Formulations for oral delivery of nucleic acids

Abstract

Provided herein are formulations for oral delivery or administration of therapeutic nucleic acids. An oral formulation can include: a nucleic acid; at least one casein protein; and a chitosan. In some embodiments, the formulations are liposome-free formulations having no cationic lipids typically used as transfection reagents in conventional nucleic acid formulations for oral delivery. The formulations provided herein find use in treating a condition associated with inflammation and / or fibrosis, or a cardiometabolic disorder by oral administration.

Description

CSMC.025WO2 PATENTFORMULATIONS FOR ORAL DELIVERY OF NUCLEIC ACIDSINCORPORATION BY REFERENCE TO PRIOR APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 681071, filed on August 8, 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. R01 HL 164588, awarded to Dr. Eduardo Marban 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 in electronic format. The Sequence Listing is provided as a file entitled CSMC025WO2SEQLIST.xml created on August 5, 2025, which is 54,080 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.BACKGROUND

[0004] Nucleic acid therapeutics offer the potential to treat diseases at a genetic level. Many conventional treatments generally induce therapeutic effects that are transient because they target proteins rather than underlying causes. In contrast, nucleic acid therapeutics have the potential for long-lasting (or even permanent, e.g., curative) effects via gene inhibition, addition, replacement, or editing. However, the successful use of nucleic acid therapeutics will hinge on delivery technologies that improve stability and / or bioavailability.SUMMARY

[0005] Provided herein is a liposome-free formulation for oral delivery of a nucleic acid, comprising: a nucleic acid; at least one casein protein; and a chitosan.

[0006] Provided herein is a method of treating a metabolic disorder or disease, comprising: identifying a subject in need of treating a metabolic disorder or disease; and coadministering to the subject therapeutically effective amounts of: an isolated nucleic acid comprising a nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12) or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA, and wherein the nucleic acid is at most 30 nt long; and a second therapeutic that is an incretin orfunctional analogue thereof, and / or is an activator of glucagon-like peptide 1 (GLP-1 ) receptor signaling, thereby treating the metabolic disorder or disease.

[0007] Also provided is a method of treating metabolic disorder or disease, comprising: identifying a subject in need of treating metabolic disorder or disease; and coadministering to the subject therapeutically effective amounts of: an anti-inflammatory, cardioprotective nucleic acid therapeutic, wherein the nucleic acid therapeutic is RNA; and a second therapeutic that is an incretin or functional analogue thereof, and / or an activator of glucagon-like peptide 1 (GLP-1) receptor signaling, thereby treating the metabolic disorder or disease.

[0008] Further provided is an isolated nucleic acid for co-administering with a second therapeutic to treat a metabolic disorder or disease, the isolated nucleic acid comprising a nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12) or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA, and wherein the nucleic acid is at most 30 nt long, wherein the second therapeutic is an incretin or functional analogue thereof, and / or is an activator of glucagon-like peptide 1 (GLP-1) receptor signaling.

[0009] Also provided is anti-inflammatory, cardioprotective nucleic acid therapeutic for co-administering with a second therapeutic to treat a metabolic disorder or disease, wherein the nucleic acid therapeutic is RNA, and wherein the nucleic acid is at most 30 nt long, wherein the second therapeutic is an incretin or functional analogue thereof, and / or is an activator of glucagon-like peptide 1 (GLP-1) receptor signaling.

[0010] Provided herein is a composition comprising: an isolated nucleic acid comprising a nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12) or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA, and wherein the nucleic acid is at most 30 nt long; a second therapeutic that is an incretin or functional analogue thereof, and / or is an activator of glucagon-like peptide 1 (GLP- 1 ) receptor signaling; and a pharmaceutically acceptable excipient.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a schematic diagram showing a “two-hit” mouse HFpEF study design and timeline.

[0012] FIG. 2 is a collection of graphs showing non-limiting embodiments of food intake and body weight over time of animals in the “two-hit” HFpEF study.

