Methods of RNA sorting and tissue accumulation

Abstract

This disclosure relates to compositions and methods for sorting and accumulation of RNA in different tissues. Described herein are nucleotide motifs that direct the sorting and tissue accumulation of non-coding RNAs.

Description

METHODS OF RNA SORTING AND TISSUE ACCU ULATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 654,306, filed May 31, 2024, the disclosure of which is hereby incorporated by reference in its entirety.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on May 28, 2025, is named “01123-0020-00PCT.xml” and is 60,756 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.DESCRIPTION

[0003] This invention was made with government support under Research Grant No. DK082659 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0004] This disclosure relates to compositions and methods for sorting of RNAs and their accumulation in different tissues. Described herein, in some embodiments, are nucleotide motifs that direct the sorting and tissue accumulation of non-coding RNAs.BACKGROUND

[0005] microRNAs (miRNAs) are important bioactive molecules that regulate the expression of specific target genes in tissues throughout the body. miRNAs occur within cells and are also secreted into the circulation and extracellular fluid. miRNAs may be secreted by at least two mechanisms: 1) via small extracellular vesicles (sEVs) called exosomes or 2) bound to proteins such as argonaute or lipoproteins (HDL).

[0006] Adipose tissue is a major source of circulating miRNAs1'6. These secreted miRNAs can be taken up and regulate gene expression in distal tissues, and thus regulate metabolic and other functions of organs and tissues at a distance. Importantly, the process of miRNA secretion into sEVs or retention in cells is highly regulated7, such that only some miRNAs containing specific nucleotide codes (motifs) are preferentially secreted into sEVs, whereas other miRNAs are retained in the cell or possibly secreted by other mechanisms. Understanding these codes is not only important for development of potential miRNA therapeutics, but it also provides insights that can be used in the development of other RNA therapeutics, such as interfering RNAs (RNAi), since this could help design therapeutic small RNAs that can be programmed for secretion via sEVs or programmed for cellular retention.

[0007] Described herein in some embodiments are means to label miRNAs and other RNAs specifically produced in fat or other tissues and track their appearance in the plasma in either sEVs or bound to proteins and taken up by peripheral tissues, including the liver, muscle, and brain / hypothalamus. Data presented herein shows that different classes of non-coding RNAs (ncRNAs), including miRNAs, tRNAs and tRNA fragments are 1) labeled in fat; 2) secreted into plasma via sEV or non-sEV carriers; and 3) transferred to the liver, muscle, or brain, with remarkable specificity. The fact that these miRNAs and other ncRNAs originating from one tissue can be secreted by multiple mechanisms and can accumulate in specific organs, even if they were in the same carrier, was a totally unexpected finding and showed that the ncRNA accumulation in the liver, muscle, or brain must carry information encoded within the RNA molecules themselves that determines their fate. Indeed, described herein are nucleotide sequences (motifs) that not only direct miRNAs (and other ncRNAs) to be secreted in sEVs versus non-sEV plasma carriers, but also motifs that target miRNAs (and other ncRNAs) to liver, muscle, and brain, and potentially all other tissues.

[0008] A strategy is described for the design of miRNA / RNA-based therapies by incorporating nucleotide motifs that drive exosomal and non-exosomal secretion (i.e., sorting motifs). Also described in a strategy for design of miRNA / RNA-based therapeutic that can beused to enable tissue-specific accumulation via incorporating nucleotide motifs (i.e., accumulation motifs). These strategies can be in new or optimized RNA-based therapies.SUMMARY

[0009] Described herein is a method for identifying novel nucleotide motifs within miRNAs and other ncRNAs and therapeutic RNAs that may be used therapeutically to enhance RNA secretion or retention in cells, secretion by vesicular vs. non-vesicular routes, and / or accumulation in specific tissues and / or cell types. The accumulation motifs described herein can improve the tissue-specific effects of RNA / miRNA-based therapies. In some embodiments, the introduction of the nucleotide motifs described herein into newly developed or existing RNA / miRNA-based therapies can target them to the muscle, liver, or brain, via small extracellular vesicles (sEV) or non-sEV routes. Each of the following embodiments are provided:

[0010] Embodiment 1. A composition comprising an RNA comprising (a) a sorting motif comprising a sequence selected from any one of SEQ ID NOs: 46-55 or 59 -71; and (b) a tissue accumulation motif.

[0011] Embodiment 2. The composition of embodiment 1, wherein the tissue accumulation motif comprises a sequence selected from any one of SEQ ID NOs: 1-39.

[0012] Embodiment 3. A composition comprising an RNA comprising a tissue accumulation motif comprising a sequence selected from any one of SEQ ID NOs: 1-39.

[0013] Embodiment 4. The composition of embodiment 3, wherein the RNA is packaged within extracellular vesicles and / or lipid nanoparticles.

[0014] Embodiment 5. The composition of embodiment 4, wherein the extracellular vesicles comprise small extracellular vesicles (sEVs), exosomes, microvesicles, large oncosomes, apoptotic bodies, chylomicrons, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and / or high-density lipoprotein (HDL).

[0015] Embodiment 6. The composition of embodiment 4, wherein the lipid nanoparticles comprise one or more of polymersomes, dendrimers, polymer micelles,nanospheres, silica nanoparticles, quantum dots, iron oxide nanoparticles, gold nanoparticles, liposomes, lipid nanoparticles, or emulsions.

[0016] Embodiment 7. The composition of any one of embodiments 1-6, wherein the RNA comprises non-coding RNA.

[0017] Embodiment 8. The method of embodiment 7, wherein the non-coding RNA is a micro RNA (miRNA), transfer RNA (tRNA), tRNA fragment, tRNA half, small nucleolar RNAs (snoRNA), piwi-interacting RNAs (piRNA), small nuclear RNAs (snRNA), ribosomal RNAs (rRNA), small cajal body-specific RNAs (scaRNA), or mitochondrial-encoded non-coding RNA, or the complement thereof.

[0018] Embodiment 9. The composition of any one of embodiments 1-8, wherein the RNA comprises a naturally-occurring RNA sequence or the complement of a naturally-occurring RNA sequence.

[0019] Embodiment 10. The composition of any one of embodiments 1-9, wherein the tissue accumulation motif is capable of producing accumulation of the RNA in (a) the brain, optionally the hypothalamus, if the sequence is any of SEQ ID NOs: 1-25; (b) the liver if the sequence is any one of SEQ ID NOs: 26-33; or (c) the muscle if the sequence is any of SEQ ID NOs: 34-39.

[0020] Embodiment 11. A method of accumulating a RNA in a specific tissue and / or cell type in vivo comprising administering to a subject the RNA, wherein the RNA comprises a tissue accumulation motif comprising a sequence selected from any one of SEQ ID NOs: 1-39.

[0021] Embodiment 12. The method of embodiment 11, wherein the RNA further comprises a sorting motif comprising a sequence selected from any one of SEQ ID NOs: 46-55 or 59-71.

[0022] Embodiment 13. The method of any one of embodiments 11-12 , wherein the RNA comprises non-coding RNA.

[0023] Embodiment 14. The method of embodiment 13, wherein the non-coding RNA is a miRNA, tRNA, tRNA half, tRNA fragment, snoRNA, piRNA, snRNA, rRNA, scaRNA, or mitochondrial-encoded non-coding RNA, or the complement thereof.

[0024] Embodiment 15. The method of any one of embodiments 11-14, wherein the RNA comprises a naturally-occurring RNA sequence or the complement of a naturally-occurring RNA sequence.

