Therapeutic TRNA-based nucleic acids and methods of use thereof

Abstract

A phosphorothioate-containing RNA molecule that includes the nucleotide sequence: 5'-NCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3' (SEQ ID NO: 1), where N is uracil or thymine, is provided. The RNA molecule can include one or more phosphorothioate bonds between nucleic acid residues in one or more pairs (e.g., between nucleic acid residues in each pair) of consecutive nucleic acid residues within SEQ ID NO: 1. The RNA molecule and compositions thereof find use in treating muscle disorders, such as muscular dystrophy, heart conditions, such as heart failure or myocardial infarction, and / or conditions associated with inflammation and / or fibrosis.

Description

THERAPEUTIC TRNA-BASED NUCLEIC ACIDS AND METHODS OF USETHEREOFINCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 572561, filed April 1, 2024, which is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED R&D

[0002] This invention was made with government support under Grant No. R01HL124074 and R01HL155346, awarded by the National Institutes of Health. The Government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] The present application is being filed along with a Sequence Listing XML in electronic format. The Sequence Listing XML is provided as a file entitled CSMC023WOSequenceListing.xml, created March 27, 2025, which is 28,672 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.BACKGROUND

[0004] The present disclosure relates to chemically modified, therapeutic RNA molecules, and treatment of muscle disorders and / or heart conditions and / or inflammatory conditions and / or fibrosis using same.SUMMARY

[0005] Provided herein is a phosphorothioate-containing RNA molecule comprising 5’-NCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 1), wherein N is uracil or thymine, wherein the RNA molecule comprises one or more phosphorothioate bonds between nucleic acid residues in one or more pairs of consecutive nucleic acid residues within SEQ ID NO: 1. Also provided is a phosphorothioate-containing RNA molecule comprising a sequence at least 90% identical to 5’- NCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 1), wherein N is uracil or thymine, wherein the RNA molecule comprises one or more phosphorothioate bondsbetween nucleic acid residues in one or more pairs of consecutive nucleic acid residues within the sequence. In some embodiments, the RNA molecule includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 phosphorothioate bonds within SEQ ID NO: 1. In some embodiments, N is uracil. In some embodiments, the one or more phosphorothioate bonds comprise: a phosphorothioate bond between at least one pair of consecutive nucleic acid residues within positions 1-6 of SEQ ID NO: 1; and a phosphorothioate bond between at least one pair of consecutive nucleic acid residues within positions 27-32 of SEQ ID NO: 1. In some embodiments, the one or more phosphorothioate bonds comprise: a phosphorothioate bond between at least one pair of consecutive nucleic acid residues within positions 1-3 of SEQ ID NO: 1; and a phosphorothioate bond between at least one pair of consecutive nucleic acid residues within positions 30-32 of SEQ ID NO: 1. In some embodiments, the RNA molecule includes a phosphorothioate bond between nucleic acid residues in each of the following pairs of consecutive nucleic acid residues within SEQ ID NO: 1 : N1 / C2; C2 / C3; C30 / G31; G31 / C32, optionally wherein the RNA molecule comprises a phosphodiester bond between nucleic acid residues in all remaining pairs of consecutive nucleic acid residues within SEQ ID NO : 1. In some embodiments, the RNA molecule includes a nucleotide sequence set forth in SEQ ID NO: 8 (5’-UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’). In some embodiments, the RNA molecule includes a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1.

[0006] In several embodiments, the RNA molecule includes one or more backbone sugar modifications within SEQ ID NO: 1. In some embodiments, the RNA molecule includes the one or more backbone sugar modifications at one or more nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1. In some embodiments, up to 5 consecutive nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise the one or more backbone sugar modifications, optionally wherein 1-3 nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise the one or more backbone sugar modifications. In some embodiments, the one or more backbone sugar modifications comprises a locked nucleic acid (LNA) and / or a 2’-O-methylation. In some embodiments, up to 5 consecutive nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise a 2’-O-methylation, optionally wherein 1-3 nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1comprise a 2 ~-O-m ethylation. Tn several embodiments, the RNA molecule includes one or more backbone sugar modifications within SEQ ID NO: 1, and the RNA molecule includes a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1. In some embodiments, the RNA molecule includes the one or more backbone sugar modifications at one or more nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1, and the RNA molecule includes a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1. In some embodiments, up to 5 consecutive nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise the one or more backbone sugar modifications, and the RNA molecule includes a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1, optionally wherein 1-3 nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise the one or more backbone sugar modifications. In some embodiments, the one or more backbone sugar modifications comprises a locked nucleic acid (LNA) and / or a 2’-(9-methylation, and the RNA molecule includes a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1. In some embodiments, up to 5 consecutive nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise a 2’-O-methylation, and the RNA molecule includes a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO:1, optionally wherein 1-3 nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise a 2’-O-methylation.

[0007] In several embodiments, the RNA molecule includes a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1, and wherein nucleic acid residues at the following positions of SEQ ID NO: 1 comprises a 2’-O- methylation: (a) 1 and 32; (b) 1, 2, 31, and 32; or (c) 1, 2, 3, 30, 31, and 32. In some embodiments, up to 5 consecutive nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise a LNA, wherein if the nucleic acid residue at position 1 of SEQ ID NO: 1 comprises the LNA, N is thymine, optionally wherein 1-3 nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise a LNA. In some embodiments, the RNA molecule includes a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1, wherein N is thymine, and wherein nucleic acid residues at the following positions of SEQ ID NO: 1 comprises a LNA: (a) 1 and 32; or (b) 1, 2, 3, 30, 31, and 32. In several embodiments, the RNA molecule includes a phosphorothioate bond betweeneach pair of consecutive nucleic acid residues of SEQ ID NO: 1, and wherein nucleic acid residues at the following positions of SEQ ID NO: 1 comprises a 2’-(?-methylation: (a) 1 and 32; (b) 1, 2, 31, and 32; or (c) 1, 2, 3, 30, 31, and 32, and the RNA molecule includes a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1. In some embodiments, up to 5 consecutive nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise a LNA, wherein if the nucleic acid residue at position 1 of SEQ ID NO: 1 comprises the LNA, N is thymine, and the RNA molecule includes a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1, optionally wherein 1-3 nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise a LNA. In some embodiments, the RNA molecule includes a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1, wherein N is thymine, and wherein nucleic acid residues at the following positions of SEQ ID NO: 1 comprises a LNA: (a) 1 and 32; or (b) 1, 2, 3, 30, 31, and 32, and the RNA molecule includes a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1.

[0008] In some embodiments, the RNA molecule is at most 60 bases long, optionally, wherein the RNA molecule is about 32 bases long. In some embodiments, the RNA molecule consists of or consists essentially of the sequence of SEQ ID NO: 1. In some embodiments, the RNA molecule includes any one of SEQ ID NOs: 2-7. In some embodiments, the RNA molecule consists of or consists essentially of any one of SEQ ID NOs: 2-7.

[0009] Provided herein is a phosphorothioate-containing RNA molecule comprising, consisting essentially of, or consisting of 5’- UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 9), wherein the RNA molecule comprises a phosphorothioate bond between nucleic acid residues in each of the following pairs of consecutive nucleic acid residues: U1 / C2; C2 / C3; C30 / G31; G31 / C32, optionally wherein the RNA molecule comprises a phosphodiester bond between nucleic acid residues in all remaining pairs of consecutive nucleic acid residues.

[0010] Also provided is a composition comprising: the RNA molecule of the present disclosure; and a pharmaceutically acceptable excipient. In some embodiments, the composition includes a transfection reagent. In some embodiments, the transfection reagentcomprises one or more of a liposome, an extracellular vesicle (EV), and / or a polyethylene glycol (PEG)-cationic lipid complex (PCLC). In some embodiments, the transfection reagent comprises an EV derived from cardiosphere-derived cells (CDC).

[0011] In some embodiments, the composition includes comprising a casein phosphoprotein. In some embodiments, the composition includes casein micelles. In some embodiments, the composition includes chitosan. In some embodiments, the RNA molecule is encapsulated in a casein-chitosan complex. In some embodiments, the composition includes casein-chitosan micelles.

[0012] In several embodiments, the composition includes an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide comprises an exonskipping agent that targets a dystrophin transcript.

[0013] Also provided herein is method of treating a condition associated with inflammation and / or fibrosis, comprising administering to a subject in need of treating a condition associated with inflammation and / or fibrosis a therapeutically effective amount of the RNA molecule or the composition of the present disclosure. In some embodiments, the condition associated with inflammation and / or fibrosis comprises inflammation and / or fibrosis of the heart or skeletal muscle. In some embodiments, the condition associated with inflammation and / or fibrosis comprises a symptom and / or sequelae of heart failure, myocardial infarction, or muscular dystrophy. In some embodiments, the subject has suffered heart failure or myocardial infarction. In some embodiments, the subject has or is predisposed to having muscular dystrophy. In some embodiments, the subject has or is predisposed to having Duchenne muscular dystrophy. In some embodiments, the method includes administering a second therapy for the muscle disorder. In some embodiments, the second therapy comprises an exon-skipping agent and / or gene therapy. In some embodiments, the method includes orally administering the therapeutically effective amount of the RNA molecule or of the composition to the subject. In some embodiments, the method includes parenterally administering the therapeutically effective amount of the RNA molecule or of the composition to the subject. In some embodiments, the method includes intravenously, intramuscularly, intramyocardially, or intracardially administering the therapeutically effective amount of the RNA molecule or of the composition to the subject.

[0014] Also provided is a method of treating a muscle disorder or symptom thereof, comprising administering to a subject in need of treating a muscle disorder or symptom thereof a therapeutically effective amount of the RNA molecule or the composition of the present disclosure.

[0015] Also provided is a method of immunomodulation, comprising contacting an effective amount of the RNA molecule or of the composition of the present disclosure with a population of macrophages. In some embodiments, the contacting comprises administering to a subject in need of treating a condition associated with inflammation and / or fibrosis the effective amount of the RNA molecule or of the composition. In some embodiments, the contacting is done in vitro. In some embodiments, the macrophage is a human macrophage. In some embodiments, contacting the effective amount of the RNA molecule or of the composition increases expression of one or more of IL-10, ILla, ILip, TGFpi, NFKB, TNF, CCL3 and VGEFA, in the population of macrophages. In some embodiments, expression is increased to a greater extent than an increase in expression achieved by a comparable amount of a reference RNA molecule comprising 5’- UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 8), wherein the reference RNA molecule does not comprise a phosphorothioate bond within SEQ ID NO: 8.

[0016] Also provided is a kit comprising: the RNA molecule of the present disclosure; and optionally a transfection reagent. In some embodiments, the transfection reagent comprises one or more of a lipid, PEGylated lipid, and an extracellular vesicle (EV). In some embodiments, the kit includes a pharmaceutically acceptable excipient. In some embodiments, the kit includes a casein phosphoprotein. In some embodiments, the kit includes chitosan. In some embodiments, the kit includes an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide comprises an exon-skipping agent that targets a dystrophin transcript.

[0017] Also provided is use of the RNA molecule of the present disclosure or the composition of the present disclosure for treatment of a condition associated with inflammation and / or fibrosis in a subject in need thereof. Also provided is use of the RNA molecule of the present disclosure or the composition of the present disclosure for preparation of a medicament for treatment of a condition associated with inflammation and / or fibrosis a subject in need thereof. In some embodiments, the condition associated with inflammation and / or fibrosiscomprises inflammation and / or fibrosis of the heart and / or skeletal muscle. In some embodiments, the condition associated with inflammation and / or fibrosis comprises a symptom and / or sequelae of heart failure, myocardial infarction, or muscular dystrophy.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1A-1D show the chemical structures of modifications selected for incorporating into tREX-1, according to some non-limiting embodiments.

[0019] FIG. IE shows some non-limiting embodiments of sequence and / or chemical variants of tREX-1.

[0020] FIG. 2 shows the effects of unmodified tREX-1 on in vitro gene expression in macrophages compared to unmodified scramble RNA control.

[0021] FIG. 3 shows the effect of chemically modified tREX-1 on in vitro gene expression in macrophages compared to unmodified tREX-1, according to some non-limiting embodiments of the present disclosure.

[0022] FIG. 4A shows some non-limiting embodiments of chemically modified variants of tREX-1.

[0023] FIG. 4B shows the effect of chemically modified tREX-1 on in vitro gene expression in macrophages compared to scramble RNA (unmodified and TT9-modified), according to some non-limiting embodiments of the present disclosure.

[0024] FIG. 5 shows the effect of TT1 -modified and TT8-modified scramble RNA on in vitro gene expression in macrophages compared to unmodified scramble tREX-1, according to some non-limiting embodiments of the present disclosure.

[0025] FIG. 6 shows the effect of oral administration of vehicle, TT1 and TT1- modified scramble RNA on infarct size after undergoing ischemia / reperfusion, according to some non-limiting embodiments of the present disclosure.

[0026] FIG. 7 shows the effect of oral administration of TT8 and TT8-modified scramble RNA on infarct size after undergoing ischemia / reperfusion, according to some nonlimiting embodiments of the present disclosure.DETAILED DESCRIPTION

[0027] tREX-1 represents a new class of defined non-coding RNAs (ncRNAs) mined from extracellular vesicles (EV) secreted by cardiosphere-derived cells (CDCs). CDCs are cardiac progenitor / stromal cells with immunomodulatory, anti-fibrotic, and pro-regenerative properties. These therapeutic actions antagonize crucial pathways central to the pathology of Duchenne muscular dystrophy (DMD). Without being bound to theory, mechanistic studies in preclinical models demonstrate CDCs work indirectly by secreting extracellular vesicles (CDC-EVs), which are lipid nanoparticles laden with a rich repertoire of bioactive molecules. Inventories of CDC-EV contents, generated by RNA-sequencing, now serve as Rosetta stones to decipher and exploit the complex biology of EVs. In CDC-EVs, the largest percentage of mapped reads are transfer RNA (tRNA) fragments. These molecular entities were previously thought to be nonspecific degradation products, but they are increasingly recognized as comprising a novel class of small ncRNAs with potential therapeutic bioactivity. In CDC-EVs, a species comprising the 5’ half of one specific tRNA is particularly plentiful. This entity, tREX-1, showed disease-modifying bioactivity in models of skeletal muscle and heart disease, including muscular dystrophy and myocardial infarction, for example, as described in International Application No. PCT / US2023 / 075437 (filed September 28, 2023), which is hereby incorporated by reference in its entirety. As used herein, “tREX- 1” refers to an RNA having the sequence 5’- UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 8), without the modifications described herein. “tREX-1” may be used herein interchangeably with “unmodified tREX-1.”