[0013] FIG. 3 is a graph showing non-limiting embodiments of non-fasting blood glucose of animals in the “two-hit” HFpEF study.

[0014] FIGs. 4A-4C are a collection of graphs showing non-limiting embodiments of echocardiography (FIG. 4A), treadmill exercise performance (FIG. 4B), and left ventricular function (FIG. 4C) of animals in the “two-hit” HFpEF study.

[0015] FIG. 5 is a graph showing non-limiting embodiments of exercise capacity of animals in the “two-hit” HFpEF study.

[0016] FIG. 6 is a graph showing non-limiting embodiments of left ventricular function of animals in the “two-hit” HFpEF study.

[0017] FIG. 7 is a collection of graphs showing non-limiting embodiments of hang wire test of animals in the “two-hit” HFpEF study, at 15 weeks.

[0018] FIG. 8 is a collection of graphs showing non-limiting embodiments of blood pressure of animals in the “two-hit” HFpEF study, at 15 weeks.

[0019] FIG. 9 is a collection of graphs showing non-limiting embodiments of time constant of isovolumic left ventricular pressure decay of animals in the “two-hit” HFpEF study, at 15 weeks.

[0020] FIG. 10 is a schematic diagram showing a genetic model of rat HFpEF study design and timeline.

[0021] FIG. 11 is a collection of graphs showing non-limiting embodiments of food intake and body weight over time of animals in the genetic HFpEF study.

[0022] FIGs. 12A-12C are a collection of graphs showing non-limiting embodiments of non-fasting blood glucose (FIG. 12A), echocardiography (FIG. 12B), and left ventricular function (FIG. 12C) of animals in the genetic HFpEF study.

[0023] FIG. 13 is a collection of graphs showing non-limiting embodiments of exercise capacity of animals in the genetic HFpEF study.

[0024] FIG. 14 is a graph showing non-limiting embodiments of left ventricular function of animals in the genetic HFpEF study.

[0025] FIGs. 15A-15D are a collection of graphs showing non-limiting embodiments of ex vivo endothelium-dependent and independent vascular responses of animals in the genetic HFpEF study. FIGs. 15A and 15B are graphs showing vasorelaxationresponses to increasing concentrations of acetylcholine (ACh) and sodium nitroprusside (SNP), respectively. FIGs. 15C and 15D are graphs showing respective EC50 values.

[0026] FIG. 16 is a graph showing non-limiting embodiments of circulating 8- isoprostane levels of animals in the genetic HFpEF study.

[0027] FIGs. 17A-17C are a collection of schematic diagrams and graphs showing a study scheme for pilot toxicology study of oral TY1 in healthy animals (n=5), (FIG. 17A), weight in animals fed oral TY1 (FIG. 17B), and changes in exercise endurance (FIG. 17C).

[0028] FIG. 18A is a schematic diagram showing non-limiting embodiments of dose-ranging studies for therapeutic optimization in HFpEF.

[0029] FIG. 18B is a schematic diagram showing non-limiting embodiments of efficacy studies in a translational model of cardiometabolic HFpEF.

[0030] FIG. 18C is a schematic diagram showing dose-ranging studies for toxicology.

[0031] FIG. 19 is a schematic diagram showing the structure of semaglutide.

[0032] FIGs. 20A-20D show non-limiting examples of second therapeutics.

[0033] FIG. 21 is a schematic showing non-limiting embodiments of a TY1 formulation in casein-chitosan micelles.

[0034] FIGs. 22A-22C are a collection of graphs showing non-limiting embodiments of total TY1-C2 copy number (FIG. 22A), mean and mode micelle particle size (FIG. 22B), and TY1-C2 loading efficiency (FIG. 22C) across three different batches. TY1 abundance (by qPCR; FIG. 22C).

[0035] FIG. 23 is a schematic for a non-limiting example of an oral RNA formulation.