[0025] Embodiment 16. A method of blocking the effect of an endogenous non-coding RNA in a specific tissue and / or cell type in vivo comprising (a) administering to the subject an exogenous RNA comprising (i) the complement of the endogenous non-coding RNA; and (ii) a tissue accumulation motif comprising a sequence selected from any one of SEQ ID NOs: 1-39, and (b) allowing binding of the exogenous RNA to the endogenous non-coding RNA, wherein the binding preferentially occurs in the specific tissue and / or cell type.

[0026] Embodiment 17. A method of blocking the effect of an administered non-coding RNA sequence in a specific tissue and / or cell type in vivo comprising (a) administering to the subject an exogenous RNA comprising the complement of the non-coding RNA sequence, wherein the exogenous RNA further comprises a tissue accumulation motif comprising a sequence selected from any one of SEQ ID NOs: 1-39; (b) administering the non-coding RNA sequence to the subject; and (c) allowing binding of the exogenous RNA to the administered non-coding RNA sequence, wherein the binding preferentially occurs in the specific tissue and / or cell type.

[0027] Embodiment 18. The method of embodiment 17, wherein the non-coding RNA sequence further comprises a tissue accumulation motif that directs accumulation in a different tissue than the tissue accumulation motif in the exogenous RNA.

[0028] Embodiment 19. The method of embodiment 17 or 18, wherein administering the non-coding RNA sequence to the subject occurs simultaneously or after administering the exogenous RNA.

[0029] Embodiment 20. The method of any one of embodiments 16-19, wherein the exogenous RNA sequence further comprises a sorting motif comprising a sequence selected from any one of SEQ ID NOs: 46-55 or 59-71.

[0030] Embodiment 21. The method of any one of embodiments 16-20, wherein the binding blocks activity of the endogenous non-coding RNA or the administered non-coding RNA sequence preferentially in the specific tissue and / or cell type.

[0031] Embodiment 22. The method of embodiment 16-21, wherein the endogenous noncoding RNA or the administered non-coding RNA sequence retains activity when not bound to the exogenous RNA.

[0032] Embodiment 23. The method of any one of embodiments 16-22, wherein the specific tissue and / or cell type in vivo is (a) brain, optionally the hypothalamus, and the tissue accumulation motif comprises a sequence selected from any one of SEQ ID NOs: 1-25; (b) liver, and the tissue accumulation motif comprises a sequence selected from any one of SEQ ID NOs: 26-33; or (c) muscle, and the tissue accumulation motif comprises a sequence selected from any one of SEQ ID NOs: 34-39.

[0033] Embodiment 24. The method of any one of embodiments 16-23, wherein the administered non-coding RNA sequence or the exogenous RNA is administered into fat or administered by intravenous, intraperitoneal, intraocular, or intrathecal injection.

[0034] Embodiment 25. The method of any one of embodiments 16-24, wherein the administered non-coding RNA sequence or the exogenous RNA is packaged within extracellular vesicles and / or lipid nanoparticles.

[0035] Embodiment 26. The method of embodiment 25, wherein the extracellular vesicles comprise small sEVs, exosomes, microvesicles, large oncosomes, apoptotic bodies, chylomicrons, very VLDL, LDL, and / or HDL.

[0036] Embodiment 27. The method of embodiment 25, wherein the lipid nanoparticles comprise one or more of polymersomes, dendrimers, polymer micelles, nanospheres, silica nanoparticles, quantum dots, iron oxide nanoparticles, gold nanoparticles, liposomes, lipid nanoparticles, or emulsions.

[0037] Embodiment 28. The method of any one of embodiments 16-27, wherein the noncoding RNA is a miRNA, tRNA, tRNA half, tRNA fragment, snoRNA, piRNA, snRNA, rRNA, scaRNA, or mitochondrial-encoded non-coding RNA, or the complement thereof.

[0038] Embodiment 29. A method of enhancing secretion of an RNA from cells, comprising adding any one of SEQ ID NOs: 46-55 or 59-71 to the RNA.

[0039] Embodiment 30. A method of enhancing secretion of an RNA via an extracellular vesicle, comprising adding any one of SEQ ID NOs: 46-55 to the RNA.

[0040] Embodiment 31. A method of enhancing secretion of an RNA via a non-vesicle route, comprising adding any of SEQ ID NOs: 59-71 to the RNA.

[0041] Embodiment 32. The method of embodiment 31, wherein the non-vesicle route comprises binding to one or more RBP.

[0042] Embodiment 33. The method of embodiment 32, wherein the one or more RNA- binding protein comprises argonaute, a heterogeneous nuclear ribonucleoprotein, or a lipoprotein.

[0043] Embodiment 34. The method of embodiment 33, wherein the one or more RNA- binding protein is selected from one or more of those listed in Table 10.

[0044] In some instances, in each of the embodiments, the subject is human.

[0045] Additional objects and advantages will be set forth in part in the description which follows, and in part will be understood 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.

[0046] 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.

[0047] 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

[0048] Figure 1 shows inter-organ transfer of miRNAs from brown fat to muscle, liver, and hypothalamus via small extracellular vesicle (sEV) or non-sEV routes. Brown fat secreted miRNAs (left heatmaps) were abundantly labeled in plasma. These were separated into threegroups; miRNAs that were sEV-enriched (top left heatmap), miRNAs that were enriched in non- sEV compartment (enriched in RBPs; bottom left heatmap), or miRNAs that were equally abundant in both compartments (middle left heatmap). These labeled miRNAs were then detected in recipient organs like the hypothalamus, liver, and muscle (right heatmaps). Notably, brown fat-derived miRNAs exhibit remarkable tissue specificity. As described herein, nucleotide motifs within brown fat-secreted miRNAs determined their secretion into different compartments of the plasma, and their accumulation in specific tissues and / or cell types. This enables the design of RNA-based therapeutics that can be targeted to specific organs via different routes of administration.

[0049] Figure 2 shows sequence analysis of brown fat-secreted miRNAs. Dendrogram depicting sequence similarity of the brown fat-secreted miRNAs shows remarkable overlap between miRNA sequence and tissue enrichment of miRNAs. Specifically, hypothalamus enriched miRNAs tended to cluster together (top and bottom third of dendrogram). This is also true for liver and muscle-enriched miRNAs, which clustered together in the middle panel. Thus, miRNA sequence is a major predictor of tissue-specificity of miRNAs.

[0050] Figure 3 shows sequences and novel nucleotide motifs in sEV and non-sEV miRNAs. Brown fat secretes miRNAs into the plasma via two main routes: via sEVs (top panel) or RNA-binding proteins (RBPs; bottom panel). The sorting of these miRNAs into these different extracellular compartments is determined by RNA-protein interactions driven by the nucleotide motifs boxed in each sequence (guanine (G) bolded, cytosine (C) outlined). Sequences shown in this Figure correspond to SEQ ID NOs: 76-112 in order from top to bottom.