[0028] Provided herein are tREX-1 variants obtained by chemically modifying tREX-1. The tREX-1 variants can have enhanced bioactivity compared to unmodified tREX- 1. In some embodiments, chemically modifying tREX-1 by incorporating a phosphorothioate bond between nucleic acid residues in one or more pairs of consecutive nucleic acid residues enhances bioactivity compared to unmodified tREX-1. In some embodiments, the number and / or distribution of phosphorothioate bonds in the tREX-1 variant affects non-specific bioactivity of the RNA molecule (e.g., bioactivity that is independent of the nucleotide sequence of tREX-1). In some embodiments, including a phosphorothioate bond between each pair of consecutive nucleic acid residues in a scramble RNA of tREX-1 increases non-specific bioactivity. In some embodiments, specific bioactivity (e.g., sequence-specific bioactivity) of tREX-1, such as inducing a gene expression signature in macrophages as described herein, is preserved without inducing non-specific bioactivity by restricting the position of the phosphorothioate bond along the tREX-1 variant to the 5’ and / or 3’ ends, while keeping thephosphodiester bond between nucleic acid residues in the rest of the molecule. In some embodiments, the tREX-1 variant includes a phosphorothioate bond up to the third residue from the 5’ end of tREX-1 and up to the third residue from the 3’ end of tREX-1.Terms

[0029] As used herein “nucleic acid” and “oligonucleotide” have their customary and ordinary meaning as understood by one of ordinary skill in the art in view of the present disclosure. “Nucleic acid” and “oligonucleotide” refer to multiple nucleotides (e.g., molecules comprising a sugar (e.g. ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g. cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G))), or analogues thereof. Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are nucleic acids in which the nucleotides are linked through a 3 ',5 '-phosphodiester bond to form a backbone of alternating phosphate groups and sugar moiety. RNA is composed of ribose sugar groups and DNA is composed of deoxyribose sugar groups. A nucleic acid includes polynucleosides (e.g., a polynucleotide minus the phosphate) and any other organic basecontaining polymer. Purines and pyrimidines include but are not limited to adenine, cytosine, guanine, thymidine, inosine, 5 -methyl cytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6- diaminopurine, hypoxanthine, and other naturally and non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties. Chemical modifications can be introduced at the site of the phosphodiester bond, sugar moiety, nucleobase, or combined sites thereof. Phosphorothioate (PS) oligonucleotides, are nucleic acids with a modified phosphate in which one of the non-bridging oxygens of the phosphate-ester group is changed to sulfur. This modification retains the negative charge of the backbone. They can have highly increased nuclease resistance relative to the natural nucleic acids. In some embodiments, a change in electronegativity due to the change from phosphodiester to phosphorothioate bonds provides nuclease resistance. 2’-( -methylation is a modification on the ribose sugar of RNA in which a methyl group is added to the 2’ hydroxyl group of the native RNA. In “locked” nucleic acids (LNAs) the 2'-oxygen is attached to the 4'-carbon of the ribose sugar via a methylene bridge that locks in an RNA-like C3’-endo conformation. These derivatives are also known as 2', d'bridged nucleic acids (BNA). The methylene bridge fixes the furanose ring in the 3'-endo conformation that is highly advantageous for base pairing. LNA can form very stable duplexeswith RNA and DNA according to the Watson-Crick rule with excellent selectivity. The incorporation of LNA monomers into DNA oligomer can increase the strength of hybridization. A nucleic acid can include any other suitable modifications. Thus, the term nucleic acid also encompasses nucleic acids with substitutions or modifications, such as in the bases and / or sugars. Nucleic acid includes nucleotide analogues and polymers thereof, including, but not limited to, a polynucleoside in which two nucleosides are linked by a non- phosphodiester bond, e.g., linked by a phosphorothioate bond. “RNA molecule” as used herein denotes a nucleic acid that includes ribose as the sugar component, and derivatives thereof, including a nucleic acid that has been modified at the site of the phosphodi ester bond, sugar moiety, nucleobase, or combined sites thereof.

[0030] Nucleic acid molecules of the present disclosure may share a certain degree of sequence similarity or identity with the reference molecules (e g., reference polypeptides or reference nucleic acid), for example, with art-described molecules (e g., engineered or designed molecules or wild-type molecules). The term “identity” as known in the art, refers to a relationship between the sequences of two or more polypeptides or nucleic acids, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between them as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related peptides can be readily calculated by known methods. “% identity” as it applies to polypeptide or nucleic acid sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Any suitable methods and computer programs for the alignment can be used. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Generally, sequence variants of a particular nucleic acid or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference nucleic acid or polypeptide asdetermined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”. Nucleic Acids Res. 25:3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147: 195-197.) A general global alignment technique based on dynamic programming is the Needleman- Wunsch algorithm (Needleman, S. B. & Wunsch. C. D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443- 453.). More recently a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed that purportedly produces global alignment of nucleobase and protein sequences faster than other optimal global alignment methods, including the Needleman- Wunsch algorithm. Other tools are described herein, specifically in the definition of “identity” below.

[0031] The term “identity” refers to the overall relatedness between polymeric molecules, for example, between nucleic acid molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent identity of two polynucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleobase at corresponding positions are then compared. When a position in the first sequence is occupied by the same nucleobase as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a suitable mathematical algorithm. For example, the percent identity between two nucleic acid sequences can bedetermined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects. Smith. D. W., ed., Academic Press. New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleic acid sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0032] The term “Watson-Crick base-pairing”, or “base-pairing” refers to the formation of hydrogen bonds between specific pairs of bases (“complementary base pairs”). For example, two hydrogen bonds form between adenine (A) and uracil (U), and three hydrogen bonds form between guanine (G) and cytosine (C). One method of assessing the strength of bonding between two nucleic acids is by quantifying the percentage of bonds formed between the guanine and cytosine bases of the two nucleic acids (“GC content”). The formation of a nucleic acid duplex via bonding of complementary base pairs can also be referred to as “hybridization”. Generally, two nucleic acids sharing a region of complementarity are capable, under suitable conditions, of hybridizing (e.g., via nucleic acid base pairing) to form a duplex structure. A region of complementarity can vary in size. In some embodiments, a region of complementarity ranges in length from about 2 base pairs to about 100 base pairs. In some embodiments, a region of complementarity ranges in lengthfrom about 5 base pairs to about 75 base pairs. In some embodiments, a region of complementarity ranges in length from about 10 base pairs to about 50 base pairs. In some embodiments, a region of complementarity ranges in length from about 20 base pairs to about 30 base pairs.

[0033] “Isolated” as used herein with reference to an isolated biomolecule, e.g., a nucleic acid, has the ordinary and customary meaning to one of ordinary skill in the art in view of the present disclosure. An isolated biomolecule, e.g., an isolated nucleic acid, is generally in a non-natural environment, or in an environment that the biomolecule would otherwise not have been without human intervention of the biomolecule or its environment. In some embodiments, an isolated biomolecule is not inside a cell or an organism.

[0034] “Extracellular vesicle” or “EV” as used herein have their ordinary and customary meaning as understood by one of ordinary skill in the art, in view of the present disclosure. EVs include lipid bilayer structures generated by cells, and include exosomes, microvesicles, epididimosomes, argosomes, exosome-like vesicles, microparticles, promininosomes, prostasomes, dexosomes, texosomes, dex, tex, archeosomes and oncosomes.

[0035] “Micelle,” as used herein with reference to casein micelles, has its customary and ordinary meaning as understood by one of ordinary skill in the art, in view of the present disclosure. Casein micelles are colloidal particles that can include aggregates of one or more casein phosphoproteins (e.g., one or more, two or more, three or more, or all four of alpha si casein, alpha s2 casein, beta casein, and kappa casein). “Micelle” as used herein with reference to lipid micelles has its customary and ordinary meaning as understood by one of ordinary skill in the art, in view of the present disclosure.

[0036] “Subject,” as used herein refers to any vertebrate animal, including mammals and non-mammals. A subject can include primates, including humans, and nonprimate mammals, such as rodents, domestic animals or game animals. Non-primate mammals can include mouse, rat, hamster, rabbit, dog, fox, wolf, cat, horse, cow, pig, sheep, goat, camel, deer, buffalo, bison, etc. Non-mammals can include bird (e.g., chicken, ostrich, emu, pigeon), reptile (e.g., snake, lizard, turtle), amphibian (e.g., frog, salamander), fish (e.g., salmon, cod, pufferfish, tuna), etc. The terms, “individual,” “patient,” and “subject” are used interchangeably herein.

[0037] “Administering” as used herein can include any suitable routes of administering a therapeutic agent or composition as disclosed herein. Suitable routes of administration include, without limitation, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, injection or topical administration. Administration can be local or systemic.

[0038] As used herein, “treat” and “treatment” includes curing, improving, ameliorating, reducing the severity of, preventing, slowing the progression of, and / or delaying the appearance of a disease, condition and / or symptoms thereof.

[0039] A treatment can be considered “effective,” or “therapeutically effective” as used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response or outcome is induced e.g., by at least 2%, 3%, 4%, 5%, 10%, or more, following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. exercise endurance. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (e.g., progression of the disease is halted). Treatment includes any treatment of a disease or condition in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease or condition, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease or condition, e.g., causing regression of symptoms. An effective amount for the treatment of a disease or condition means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease or condition. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response or outcome, (e.g., muscle function, mass, or volume, such as heart function, mass, or volume). One skilled in the art can monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters.

[0040] The term “effective amount” or “therapeutically effective amount” as used herein refers to the amount of a composition or an agent needed to alleviate at least one or more symptom of the disease or condition, and relates to a sufficient amount of therapeuticcomposition to provide the desired effect. The term “effective amount” or “therapeutically effective amount” can refer to an amount of a composition or therapeutic agent that is sufficient to provide a particular anti-inflammatory, anti-fibrotic, immunomodulatory, myoprotective and / or cardioprotective effect when administered to a typical subject. An effective amount as used herein, in various contexts, can include an amount sufficient to delay the development of a symptom of the disease or condition, alter the course of a symptom disease or condition (for example but not limited to, slowing the progression of a symptom of the disease or condition), or reverse a symptom of the disease or condition. In some embodiments, the therapeutically effective amount is administered in one or more doses of the therapeutic agent. In some embodiments, the therapeutically effective amount is administered in a single administration, or over a period of time in a plurality of doses.

[0041] As used herein, the phrase “physiologically compatible” and “pharmaceutically acceptable” are employed interchangeably herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0042] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The abbreviation, “e.g.” is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” The term “about” as used herein to, for example, define the values and ranges of molecular weights means that the indicated values and / or range limits can vary within ±20%, e.g., within ±10%, including within ±5%. The use of “about” before a number includes the number itself. For example, “about 5” provides express support for “5.” Numbers provided in ranges include overlapping ranges and integers in between; for example a range of 1-4 and 5-7 includes for example, 1-7, 1-6, 1-5, 2-5, 2-7, 4-7, 1, 2, 3, 4, 5, 6 and 7.NUCLEIC ACIDS

[0043] Provided herein is a phosphorothioate-containing RNA molecule that includes 5’-NCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 1), where N is uracil or thymine, and the RNA molecule includes one or more phosphorothioatebonds between nucleic acid residues in one or more pairs of consecutive nucleic acid residues within SEQ ID NO: 1. Also provided is a phosphorothioate-containing RNA molecule that includes a sequence at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, or about 100% identical to 5’-NCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 1), where N is uracil or thymine, and the RNA molecule includes one or more phosphorothioate bonds between nucleic acid residues in one or more pairs of consecutive nucleic acid residues within the sequence (e.g., the sequence having the noted % sequence identity to SEQ ID NO: 1). In some embodiments, the phosphorothioate-containing RNA molecule includes 5’-NCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 1) with a sequence variation at up to 1, 2, 3, 4, or 5 nucleobases in the sequence relative to SEQ ID NO:1. In some embodiments, the RNA molecule is an isolated RNA molecule. In some embodiments, the introduction of a phosphorothioate group as linker between nucleic acid residues in the RNA molecule improves stability against a nuclease attack.

[0044] “Consecutive nucleic acid residues” as used herein denotes a sequence of nucleic acid residues with no other residues within the sequence. A “pair of consecutive nucleic acid residues” denotes two nucleic acid residues linked to each other (e g., through a phosphodiester or phosphorothioate bond) with no intervening residue. For example, without limitation, a pair of consecutive nucleic acid residues within SEQ ID NO:1 may be N (uracil or thymine) at position 1, and C at position 2, and a phosphorothioate bond may link the N at position 1 and the C at position 2 (in lieu of a phosphodiester bond). As used herein, a “position” within a nucleic acid sequence is defined relative to the 5’ end of the nucleic acid sequence (e.g., a sequence within a RNA molecule), unless indicated otherwise.

[0045] The RNA molecule can include any suitable number of phosphorothioate bonds within SEQ ID NO: 1. In some embodiments, the RNA molecule includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 phosphorothioate bonds within SEQ ID NO: 1. In some embodiments, the RNA molecule includes 2-6 phosphorothioate bonds within SEQ ID NO: 1. In some embodiments, the RNA molecule includes 4 phosphorothioate bonds within SEQ ID NO: 1.