[0036] FIG. 24 is a non-limiting study design for assessing the therapeutic efficacy of orally delivered therapeutic RNA formulated without lipid transfection reagent in the cardiac ischemia / reperfusion injury model.

[0037] FIGs. 25A-25C are a collection of graphs and images showing non-limiting embodiments of oral formulations of a therapeutic RNA (without the lipid transfection reagents) in reducing scar size (FIG. 25A), infarct (FIG. 25B), and cardiac troponin levels (cTnl) (FIG. 25C) in the cardiac ischemia / reperfusion injury model.

[0038] FIGs. 26A-26C are a collection of graphs and images showing non-limiting embodiments of the effectiveness of oral formulations of a therapeutic RNA (without the lipid transfection reagents) in reducing infarct size (FIG. 26A), infarct (FIG. 26B), and cardiac troponin levels (cTnl) (FIG. 26C) in the cardiac ischemia / reperfusion injury model.

[0039] FIGs. 27A and 27B are a collection of a graph and images showing nonlimiting embodiments of an oral formulation of a therapeutic RNA without a lipid transfection reagent in reducing infarct size (%) in the cardiac ischemia / reperfusion injury model.

[0040] FIG. 28 is a graph showing a non-limiting embodiment of an oral formulation of a therapeutic RNA without a lipid transfection reagent in reducing infarct mass (g) in the cardiac ischemia / reperfusion injury model.DETAILED DESCRIPTION

[0041] The present disclosure relates to formulations for oral delivery of nucleic acids, such as therapeutic RNA (e.g., non-coding or coding RNA). In several embodiments, the formulations enhance the oral bioavailability of such RNAs such that oral delivery, rather than delivery by, for example, injection allows for treatment of various diseases, in particular (but not limited to) those marked by inflammation and / or fibrosis.

[0042] In several embodiments, the formulations provided for herein allow the enhanced delivery (e.g., oral delivery) of nucleic acids to a subject. Provided herein are formulations for oral delivery (or administration) of a nucleic acid (e.g., an RNA therapeutic such as but not limited to TY1, miR-1246, uREXl, TT1, TT8, and yREX3). The oral formulations can be a simplified composition, having fewer excipients relative to previous formulations. In some embodiments, the formulations are liposome-free formulations having no cationic lipids typically used as transfection reagents in conventional nucleic acid formulations for oral delivery.

[0043] The oral formulations of the present disclosure find use in treating various diseases, including and without limitation those marked by inflammation and / or fibrosis. In some embodiments, the oral formulations are suitable for oral delivery of a therapeutic nucleic acid that acts on or targets macrophages to a subject. In some embodiments, a therapeutic nucleic acid contained in an oral formulation modulates macrophage function or activity (e.g., promotes anti-inflammatory activity of macrophages) to treat a condition or disease associated with inflammation and / or fibrosis. In some embodiments, an oral formulation of the presentdisclosure (e.g., a formulation that includes TY1 , or any suitable therapeutic nucleic acid described herein) finds use in treating a heart condition associated with inflammation and / or fibrosis, or a cardiometabolic disorder, such as but not limited to heart failure with preserved ejection fraction (HFpEF).

[0044] The majority of attempts to treat HFpEF have involved the repurposing of traditional heart failure drugs that have been shown to be effective in heart failure with reduced ejection fraction (HFrEF). These include drugs that modulate the renin-agiotensin system, beta blockers, mineralocorticoid receptor antagonists, PDE5 inhibitors, and nitrates.

[0045] More recently, SGLT2 inhibitors and GLP-1 receptor agonists (semaglutide and terzepatide) have been shown to reduced hospitalizations, improve exercise performance (GLP-1), and improve quality of life in HFpEF patients. These 2 classes of drugs do improve outcomes, but fail to reduce mortality or halt the progression of HFpEF.