[0051] Figure 4 shows sequences and novel nucleotide motifs in miRNAs that exhibit tissue-specificity. The brown fat-secreted miRNAs discovered in the present studies became enriched in the hypothalamus (top panel), liver (middle panel), or muscle (bottom panel). Highlighted in gray (with guanine (G) bolded) are newly discovered nucleotide motifs specifically enriched in miRNAs targeted to the hypothalamus, liver, or muscle. Sequences shown in this Figure correspond to SEQ ID NOs: 76-126.DESCRIPTION OF THE SEQUENCES

[0052] Table 1 provides a listing of certain sequences referenced herein. Bold letters highlight C or G nucleotides that were consistently present within given consensus motifs.DESCRIPTION OF THE EMBODIMENTSI. Compositions comprising RNA

[0053] While RNA-based therapies have a wide range of clinical applications, one drawback is unwanted side-effects of such therapies outside of a desired site of action. For example, an RNA-based therapy may have a desired site of action in the brain but unwanted side-effects in other tissues of the body.

[0054] Described herein are tissue accumulation motifs that have an unexpected effect to increase the presence of a given RNA in a particular tissue. Figure 1 shows data on the ability of an RNA to accumulate in specific tissues and / or cell types, which was evident in the present unbiased screen for accumulation of miRNAs produced by fat as described in Examples 1 and 2. Also described herein are sorting motifs that can direct a given RNA for secretion via a carrier, such as an exosome or non-vesicle route. In some embodiments, the non-vesicle route is via binding to RNA-binding proteins (RBPs).

[0055] In some embodiments, a composition comprises an RNA comprising (a) a sorting motif comprising a sequence selected from any one of SEQ ID NOs: 46-55 or 59 -71; and (b) a tissue accumulation motif. In some embodiments, the tissue accumulation motif comprises a sequence selected from any one of SEQ ID NOs: 1-39.

[0056] In some embodiments, a composition comprises an RNA comprising a tissue accumulation motif comprising a sequence selected from any one of SEQ ID NOs: 1-39.

[0057] In some embodiments, the sorting motif comprises a sequence selected from any one of SEQ ID NOs: 46-55 or 59 -71 with a change of 1, 2, 3, or 4 nucleotides. In some embodiments, the sorting motif comprises a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 46-55 or 59 -71.

[0058] In some embodiments, the targeting motif comprises a sequence selected from any one of SEQ ID NOs: 1-39 with a change of 1, 2, 3, or 4 nucleotides. In some embodiments,the targeting motif comprises a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 1-39.A. RNA

[0059] The RNA described herein can be any type of RNA, including coding or noncoding RNA. In some embodiments, the RNA comprises an RNA used as a therapy, such as a small interfering RNA (siRNA) or an anti-sense oligonucleotide (ASO).

[0060] In some embodiments, the RNA is non-coding RNA. In some embodiments, a non-coding RNA (ncRNA) is secreted from one cell and has activity at a different cell. For example, a ncRNA can be secreted from fat but can have biological activity in liver.

[0061] Examples of non-coding RNA include miRNA, transfer RNA (tRNA), tRNA fragments, ribosomal RNA, or mitochondrial-encoded non-coding RNA, or the complement thereof. In some embodiments, the non-coding RNA is a miRNA, tRNA, tRNA half, tRNA fragment, snoRNA, piRNA, snRNA, rRNA, scaRNA, or mitochondrial-encoded non-coding RNA, or the complement thereof. tRNA fragments are produced by enzymatic cleavage of tRNAs (and comprise less than a half or less than a half of the full tRNA) and can have similar functions as miRNA. Altered expression of tRNA fragments are associated with cell proliferation and invasive metastasis, and tRNAs are considered biomarkers of progression in several types of malignancies (see, for example, Yu et al., Journal of Hematology & Oncology 13:121 (2020).

[0062] The motifs described herein can be used to modify the accumulation of a variety of different non-coding RNAs (ncRNAs) originating from nuclear DNA.

[0063] A variety of ncRNAs may be sorted for extracellular delivery and / or accumulated in a specific tissue and / or cell type using the presently described methods. Exemplary ncRNAs include microRNAs (miRNAs), transfer RNAs (tRNAs), tRNA fragments (tRFs, including tRNA halves), small nucleolar RNAs (snoRNAs), piwi -interacting RNAs (piRNAs), small nuclear RNAs (snRNAs), ribosomal RNAs (rRNAs), Y-RNAs, small cajal body-specific RNAs (scaRNAs), long non-coding RNAs (IncRNAs), IncRNA fragments, long intergenic / intervening non-coding RNAs (lincRNAs), and lincRNA fragments. In addition, miRNA inhibitors(antimiRs) and other non-coding RNA inhibitors are reverse complementary sequence to any type of ncRNAs. Use of a representative miRNA is described below in Example 3.

[0064] In some embodiments, the RNA comprises a naturally-occurring RNA sequence or the complement of a naturally-occurring RNA sequence. In some embodiments, the naturally occurring RNA sequence has a biological effect. In some embodiments, the RNA sequence is the complement of a naturally occurring RNA sequence and thus can have effects by blocking the biological effect of the naturally occurring RNA sequence. In this way, RNA described herein may be an inhibitor of a naturally occurring RNA sequence.1. microRNA

[0065] “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.

[0066] In some embodiments, a cell already expresses a miRNA and that miRNA is modified in vitro to contain a motif.

[0067] 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.

[0068] In some embodiments, the miRNA is non-naturally prepared ex vivo. In some embodiments, the miRNA alters gene function.

[0069] 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.

[0070] All descriptions of miRNAs can be extended to other types of ncRNAs, as well as coding RNAs and therapeutic RNAs.B. Consensus motifs

[0071] In some embodiments, a motif described herein comprises a consensus motif. This consensus motif can be used to describe a list of unique motif variations. In some embodiments, The nucleotide motifs described herein can be written as consensus motifs using the nucleotide alphabet below:RNA Nucleotide AlphabetA "Adenine"C "Cytosine"G "Guanine"U "Uracil"N "Any base" = ACGU. = ACGUX = ACGUV "Not U" = ACGH "Not G" = ACUD "Not C" = AGUB "Not A" = CGUM "Amino" = ACR "Purine" = AGW "Weak" = AUS "Strong" = CGY "Pyrimidine" = CUK "Keto" = GUT = U

[0072] For sequences involving multiple nucleotides, each base / position is separated by a comma, with the consensus sequence appearing in quotations. To represent nucleotide degeneracy the dominant nucleotide(s) is listed first, followed by the less dominant nucleotide(s), separated by a forwardC. Sorting motifs

[0073] “Sorting motif,” as used herein, refers to a sequence of nucleotides that when naturally or artificially present or appended to an RNA cause the RNA to be substantially directed for secretion via one or more carrier. In some embodiments, the RNA comprising a sorting motif is an RNA that is normally secreted, such as a non-coding RNA including miRNA, tRNA, or tRNA fragments. In some embodiments, the carrier is an exosome or a protein, such as an RNA-binding protein (RBP). When a sorting motif causes an RNA to be substantially directed for secretion by exosomes, the sorting motif may be referred to herein as “an exosomal sorting motif.”

[0074] Secreted miRNAs and other ncRNAs are typically bound to or encapsulated in carrier particles which include small extracellular vesicles (sEVs) or other protein / lipoprotein carriers including RNA-binding proteins (RBPs). The consensus motifs of miRNAs associated with sEV or non-sEV carriers (which may be termed as “extracellular motifs”) allow design of miRNA / RNA-based therapies that can be delivered by various carriers including, but not limited to sEVs or RBP / lipoproteins.

[0075] Identified herein are motifs associated with miRNAs that were preferentially loaded onto sEVs or non-sEV carriers like RBPs / lipoproteins. The sEV-associated motifs are “S,W,W,G” and “G,U / W,C / S,W,S”. Exemplary “S,W,W,G” motifs include SEQ ID NOs: 51-55. Exemplary “G,U / W,C / S,W,S” motifs include SEQ ID NOs: 46-50.