[0046] Any suitable pair of consecutive nucleic acid residues within SEQ ID NO: 1 can be linked by a phosphorothioate bond. In some embodiments, a phosphorothioate bond links nucleic acid residues of any one or more of pairs of consecutive nucleic acid residuesstarting at the following positions within SEQ ID NO: 1 : 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and 31. In some embodiments, unless indicated otherwise, any pair of consecutive nucleic acid residues that are not specified as being linked by a phosphorothioate bond are linked by phosphodiester bond. The phosphorothioate bond(s) can be distributed along the RNA molecule in any suitable manner. In some embodiments, the RNA molecule includes at least one chemically modified residue within the first half of the nucleic acid, e.g., the 5’ half of the nucleic acid. In some embodiments, the RNA molecule includes at least one phosphorothioate bond within the second half of the nucleic acid, e.g., the 3’ half of the nucleic acid. In some embodiments, the RNA molecule includes at least one phosphorothioate bond within the first half of the nucleic acid, e.g., the 5’ half of the nucleic acid, and at least one phosphorothioate bond within the second half of the nucleic acid, e.g., the 3’ half of the nucleic acid. In some embodiments, the RNA molecule includes one or more phosphorothioate bonds within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues from the 5’ end of the nucleic acid. In some embodiments, the RNA molecule includes one or more phosphorothioate bonds within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues from the 3’ end of the nucleic acid. In some embodiments, the one or more phosphorothioate bonds are between at least one pair of consecutive nucleic acid residues within positions 1-6 of SEQ ID NO: 1 (e.g., between positions 1 and 2, 2 and 3, 3 and 4, 4 and 5, and / or 5 and 6 of SEQ ID NO: 1). In some embodiments, the one or more phosphorothioate bonds are between at least one pair of consecutive nucleic acid residues within positions 27-32 of SEQ ID NO: 1 (e.g., between positions 27 and 28, 28 and 29, 29 and 30, 30 and 31, and / or 31 and 32 of SEQ ID NO: 1). In some embodiments, the one or more phosphorothioate bonds are between at least one pair of consecutive nucleic acid residues within positions 1-6 of SEQ ID NO: 1, and at least one pair of consecutive nucleic acid residues within positions 27-32 of SEQ ID NO: 1. In some embodiments, the one or more phosphorothioate bonds are between at least one pair of consecutive nucleic acid residues within positions 1-3 of SEQ ID NO: 1. In some embodiments, the one or more phosphorothioate bonds are between at least one pair of consecutive nucleic acid residues within positions 30-32 of SEQ ID NO: 1. In some embodiments, the one or more phosphorothioate bonds are between at least one pair of consecutive nucleic acid residues within positions 1-3 of SEQ ID NO: 1, and at least one pair of consecutive nucleic acid residues within positions 30-32 of SEQ ID NO: 1. In someembodiments, the RNA molecule includes a phosphorothioate bond between nucleic acid residues in each of the following pairs of consecutive nucleic acid residues within SEQ ID NO: 1 : N1 / C2; C2 / C3; C30 / G31; and G31 / C 32 (where, for example, “Nl” denotes N at position 1 in SEQ ID NO: 1, “C2” denotes C at position 2 in SEQ ID NO: 1, and the phosphorothioate bond is between positions 1 and 2 in SEQ ID NO:1, etc.). In some embodiments, the RNA molecule includes a phosphorothioate bond between nucleic acid residues in each of the following pairs of consecutive nucleic acid residues within SEQ ID NO: 1 : N1 / C2; C2 / C3; C30 / G31; and G31 / C32, and nucleic acid residues in all remaining pairs of consecutive nucleic acid residues within SEQ ID NO:1 are linked by a phosphodiester bond. In some embodiments, the RNA molecule includes a nucleotide sequence set forth in SEQ ID NO: 8 (5’-UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’). In some embodiments, the RNA molecule includes a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1. In some embodiments, the RNA molecule does not include any phosphodiester bond within SEQ ID NO: 1.

[0047] In some embodiments, the RNA molecule includes one or more backbone sugar modifications (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or a number of backbone sugar modifications in a range defined by any two of the preceding values (e.g., 1-10, 2-8, 2-6, 4-6, 3-8, etc.)) within SEQ ID NO: 1. In some embodiments, the RNA molecule includes one or more backbone sugar modifications at one or more nucleic acid residues (or consecutive nucleic acid residues if two or more) from the 5’ end and / or 3’ end of SEQ ID NO: 1. For example, and without limitation, 3 consecutive nucleic acid residues from the 5’ end of SEQ ID NO: 1 include N (uracil or thymine) at position 1, C at position 2, and C at position 3, each of which may include a backbone sugar modification. For example, and without limitation, 3 consecutive nucleic acid residues from the 3’ end of SEQ ID NO: 1 include C at position 30, G at position 31, and C at position 32, each of which may include a backbone sugar modification. In some embodiments, up to 5, up to 4, or up to 3 consecutive nucleic acid residues from the 5’ end of SEQ ID NO: 1 include a backbone sugar modification. In some embodiments, up to 5, up to 4, or up to 3 consecutive nucleic acid residues from the 3’ end of SEQ ID NO: 1 include a backbone sugar modification. In some embodiments, up to 5, up to 4, or up to 3 consecutive nucleic acid residue from the 5’ end of SEQ ID NO: 1, and up to 5, up to 4, or up to 3 consecutive nucleic acid residues from the 3’ end of SEQ ID NO: 1 includea backbone sugar modification. In some embodiments, 1, 2, or 3 nucleic acid residues (or consecutive nucleic acid residues if 2 or 3) from the 5’ end of SEQ ID NO: 1 include a backbone sugar modification. In some embodiments, 1, 2, or 3 nucleic acid residues (or consecutive nucleic acid residues if 2 or 3) from the 3’ end of SEQ ID NO: 1 include a backbone sugar modification. In some embodiments, 1, 2, or 3 nucleic acid residues (or consecutive nucleic acid residues if 2 or 3) from the 5’ end of SEQ ID NO: 1, and 1, 2, or 3 nucleic acid residues (or consecutive nucleic acid residues if 2 or 3) from the 3’ end of SEQ ID NO: 1 include a backbone sugar modification. In some embodiments, the RNA molecule does not include any backbone sugar modification within SEQ ID NO: 1.

[0048] The RNA molecule can include any suitable backbone sugar modification. In some embodiments, the backbone sugar modification includes a locked nucleic acid (LNA) and / or a 2’ -O-m ethylation. In some embodiments, a 2’-O-methylation includes introduction of a 2'-O-methyl on the ribose sugar of the nucleic acid to improve nuclease resistance and / or binding affinity. In some embodiments, a LNA modification includes introduction of a methylene bridge connecting the 2’-0 atom and the 4’-C atom of the ribose sugar of the nucleic acid to lock the conformation. In some embodiments, when the nucleic acid residue at position 1 of SEQ ID NO: 1 includes a LNA, and N is thymine. In some embodiments, the backbone sugar modification includes a locked nucleic acid (LNA) and a 2’-O-methylation (e.g., in the same residue). In some embodiments, the backbone sugar modification does not include a 2’- O-methylation. In some embodiments, the RNA molecule does not include a 2’-O-methylation of a nucleic acid residue. In some embodiments, the backbone sugar modification does not include a LNA.

[0049] The backbone sugar modification present at one residue of SEQ ID NO: 1 may or may not be the same as the backbone sugar modification present at another residue. In some embodiments, the backbone sugar modifications present in SEQ ID NO: 1 are all the same.

[0050] In some embodiments, up to 5, up to 4, or up to 3 consecutive nucleic acid residues from the 5’ end of SEQ ID NO: 1 include a 2’ -O-m ethylation. In some embodiments, up to 5, up to 4, or up to 3 consecutive nucleic acid residues from the 3’ end of SEQ ID NO: 1 includes a 2’ -O-m ethylation. In some embodiments, up to 5, up to 4, or up to 3 consecutive nucleic acid residues from the 5’ end of SEQ ID NO: 1, and up to 5, up to 4, or up to 3consecutive nucleic acid residues from the 3’ end of SEQ ID NO: 1 include a 2’-O-methylation. In some embodiments, 1, 2, or 3 nucleic acid residues (or consecutive nucleic acid residues if 2 or 3) from the 5’ end of SEQ ID NO: 1 include a 2’-O-methylation. In some embodiments, 1, 2, or 3 nucleic acid residues (or consecutive nucleic acid residues if 2 or 3) from the 3’ end of SEQ ID NO: 1 include a 2’-O-methylation. In some embodiments, 1, 2, or 3 nucleic acid residues (or consecutive nucleic acid residues if 2 or 3) from the 5’ end of SEQ ID NO: 1, and 1, 2, or 3 nucleic acid residues (or consecutive nucleic acid residues if 2 or 3) from the 3’ end of SEQ ID NO: 1 include a 2’-( -methylation. In some embodiments, the RNA molecule includes a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1, and nucleic acid residues at the following positions of SEQ ID NO: 1 includes a 2’-O-methylation: (a) 1 and 32, (b) 1, 2, 31, and 32; or (c) 1, 2, 3, 30, 31, and 32. In some embodiments, the RNA molecule includes a phosphorothioate bond between nucleic acid residues in each of the following pairs of consecutive nucleic acid residues within SEQ ID NO: 1 : N1 / C2; C2 / C3; C30 / G31; G31 / C32, and nucleic acid residues at the following positions of SEQ ID NO: 1 includes a 2’-O-methylation: 1, 2, 3, and 30, 31, and 32. In some embodiments, the RNA molecule does not include a 2’-( -methylation of a nucleic acid residue. In some embodiments, the RNA molecule does not include a 2’-O-methylation within SEQ ID NO: 1.

[0051] In some embodiments, up to 5, up to 4, or up to 3 consecutive nucleic acid residues from the 5’ end of SEQ ID NO: 1 include a LNA, where N is thymine. In some embodiments, up to 5, up to 4, or up to 3 consecutive nucleic acid residues from the 3’ end of SEQ ID NO: 1 includes a LNA. In some embodiments, up to 5, up to 4, or up to 3 consecutive nucleic acid residues from the 5’ end of SEQ ID NO: 1, and up to 5, up to 4, or up to 3 consecutive nucleic acid residues from the 3’ end of SEQ ID NO: 1 include a LNA, where N is thymine. In some embodiments, 1, 2, or 3 nucleic acid residues (or consecutive nucleic acid residues if 2 or 3) from the 5’ end of SEQ ID NO: 1 include a 2’-O-methylation, where N is thymine. In some embodiments, 1, 2, or 3 nucleic acid residues (or consecutive nucleic acid residues if 2 or 3) from the 3’ end of SEQ ID NO: 1 include a 2’-O-methylation. In some embodiments, 1, 2, or 3 nucleic acid residues (or consecutive nucleic acid residues if 2 or 3) from the 5’ end of SEQ ID NO: 1, and 1, 2, or 3 nucleic acid residues (or consecutive nucleic acid residues if 2 or 3) from the 3’ end of SEQ ID NO: 1 include a LNA, where N is thymine.In some embodiments, the RNA molecule includes a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1, and nucleic acid residues at the following positions of SEQ ID NO: 1 includes a LNA: (a) 1 and 32, or (b) 1, 2, 3, 30, 31, and 32, where N is thymine. In some embodiments, the RNA molecule does not include a LNA within SEQ ID NO: 1. In some embodiments, up to 5, up to 4, or up to 3 consecutive nucleic acid residues from the 5’ end of SEQ ID NO: 1 include a 2’ -O-m ethylation and a LNA, where N is thymine.

[0052] In some embodiments, the RNA molecule includes any one of SEQ IDNOs:2-7 and 9-11, as set forth in Table 1 :Table 1Residue with 2 ’-(J-m ethylation is underlined; residue with LNA is italicized

[0053] In some embodiments, the RNA molecule is or is essentially the sequence of SEQ ID NO: 1. In some embodiments, the RNA molecule is or is essentially any one of SEQ ID NOs: 2-7. In some embodiments, the RNA molecule includes SEQ ID NO: 2. In some embodiments, the RNA molecule is or is essentially SEQ ID NO: 2 (TT1). In someembodiments, the RNA molecule includes SEQ ID NO: 3. In some embodiments, the RNA molecule is or is essentially SEQ ID NO: 3 (TT2). In some embodiments, the RNA molecule includes SEQ ID NO: 4. In some embodiments, the RNA molecule is or is essentially SEQ ID NO: 4 (TT3). In some embodiments, the RNA molecule includes SEQ ID NO: 5. In some embodiments, the RNA molecule is or is essentially SEQ ID NO: 5 (TT4). In some embodiments, the RNA molecule includes SEQ ID NO: 6. In some embodiments, the RNA molecule is or is essentially SEQ ID NO: 6 (TT5). In some embodiments, the RNA molecule includes SEQ ID NO: 7. In some embodiments, the RNA molecule is or is essentially SEQ ID NO: 7 (TT7). In some embodiments, the RNA molecule includes SEQ ID NO: 9. In some embodiments, the RNA molecule is or is essentially SEQ ID NO: 9 (TT8). In some embodiments, the RNA molecule includes SEQ ID NO: 10. In some embodiments, the RNA molecule is or is essentially SEQ ID NO: 10 (TT9). In some embodiments, the RNA molecule includes SEQ ID NO: 11. In some embodiments, the RNA molecule is or is essentially SEQ ID NO: 11 (TT10). In some embodiments, the phosphorothioate-containing RNA molecule includes, consists essentially of, or consists of 5’- UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 9), wherein the RNA molecule comprises a phosphorothioate bond between nucleic acid residues in each of the following pairs of consecutive nucleic acid residues: U1 / C2; C2 / C3; C30 / G31; G31 / C32. In some embodiments, the RNA molecule includes a phosphodiester bond between nucleic acid residues in all remaining pairs of consecutive nucleic acid residues. In some embodiments, the phosphorothioate-containing RNA molecule consists essentially of, or consists of 5’-UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 9), wherein the RNA molecule comprises a phosphorothioate bond between nucleic acid residues in each of the following pairs of consecutive nucleic acid residues: U1 / C2; C2 / C3; C30 / G31; G31 / C32.