[0046] Embodiments of the present disclosure demonstrate strong synergy with the GLP-1 receptor agonist, semaglutide. Orally administered TY1 provides highly effective antiinflammatory, anti-fibrotic, and cytoprotective actions in multiple organ systems including the heart. The beneficial effects of orally administered TY1 are synergistic with the weight loss and anti-diabetic effects of semaglutide. The present disclosure provides a novel combination therapy that affords profound metabolic and anti-inflammatory benefits in the setting of HFpEF. In some embodiments, embodiments of the present disclosure (e.g., oral TY1 + Semaglutide) can be targeted to treat obesity, diabetes, and HFpEF.

[0047] Non-limiting embodiments of the present disclosure (e.g., oral TY1 + Semaglutide) provide a combination therapy to patients that suffer from heart failure, including HFrEF and HFpEF. The present disclosure provides options to treat obesity and diabetes as well as fatty liver diseases (NASH, NAFLD). Furthermore, this novel approach can be applied to new anti-obesity that are currently under development.Terms

[0048] As used herein the term “nucleic acid” or “oligonucleotide” has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and refers 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 substitutedpurine (e.g. adenine (A) or guanine (G)). The term includes polynucleosides (i.e. a polynucleotide minus the phosphate) and any other organic base containing polymer. Purines and pyrimidines include but are not limited to adenine, cytosine, guanine, thymidine, inosine, 5-methylcytosine, 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. 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',4'-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 duplexes with 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 phosphodiester bond, sugar moiety, nucleobase, or combined sites thereof. As used herein, a “nucleobase sequence” denotes a sequence of nucleobases (e.g., cytosine (C), thymidine (T) / uracil (U), adenine (A) or guanine (G)) consecutively ordered along a backbone structure in a nucleic acid, e.g., along a sugar-phosphate backbone structure.

[0049] Polypeptide or nucleic acid molecules of the present disclosure may share a certain degree of sequence similarity or identity with the reference molecules (e.g., referencepolypeptides or reference polynucleotides), 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 polynucleotides, 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 polynucleotide 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, variants of a particular polynucleotide 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 polynucleotide or polypeptide as determined 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 PSLBLAST: 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 nucleotide and protein sequences faster than other optimal global alignment methods, including theNeedleman-Wunsch algorithm. Other tools are described herein, specifically in the definition of “identity” below.

[0050] The term “identity” refers to the overall relatedness between polymeric molecules, for example, between polynucleotide 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 nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide 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 be determined 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.CMPmatrix. 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)).

[0051] The term “Watson-Crick base-pairing”, or “base-pairing” refers to the formation of hydrogen bonds between specific pairs of nucleotide 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 polynucleotides is by quantifying the percentage of bonds formed between the guanine and cytosine bases of the two polynucleotides (“GC content”). In some embodiments, the GC content of bonding between two nucleic acids of a multimeric molecule (e.g., a multimeric mRNA molecule) is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%. In some embodiments, the GC content of bonding between two nucleic acids of a multimeric molecule (e.g., a multimeric mRNA molecule) is between 10% and 70%, about 20% to about 60%, or about 30% to about 60%. 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 length from 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.

[0052] 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 nothave been without human intervention of the biomolecule or its environment. In some embodiments, an isolated biomolecule is not inside a cell or an organism.

[0053] “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.

[0054] 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).

[0055] “Liposome” as used herein has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and denotes a vesicle or particle which possesses a lipid bilayer enclosing an aqueous compartment. Lipids that form liposomes include amphiphilic lipids, such as but not limited to, fatty acids, phospholipids, and cationic lipids. Cationic lipids include a basic, ionizable functional group (e.g., an amine or a nitrogen-containing heteroaryl), which may be present in neutral or charged form. In some embodiments, a cationic lipid includes a hydrazine, hydrazine, hydroxylamine, ethanolamine or diethylene diamine moiety linked to at least one saturated or unsaturated fatty acid residue.

[0056] “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.