[0076] The RBP / lipoprotein-associated motifs are “U / W,G / S,U / H,A / D,U / N,G / D,G / S” and “G,A / S,C,C,C” Exemplary “UAV,G / S,U / H,A / D,U / N,G / D,G / S” motifs include SEQ ID NOs: 59-69. Exemplary “G,A / S,C,C,C” motifs include SEQ ID NOs: 70 and 71.

[0077] The unique motifs are listed below in Tables 2-5.

[0078] In some embodiments, the sorting motif comprises a sequence selected from any one of SEQ ID NOs: 46-55 or 59 -71. In some embodiments, the sorting motif comprises a sequence selected from any one of SEQ ID NOs: 46-55 or 59 -71 with a change of 1, 2, 3, or 4 nucleotides. In some embodiments, the sorting motif comprises a sequence having 80% orgreater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs:46-55 or 59 -71.D. Accumulation motifs

[0079] “A accumulation motif,” as used herein, refers to a sequence of nucleotides that when naturally or artificially present or appended to an RNA cause the RNA to accumulate in one or more tissue. In some embodiments, inclusion of an accumulation motif in an RNA increases the amount of said RNA present in one or more tissue as compared to other tissues.

[0080] While it might have been expected that the tissue where a given RNA accumulated was based on the carrier with which it was associated after being secreted from its source cell, the present disclosure identified instead that there are distinct accumulation motifs that are drive the location where the RNA accumulates, irrespective of the carrier associated with the RNA. For example, secreted miRNA that were directed to small extracellular vesicles (sEVs, also known as exosomes) as carriers could be found localized (i.e., accumulated) in the muscle, liver, or hypothalamus (as shown in Figure 1). The same situation was true for secreted miRNA that were directed to secretion with non-sEV carriers (i.e., not using exosomes and likely associated with RNA-binding proteins), and these non-sEV miRNAs could accumulate in the muscle, liver, or hypothalamus. Thus, there was unexpected homology in miRNAs that accumulated in different organs, even though all the miRNAs in the study originated in thebrown fat (Figure 2). Analysis of these homologous regions led to the identification of the presently described tissue accumulation motifs (Figure 4).

[0081] In some embodiments, the tissue accumulation motif comprises a sequence selected from any one of SEQ ID NOs: 1-39. In some embodiments, the targeting motif comprises a sequence selected from any one of SEQ ID NOs: 1-39 with a change of 1, 2, 3, or 4 nucleotides. In some embodiments, the targeting motif comprises a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 1-39.

[0082] In some embodiments, the tissue accumulation motif is capable of producing accumulation of the RNA in (a) the brain, optionally the hypothalamus, if the sequence is any of SEQ ID NOs: 1-25; (b) the liver if the sequence is any one of SEQ ID NOs: 26-33; or (c) the muscle if the sequence is any of SEQ ID NOs: 34-39.

[0083] In some embodiments, the tissue accumulation motif is capable of producing accumulation of the RNA in (a) the brain, optionally the hypothalamus, if the sequence is any of SEQ ID NOs: 1-25 with a change of 1, 2, 3, or 4 nucleotides; (b) the liver if the sequence is any one of SEQ ID NOs: 26-33 with a change of 1, 2, 3, or 4 nucleotides; or (c) the muscle if the sequence is any of SEQ ID NOs: 34-39 with a change of 1, 2, 3, or 4 nucleotides.

[0084] In some embodiments, the tissue accumulation motif is capable of producing accumulation of the RNA in (a) the brain, optionally the hypothalamus, if the motif comprises a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 1-25; (b) the liver if the motif comprises a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 26-33; or (c) the muscle if the motif comprises a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 34-39.1. Brain / hypothalamus motifs

[0085] There were two six-nucleotide motifs found in miRNAs that accumulated in the hypothalamus: (1) “G,M / H,G,G,N,N / Y” (including representative SEQ ID NOs: 1-18); and (2) "G / K,U / Y,A / R,U / Y,D,G" (including representative SEQ ID NOs: 19-25). The majority ofmiRNAs contained the first motif and some of them also contained the second motif. Thus, these motifs could be found in combination with each other. Tables 6 and 7 present lists of the unique motifs in miRNAs that accumulate in the hypothalamus.

[0086] In some embodiments, the tissue accumulation motif is capable of producing accumulation of the RNA in (a) the brain, optionally the hypothalamus, if the sequence is any of SEQ ID NOs: 1-25 with a change of 1, 2, 3, or 4 nucleotides. In some embodiments, the tissue accumulation motif is capable of producing accumulation of the RNA in (a) the brain, optionally the hypothalamus, if the motif comprises a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 1-25.2. Liver motifs

[0087] In miRNAs that accumulated in the liver, a six-nucleotide motif was discovered as "G,A,A / H,Y / N,U / D,Y / N,G" (including representative SEQ ID NOs: 26-32).

[0088] In some embodiments, the tissue accumulation motif is capable of producing accumulation of the RNA in (a) the liver if the sequence is any of SEQ ID NOs: 26-32 with a change of 1, 2, 3, or 4 nucleotides. In some embodiments, the tissue accumulation motif is capable of producing accumulation of the RNA in (a) the liver if the motif comprises a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 26-32.

[0089] Table 8 shows a list of the unique occurrences of this motif in miRNAs that accumulate in the liver.3. Muscle motifs

[0090] In miRNAs that accumulated in muscle, a 5-nucleotide motif comprising "U / W,G,U,W / H,W / H" was identified. This motif often exists in combination with a second three-nucleotide motif "A,W,S", two nucleotides downstream of the first motif. Thus, the consensus motif identified in miRNAs that accumulate in the muscle is "U / W,G,U,W / H,W / H,N,N,A,W,S" (including representative SEQ ID NOs: 34-39).

[0091] In some embodiments, the tissue accumulation motif is capable of producing accumulation of the RNA in (a) the muscle if the sequence is any of SEQ ID NOs: 34-39 with a change of 1, 2, 3, or 4 nucleotides. In some embodiments, the tissue accumulation motif is capable of producing accumulation of the RNA in (a) the muscle if the motif comprises a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 34-39.

[0092] Table 9 shows a list of the unique occurrences of this motif in miRNAs that accumulate in the muscle.E. Packaging and Carriers

[0093] In some embodiments, RNA is packaged within extracellular vesicles and / or lipid nanoparticles. Packaging into an extracellular vesicle may occur during secretion from a cell based on a sorting motif described herein. RNA may also be packaged in vitro within extracellular vesicles and / or lipid nanoparticles.

[0094] In some embodiments, compositions comprising RNA are packaged within carriers, such as extracellular vesicles, based on sorting motifs. Small non-coding RNAs (ncRNAs) are found in the nucleus, cytoplasm, various organelles, and are also secreted via various extracellular carriers. These can include small extracellular vesicles (sEVs) which are 50-200nm in diameter and include exosomes that are also marked by surface proteins like CD63, CD9, and CD81.

[0095] Naturally occurring extracellular vesicle miRNA / ncRNA carriers include extracellular vesicles comprising small extracellular vesicles (sEVs), exosomes, microvesicles, large oncosomes, apoptotic bodies, chylomicrons, very low-density lipoprotein (VLDL), low- density lipoprotein (LDL), and / or high-density lipoprotein (HDL). Although sEVs / exosomes are the most-studied carriers of extracellular miRNAs and ncRNAs, other particles including microvesicles, oncosomes, and lipoproteins have also been reported to contain miRNAs.