[0054] A nucleic acid (e.g., RNA molecule) of the present disclosure can be single stranded or double stranded (e.g., RNA / DNA hybrid). In some embodiments, the RNA molecule is single stranded.

[0055] The RNA molecule can be any suitable length. In some embodiments, the RNA molecule is or is about 32 bases long. In some embodiments, the RNA molecule is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 bases long, or longer. In some embodiments, the RNA molecule is at most 60 bases long. In some embodiments, the RNA molecule is at most 40 bases long. In some embodiments, the nucleic acid is 20-35 bases long, or 30-35 bases long.

[0056] In some embodiments, the RNA molecule (e.g., tREX-1 variant) has increased in vitro and / or in vivo stability compared to the unmodified counterpart (e.g., unmodified tREX-1). In some embodiments, the RNA molecule (e.g., tREX-1 variant) has increased therapeutic potency, e.g., for treating an inflammatory condition, cardiac injury, or muscular dystrophy, compared to the unmodified counterpart (e.g., unmodified tREX-1).

[0057] The RNA molecules of the present disclosure can be prepared using any suitable option. Suitable options include, without limitation, chemical synthesis, enzymatic production and / or biological production. In some embodiments, the RNA molecules are prepared using chemical synthesis. Any suitable option for chemical synthesis of RNA molecules can be used. In some embodiments, the RNA molecules are prepared by chemically modifying an unmodified RNA molecule having a nucleotide sequence of interest. COMPOSITIONS

[0058] Also provided herein are compositions that include the RNA molecule of the present disclosure. In some embodiments, the composition is a pharmaceutical or therapeutic composition. In some embodiments, the composition includes a therapeutically effective amount of the RNA molecule. In some embodiments, the therapeutically effective amount of the RNA molecule is an amount that, when administered to a subject, can bring about a desired outcome in the subject in need of treatment of a condition or disease (e.g., a condition associated with inflammation and / or fibrosis, a muscle disorder or symptom thereof, etc.) as described herein. In some embodiments, the therapeutically effective amount of the RNA molecule is sufficient on its own to bring about the desired outcome (e.g., without administering another therapeutic agent for the same condition or disease). In some embodiments, the composition includes any one or more of the RNA molecules selected from: TT1, TT2, TT3, TT4, TT5, TT7, TT8, TT9, and TT10. In some embodiments, the composition includes TT1 (SEQ ID NO:2). In some embodiments, the composition includes TT2 (SEQ ID NO:3). In some embodiments, the composition includes TT3 (SEQ ID NO:4). In some embodiments, the composition includes TT4 (SEQ ID NO: 5). In some embodiments, thecomposition includes TT5 (SEQ ID NO:6). In some embodiments, the composition includes TT7 (SEQ ID NO:7). In some embodiments, the composition includes TT8 (SEQ ID NO:9). In some embodiments, the composition includes TT9 (SEQ ID NO: 10). In some embodiments, the composition includes TT10 (SEQ ID NO: 11). In some embodiments, the composition includes any two or more of the RNA molecules selected from: TT1, TT2, TT3, TT4, TT5, TT7, TT8, TT9, and TT10. In some embodiments, the composition includes any three or more of the RNA molecules selected from: TT1, TT2, TT3, TT4, TT5, TT7, TT8, TT9, and TT10.

[0059] In some embodiments the composition includes a pharmaceutically acceptable excipient. In some embodiments, the composition consists of, or consists essentially of the therapeutically effective amount of the RNA molecule and the pharmaceutically acceptable excipient. In some embodiments, the composition is a cell-free composition, e.g., the composition is substantially free of cells such as CDC. In some embodiments, the composition is an extracellular vesicle-free composition, e g., the composition is substantially free of extracellular vesicles, such as exosomes.

[0060] Some non-limiting examples of materials which can serve as pharmaceutically-acceptable excipients include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations.

[0061] In some embodiments, the composition includes a transfection reagent, e.g., to promote delivery of the RNA molecule to a cellular target (in vitro or in vivo). Any suitable transfection reagent can be included in the composition. Suitable transfection reagents include, without limitation, a liposome, extracellular vesicle (EV), and / or a polyethylene glycol (PEG)- cationic lipid complex (PCLC). In some embodiments, the transfection reagent includes a lipid (e.g., a liposome-forming lipid), or a PEGylated lipid. In some embodiments, the lipid is a cationic lipid, as provided herein. In some embodiments, the transfection reagent includes DharmaFECT® or Lipofectamine®. In some embodiments, the RNA molecule of the present disclosure is formulated with the transfection reagent in the composition so as to promote cellular uptake and / or pharmacokinetics of the RNA molecule.

[0062] Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV), which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV), which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV), which may be between 50 and 500 nm in diameter. Liposome design may include, without limitation, opsonins or ligands in order to improve the attachment of liposomes to target tissue / cells, or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the cargo, e.g., a nucleic acid of the present disclosure.

[0063] In some embodiments, the composition includes, without limitation, liposomes such as those formed from l,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), l,2-dilinoleyloxy-3- dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]- dioxolane (DLin-KC2-DMA), and MC3 and liposomes such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

[0064] In some embodiments, the composition includes a cationic lipid. Any suitable cationic lipid may be used in the present compositions. Suitable cationic lipids include, without limitation, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. In some embodiments, the composition includes a cationiclipid complex, e.g., a polyethylene glycol (PEG)-cationic lipid complex (PCLC). In some embodiments, the cationic lipid is PEGylated, e.g., 2 kDa PEG (“PEG2000”). Any suitable option can be used to PEGylate the cationic lipid. In some embodiments, PCLC is formed by exposing a mixture of PEG and the cationic lipid to one or more freeze / thaw cycles, e.g., 1, 2, 3, 4, 5 or more freeze / thaw cycles. In some embodiments, a freeze / thaw cycle includes freezing the mixture with liquid nitrogen (e.g., around -190 °C) for about 5 minutes, and thawing at about 60 °C for about 5 minutes. An RNA molecule of the present disclosure can be mixed with the PCLC to generate a complex of the RNA molecule and the PCLC.

[0065] In some embodiments, the composition includes extracellular vesicles (EV), e.g., exosomes. The extracellular vesicles (EV) can be those from any suitable source, e.g., EV derived from cardiosphere-derived cells (CDC), or from fibroblasts. Suitable EV, such as CDC-derived EV, are provided in, e.g., U.S. Application Publication Nos. 20080267921, 20160158291 and 20160160181; Smith et al., Circulation. 2007. 115:896-908; Aminzadeh, M. A. et al. Stem Cell Reports 10, 942-955 (2018); and Ibrahim et al., Stem Cell Reports. 2014 May 8;2(5):606-19, Ibrahim, A. G. et al. Nanomedicine 33, 102347 (2020), each of which is incorporated by reference in its entirety. In some embodiments, the EVs are those isolated from serum-free media conditioned by human CDCs in culture. In some embodiments, the composition includes EV and liposomes and / or PCLC as transfection reagents. In some embodiments, the composition is substantially free of CDC-derived EV.

[0066] EVs, e.g., exosomes, disclosed herein can vary in size, depending on the embodiment. Depending on the embodiment, the size of the EVs ranges in diameter from about 15 nm to about 95 nm in diameter, including about 15 nm to about 20 nm, about 20 nm to about 30 nm, about 30 nm to about 40 nm, about 40 nm to about 50 nm, about 50 nm to about 60 nm, about 60 nm to about 70 nm, about 70 nm to about 80 nm, about 80 nm to about 90 nm, about 90 nm to about 95 nm, and overlapping ranges thereof. In several embodiments, EVs are larger (e.g., those ranging from about 140 to about 210 nm, including about 140 nm to about 150 nm, about 150 nm to about 160 run, about 160 nm to about 170 nm, about 170 nm to about 180 nm, about 180 nm to about 190 nm, 190 nm to about 200 nm, about 200 nm to about 210 nm, and overlapping ranges thereof). In some embodiments, the EV diameter is in a range of about 15 nm to about 200 nm in diameter, including about 15 nm to about 20 nm, about 20 nm to about 30 nm, about 30 nm to about 40 nm, about 40 nm to about 50 nm, about50 nm to about 60 nm, about 60 nm to about 70 nm, about 70 nm to about 80 nm, about 80 nm to about 90 nm, about 90 nm to about 100 nm, about 100 nm to about 110 nm, about 110 nm to about 120 nm, about 120 nm to about 130 nm, about 130 nm to about 140 nm, about 140 nm to about 150 nm, about 150 nm to about 160 nm, about 160 nm to about 170 nm, about 170 nm to about 180 nm, about 180 nm to about 190 nm, about 190 nm to about 200 nm, and overlapping ranges thereof. In some embodiments, the EVs that are generated from the original cellular body are 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 5,000, or 10,000 times smaller in at least one dimension (e.g., diameter) than the original cellular body.

[0067] The composition containing the EV and RNA molecules of the present disclosure can be prepared using any suitable option. In some embodiments, loading the RNA molecules into the EV includes: formulating the RNA molecules with liposomes and / or PCLC, e g., as provided above, to generate a RNA molecule-liposome mixture; combining the RNA molecule-liposome mixture with the EV; and enriching for EV associated with exosome markers to generate a population of EV enriched for the RNA molecule. Combining the RNA molecule-liposome mixture with the EV can be done using any suitable option. In some embodiments, the RNA molecule-liposome mixture is combined with the EV at 37 °C with shaking for about 30 minutes or more. Enriching to generate a population of EV enriched for the RNA molecule can be done using any suitable option. In some embodiments, enriching for EV associated with exosome markers includes immunoprecipitating EV associated with exosome markers using antibodies specific to an exosome marker. In some embodiments, the exosome marker is one or more of CD9, CD63 and CD81. In some embodiments, enriching for EV associated with exosome markers includes immunoprecipitating EV associated with all the exosome markers, CD9, CD63 and CD81. In some embodiments, the size distribution of the population of EV enriched for the RNA molecule is substantially unimodal. In some embodiments, at least 80%, 85%, 90%, 95%, 97%, 99% of the population has a diameter under a single peak in the size distribution. In some embodiments, the population of EV enriched for the RNA molecule has an average diameter of about 50-180 nm, e.g., 60-170 nm, 70-160 nm, 80-150 nm, 90-140 nm, 100-130 nm, or about 110-130 nm.

[0068] In some embodiments, the composition includes casein, e.g., a casein micelle. In some embodiments, the composition includes chitosan. In some embodiments, the composition includes casein and chitosan, e.g., a casein-chitosan micelle. In someembodiments, the composition includes a casein-chitosan complex. In some embodiments, the RNA molecule in the composition is encapsulated in a casein-chitosan complex. In some embodiments, the composition includes one or more of phosphoproteins: alpha si casein, alpha s2 casein, beta casein, and kappa casein. In some embodiments, the at least one casein protein includes one or more of phosphoproteins: alpha si casein, alpha s2 casein, beta casein, and kappa casein. In some embodiments, the composition includes two or more, three or more, or all four phosphoproteins: alpha si casein, alpha s2 casein, beta casein, and kappa casein. In some embodiments, the at least one casein protein includes all four phosphoproteins: alpha si casein, alpha s2 casein, beta casein, and kappa casein. The phosphoproteins may be present in the composition at any suitable concentration (relative to each other, and relative to the total volume of the composition), and in some embodiments, is present in an amount suitable for forming casein micelles. In some embodiments, the casein phosphoproteins are collectively present in the composition at about 5-10 % (weight by volume). In some embodiments, the casein phosphoproteins are collectively present in the composition at about 8 % (weight by volume). In some embodiments, the casein phosphoproteins are collectively present in the composition at about 5 % (weight by volume). The casein phosphoproteins can be those from any suitable animal, e.g., mammal such as, but not limited to, human, non-human primate, cow, pig, horse, camel, goat, and sheep. In some embodiments, the casein phosphoproteins are bovine alpha si casein, alpha s2 casein, beta casein, and kappa casein. Suitable casein formulations with EV are provided in, e g., Aminzadeh et al., J Extracell Vesicles. 2021 Jan;10(3):el2045, the entirety of which is incorporated herein by reference. In some embodiments, a composition, e.g., pharmaceutical composition, of the present disclosure formulated with casein, as provided herein, is suitable for oral administration to the subject. Suitable oral formulations for the nucleic acids (e.g., RNA molecules) of the present disclosure are provided in, e.g., International Application Nos. PCT / US2022 / 035866 (filed June 30, 2022) and PCT / US2022 / 035870 (filed June 30, 2022), each of which is incorporated herein by reference in its entirety. In some embodiments, an oral formulation of the present disclosure includes any one or more RNA molecules described herein; at least one casein protein; and a chitosan. In some embodiments, an oral formulation of the present disclosure includes any one or more RNA molecules described herein; a cationic lipid; at least one casein protein; and a chitosan. In some embodiments, an oral formulation of the present disclosure includes anyone or more of the RNA molecules selected from: TT1, TT2, TT3, TT4, TT5, TT7, TT8, TT9, and TT10; at least one casein protein; and a chitosan. In some embodiments, the oral formulation includes an RNA molecule that includes 5’- NCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 1); at least one casein protein; and a chitosan. In some embodiments, the oral formulation includes an RNA molecule that includes 5’-NCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 1); a cationic lipid; at least one casein protein; and a chitosan. . In some embodiments, the oral formulation includes TT8 (SEQ ID NO:9); at least one casein protein; and a chitosan. In some embodiments, an oral formulation includes an artificial lipid micelle or a liposome; any one or more nucleic acids described herein, wherein the one or more nucleic acids is encapsulated within the artificial lipid micelle or the liposome; and a coating on the artificial lipid micelle or the liposome, wherein the coating comprises a mixture of casein proteins and chitosan polymers. In some embodiments, an oral formulation includes an artificial lipid micelle or a liposome; an RNA molecule that includes 5’- NCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 1), wherein the RNA molecule is encapsulated within the artificial lipid micelle or the liposome; and a coating on the artificial lipid micelle or the liposome, wherein the coating comprises a mixture of casein proteins and chitosan polymers. In some embodiments, the artificial lipid micelle includes a cationic lipid micelle. In some embodiments, the liposome includes cationic lipids. In some embodiments, the liposome includes DharmaFECT® or Lipofectamine®.