[0057] The term “substantially free” as used herein denotes a lack of a substance or component in a formulation or composition, or use thereof in a method, or the presence ofthe substance or component in the formulation or composition, or use thereof in a method, at a level below the amount or concentration at which the substance or component is or is known to be effective (e.g., for promoting oral delivery of a nucleic acid). In some embodiments, the substance or component is absent from the composition or formulation, or is present in the composition or formulation below a detection limit for the substance or component. For example and without limitation, the formulation or composition may have less than a specific percentage of a component as measured by weight / volume (% w / v). In some embodiments, the formulation or composition is substantially free of a substance or component when the formulation or composition includes, includes about, or includes at most (or the method includes using, using about, or using at most) 0.1% w / v, 0.05% w / v, 0.045% w / v, 0.040% w / v, 0.035% w / v, 0.03% w / v, 0.025% w / v, 0.02% w / v, 0.015% w / v, 0.01% w / v, 0.005% w / v, 0.0025% w / v, or 0.001% w / v of the substance or component, optionally the substance or component included in the formulation or used in the method is in a range defined by any two of the preceding values (e.g., 0.001%-0.1% w / v, 0.001%-0.01% w / v, 0.001%-0.005% w / v, 0.01%-0.1% w / v, 0.005%-0.025% w / v, etc.).

[0058] “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.

[0059] 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.

[0060] 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 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 asassessed 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, (e.g. muscle 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.

[0061] 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 symptom of the disease or condition, and relates to a sufficient amount of therapeutic composition 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 therapeutic effect (e.g., cardioprotective effect, anti-inflammatory effect, anti-diabetic effect, etc.) 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.

[0062] 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.

[0063] Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-91 1910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632- 02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN- 10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081- 569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

[0064] 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.COMPOSITIONS

[0065] Provided herein are formulations or compositions that are configured for oral administration and comprise a therapeutic nucleic acid (e.g., an RNA (coding or noncoding RNA) molecule). As used herein, “composition” and “formulation” are used interchangeably, unless the context indicates otherwise. In several embodiments, the formulation or composition is a pharmaceutical or therapeutic composition. In some embodiments, the formulation or composition includes a pharmaceutically acceptable excipient. In some embodiments, the formulation or composition is a cell-free formulation or composition, e.g., the formulation or composition is substantially free of cells such as cardiosphere-derived cells (CDC). In several embodiments, the formulation or compositions is free or substantially free of cell-derived materials, such as exosomes or extracellular vesicles(EV). In some embodiments, the formulation does not include exosomes or extracellular vesicles (EV). In some embodiments, the formulation does not include exosomes or extracellular vesicles as a transfection reagent (e.g., includes less than an amount effective to promote delivery of a nucleic acid to a cell or tissue). In several embodiments, the formulation or composition comprises an artificial vesicle (e.g., a vesicle formed from lipids, such as cationic lipids). In several embodiments, the composition or formulation is substantially free of liposomes and / or lipids, such as cationic lipids, and / or lipid-based transfection reagents. In any formulation or composition herein, in some embodiments, the formulation or composition is an EV-free formulation or composition. In any formulation or composition herein, in some embodiments, the formulation or composition is an EV-free, liposome-free formulation or composition.