[0096] Other carriers include large lipoproteins and other larger vesicles, as well as synthetic nanoparticle carriers. Several synthetic nanoparticle carriers have been described, including liposomes and lipid nanoparticles, as well as newer formulations that include polymersomes, nanospheres, and quantum dots, that are used to deliver RNA-based and other therapies (described in greater detail in (Mitchell et al., 2021). In some embodiments, lipid nanoparticles comprise one or more of polymersomes, dendrimers, polymer micelles, nanospheres, silica nanoparticles, quantum dots, iron oxide nanoparticles, gold nanoparticles, liposomes, lipid nanoparticles, or emulsions.

[0097] In addition, RNA-binding proteins are known to bind miRNAs via specific nucleotide motifs (Ray et al., Nature 499(7457): 172-177 (2013); Treiber et al., Mol Cell66(2):270-284 e213 (2017)). These include the Argonaute proteins (AGO1 - 4), which are important to miRNA function, but can also be secreted and are thus extracellular miRNA carriers (Arroyo et al., Proc Natl Acad Set USA 108(12):5003-5008 (2011)).

[0098] In some embodiments, the one or more RNA-binding protein comprises argonaute, a heterogeneous nuclear ribonucleoprotein, or a lipoprotein. In some embodiments, the one or more RNA-binding protein is selected from one or more of those listed in Table 10.

[0099] Table 10 provides a list of representative RNA-binding proteins (RBPs) that can function as miRNA / ncRNA carriers. Several reports have demonstrated that the hnRNP family of RBPs as well as AGO proteins can bind to miRNAs and other ncRNAs. These interactions are nucleotide sequence-specific and mediate nucleo-cytoplasmic shuttling as well as secretion of miRNAs / ncRNAs.

[0100] In some embodiments, extracellular vesicles may be produced in vivo by methods described herein via sorting motifs that direct an RNA to be secreted via the vesicles. Alternatively, compositions comprising RNA may be packaged within extracellular vesicles produced in vitro or within synthetic exosomes (i.e., lipid layers that mimic the properties of exosomes).

[0101] In some embodiments, the extracellular vesicles are produced from fat. In some embodiments, the fat is white adipose tissue (white fat) or brown adipose tissue (brown fat).

[0102] In some embodiments, RNA is produced in vitro and packaged within extracellular vesicles and / or lipid nanoparticles. In some embodiments, RNA packaged in extracellular vesicles and / or lipid nanoparticles does not comprise a sorting motif.

[0103] In some embodiments, when an RNA packaged in vitro in extracellular vesicles and / or lipid nanoparticles is administered to a subject, the extracellular vesicles and / or lipid nanoparticles are treated in the same or similar manner as endogenously produced RNA that is packaged in endogenous exosomes. In other words, packaging of RNA in vitro in extracellular vesicles and / or lipid nanoparticles can bypass a requirement for a sorting motif. In some embodiments, RNA packaged in vitro in extracellular vesicles and / or lipid nanoparticles can accumulate in one or more tissue.1. Exosomes

[0104] “Exosomes” or “extracellular vesicles” or “EVs” 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. In some embodiments, a carrier for ncRNAs is an exosome.

[0105] 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- 150nm (which contain exosomal markers), and / or larger exosome-like vesicles of 100-600nm.

[0106] Autologous or heterologous exosomes may be prepared. 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. 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.

[0107] 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 multivesicular 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.

[0108] 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.

[0109] 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.IL Methods of accumulating RNA in a specific tissue and / or cell type

[0110] In some embodiments, a method allows for accumulating an RNA in a specific tissue and / or cell type. In some embodiments, the tissue is in vivo, i.e., the method allows for accumulating an RNA in a specific tissue and / or cell type of a subject. In some embodiments, the RNA comprises a tissue accumulation motif, wherein the accumulation motif increases levels of an RNA in a desired tissue. As described herein, an accumulation motif may lead to accumulation of a non-coding RNA in a different tissue than it was secreted from (as shown in Figure 1, wherein miRNAs secreted from the fat were found accumulated in other targets).

[0111] In some embodiments, a method of accumulating a RNA in a specific tissue and / or cell type in vivo comprises administering to a subject the RNA, wherein the RNA comprises a tissue accumulation motif comprising a sequence selected from any one of SEQ ID NOs: 1-39. In some embodiments, a method of accumulating a RNA in a specific tissue and / or cell type in vivo comprises administering to a subject the RNA, wherein the RNA comprises a tissue accumulation motif comprising a sequence selected from any one of SEQ ID NOs: 1-39 with a change of 1, 2, 3, or 4 nucleotides. In some embodiments, a method of accumulating a RNA in a specific tissue and / or cell type in vivo comprises administering to a subject the RNA, wherein the RNA comprises a tissue accumulation motif comprising a sequence sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 1-39.

[0112] In some embodiments, the RNA administered to the subject further comprises a sorting motif comprising a sequence selected from any one of SEQ ID NOs: 46-55 or 59-71. In some embodiments, the RNA administered to the subject further comprises a sorting motif comprises a sequence selected from any one of SEQ ID NOs: 46-55 or 59 -71 with a change of 1, 2, 3, or 4 nucleotides. In some embodiments, the RNA administered to the subject further comprises a sorting motif comprises a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 46-55 or 59 -71.

[0113] In some embodiments, the RNA administered to the subject comprises non-coding RNA. In some embodiments, the RNA administered to the subject comprises miRNA, tRNA, tRNA half, tRNA fragment, snoRNA, piRNA, snRNA, rRNA, scaRNA, or mitochondrial-encoded non-coding RNA, or the complement thereof. In some embodiments, the RNA administered to the subject comprises a naturally-occurring RNA sequence or the complement of a naturally-occurring RNA sequence.

[0114] In some embodiments, the hypothalamus, liver, or muscle consensus motifs described above can be used to confer tissue-specific accumulation of miRNA / RNA- based therapies by promoting their accumulation in the hypothalamus, liver, or muscle,respectively. These accumulation motifs may also be used in combination to drive miRNA / RNA accumulation in any combination of the tissues described above.

[0115] Accumulating a RNA in a specific tissue and / or cell type can have numerous advantages, particularly to decrease off-target or unwanted effects in other tissues. For example, miRNAs have been implicated in a variety of disease states, including diabetes. Certain miRNAs present in the liver appear to have a crucial role in glucose homeostasis and are altered in subjects with diabetes and / or animal models of diabetes. Using methods described herein to increase accumulation of an miRNA specifically in the liver may therefore be an attractive treatment option (see, for example, Karolina et al., Expert Rev. Endocrinol. Metab. 7(3), 281— 300 (2012)). One skilled in the art would be able to identify an RNA of interest with a tissuespecific accumulation pattern and use the present methods to increase the amount of the RNA in a desired tissue.

[0116] In some embodiments, the RNA is administered into fat or administered by intravenous, intraperitoneal, intraocular, or intrathecal injection. In some embodiments, the fat is white fat or brown fat.1. Means of administering RNA

[0117] RNA constructs, such as miRNA and other non-coding RNA, 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 an RNA 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 RNA 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 RNA 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 al., Expert Opin. DrugDeliv. (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.2. Exosomes from brown adipose tissue

[0118] In some embodiments, an exosome is derived from brown adipose tissue (BAT). 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.III. Methods of decreasing levels or physiologic effects of an RNA in a specific tissue and / or cell type

[0119] In some embodiments, methods described herein are used to decrease the levels or physiologic effects of an RNA in a specific tissue and / or cell type. The use of antimiRs (miRNA inhibitors that have the reverse complementary sequence of a miRNA) is a well-known approach to inhibit specific miRNAs. The present disclosure provides new insights into how antimiRs could be accumulated specifically in a tissue of interest.