[0069] In some embodiments, the composition is in a parenteral dosage form. In some embodiments, the parenteral dosage form is sterile or capable of being sterilized before administering to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration to a subject. Suitable excipients that can be used to provide parenteral dosage forms of the RNA molecule include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol,polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0070] In some embodiments, the composition includes an antisense oligonucleotide, such as those targeting one or more exons of a dystrophin transcript. In some embodiments, the composition includes an antisense oligonucleotide that includes an exonskipping agent that targets a dystrophin transcript.METHODS

[0071] Provided herein are methods of treating a subject in need thereof using the RNA molecules of the present disclosure (also referred to herein as “treatment methods”). Conditions that may be treated by the treatment methods include, without limitation, muscle disorders, heart conditions, fibrotic conditions, and inflammatory conditions. In some embodiments, the conditions include, without limitation, muscular disorders, myocardial infarction, cardiac disorders, myocardial alterations, muscular dystrophy, fibrotic disease, inflammatory disease, viral infection, sepsis or wound healing. In some embodiments, the conditions include acute myocardial infarction. In some embodiments, conditions treated by the treatment methods include, without limitation, conditions associated with inflammation and / or fibrosis. In some embodiments, a subject treated by administering the RNA molecules of the present disclosure, according to the treatment methods herein, are in need of treatment for conditions associated with inflammation and / or fibrosis. The conditions associated with inflammation and / or fibrosis can include, without limitation, inflammation and / or fibrosis of the heart or skeletal muscle. In some embodiments, the conditions treated by the present treatment methods are a symptom and / or sequelae of an infection. In some embodiments, the infection is a viral infection, e.g., a respiratory virus infection, such as COVID-19, infections due to other coronaviruses, or other viral pathogens (e.g., flu, H1N1, Hepatitis C, HIV, etc.). In some embodiments, the method includes identifying a subject having or diagnosed with a muscle disorder, heart condition, or inflammatory condition, as described herein, and administering a therapeutically effective amount of the RNA molecules of the present disclosure to the subject. In some embodiments, the method includes administering a therapeutically effective amount of any one or more of the RNA molecules selected from: TT1, TT2, TT3, TT4, TT5, TT7, TT8, TT9, and TT10. In some embodiments, the method includesadministering a therapeutically effective amount of TT1 (SEQ ID NO:2). In some embodiments, the method includes administering a therapeutically effective amount of TT2 (SEQ ID NO:3). In some embodiments, the method includes administering a therapeutically effective amount of TT3 (SEQ ID NO:4). In some embodiments, the method includes administering a therapeutically effective amount of TT4 (SEQ ID NO:5). In some embodiments, the method includes administering a therapeutically effective amount of TT5 (SEQ ID NO:6). In some embodiments, the method includes administering a therapeutically effective amount of TT7 (SEQ ID NO: 7). In some embodiments, the method includes administering a therapeutically effective amount of TT8 (SEQ ID NO:9).

[0072] A treatment method of the present disclosure can include administering to a subject in need of treatment a therapeutically effective amount of the RNA molecule of the present disclosure (or a composition containing the same, as described herein) to thereby treat the subject. In some embodiments, the therapeutically effective amount of the RNA molecule is an amount that is less than the therapeutically effective amount of an unmodified counterpart RNA molecule (e.g., unmodified tREX-1). In some embodiments, the therapeutically effective amount of the RNA molecule is less than the therapeutically effective amount of an unmodified counterpart RNA molecule (e.g., unmodified tREX-1) by, by about, or by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80% or more, or by a percentage in a range defined by any two of the preceding values (e.g., 5-80%, 10-75%, 20-50%, 30-70%, etc.). In some embodiments, the therapeutically effective amount of the RNA molecule provides a greater therapeutic result than a comparable amount (or greater amount) of an unmodified counterpart RNA molecule (e.g., unmodified tREX-1). In some embodiments, the therapeutically effective amount of the RNA molecule provides a greater therapeutic result than a comparable amount (or greater amount) of an unmodified counterpart RNA molecule (e.g., unmodified tREX-1) by, by about, or by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100% or more, or by a percentage in a range defined by any two of the preceding values (e.g., 5- 100%, 10-90%, 20-80%, 50-75%, etc.).

[0073] In some embodiments, a method of treating a muscle disorder or symptom thereof includes administering to a subject in need of treating a muscle disorder or symptom thereof a therapeutically effective amount of the RNA molecule of the present disclosure (or a composition containing the same, as described herein), thereby treating the muscle disorder orsymptom thereof. In some embodiments, the muscle disorder comprises a skeletal muscle disorder and / or a heart condition. In some embodiments, the muscle disorder comprises muscular dystrophy (e.g., Duchenne muscular dystrophy). In some embodiments, the muscular disorder comprises both the heart (cardiomyopathy) and skeletal muscle (myopathy) in Duchenne muscular dystrophy. In some embodiments, a method of treating muscular dystrophy (e.g., Duchenne muscular dystrophy) further includes administering a second therapy (e.g., an exon-skipping agent and / or gene therapy) for the muscle disorder. In some embodiments, an exon-skipping agent includes an antisense oligonucleotide that targets a dystrophin transcript. In some embodiments, the heart condition includes myocardial infarction, heart failure, or a symptom or sequelae thereof (e.g., reduced heart function, cardiac tissue fibrosis, etc.). In some embodiments, the heart condition includes acute myocardial infarction. In some embodiments, heart function includes left ventricle function, which can be represented by any suitable option such as, without limitation, ejection fraction. In some embodiments, the method includes identifying a subject having or diagnosed with a muscle disorder or symptom thereof, as described herein, and administering a therapeutically effective amount of the RNA molecule of the present disclosure to the subject. In some embodiments, the method includes identifying a subject having or diagnosed with muscular dystrophy (e.g., Duchenne muscular dystrophy) or a symptom thereof, as described herein, and administering a therapeutically effective amount of the RNA molecule of the present disclosure to the subject. In some embodiments, the method includes identifying a subject having or diagnosed with heart failure, or a symptom or sequelae thereof, as described herein, and administering a therapeutically effective amount of the RNA molecule of the present disclosure to the subject. In some embodiments, the method includes identifying a subject who is at risk of, or has suffered myocardial infarction, or a symptom or sequelae thereof, as described herein, and administering a therapeutically effective amount of the RNA molecule of the present disclosure to the subject. In some embodiments, the method includes identifying a subject who is at risk of, or has suffered acute myocardial infarction, or a symptom or sequelae thereof, as described herein, and administering a therapeutically effective amount of the RNA molecule of the present disclosure to the subject.

[0074] In some embodiments, a method of treating a condition associated with inflammation and / or fibrosis includes administering to a subject in need of treating a conditionassociated with inflammation and / or fibrosis a therapeutically effective amount of the RNA molecule of the present disclosure (or a composition containing the same, as described herein), thereby treating the condition associated with inflammation and / or fibrosis. In some embodiments, the condition associated with inflammation and / or fibrosis comprises inflammation and / or fibrosis of the heart or skeletal muscle, or a symptom and / or sequelae of myocardial infarction, heart failure, hypertrophic cardiomyopathy, heart failure with preserved ejection fraction (HFpEF), or muscular dystrophy, or a symptom or sequelae of an infectious disease (e.g., a viral infection) or is associated with immunotherapy, or a symptom or sequelae of an infectious disease, idiopathic pulmonary fibrosis or cirrhosis of the liver. In some embodiments, the condition associated with inflammation and / or fibrosis comprises inflammation and / or fibrosis of the heart. In some embodiments, the condition associated with inflammation and / or fibrosis comprises inflammation and / or fibrosis of skeletal muscle. In some embodiments, the condition associated with inflammation and / or fibrosis comprises a symptom and / or sequelae of myocardial infarction or heart failure (e.g., reduced heart function, cardiac tissue fibrosis, etc.). In some embodiments, the condition associated with inflammation and / or fibrosis comprises a symptom and / or sequelae of muscular dystrophy (e.g., reduced muscle function, tissue fibrosis, etc.). In some embodiments, the method includes identifying a subject having or diagnosed with inflammation and / or fibrosis of the heart, as described herein, and administering a therapeutically effective amount of the RNA molecule of the present disclosure to the subject.

[0075] In some embodiments, the subject is a subject who has or has suffered heart failure. In some embodiments, the subject is a subject who has or has suffered myocardial infarction. In some embodiments, the subject is a subject who has or has suffered acute myocardial infarction. In some embodiments, the subject is at risk of having heart failure and / or myocardial infarction (e.g., acute myocardial infarction). In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject prevents reduction in ejection fraction due to heart failure. In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject prevents reduction in ejection fraction after myocardial infarction (e.g., acute myocardial infarction). In some embodiments, the subject’s ejection fraction does not substantively decrease due to heart failure or myocardial infarction after administering the therapeutically effective amount of theRNA molecule to the subject. In some embodiments, the subject’s ejection fraction does not substantively decrease due to acute myocardial infarction after administering the therapeutically effective amount of the RNA molecule to the subject. In some embodiments, the subject’s ejection fraction does not decrease, or decreases by, by about, or by at most 1, 2, 3, 4, 5%, or by a percentage in a range defined by any two of the preceding values (e.g., 0-5%, 0-3%, 1-4%, etc.) due to heart failure or myocardial infarction after administering the therapeutically effective amount of the RNA molecule to the subject. In some embodiments, the subject’s ejection fraction does not decrease, or decreases by, by about, or by at most 1, 2, 3, 4, 5%, or by a percentage in a range defined by any two of the preceding values (e.g., 0-5%, 0-3%, 1-4%, etc.) due to acute myocardial infarction after administering the therapeutically effective amount of the RNA molecule to the subject. In some embodiments, the subject’s ejection fraction increases after administering the therapeutically effective amount of the RNA molecule to the subject, where the subject has suffered heart failure and / or myocardial infarction. In some embodiments, the subject’s ejection fraction increases after administering the therapeutically effective amount of the RNA molecule to the subject, where the subject has suffered acute myocardial infarction. In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject reduces myocardial fibrosis due to heart failure and / or myocardial infarction. In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject reduces myocardial fibrosis due to acute myocardial infarction.

[0076] In some embodiments, the subject has or is predisposed to having muscular dystrophy, e.g., Duchenne muscular dystrophy. In some embodiments, the subject is genetically predisposed to having muscular dystrophy, e.g., Duchenne muscular dystrophy. In some embodiments, the method includes identifying a subject who is predisposed to having muscular dystrophy, and administering a therapeutically effective amount of the RNA molecule of the present disclosure to the subject. In some embodiments, the subject has one or more mutations in a dystrophin gene that predisposes the subject to developing muscular dystrophy, e.g., Duchenne muscular dystrophy. In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject prevents or attenuates the reduction in skeletal muscle function e g., the force or torque exerted by a skeletal muscle group, due to muscular dystrophy. In some embodiments, administering the therapeuticallyeffective amount of the RNA molecule to the subject suffering from muscular dystrophy increases or restores skeletal muscle function e.g., the force or torque exerted by an affected skeletal muscle group. In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject suffering from muscular dystrophy increases the force or torque exerted by an affected skeletal muscle group by, by about, or by at least 5, 7, 10, 12, 15, 17, 20%, or by a percentage defined by any two of the preceding values (e.g., 5- 20%>, 5-15%, 7-17%, etc.), compared to before administering. In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject reduces skeletal muscle fibrosis due to muscular dystrophy. In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject reduces the percentage of skeletal muscle fibrosis due to muscular dystrophy by, by about, or by at least 2, 5, 7, 10, 12, or 15 percentage points, or by a percentage point in a range defined by any two of the preceding values (e g., 2-15%, 5-15%, 10-12%, 7-15%, etc.) compared to a suitable reference (e.g., an average percentage of skeletal muscle fibrosis due to muscular dystrophy in a subject who does not receive the therapeutically effective amount of the RNA molecule). In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject having muscular dystrophy increases myofiber number in skeletal muscle. In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject having muscular dystrophy increases myofiber number in skeletal muscle by, by about, or by at least 5, 10, 12, 15, 18, 20, 25%>, or by a percentage in a range defined by any two of the preceding values (e.g., 5-25%>, 10-20%>, 12018%, 10-15%, etc.) compared to before the administering. In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject having muscular dystrophy increases skeletal muscle mass or volume. In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject having muscular dystrophy increases skeletal muscle mass or volume by, by about, or by at least 5, 10, 12, 15, 18, 20, 25%, or by a percentage in a range defined by any two of the preceding values (e.g., 5-25%o, 10-20%, 12018%), 10-15%, etc.) compared to before the administering.

[0077] In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject alters the composition of macrophage subtypes in a muscle tissue, e g., in skeletal muscle or cardiac muscle. In some embodiments, administering thetherapeutically effective amount of the RNA molecule to the subject increases the number and / or abundance of CD68+ / CD206+ cells in a muscle tissue, e.g., skeletal muscle, compared to a suitable reference (e.g., before the administering, other subjects treated with a scramble oligonucleotide, etc.). In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject increases the number and / or abundance of CD68+ / CD206+ cells in a muscle tissue, e.g., skeletal muscle, by, by about, or by at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.2, 2.5, 3, 3.5, 4, 4.5, 5 fold, or by a fold amount in a range defined by any two of the preceding values (e.g., 1.1-5 fold, 1.5-4.5 fold, 2-4 fold, 1.2-5 fold, etc.), compared to a suitable reference (e.g., before the administering, other subjects treated with a scramble oligonucleotide, etc.). In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject increases the number and / or abundance of CD68+ / CD206+ cells in a muscle tissue, e.g., skeletal muscle, compared to a suitable control. In some embodiments, the number and / or abundance of CD68+ / CD206+ cells in a muscle tissue, e.g., in skeletal muscle or cardiac muscle, is determined relative to the number and / or abundance of CD68+ / CD206- cells in the same tissue.