[0066] Provided herein is a liposome-free formulation (e.g., liposome-free therapeutic formulation) for oral delivery of a nucleic acid, comprising: a nucleic acid; at least one casein protein; and a chitosan. As used herein “liposome-free” denotes a formulation or composition that is at least substantially free of liposomes. In some embodiments, a liposome- free formulation or composition is free of or is substantially free of a lipid reagent (e.g., a cationic lipid). In some embodiments, a liposome-free formulation or composition is free of or is substantially free of a transfection reagent (e.g., lipid transfection reagent, such as a cationic lipid transfection reagent). In some embodiments, a liposome-free formulation or composition is free of or is substantially free of lipid additives. In some embodiments, a liposome-free formulation or composition is prepared without the need for prior formation of lipid nanoparticles. In some embodiments, liposomes are not present in detectable amounts in a liposome-free composition or formulation. In some embodiments, the liposome-free formulation does not include a cationic lipid as a transfection reagent (e.g., includes less than an amount effective to promote delivery of a nucleic acid to a cell or tissue). Examples of cationic lipid transfection reagents include, without limitation, N-[l-(2,3-dioleyloxy)propyl]- N,N,N-trimethylammonium chloride or “DOTMA”. DOTMA can be formulated alone or can be combined with the neutral lipid, dioleoylphosphatidyl-ethanolamine or “DOPE” or other cationic or non-cationic lipids into a transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells. Other cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or “DOGS,” 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propanaminium or “DOSPA”, 1 ,2- Dioleoyl-3 -Dimethylammonium -Propane or “DODAP,” l,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP.” Cationic lipids can also include 1,2-distearyloxy- N,N-dimethyl-3 -aminopropane or “DSDMA”, l,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA,” l,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA,” 1,2- dilinolenyloxy-N,N-dimethyl-3-aminopropane or “DLenDMA,” N-dioleyl-N,N- dimethylammonium chloride or “DODAC,” N,N-distearyl-N,N-dimethylammonium bromide or “DDAB,” N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide or “DMRIE,” 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis- 9,12-octadecadienoxy)propane or “CLinDMA,” 2-[5'-(cholest-5-en-3-beta-oxy)-3'- oxapentoxy)-3-dimethy l-l-(cis,cis-9', 1-2 '-octadecadi enoxy)propane or “CpLinDMA,” N,N- dimethyl-3,4-dioleyloxybenzylamine or “DMOBA,” l,2-N,N'-dioleylcarbamyl-3- dimethylaminopropane or “DOcarbDAP,” 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP,” l,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP,” 1,2- Dilinoleoylcarbamyl-3 -dimethylaminopropane or “DLinCDAP,” 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane or “DLin-K-DMA,” 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane or “DLin-K-XTC2-DMA,” and 2-(2,2-di((9Z,12Z)- octadeca-9,12-dien-l-yl)-l,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA), or mixtures thereof. Examples of cationic lipids include, without limitation, DLin-DMA, DLin- D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids.

[0067] Also provided is a therapeutic formulation for oral delivery of a nucleic acid, comprising: a nucleic acid; at least one casein protein; and a chitosan, wherein the formulation is substantially free of cationic lipids (e.g., substantially free of cationic lipids used as a transfection reagent). Also provided is a therapeutic formulation for oral delivery of a nucleic acid, comprising: a nucleic acid; at least one casein protein; and a chitosan, wherein the formulation (e.g., liposome-free formulation) does not comprise a cationic lipid as a transfection reagent.

[0068] In some embodiments, the formulation (e.g., liposome-free formulation) does not comprise a PEGylated cationic lipid. In some embodiments, the formulation (e.g., liposome-free formulation) does not comprise a liposome-based transfection reagent. In some embodiments, the formulation (e.g., liposome-free formulation) does not include Dharmafect®or Lipofectamine® (or any other cationic lipid typically used as a transfection reagent). In some embodiments, the formulation includes less than 0.1 microliters of a cationic lipid (e.g., Dharmafect® or Lipofectamine®) for each microgram of the nucleic acid in the formulation. In some embodiments, the formulation includes less than 0.1, 0.05, 0.02, 0.01, 0.005, 0.002, or 0.001 microliters or less of a cationic lipid (e.g., Dharmafect® or Lipofectamine®) for each microgram of the nucleic acid in the formulation, or comprises no cationic lipid. In some embodiments, the formulation includes less than 50 mole percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 40 mole percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 30 mole percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 20 mole percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 10 mole percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 5 mole percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 4 mole percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 3 mole percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 2 mole percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 1 mole percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.1 mole percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.01 mole percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.001 mole percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.0001 mole percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments,the formulation includes, includes about, or includes less than 0.00001 mole percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.000001 mole percent of a cationic lipid relative to the nucleic acid in the formulation.