[0120] In some embodiments, the tissue accumulation motifs described above can also be incorporated into the design of antimiRs or other non-coding RNA inhibitors. This would lead to accumulation of these RNA inhibitors in specific tissues and / or cell types, thus inhibiting their target miRNA / ncRNA primarily in the target tissue. In some embodiments, antimiRs or other non-coding RNA inhibitors (i.e., miRNA / ncRNA inhibitor) with accumulation motifs can be used to inhibit / silence a specific miRNA or ncRNA in a desired tissue. In some embodiments,administration of a miRNA / ncRNA inhibitor prior to or simultaneously with a miRNA / ncRNA therapy provides an additional layer of specificity. Example 3 below provides a description of a representative use of a miRNA / ncRNA therapy.

[0121] In some embodiments, a method of blocking the effect of an endogenous non-coding RNA in a specific tissue and / or cell type in vivo comprises (a) administering to the subject an exogenous RNA comprising (i) the complement of the endogenous non-coding RNA; and (ii) a tissue accumulation motif comprising a sequence selected from any one of SEQ ID NOs: 1-39, and (b) allowing binding of the exogenous RNA to the endogenous non-coding RNA, wherein the binding preferentially occurs in the specific tissue and / or cell type.

[0122] In some embodiments, a method of blocking the effect of an administered non-coding RNA sequence in a specific tissue and / or cell type in vivo comprises (a) administering to the subject an exogenous RNA comprising the complement of the non-coding RNA sequence, wherein the exogenous RNA further comprises a tissue accumulation motif comprising a sequence selected from any one of SEQ ID NOs: 1-39; (b) administering the noncoding RNA sequence to the subject; and (c) allowing binding of the exogenous RNA to the administered non-coding RNA sequence, wherein the binding preferentially occurs in the specific tissue and / or cell type. In some embodiments, the non-coding RNA sequence further comprises a tissue accumulation motif that directs accumulation in a different tissue than the tissue accumulation motif in the exogenous RNA. In some embodiments, administering the noncoding RNA sequence to the subject occurs simultaneously or after administering the exogenous RNA.

[0123] In some embodiments, the targeting motif comprises a sequence selected from any one of SEQ ID NOs: 1-39 with a change of 1, 2, 3, or 4 nucleotides. In some embodiments, the targeting motif comprises a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 1-39.

[0124] In some embodiments, the exogenous RNA sequence further comprises a sorting motif comprising a sequence selected from any one of SEQ ID NOs: 46-55 or 59-71. In some embodiments, the exogenous RNA sequence further comprises a sorting motif comprises asequence selected from any one of SEQ ID NOs: 46-55 or 59 -71 with a change of 1, 2, 3, or 4 nucleotides. In some embodiments, the exogenous RNA sequence further comprises a sorting motif comprises a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 46-55 or 59 -71.

[0125] In some embodiments, the binding blocks activity of the endogenous noncoding RNA or the administered non-coding RNA sequence preferentially in the specific tissue and / or cell type. In other words, binding of the exogenous RNA can inhibit the effects of the endogenous non-coding RNA or the administered non-coding RNA sequence. In some embodiments, the endogenous non-coding RNA or the administered non-coding RNA sequence retains activity when not bound to the exogenous RNA.

[0126] In some embodiments, the specific tissue and / or cell type in vivo is (a) brain, optionally the hypothalamus, and the tissue accumulation motif comprises a sequence selected from any one of SEQ ID NOs: 1-25; (b) liver, and the tissue accumulation motif comprises a sequence selected from any one of SEQ ID NOs: 26-33; or (c) muscle, and the tissue accumulation motif comprises a sequence selected from any one of SEQ ID NOs: 34-39. In some embodiments, the specific tissue and / or cell type in vivo is (a) the brain, optionally the hypothalamus, if the sequence is any of SEQ ID NOs: 1-25 with a change of 1, 2, 3, or 4 nucleotides; (b) the liver if the sequence is any one of SEQ ID NOs: 26-33 with a change of 1, 2, 3, or 4 nucleotides; or (c) the muscle if the sequence is any of SEQ ID NOs: 34-39 with a change of 1, 2, 3, or 4 nucleotides. In some embodiments, the specific tissue and / or cell type in vivo is (a) the brain, optionally the hypothalamus, if the motif comprises a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 1-25; (b) the liver if the motif comprises a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 26-33; or (c) the muscle if the motif comprises a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 34-39.

[0127] In some embodiments, the non-coding RNA sequence or the exogenous RNA can be administered systemically or into an organ. In some embodiments, theadministration is intravenous, intraperitoneal, intraocular, or intrathecal injection. In some embodiments, the administered non-coding RNA sequence or the exogenous RNA is administered into fat or administered by intravenous, intraperitoneal, intraocular, or intrathecal injection. In some embodiments, the administered non-coding RNA sequence or the exogenous RNA is administered into fat. In some embodiments, the administered non-coding RNA sequence or the exogenous RNA is administered into the eye.

[0128] In some embodiments, the administered non-coding RNA sequence or the exogenous RNA is packaged within extracellular vesicles and / or lipid nanoparticles. In some embodiments, the extracellular vesicles comprise small sEVs, exosomes, microvesicles, large oncosomes, apoptotic bodies, chylomicrons, very VLDL, LDL, and / or HDL. In some embodiments, the lipid nanoparticles comprise one or more of polymersomes, dendrimers, polymer micelles, nanospheres, silica nanoparticles, quantum dots, iron oxide nanoparticles, gold nanoparticles, liposomes, lipid nanoparticles, or emulsions.

[0129] In some embodiments, the non-coding RNA is a miRNA, tRNA, tRNA half, tRNA fragment, snoRNA, piRNA, snRNA, rRNA, scaRNA, or mitochondrial -encoded noncoding RNA, or the complement thereof.

[0130] One skilled in the art would be able to identify an RNA of interest with a tissue-specific accumulation pattern and use the present methods to decrease its activity in a desired tissue. Such methods can use means of delivering RNA as described above.IV. Methods of enhancing secretion

[0131] In some embodiments, secretion of an RNA is enhanced by a sorting motif. In some embodiments, a method of enhancing secretion of an RNA from cells comprises adding any one of SEQ ID NOs: 46-55 or 59-71 to the RNA. In some embodiments, a method of enhancing secretion of an RNA from cells comprises adding any one of SEQ ID NOs: 46-55 or 59 -71 with a change of 1, 2, 3, or 4 nucleotides. In some embodiments, a method of enhancing secretion of an RNA from cells comprises adding a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 46-55 or 59 - 71.

[0132] In some embodiments, a method of enhancing secretion of an RNA via an extracellular vesicle comprises adding any one of SEQ ID NOs: 46-55 to the RNA. In some embodiments, a method of enhancing secretion of an RNA via an extracellular vesicle comprises adding any one of SEQ ID NOs: 46-55 with a change of 1, 2, 3, or 4 nucleotides. In some embodiments, a method of enhancing secretion of an RNA via an extracellular vesicle comprises adding a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 46-55.

