein modifying the miRNA with an exosomal sorting motif results in more miRNA in the exosome as compared to an exosome produced with a miRNA not modified with an exosomal sorting motif.

[00106] Item 7. The method of item 1 or 2, wherein the removal of the cellular retention motif results in more miRNA in the exosome as compared to an exosome produced with a miRNA comprising a cellular retention motif.

[00107] Item 8. The method of item 1 or 2, wherein the miRNA contains a cell retention motif and wherein the cell retention motif is removed.

[00108] Item 9. A method for retaining miRNA inside a cell in vitro comprising:

modifying a miRNA to include at least one cell retention motif and/or removing any exosomal sorting motifs; and

introducing the modified miRNA into a cell capable of producing an exosome or exosome-like vesicle under conditions that will result in expression of the modified miRNA, wherein the cell retention motif is CAGU, ACAG, AUUG, UAGC, or CCCG, and the exosomal sorting motif, if present is UGUG, GGAG, CAUG, GGCA/G, A/CGGG, CUGG, or CGGGAG.

[00109] Item 10. A method for treating a subject in need of gene silencing comprising:

collecting the subject’s cells and manipulating them ex vivo to express a miRNA having at least one cellular retention motif and/or removing any exosomal sorting motifs, and administering the ex vivo manipulated cell comprising the modified miRNA to the same or different subject from which it was collected, wherein the cellular retention motif is selected from CAGU, ACAG, AUUG, UAGC, and CCCG, and the exosomal sorting motif, if present, is selected from UGUG, GGAG, CAUG, GGCA/G, A/CGGG, CUGG, and CGGGAG.

[00110] Item 11. The method of item 9 or 10, wherein the miRNA comprises one cellular retention motif.

[00111] Item 12. The method of item 9 or 10, wherein the miRNA comprises more than one cellular retention motif. [00112] Item 13. The method of item 9 or 10, wherein the addition of the cellular retention motif reduces the export of the miRNA into an exosome or exosomal- like vesicle.

[00113] Item 14. The method of item 9 or 10, wherein the removal of the exosomal sorting motif reduces the export of the miRNA into an exosome or exosomal- like vesicle.

[00114] Item 15. The method of item 9, further comprising administering the cell to a subject.

[00115] Item 16. The method of item 10 or 15, wherein the miRNA levels in non-implanted cell-types after administration to the subject are reduced as compared to levels in subject administered a non-modified miRNA containing cell.

[00116] Item 17. The method of any one of the preceding items, wherein the cell is an adipocyte, muscle cell, hepatocyte, or vascular endothelial cell.

[00117] Item 18. The method of item 17, wherein the adipocyte is a white adipocyte or brown adipocyte.

[00118] Item 19. The method of item 18, wherein the white adipocyte is a 3T3-L1 cell.

[00119] Item 20. The method of item 18, wherein the brown adipocyte is a BAT cell.

[00120] Item 21. The method of item 17, wherein the muscle cell is a C2C12 cell.

[00121] Item 22. The method of item 17, wherein the hepatocyte is an AML 12 cell.

[00122] Item 23. The method of item 17, wherein the vascular endothelial cell is a SVEC cell.

[00123] Item 24. The method of item 1 or 2, wherein the cell is a hepatocyte or endothelial cell and the exosomal sorting motif is A/CGGG; CUGG;

GGAG; or CGGGAG. [00124] Item 25. The method of item 1 or 2, wherein the cell is a brown or white adipocyte or muscle cell and the exosomal sorting motif is UGUG; CAUG; CUGG; or CGGGAG.

[00125] Item 26. The method of any one of the preceding items, wherein the miRNA is any one of the miRNAs of SEQ ID Nos: 1-704.

EQUIVALENTS

[00126] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[00127] As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., +/-5-l0% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.