[0078] In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject reduces the number and / or abundance of CD68+ / CD206+ cells in a muscle tissue, e.g., cardiac muscle, compared to a suitable reference (e.g., before the administering, other subjects treated with a scramble oligonucleotide, etc.). In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject reduces the number and / or abundance of CD68+ / CD206+ cells in a muscle tissue, e.g., cardiac muscle, by, by about, or by at least 0.1, 0.2, 0.3, 0.4 or 0.5 fold, or by a fold amount in a range defined by any two of the preceding values (e.g., 0.1-0.5 fold, 0.1-0.4 fold, 0.2-0.4 fold, 0.1-0.3 fold, etc.), compared to a suitable reference (e.g., before the administering, other subjects treated with a scramble oligonucleotide). In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject reduces the number and / or abundance of CD68+ / CD206+ cells in a muscle tissue, e.g., cardiac muscle, compared to a suitable control. In some embodiments, the number and / or abundance of CD68+ / CD206+ cells in a muscle tissue, e.g., in skeletal muscle or cardiac muscle, is determined relative to the number and / or abundance of CD68+ / CD206- cells in the same tissue.

[0079] In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject increases or reduces the number and / or abundance of CD68+ / CD206+ cells in a muscle tissue, e.g., cardiac muscle, compared to a suitable reference (e.g., before the administering, other subjects treated with a scramble oligonucleotide, etc.). In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject increases or reduces the number and / or abundance of CD68+ / CD206+ cells in a muscle tissue, e.g., cardiac muscle, by, by about, or by at least 0.1, 0.2, 0.3, 0.4 or 0.5 fold, or by a fold amount in a range defined by any two of the preceding values (e.g., 0.1-0.5 fold, 0.1- 0.4 fold, 0.2-0.4 fold, 0.1-0.3 fold, etc.), compared to a suitable reference (e.g., before the administering, other subjects treated with a scramble oligonucleotide). In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject increases or reduces the number and / or abundance of CD68+ / CD206+ cells in a muscle tissue, e.g., cardiac muscle, compared to a suitable control. In some embodiments, the number and / or abundance of CD68+ / CD206+ cells in a muscle tissue, e.g., in skeletal muscle or cardiac muscle, is determined relative to the number and / or abundance of CD68+ / CD206- cells in the same tissue.

[0080] In some embodiments, administering the therapeutically effective amount of the RNA molecule to the subject does not alter the number and / or abundance of CD68+ / C80+ cells in a muscle tissue, e.g., in skeletal muscle or cardiac muscle. In some embodiments, the number and / or abundance of CD68+ / CD80+ cells in a muscle tissue, e.g., in skeletal muscle or cardiac muscle, is determined relative to the number and / or abundance of CD68+ / CD80- cells in the same tissue.

[0081] The RNA molecule can be administered to the subject at any suitable amount. In some embodiments, the therapeutically effective amount of the RNA molecule includes about 0.01 pg, 0.02 pg, 0.05 pg, 0.1 pg, 0.2 pg, 0.5 pg, 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 15 pg, 20 pg, 25 pg, 30 pg, 40 pg, 50 pg, 75 pg, 100 pg, 125 pg, 150 pg, 175 pg, 200 pg, 250 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg or more, or an amount in a range defined by any two of the preceding values (e.g., 0.01 pg-0.1 pg, 0.1 pg-1 pg, 1 pg-10 pg, 10 pg-100 pg, 100 pg-1 mg, 1 mg-lOmg, lOmg-lOOmg). In some embodiments, the therapeutically effective amount of the RNA molecule is in a range of 0.1mg to 100 mg. In some embodiments, the therapeutically effective amount of the RNA molecule is in a range of 1 mg to 100 mg. In some embodiments, the therapeutically effective amount of the RNA molecule includes about 0.001 pg / g, 0.002 pg / g, 0.005 pg / g, 0.01 pg / g, 0.02 pg / g, 0.05 pg / g, 0.1 pg / g, 0.15 pg / g, 0.2 pg / g, 0.5 pg / g, 1 pg / g, 2 pg / g, 3 pg / g, 4 pg / g, 5 pg / g, 6 pg / g, 7 pg / g, 8 pg / g, 9 pg / g, 10 pg / g, 15 pg / g, 20 pg / g, 25 pg / g, 30 pg / g, 35 pg / g, 40 pg / g, 45 pg / g, 50 pg / g, 60 pg / g, 70 pg / g, 80 pg / g, 90 pg / g, 100 pg / g of body weight, or more, or an amount in a range defined by any two of the preceding values (e.g., 0.001 pg / g-0.01 pg / g, 0.01 pg / g-0.1 pg / g, 0.1 pg / g-1 pg / g, 1 pg / g-10 pg / g, 10 pg / g-100 pg / g). In some embodiments, the therapeutically effective amount of the RNA molecule is about 0.001 pg / g, 0.002 pg / g, 0.005 pg / g, 0.01 pg / g, 0.02 pg / g, 0.05 pg / g, 0.1 pg / g, 0.2 pg / g, 0.5 pg / g, or about 1 pg / g of body weight, or more, or an amount in a range defined by any two of the preceding values (e.g., 0.001 pg / g-0.01 pg / g, 0.01 pg / g-0.05 pg / g, 0.05 pg / g-0.1 pg / g, 0.1 pg / g-0.2 pg / g, 0.2 pg / g-0.5 pg / g, or 0.5 pg / g-1 pg / g). In some embodiments, the therapeutically effective amount of the RNA molecule includes about 0.001 mg / kg, 0.002 mg / kg, 0.005 mg / kg, 0.01 mg / kg, 0.02 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg of body weight, or more, or an amount in a range defined by any two of the preceding values (e.g., 0.001 mg / kg-0.01 mg / kg, 0.01 mg / kg-0.1 mg / kg, 0.1 mg / kg-1 mg / kg, 1 mg / kg-10 mg / kg, 10 mg / kg-100 mg / kg). In some embodiments, the therapeutically effective amount of the RNA molecule is about 0.001 mg / kg, 0.002 mg / kg, 0.005 mg / kg, 0.01 mg / kg, 0.02 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, or about 1 mg / kg of body weight, or more, or an amount in a range defined by any two of the preceding values (e.g., 0.001 mg / kg-0.01 mg / kg, 0.01 mg / kg-0.05 mg / kg, 0.05 mg / kg-0.1 mg / kg, 0.1 mg / kg-0.2 mg / kg, 0.2 mg / kg-0.5 mg / kg, or 0.5 mg / kg-1 mg / kg). In some embodiments, the therapeutically effective amount of the RNA molecule is in a range of 0.1 mg / kg to 10 mg / kg of body weight.

[0082] In any method of the present disclosure, in some embodiments, the therapeutically effective amount of the RNA molecule is sufficient on its own to bring about the desired outcome (e.g., without administering another therapeutic agent for the same condition or disease). In any method of the present disclosure, in some embodiments, themethod includes administering a therapeutically effective amount of a composition that consists of, or consists essentially of the RNA molecule and a pharmaceutically acceptable excipient. In some embodiments, the method includes administering a therapeutically effective amount of a cell-free composition (e.g., a composition substantially free of cells such as CDC) that includes the RNA molecule. In some embodiments, the method includes administering a therapeutically effective amount of an extracellular vesicle-free composition (e.g., a composition substantially free of extracellular vesicles such as exosomes) that includes the RNA molecule.

[0083] The RNA molecule or composition can be administered to the subject at any suitable dosing schedule. In some embodiments, the therapeutically effective amount of the RNA molecule or the composition is administered to the subject at a frequency of, of about, or no more frequently than once a day, once every other day, three times a week, twice a week, once a week (QW), once every two weeks (Q2W), once every month (QM), once every two months (Q2M), once every three months (Q3M), once every four months (Q4M) or longer, or at a frequency in a range defined by any two of the preceding values (e.g., three times a week to once every four months (Q4M), twice a week to once every two months (Q2M), or twice a week to once a month (QM)). In some embodiments, the therapeutically effective amount of the RNA molecule or the composition is administered to the subject at a frequency in a range of once a day to once a month. In some embodiments, the therapeutically effective amount of the RNA molecule or the composition is administered to the subject at a frequency in a range of once a day to once every two weeks (Q2W). In some embodiments, the therapeutically effective amount of the RNA molecule or the composition is administered to the subject at a frequency in a range of once a day to once a week (QW). In some embodiments, the therapeutically effective amount of the RNA molecule or the composition is administered to the subject at a frequency of or of about once a day. In some embodiments, the therapeutically effective amount of the RNA molecule or the composition is administered to the subject at a frequency of or of about twice a week. In some embodiments, the nucleic acid is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more times, or a number of times in a range defined by any two of the preceding values (e.g., 1-30 times, 2-20 times, 5-15 times, 1-20 times, etc.). In some embodiments, the RNA molecule is administered to the subject atregular intervals. In some embodiments, the RNA molecule is administered to the subject chronically.

[0084] In some embodiments, the therapeutically effective amount of the RNA molecule or the composition is administered to the subject at a dosing schedule that is less frequent than the dosing schedule for administering a therapeutically effective amount of an unmodified counterpart RNA molecule, to achieve comparable (or enhanced) therapeutic results. In some embodiments, the therapeutically effective amount of the RNA molecule or the composition is administered to the subject at a dosing interval that is longer than the dosing interval for an unmodified counterpart RNA molecule, by, by about, or by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100% or more, or by a percentage in a range defined by any two of the preceding values (e.g., 5-100%, 10-90%, 20-80%, 50-75%, etc ), to achieve comparable (or enhanced) therapeutic results.

[0085] The RNA molecule or composition can be administered using any suitable route. Administration can be local or systemic. In some embodiments, administration is parenteral. Suitable options for administration include, without limitation, intravenous, intramuscular, subcutaneous, intra-arterial, intraperitoneal, or oral administration. In some embodiments, the RNA molecule or composition is administered orally. In some embodiments, the RNA molecule or composition is administered by oral gavage. In some embodiments, the RNA molecule or composition is administered intravenously. In some embodiments, the RNA molecule or composition is administered by infusion.

[0086] Also provided herein is a method of immunomodulation (“immunomodulation method”). The immunomodulation method can include contacting an effective amount of the RNA molecule of the present disclosure (or a composition containing the same, as described herein), with a population of macrophages, e.g., human macrophages. In some embodiments, the contacting comprises administering to a subject in need of treating a condition characterized by inflammation and / or fibrosis an effective amount of the RNA molecule or the composition. In some embodiments, contacting the effective amount of the RNA molecule of the present disclosure with a population of macrophages increases expression of one or more anti-inflammatory cytokines. In some embodiments, contacting the effective amount of the RNA molecule of the present disclosure with a population of macrophages increases expression of IL-10, ILla, ILip, TGFpi, NFKB, TNF, CCL3 and / orVGEF-A in the population of macrophages. In some embodiments, contacting the effective amount of the RNA molecule of the present disclosure with a population of macrophages increases expression ofIL-10, ILla, ILip, TGFpi, NFKB, TNF, and / or CCL3 in the population of macrophages. In some embodiments, contacting the effective amount of the RNA molecule of the present disclosure with a population of macrophages reduces expression of VGEF-A in the population of macrophages. In some embodiments, contacting the effective amount of the RNA molecule of the present disclosure with a population of macrophages increases expression of IL-10, ILla, ILip, TGFP1, NFKB, TNF, CCL3 and / or VGEF-A in the population of macrophages by, by about, or by at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 fold, or by a fold amount in a range defined by any two of the preceding values (e.g., 1.1-10 fold, 1.5-9 fold, 2-8 fold, 1.2-5 fold, etc.). In some embodiments, contacting the effective amount of the RNA molecule of the present disclosure with a population of macrophages increases expression of IL-10, ILla, ILip, TNF, and / or CCL3 in the population of macrophages. In some embodiments, contacting the effective amount of the RNA molecule of the present disclosure with a population of macrophages increases expression of IL-10, ILla, ILip, TNF, and / or CCL3 in the population of macrophages by, by about, or by at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 fold, or by a fold amount in a range defined by any two of the preceding values (e.g., 1.1-10 fold, 1.5-9 fold, 2-8 fold, 1.2-5 fold, etc.). In some embodiments, expression of IL-10, ILla, ILip, TGFpi, NFKB, TNF, CCL3 and / or VGEFA is mRNA expression of IL- 10, ILla, ILip, TGFpi, NFKB, TNF, CCL3 and / or VGEFA.