[0133] In some embodiments, a method of enhancing secretion of an RNA via a non-vesicle route comprises adding any of SEQ ID NOs: 59-71 to the RNA. In some embodiments, a method of enhancing secretion of an RNA via a non-vesicle route comprises adding any one of SEQ ID NOs: 59-71 with a change of 1, 2, 3, or 4 nucleotides. In some embodiments, a method of enhancing secretion of an RNA via a non-vesicle route comprises adding a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater homology to any one of SEQ ID NOs: 59-71.

[0134] In some embodiments, the non-vesicle route comprises binding to one or more RBP. In some embodiments, the one or more RNA-binding protein comprises argonaute or a lipoprotein.

[0135] 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.

[0136] 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.EXAMPLESExample 1. Analysis of non-coding RNA secretion and accumulation

[0137] Adipose tissue was chosen to study the mechanisms of miRNA secretion and accumulation in other tissues, since it is a major source of circulating miRNAs and tRNAs. To this end, biosynthetic RNA labeling by thiouracil using mice with tissue specific expression of the bacterial enzyme uracil phosphoribosyltransferase (UPRT) was used to track and purify RNAs originating specifically from brown fat in mice (following general protocol of Gay et al. Nature Protocols 9:410-420 (2014)). These labeled miRNAs (as well as other non-coding RNAs including tRNA, rRNA, snoRNA, snRNA, and piRNA) were then analyzed by fraction of the plasma and immunoprecipitation coupled with small RNAseq. This revealed different miRNAs / ncRNAs in different plasma compartments (vesicular versus non-vesicular), as well as accumulation of different miRNAs to the liver, muscle, and hypothalamus.

[0138] An important aspect of the present Examples is that the screen was unbiased. In other words, all non-coding RNAs produced by brown fat could be tracked and identified using the present screening assay. In this way, the sequence of non-coding RNA found in different locations of the mice could be analyzed, knowing that all the labeled non-coding RNA had been produced from the brown fat (i.e., the labeled non-coding RNA could not have been produced locally or by other tissues than brown fat). Then the sequences of the non-coding RNA that originated in the fat and that accumulated in different locations in the body could be analyzed, as described in Example 2, to look for common motifs.

[0139] For example, as shown in Figure 1, of the 53 miRNAs assessed that were produced in brown fat, 19 were enriched in exosomes (sEV), 20 enriched in non-sEV fractions (i.e., protein bound), and 14 were found in both compartments. These labeled miRNAs then wereselectively accumulated in skeletal muscle, liver, and hypothalamus in very tissue specific manners (Figure 1).Example 2. Identification of sequences responsible for secretion and accumulation

[0140] To better understand the mechanism underlying the results in Example 1, the physicochemical properties of these miRNAs were studied for an association with tissuespecificity. Using unsupervised sequence analysis and clustering (ClustalW), a strong association between miRNA sequence and tissue accumulation was identified (Figure 2).

[0141] Thus, a search was made for novel nucleotide motifs (using the MEME Suite; meme-suite.org) in miRNAs from different plasma compartments (Figure 3), and miRNAs targeted to different tissues (Figure 4). This led to the identification of new nucleotide motifs embedded within these miRNAs. For instance, 2 unique motifs were found in 17 / 18 (94%) of sEV-miRNAs, and 2 different motifs were found in 15 / 19 (79%) of non-sEV miRNAs (Figure 3). Meanwhile, 24 / 30 (80%) of miRNAs targeted to the hypothalamus contained a unique motif that was nearly absent in miRNAs targeted to the liver or hypothalamus (Figure 4). Thus, these experiments identify a strong association between the nucleotide motifs described here and the plasma carrier and tissue preferences of miRNAs and other ncRNAs.

[0142] In summary, nucleotide sequences within miRNAs and other ncRNAs determine how they will be secreted from a tissue, such as fat, in a differential manner via sEV or non-sEV carriers. Further, nucleotide sequences determine ncRNA / miRNA accumulation in distal organs like the liver, muscle, and brain. This work describes a new method that can be used to design new or improve upon existing RNA / miRNA-based therapies that can be targeted to different individual tissues, such as liver, muscle, or brain, via either sEV or non-sEV carriers. Example 3. Use of a miRNA inhibitor

[0143] A representative use of a miRNA inhibitor would be to enhance effects of a miRNA in the liver (as the target tissue) and reduce effects in other tissues. Such a use might be appropriate for a variety of different miRNAs where their expression in the liver would be helpful for a subject, while expression in other regions would not be helpful. In other words, thismethod can target an miRNA to have specific effects in the liver while minimizing effects in other tissues.

[0144] For example, a liver-targeted miRNA / ncRNA therapy can be designed to comprise a liver motif as described above and be delivered in combination with or following administration of an antimiR (miRNA / ncRNA inhibitor) that comprise consensus motifs for the hypothalamus and / or muscle. In this example, the antimiR / inhibitor would accumulate and inhibit the miRNA / ncRNA in the “off-targef ’ tissues, adding a secondary layer of specificity. This antimiR / inhibitor would inhibit delivery and / or physiologic effects of the miRNA / ncRNA therapy in the “off-targef ’ tissues.

[0145] Thus, in addition to delivering specific miRNA / ncRNA inhibitors to target tissues as a means of miRNA / ncRNA-based therapy, the user can incorporate any combination of miRNA / ncRNA or their specific inhibitors containing the presently identified motifs to achieve tissue-specific induction or inhibition of a specific miRNA / ncRNA.References:1. Thomou, T., Mori, M.A., Dreyfuss, J.M., Konishi, M., Sakaguchi, M., Wolfrum, C., Rao, T.N., Winnay, J.N., Garcia-Martin, R , Grinspoon, S.K., et al. (2017). Adipose-derived circulating miRNAs regulate gene expression in other tissues. Nature 542, 450-455.10.1038 / nature21365.2. Brandao, B.B., Lino, M., and Kahn, C.R. (2021). Extracellular miRNAs as mediators of obesity associated disease. J Physiol. 10.1113 / JP280910.3. Mori, M.A., Ludwig, R.G., Garcia-Martin, R., Brandao, B.B., and Kahn, C.R. (2019). Extracellular miRNAs: From Biomarkers to Mediators of Physiology and Disease. Cell Metab 30, 656-673. 10.1016 / j.cmet.2019.07.011.4. Ferrante, S.C., Nadler, E.P., Pillai, D.K., Hubal, M.J., Wang, Z., Wang, J.M., Gordish- Dressman, H., Koeck, E., Sevilla, S., Wiles, A.A., and Freishtat, RJ. (2015). Adipocyte-derived exosomal miRNAs: a novel mechanism for obesity-related disease. Pediatr Res 77, 447-454. 10.1038 / pr.2014.202.5. Hubal, M.J., Nadler, E.P., Ferrante, S.C., Barberio, M.D., Suh, J.H., Wang, J., Dohm, G.L., Pories, W.J., Mietus-Snyder, M., and Freishtat, R.J. (2017). Circulating adipocyte-derived exosomal MicroRNAs associated with decreased insulin resistance after gastric bypass. Obesity (Silver Spring) 25, 102-110. 10.1002 / oby.21709.6. Scherer, P.E. (2019). The many secret lives of adipocytes: implications for diabetes. Diabetologia 62, 223-232. 10.1007 / s00125-018-4777-x.7. Garcia-Martin, R , Wang, G., Brandao, B.B., Zanotto, T.M., Shah, S., Patel, S., Schilling, B., and Kahn, C.R. (2021). Defining the miRNA codes for small extracellular vesicle / exosome release and cellular retention. Nature.EQUIVALENTS

[0146] 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 describe 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.