[0087] In some embodiments, the contacting is done in vitro, e.g., in culture. In some embodiments, after contacting the macrophages in vitro, the method includes administering the macrophages to a subject in need of treating a muscle disorder, a heart condition, fibrosis, an inflammatory condition, as described herein.KITS

[0088] Also provided herein are kits that include the RNA molecule or a composition of the present disclosure. The present kit in some embodiments finds use in treating a muscle disorder, a heart condition, fibrosis, an inflammatory condition (e.g., associated with a muscle disorder, or a viral infection), as provided herein. A kit can include the RNA molecule of the present disclosure and optionally a transfection reagent. Thetransfections reagent can be any suitable transfection reagent, as provided herein. In some embodiments, the transfection reagent includes one or more of a lipid (e.g., a liposome-forming lipid), a PEGylated lipid, and an extracellular vesicle. In some embodiments, the kit includes a pharmaceutically acceptable excipient, as provided herein. A kit can include the RNA molecule of the present disclosure and a pharmaceutically acceptable excipient. In some embodiments, the kit includes casein and / or chitosan. In some embodiments, the kit includes an antisense oligonucleotide, such as, without limitation, an exon-skipping agent that targets a dystrophin transcript. Kits can include one or more containers (e.g., vials, ampoules, test tubes, flasks or bottles) for holding one or more components of the kits. The kits may further include instructions for using the kit to treat a condition (e.g., muscular dystrophy, heart failure, myocardial infarction, or an inflammatory condition associated therewith, or an inflammatory condition associated with a viral infection). The information and instructions may be in the form of words, pictures, or both, and the like.Additional embodiments

[0089] Additional, non-limiting embodiments of the present disclosure are provided by the following numbered embodiments:1. A phosphorothioate-containing RNA molecule comprising 5’- NCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 1), wherein N is uracil or thymine, wherein the RNA molecule comprises one or more phosphorothioate bonds between nucleic acid residues in one or more pairs of consecutive nucleic acid residues within SEQ ID NO: 1.2. A phosphorothioate-containing RNA molecule comprising a sequence at least 90% identical to 5’-NCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 1), wherein N is uracil or thymine, wherein the RNA molecule comprises one or more phosphorothioate bonds between nucleic acid residues in one or more pairs of consecutive nucleic acid residues within the sequence.3. The RNA molecule of embodiment 1 or 2, comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 phosphorothioate bonds within SEQ ID NO: 1.3a. The RNA molecule of any one of the preceding embodiments, wherein N is uracil.3b. The RNA molecule of any one of the preceding embodiments, wherein the one or more phosphorothioate bonds comprise: a phosphorothioate bond between at least one pair of consecutive nucleic acid residues within positions 1-6 of SEQ ID NO: 1; and a phosphorothioate bond between at least one pair of consecutive nucleic acid residues within positions 27-32 of SEQ ID NO: 1.3c. The RNA molecule of any one of the preceding embodiments, wherein the one or more phosphorothioate bonds comprise: a phosphorothioate bond between at least one pair of consecutive nucleic acid residues within positions 1-3 of SEQ ID NO: 1; and a phosphorothioate bond between at least one pair of consecutive nucleic acid residues within positions 30-32 of SEQ ID NO: 1.3d. The RNA molecule of embodiment 3c, comprising a phosphorothioate bond between nucleic acid residues in each of the following pairs of consecutive nucleic acid residues within SEQ ID NO: 1: N1 / C2; C2 / C3; C30 / G31; G31 / C32, optionally wherein the RNA molecule comprises a phosphodiester bond between nucleic acid residues in all remaining pairs of consecutive nucleic acid residues within SEQ ID NO: 1.3e. The RNA molecule of embodiment 3c or 3d, comprising a nucleotide sequence set forth in SEQ ID NO:8 (5’-UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’).3f. The RNA molecule of any one of the preceding embodiments, that is SEQ ID NO:9.4. The RNA molecule of embodiment 1 or 2, comprising a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1.5. The RNA molecule of any one of the preceding embodiments, comprising one or more backbone sugar modifications within SEQ ID NO: 1.6. The RNA molecule of embodiment 5, comprising the one or more backbone sugar modifications at one or more nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1.7. The RNA molecule of embodiment 5 or 6, wherein up to 5 consecutive nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise the one or more backbone sugar modifications, optionally wherein 1-3 nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise the one or more backbone sugar modifications.8. The RNA molecule of any one of embodiments 5-7, wherein the one or more backbone sugar modifications comprises a locked nucleic acid (LNA) and / or a 2’ -O- methylation.9. The RNA molecule of any one of the preceding embodiments, wherein up to 5 consecutive nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise a 2’-O-methylation, optionally wherein 1-3 nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise a 2’-(9-methylation.10. The RNA molecule of any one of the preceding embodiments, comprising a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1, and wherein nucleic acid residues at the following positions of SEQ ID NO: 1 comprises a 2’-O-methylation:(a) 1 and 32;(b) 1, 2, 31, and 32; or(c) 1, 2, 3, 30, 31, and 32.11. The RNA molecule of any one of embodiments 1-8, wherein up to 5 consecutive nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise a LNA, wherein if the nucleic acid residue at position 1 of SEQ ID NO: 1 comprises the LNA, N is thymine, optionally wherein 1-3 nucleic acid residues from the 5’ end and / or 3’ end of SEQ ID NO: 1 comprise a LNA.12. The RNA molecule of any one of embodiments 1-8 and 11, comprising a phosphorothioate bond between each pair of consecutive nucleic acid residues of SEQ ID NO: 1, wherein N is thymine, and wherein nucleic acid residues at the following positions of SEQ ID NO: 1 comprises a LNA:(a) 1 and 32; or(b) 1, 2, 3, 30, 31, and 32.13. The RNA molecule of any one of the preceding embodiments, wherein the RNA molecule is at most 60 bases long, optionally, wherein the RNA molecule is about 32 bases long.14. The RNA molecule of any one of the preceding embodiments, consisting of or consisting essentially of the sequence of SEQ ID NO: 1.15. The RNA molecule of any one of the preceding embodiments, comprising any one of SEQ ID NOs: 2-7.16. The RNA molecule of any one of the preceding embodiments, consisting of or consisting essentially of any one of SEQ ID NOs: 2-7.16a. A phosphorothioate-containing RNA molecule comprising, consisting essentially of, or consisting of 5’-UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 9), wherein the RNA molecule comprises a phosphorothioate bond between nucleic acid residues in each of the following pairs of consecutive nucleic acid residues: U1 / C2; C2 / C3; C30 / G31; G31 / C32, optionally wherein the RNA molecule comprises a phosphodiester bond between nucleic acid residues in all remaining pairs of consecutive nucleic acid residues.17. A composition comprising: the RNA molecule of any one of the preceding embodiments; and a pharmaceutically acceptable excipient.18. The composition of embodiment 17, comprising a transfection reagent.19. The composition of embodiment 18, wherein the transfection reagent comprises one or more of a liposome, an extracellular vesicle (EV), and / or a polyethylene glycol (PEG)- cationic lipid complex (PCLC).20. The composition of embodiment 18, wherein the transfection reagent comprises an EV derived from cardiosphere-derived cells (CDC).21. The composition of any one of embodiments 17-20, comprising a casein phosphoprotein.22. The composition of any one of embodiments 17-21, comprising casein micelles.23. The composition of embodiment 22, comprising chitosan.24. The composition of any one of embodiments 17-23, wherein the RNA molecule is encapsulated in a casein-chitosan complex.25. The composition of any one of embodiments 17-24, comprising casein-chitosan micelles.26. The composition of any one of embodiments 17-25, comprising an antisense oligonucleotide.27. The composition of embodiment 26, wherein the antisense oligonucleotide comprises an exon-skipping agent that targets a dystrophin transcript.28. A method of treating a condition associated with inflammation and / or fibrosis, comprising administering to a subject in need of treating a condition associated with inflammation and / or fibrosis a therapeutically effective amount of the RNA molecule or the composition of any one of the preceding embodiments.29. The method of embodiment 28, wherein the condition associated with inflammation and / or fibrosis comprises inflammation and / or fibrosis of the heart or skeletal muscle.30. The method of embodiment 28 or 29, wherein the condition associated with inflammation and / or fibrosis comprises a symptom and / or sequelae of heart failure, myocardial infarction, or muscular dystrophy.31. The method of any one of embodiments 28-30, wherein the subject has suffered heart failure or myocardial infarction.32. The method of any one of embodiments 28-31, wherein the subject has or is predisposed to having muscular dystrophy.33. The method of embodiment 32, wherein the subject has or is predisposed to having Duchenne muscular dystrophy.34. The method of any one of embodiments 28-33, further comprising administering a second therapy for the muscle disorder.35. The method of embodiment 34, wherein the second therapy comprises an exonskipping agent and / or gene therapy.36. The method of any one of embodiments 28-35, comprising orally administering the therapeutically effective amount of the RNA molecule or of the composition to the subject.37. The method of any one of embodiments 28-35, comprising parenterally administering the therapeutically effective amount of the RNA molecule or of the composition to the subj ect.38. The method of embodiment 37, comprising intravenously, intramuscularly, or intracardially administering the therapeutically effective amount of the RNA molecule or of the composition to the subject.38a. A method of treating a muscle disorder or symptom thereof, comprising administering to a subject in need of treating a muscle disorder or symptom thereof a therapeutically effective amount of the RNA molecule or the composition of any one of embodiments 1-27.38b. The method of embodiment 38a, wherein the muscle disorder comprises a skeletal muscle disorder and / or a heart condition.38c. The method of embodiment 38a or 38b, wherein the subject has suffered heart failure or myocardial infarction.38d. The method of any one of embodiments 38a-38c, wherein the muscle disorder comprises muscular dystrophy, optionally wherein the muscle disorder comprises Duchenne muscular dystrophy.38e. The method of embodiment 38d, wherein the subject has or is predisposed to having muscular dystrophy38f. The method of any one of embodiments 38a-38e, further comprising administering a second therapy for the muscle disorder.38g. The method of embodiment 38f, wherein the second therapy comprises an exon-skipping agent and / or gene therapy.38h. The method of any one of embodiments 38a-38g, comprising orally administering the therapeutically effective amount of the RNA molecule or of the composition to the subj ect.39. A method of immunomodulation, comprising contacting an effective amount of the RNA molecule or of the composition of any one of embodiments 1-27 with a population of macrophages.40. The method of embodiment 39, wherein the contacting comprises administering to a subject in need of treating a condition associated with inflammation and / or fibrosis the effective amount of the RNA molecule or of the composition.41. The method of embodiment 39, wherein the contacting is done in vitro.42. The method of any one of embodiments 39-41, wherein the macrophage is a human macrophage.43. The method of any one of embodiments 39-42, wherein contacting the effective amount of the RNA molecule or of the composition increases expression of one or more of: IL-10, ILla, ILip, TGF 1, NFKB, TNF, CCL3 and VGEFA, in the population of macrophages.44. The method of embodiment 43, wherein expression is increased to a greater extent than an increase in expression achieved by a comparable amount of a reference RNA molecule comprising 5’-UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 8), wherein the reference RNA molecule does not comprise a phosphorothioate bond within SEQ ID NO: 8.45. A kit compri si ng : the RNA molecule of any one of embodiments 1-16a; and optionally a transfection reagent.46. The kit of embodiment 45, wherein the transfection reagent comprises one or more of a lipid, PEGylated lipid, and an extracellular vesicle (EV).47. The kit of embodiment 45 or 46, comprising a pharmaceutically acceptable excipient.48. The kit of any one of embodiments 45-47, comprising a casein phosphoprotein.49. The kit of any one of embodiments 45-48, comprising chitosan.50. The kit of any one of embodiments 45-49, comprising an antisense oligonucleotide.51. The kit of embodiment 50, wherein the antisense oligonucleotide comprises an exon-skipping agent that targets a dystrophin transcript.52. Use of the RNA molecule of any one of embodiments l-16a or the composition of any one of embodiments 17-27 for treatment of a condition associated with inflammation and / or fibrosis in a subject in need thereof.53. Use of the RNA molecule of any one of embodiments l-16a or the composition of any one of embodiments 17-27 for preparation of a medicament for treatment of a condition associated with inflammation and / or fibrosis a subject in need thereof.54. The use of the RNA molecule or the composition of embodiment 52 or 53, wherein the condition associated with inflammation and / or fibrosis comprises inflammation and / or fibrosis of the heart and / or skeletal muscle.55. The use of the RNA molecule or the composition of any one of embodiments 52-54, wherein the condition associated with inflammation and / or fibrosis comprises a symptom and / or sequelae of heart failure, myocardial infarction, or muscular dystrophy.

[0090] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0091] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein or nucleic acid structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0092] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated withcertain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0093] The technology described herein is further illustrated by the following examples, which in no way should be construed as being further limiting.EXAMPLESExample 1

[0094] This non-limiting example shows the design of new tREX-1 variants, and the functional effects of the tREX-1 variants.

[0095] A non-coding RNA (ncRNA) species particularly abundant in therapeutic extracellular vesicles derived from cardiosphere-derived cells (CDC-EV) was previously identified, and was named tREX-1. tREX-1 is a 32-nucleotide long RNA that has the sequence 5’-UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 8) (FIG. IE; “Unmodified”). To improve the bioactivity of tREX-1, several variants of tREX-1 were generated by chemically modifying the RNA backbone. Examples of the chemical modifications incorporated into tREX-1 are shown in FIGs. 1B-1D. FIGs. 1A-1D show the types of modifications in tREX-1 variants.

[0096] Phosphodiester bonds are naturally occurring and link each of the nucleotides of unmodified tREX-1 (FIG. 1 A). The phosphodiester bonds in tREX-1 were replaced with phosphorothioate bonds, which replace an oxygen molecule with a sulfur molecule in the backbone of tREX-1 (FIG. IB). All new chemical variants had phosphorothioate bonds between all nucleotides.

[0097] Additional modifications included 2’-( -methylation, which adds a methyl group to the oxygen on the 2’ carbon of the ribose sugar (FIG. 1C). This modification was made at the 5’ and 3’ ends of tREX-1 on 1 or more terminal nucleotides in some variants.

[0098] Locked nucleic acids sterically lock the 2’ oxygen with the 4’ oxygen on the ribose sugar (FIG. ID). This modification was made at the 5’ and 3’ ends of tREX-1 on one or more terminal nucleotides in some variants. It should be noted that this modification cannot be added to uracil nucleobases. Thus, the uracil residues at the 5’ position of tREX-1 were mutated to a thymine nucleobase.

[0099] As shown at the bottom of FIG. IE, six new chemical variants of tREX-1 were made and tested, and the results are shown in Figure 3.

[0100] The effects of unmodified tREX-1 on the expression of macrophage associated genes in vitro was compared to an unmodified scramble RNA control (FIG. 2). Bone marrow cells were isolated from mdx mice and cultured with m-CSF for 7 days to produce macrophages. Macrophages were then exposed to 25 nM unmodified tREX-1 or an unmodified scramble RNA control (bearing the same nucleotide makeup but randomized to be non-complementary to the mouse genome) for 24 hours, n=3 per group. After 24 hours of exposure, RNA was collected from macrophages and subjected to qPCR analysis.

[0101] Gene expression change was reported as fold change relative to control (scramble) (FIG. 2). Figure 2 shows how unmodified tREX-1 alters the expression of macrophage associated genes. The profile shown in FIG. 2 is a panel of selected genes known to be expressed by macrophages. Data are reported as mean ± SEM. *P<0.05, **P<0.01, ****P<0.0001, ns: not significant. Consistent with previous results, changes in the expression of macrophage associated genes were induced by unmodified tREX-1.

[0102] The effects of the modified tREX-1 variants (FIG. IE) on the expression of macrophage associated genes in vitro was tested next (FIG. 3).

[0103] Using the same protocol as for the unmodified tREX-1 above, bone marrow cells were isolated from mdx mice and cultured with m-CSF for 7 days to produce macrophages. Macrophages were then exposed to 25 nM unmodified tREX-1 (control), or 1 of 6 chemically modified variants (referred here to as TTx, where x identifies the unique compound) for 24 hours, n=3 per group. After 24 hours of exposure, RNA was collected from macrophages and subjected to qPCR analysis.

[0104] Gene expression change was reported as fold change relative to control (unmodified tREX-1). The profile shown in FIG. 3 is a panel of selected genes known to be expressed by macrophages. Data are reported as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns: not significant.

[0105] FIG. 3 demonstrates the same signature (as shown in FIG. 2) is potentiated by each of the tREX-1 variants, in terms of up-regulation and down-regulation (and those that were unchanged by unmodified tREX-1). The difference in gene expression shown in FIG. 3is relative to unmodified tREX-1 (not scramble as shown in FIG. 2); and thus, the fold change is in excess of what occurs naturally with unmodified tREX-1.

[0106] In summary, these data show chemically modified tREX-1 variants maintained the same gene signature as unmodified tREX-1, except the magnitude of change was dramatically increased.Example 2

[0107] This non-limiting example shows treatment of a subject with a condition associated with inflammation and / or fibrosis of the heart or skeletal muscle by administering a tREX-1 variant.

[0108] A subject suffering from muscular dystrophy is identified. A therapeutically effective amount of a tREX-1 variant, (e.g., TT1, TT2, TT3, TT4, TT5, and / or TT7, as described herein), is administered to the subject intravenously or orally. After administration, the subject’s heart function (e.g., ventricular ejection fraction) and / or skeletal muscle function (e.g., muscle torque) is monitored. The subject demonstrates an improvement in (or reduced deterioration of) heart function and / or skeletal muscle function compared to before administering the tREX-1 variant.Example 3

[0109] This non-limiting example shows treatment of a subject with a condition associated with inflammation and / or fibrosis of the heart or skeletal muscle by administering a tREX-1 variant.

[0110] A subject suffering from myocardial infarction (MI) is identified. A therapeutically effective amount of a tREX-1 variant, (e.g., TT1, TT2, TT3, TT4, TT5, and / or TT7, as described herein), is administered to the subject intravenously or orally. After administration, the subject’s heart function (e.g., ventricular ejection fraction) is monitored. The subject demonstrates an improvement in (or reduced deterioration of) heart function compared to before administering the tREX-1 variant.Example 4[OlH] This non-limiting example shows the further design of new tREX-1 variants, and the functional effects of the tREX-1 variants.

[0112] Additional variants of tREX-1 were generated. Examples of the chemical modifications incorporated into tREX-1 are shown in FIG. 4A. The additional variants had phosphorothioate bonds only at the 5 ’end and the 3 ’end of the molecule. TT8 had phosphorothioate bonds over the backbone from three residues from each of the 5’ end and the 3’ end. TT9 had phosphorothioate bonds over the backbone from six residues from each of the 5’ end and the 3’ end. TT10 further modified TT8 to include 2’-O-methylation on 3 residues from each of the 5’ end and the 3’ end.

[0113] The effects of chemically modified tREX-1 variants (TT8-TT10), unmodified scramble tREX-1 and modified scramble TT9 variant on the expression of macrophage associated genes were tested in vitro (FIG. 4B). Bone marrow cells were isolated from mdx mice and cultured with m-CSF for 7 days to produce macrophages. Macrophages were then exposed to 25 nM of the chemically modified tREX-1 variants (TT8, TT9, and TT10) or scramble RNA (unmodified and TT9 modified) for 24 hours, n=3 per group. After 24 hours of exposure, RNA was collected from macrophages and subjected to qPCR analysis.

[0114] Gene expression change was reported as fold change relative to control (FIG. 4). FIG. 4 shows that TT8 had the most favorable effects on macrophage gene expression compared to TT9 and TT10, which were largely ineffective on the expression of macrophage associated genes. The profile shown in FIG. 4 is a panel of selected genes known to be expressed by macrophages. Data are reported as mean ± SEM. *P<0.05, **P<0.01, * * * *P<0.0001, ns: not significant.Example 5

[0115] This non-limiting example shows how full backbone modification with phosphorothioate bonds renders the RNA bioactive.

[0116] Bone marrow cells were isolated from mdx mice and cultured with m-CSF for 7 days to produce macrophages. Macrophages were then exposed to 25 nM unmodified tREX-1 scramble, TT1 -modified scramble RNA, or TT8-modified scramble RNA for 24hours, n=3 per group. After 24 hours of exposure, RNA was collected from macrophages and subjected to qPCR analysis.

[0117] Gene expression change was reported as fold change relative to unmodified scramble tREX-1 (FIG. 5, top panel). FIG. 5, top panel shows how the modification of the full backbone with phosphorothioate bonds (TT1 -modified scramble) alters the expression of macrophage associated genes. The profile shown in FIG. 5 is a panel of selected genes known to be expressed by macrophages. Data are reported as mean ± SEM. *P<0.05, **P<0.01, ****p<0.0001, ns: not significant.

[0118] In contrast, TT8-modified scramble RNA showed minimal bioactivity (FIG. 5, bottom panel). These results indicate that reducing the number of phosphorothioate bonds in the backbone can mitigate the unintended effects of RNA having a full backbone of phosphorothioate bonds on gene expression.Example 6

[0119] This non-limiting example shows the effects of oral administration of a tREX-1 variant in a model of myocardial infarction.

[0120] In a mouse model of acute myocardial infarction, mice underwent 45 min LAD ligation, followed by 20 min reperfusion. After reperfusion, mice were administered vehicle, TT1 or TTl-modified scramble RNA (0.20 pg / g body weight) by oral gavage. Two days following ischemia / reperfusion, hearts were extracted and processed for TTC staining to quantify infarct size (average of apical, mid-papillary, and basal infarcts) relative to the left ventricle.

[0121] The percent infarct size for animals treated with modified TT1 RNA was compared to those of animals treated with vehicle or TT1 -modified scramble RNA (FIG. 6). Data are reported as mean ± SEM. *P<0.05, **P<0.01, ****P<0.0001, ns: not significant. An independent t-test was used to determine statistical significance (a=0.05).

[0122] Figure 6 shows infarct size after MI was significantly reduced in TT1- treated animals compared to the infarct size in animals treated with vehicle RNA. However, there was no significant difference in the infarct size in TT1 -treated animals compared to the infarct size in animals treated with TTl-modified scramble RNA or between infarct size in animals treated with vehicle and animals treated with TT1 -modified scramble RNA.Example 7

[0123] This non-limiting example shows the effects of oral administration of a tREX-1 variant in a model of myocardial infarction.

[0124] In a mouse model of acute myocardial infarction, mice underwent 45 min LAD ligation, followed by 20 min reperfusion. After reperfusion, mice were administered TT8 or TT8-modified scramble RNA (0.20 pg / g body weight) by oral gavage. Two days following ischemia / reperfusion, hearts were extracted and processed for TTC staining to quantify infarct size (average of apical, mid-papillary, and basal infarcts) relative to the left ventricle.

[0125] The percent infarct size for animals treated with modified TT8 RNA was compared to those of animals treated with TT8-modified scramble RNA (FIG. 7). Data are reported as mean ± SEM. *P<0.05, **P<0.01, ****P<0.0001, ns: not significant. An independent t-test was used to determine statistical significance (a=0.05).

[0126] Figure 7 shows infarct size after MI was significantly reduced in TT8- treated animals compared to the infarct size in animals treated with TT8-modified scramble RNA.Example 8

[0127] This non-limiting example shows treatment of a subject with Duchenne muscular dystrophy by administering a tREX-1 variant of the present disclosure.

[0128] A subject suffering from Duchenne muscular dystrophy is identified. A therapeutically effective amount of a tREX-1 variant, (e.g., TT8), is administered to the subject orally. After administration, the subject’s heart function (e.g., ventricular ejection fraction) and / or skeletal function is monitored. The subj ect demonstrates an improvement in (or reduced deterioration of) heart function and / or skeletal muscle function compared to before administering the tREX-1 variant.

Claims

WHAT IS CLAIMED IS:

1. A phosphorothioate-containing RNA molecule comprising 5’- NCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 1), wherein N is uracil or thymine, wherein the RNA molecule comprises one or more phosphorothioate bonds between nucleic acid residues in one or more pairs of consecutive nucleic acid residues within SEQ ID NO: 1.

2. A phosphorothioate-containing RNA molecule comprising a sequence at least 90% identical to 5’-NCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 1), wherein N is uracil or thymine, wherein the RNA molecule comprises one or more phosphorothioate bonds between nucleic acid residues in one or more pairs of consecutive nucleic acid residues within the sequence.

3. The RNA molecule of claim 1, comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 phosphorothioate bonds within SEQ ID NO: 1.

4. The RNA molecule of claim 1, wherein N is uracil.

5. The RNA molecule of claim 1, wherein the one or more phosphorothioate bonds comprise: a phosphorothioate bond between at least one pair of consecutive nucleic acid residues within positions 1-6 of SEQ ID NO: 1; and a phosphorothioate bond between at least one pair of consecutive nucleic acid residues within positions 27-32 of SEQ ID NO: 1.

6. The RNA molecule of claim 1, wherein the one or more phosphorothioate bonds comprise: a phosphorothioate bond between at least one pair of consecutive nucleic acid residues within positions 1-3 of SEQ ID NO: 1; and a phosphorothioate bond between at least one pair of consecutive nucleic acid residues within positions 30-32 of SEQ ID NO: 1.

7. The RNA molecule of claim 6, comprising a phosphorothioate bond between nucleic acid residues in each of the following pairs of consecutive nucleic acid residues within SEQ ID NO: 1 : N1 / C2; C2 / C3; C30 / G31; G31 / C32.

8. The RNA molecule of claim 7, comprising a nucleotide sequence set forth in SEQ ID NO:8 (5’-UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’).

9. The RNA molecule of claim 1, that is SEQ ID NO:9.

10. The RNA molecule of claim 1, wherein the RNA molecule is at most 60 bases long.

11. The RNA molecule of claim 1, consisting of or consisting essentially of the sequence of SEQ ID NO: 1.

12. A phosphorothioate-containing RNA molecule comprising, consisting essentially of, or consisting of 5’-UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC-3’ (SEQ ID NO: 9), wherein the RNA molecule comprises a phosphorothioate bond between nucleic acid residues in each of the following pairs of consecutive nucleic acid residues: U1 / C2; C2 / C3; C30 / G31; G31 / C32, optionally wherein the RNA molecule comprises a phosphodiester bond between nucleic acid residues in all remaining pairs of consecutive nucleic acid residues.

13. A composition comprising: the RNA molecule of any one of the preceding claims; and a pharmaceutically acceptable excipient.

14. The composition of claim 13, comprising a casein phosphoprotein and / or chitosan.

15. The composition of claim 13, comprising an antisense oligonucleotide.

16. The composition of claim 15, wherein the antisense oligonucleotide comprises an exon-skipping agent that targets a dystrophin transcript.

17. A method of treating a condition associated with inflammation and / or fibrosis, comprising administering to a subject in need of treating a condition associated with inflammation and / or fibrosis a therapeutically effective amount of the RNA molecule of claim 1.

18. The method of claim 17, wherein the condition associated with inflammation and / or fibrosis comprises inflammation and / or fibrosis of the heart or skeletal muscle.

19. The method of claim 17, wherein the condition associated with inflammation and / or fibrosis comprises a symptom and / or sequelae of heart failure, myocardial infarction, or muscular dystrophy.

20. A method of treating a muscle disorder or symptom thereof, comprising administering to a subject in need of treating a muscle disorder or symptom thereof a therapeutically effective amount of the RNA molecule of claim 1.

21. The method of claim 20, wherein the muscle disorder comprises a skeletal muscle disorder and / or a heart condition.

22. The method of claim 20, wherein the subject has suffered heart failure or myocardial infarction.

23. The method of claim 20, wherein the muscle disorder comprises muscular dystrophy, optionally wherein the muscle disorder comprises Duchenne muscular dystrophy.

24. The method of claim 23, wherein the subject has or is predisposed to having muscular dystrophy.

25. The method of claim 20, further comprising administering a second therapy for the muscle disorder.

26. The method of claim 25, wherein the second therapy comprises an exon-skipping agent and / or gene therapy.

27. The method of claim 17, comprising orally administering the therapeutically effective amount of the RNA molecule or of the composition to the subject.

28. The method of claim 17, comprising intravenously, intramuscularly, intramyocardially, or intracardially administering the therapeutically effective amount of the RNA molecule or of the composition to the subject.

29. A method of immunomodulation, comprising contacting an effective amount of the RNA molecule of claim Iwith a population of macrophages.

30. The method of claim 29, wherein the contacting comprises administering to a subject in need of treating a condition associated with inflammation and / or fibrosis the effective amount of the RNA molecule or of the composition.

31. The method of claim 29, wherein the contacting is done in vitro.

32. The method of claim 29, wherein the macrophage is a human macrophage.

33. The method of claim 29, wherein contacting the effective amount of the RNA molecule or of the composition increases expression of one or more of: IL-10, ILla, ILip, TNF, and CCL3, in the population of macrophages.

34. A kit comprising: the RNA molecule of claim 1; and a pharmaceutically acceptable excipient.

35. The kit of claim 34, comprising a casein phosphoprotein.

36. The kit of claim 34, comprising chitosan.

37. The kit of claim 34, comprising an antisense oligonucleotide.

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