[0147] 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., + / -5-10% 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.

Claims

What is Claimed is:

1. A composition comprising an RNA comprising:(a) a sorting motif comprising a sequence selected from any one of SEQ ID NOs: 46- 55 or 59-71; and(b) a tissue accumulation motif.

2. The composition of claim 1, wherein the tissue accumulation motif comprises a sequence selected from any one of SEQ ID NOs: 1-39.

3. A composition comprising an RNA comprising a tissue accumulation motif comprising a sequence selected from any one of SEQ ID NOs: 1-39.

4. The composition of claim 3, wherein the RNA is packaged within extracellular vesicles and / or lipid nanoparticles.

5. The composition of claim 4, wherein the extracellular vesicles comprise small extracellular vesicles (sEVs), exosomes, microvesicles, large oncosomes, apoptotic bodies, chylomicrons, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and / or high-density lipoprotein (HDL).

6. The composition of claim 4, wherein the lipid nanoparticles comprise one or more of polymersomes, dendrimers, polymer micelles, nanospheres, silica nanoparticles, quantum dots, iron oxide nanoparticles, gold nanoparticles, liposomes, lipid nanoparticles, or emulsions.

7. The composition of any one of claims 1-6, wherein the RNA comprises non-coding RNA.

8. The method of claim 7, wherein the non-coding RNA is a micro RNA (miRNA), transfer RNA (tRNA), tRNA fragment, tRNA half, small nucleolar RNAs (snoRNA), piwi-interacting RNAs (piRNA), small nuclear RNAs (snRNA), ribosomal RNAs (rRNA), small cajal bodyspecific RNAs (scaRNA), or mitochondrial-encoded non-coding RNA, or the complement thereof.

9. The composition of any one of claims 1-8, wherein the RNA comprises a naturally- occurring RNA sequence or the complement of a naturally-occurring RNA sequence.

10. The composition of any one of claims 1-9, wherein the tissue accumulation motif is capable of producing accumulation of the RNA in:(a) the brain, optionally the hypothalamus, if the sequence is any of SEQ ID NOs: 1- 25;(b) the liver if the sequence is any one of SEQ ID NOs: 26-33; or(c) the muscle if the sequence is any of SEQ ID NOs: 34-39.

11. A method of accumulating a RNA in a specific tissue and / or cell type in vivo comprising administering to a subject the RNA, wherein the RNA comprises a tissue accumulation motif comprising a sequence selected from any one of SEQ ID NOs: 1-39.

12. The method of claim 11, wherein the RNA further comprises a sorting motif comprising a sequence selected from any one of SEQ ID NOs: 46-55 or 59-71.

13. The method of any one of claims 11-12 , wherein the RNA comprises non-coding RNA.

14. The method of claim 13, wherein the non-coding RNA is a miRNA, tRNA, tRNA half, tRNA fragment, snoRNA, piRNA, snRNA, rRNA, scaRNA, or mitochondrial-encoded noncoding RNA, or the complement thereof.

15. The method of any one of claims 11-14, wherein the RNA comprises a naturally- occurring RNA sequence or the complement of a naturally-occurring RNA sequence.

16. A method of blocking the effect of an endogenous non-coding RNA in a specific tissue and / or cell type in vivo comprising:(a) administering to the subject an exogenous RNA comprising:(i) the complement of the endogenous non-coding RNA; and(ii) a tissue accumulation motif comprising a sequence selected from any one of SEQ ID NOs: 1-39, and(b) allowing binding of the exogenous RNA to the endogenous non-coding RNA, wherein the binding preferentially occurs in the specific tissue and / or cell type.

17. A method of blocking the effect of an administered non-coding RNA sequence in a specific tissue and / or cell type in vivo comprising:(a) administering to the subject an exogenous RNA comprising the complement of the non-coding RNA sequence, wherein the exogenous RNA further comprises a tissue accumulation motif comprising a sequence selected from any one of SEQ ID NOs: 1-39;(b) administering the non-coding RNA sequence to the subject; and(c) allowing binding of the exogenous RNA to the administered non-coding RNA sequence, wherein the binding preferentially occurs in the specific tissue and / or cell type.

18. The method of claim 17, wherein the non-coding RNA sequence further comprises a tissue accumulation motif that directs accumulation in a different tissue than the tissue accumulation motif in the exogenous RNA.

19. The method of claim 17 or 18, wherein administering the non-coding RNA sequence to the subject occurs simultaneously or after administering the exogenous RNA.

20. The method of any one of claims 16-19, wherein the exogenous RNA sequence further comprises a sorting motif comprising a sequence selected from any one of SEQ ID NOs: 46-55 or 59-71.

21. The method of any one of claims 16-20, wherein the binding blocks activity of the endogenous non-coding RNA or the administered non-coding RNA sequence preferentially in the specific tissue and / or cell type.

22. The method of claim 16-21, wherein the endogenous non-coding RNA or the administered non-coding RNA sequence retains activity when not bound to the exogenous RNA.

23. The method of any one of claims 16-22, wherein the specific tissue and / or cell type in vivo is:(a) brain, optionally the hypothalamus, and the tissue accumulation motif comprises a sequence selected from any one of SEQ ID NOs: 1-25;(b) liver, and the tissue accumulation motif comprises a sequence selected from any one of SEQ ID NOs: 26-33; or(c) muscle, and the tissue accumulation motif comprises a sequence selected from any one of SEQ ID NOs: 34-39.

24. The method of any one of claims 16-23, wherein the administered non-coding RNA sequence or the exogenous RNA is administered into fat or cerebrospinal fluid or administered by intravenous or intraperitoneal injection.

25. The method of any one of claims 16-24, wherein the administered non-coding RNA sequence or the exogenous RNA is packaged within extracellular vesicles and / or lipid nanoparticles.

26. The method of claim 25, wherein the extracellular vesicles comprise small sEVs, exosomes, microvesicles, large oncosomes, apoptotic bodies, chylomicrons, very VLDL, LDL, and / or HDL.

27. The method of claim 25, wherein the lipid nanoparticles comprise one or more of polymersomes, dendrimers, polymer micelles, nanospheres, silica nanoparticles, quantum dots, iron oxide nanoparticles, gold nanoparticles, liposomes, lipid nanoparticles, or emulsions.

28. The method of any one of claims 16-27, wherein the non-coding RNA is a miRNA, tRNA, tRNA half, tRNA fragment, snoRNA, piRNA, snRNA, rRNA, scaRNA, or mitochondrial-encoded non-coding RNA, or the complement thereof.

29. A method of enhancing secretion of an RNA from cells, comprising adding any one of SEQ ID NOs: 46-55 or 59-71 to the RNA.

30. A method of enhancing secretion of an RNA via an extracellular vesicle, comprising adding any one of SEQ ID NOs: 46-55 to the RNA.

31. A method of enhancing secretion of an RNA via a non-vesicle route, comprising adding any of SEQ ID NOs: 59-71 to the RNA.

32. The method of claim 31, wherein the non-vesicle route comprises binding to one or more RBP33. The method of claim 32, wherein the one or more RNA-binding protein comprises argonaute, a heterogeneous nuclear ribonucleoprotein, or a lipoprotein.

34. The method of claim 33, wherein the one or more RNA-binding protein is selected from one or more of those listed in Table 10.

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