[0069] In some embodiments, the formulation includes, includes about, or includes less than 50 weight percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 40 weight percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 30 weight percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 20 weight percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 10 weight percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 5 weight percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 4 weight percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 3 weight percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 2 weight percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 1 weight percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.1 weight percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.01 weight percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.001 weight percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.0001 weight percent of a cationic lipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.00001 weight percent of a cationiclipid relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.000001 weight percent of a cationic lipid relative to the nucleic acid in the formulation.

[0070] In some embodiments, the formulation includes less than 50 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 40 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 30 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 20 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 10 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 5 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 4 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 3 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 2 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 1 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.1 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.01 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.001 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.0001 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In someembodiments, the formulation includes, includes about, or includes less than 0.00001 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.000001 mole percent of a lipid transfection reagent relative to the nucleic acid in the formulation.

[0071] In some embodiments, the formulation includes, includes about, or includes less than 50 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 40 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 30 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 20 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 10 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 5 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 4 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 3 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 2 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 1 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.1 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.01 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.001 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes lessthan 0.0001 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.00001 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation. In some embodiments, the formulation includes, includes about, or includes less than 0.000001 weight percent of a lipid transfection reagent relative to the nucleic acid in the formulation.

[0072] In some embodiments, the formulation or composition includes casein, e.g., a casein micelle. As used herein, “casein,” “casein protein(s),” and “casein phosphoprotein(s)” are used interchangeably unless the context indicates otherwise. In some embodiments, the formulation or composition includes one or more of phosphoproteins: alpha si casein, alpha s2 casein, beta casein, and kappa casein. In some embodiments, the formulation or composition includes at least an a-sl casein subunit. In some embodiments, the formulation or composition includes two or more, three or more, or all four phosphoproteins: alpha si casein, alpha s2 casein, beta casein, and kappa casein. The casein phosphoproteins may be present in the formulation or 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. As used herein, the amount of casein relative to the formulation (e.g., in precent weight per volume) denotes the total amount of casein phosphoproteins (a-sl casein subunit, a-s2 casein subunit, P casein subunit, and / or K casein subunit) as a percent (weight by volume) of the formulation. In some embodiments, the at least one casein protein (e g., casein phosphoproteins) is / are present in the formulation or composition at 0.5%-10% (weight by volume). In some embodiments, the at least one casein protein (e.g., casein phosphoproteins) is / are present in the formulation or composition at 0.5%- 5% (weight by volume). In some embodiments, the at least one casein protein is collectively present in the formulation or composition at, at about, or at no less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%. 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 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% or more, and optionally the at least one casein protein is collectively present at a percentage in a range defined by any two of the preceding values (e.g., 0.1-10%, 0.2-10%, 0.5- 10%, 0.5%-5%, 0.3-8%, 1.5-6%, etc.). In some embodiments, the at least one casein protein comprises a mixture of an a-sl casein subunit, an a-s2 casein subunit, a casein subunit, anda K casein subunit, wherein the casein subunits are collectively present in an amount in a range of 0.5% to 5% of the formulation by weight per volume. In some embodiments, the casein phosphoproteins (e.g., a mixture of an a-sl casein subunit, an a-s2 casein subunit, a casein subunit, and a K casein subunit) are present in the composition at 1-5 (weight by volume) of the formulation. In some embodiments, the casein phosphoproteins (e.g., a mixture of an a-sl casein subunit, an a-s2 casein subunit, a 0 casein subunit, and a K casein subunit) are present in the composition at 1-3% (weight by volume) of the formulation. In some embodiments, the casein phosphoproteins (e.g., a mixture of an a-sl casein subunit, an a-s2 casein subunit, a 0 casein subunit, and a K casein subunit) are present in the composition collectively at 1-2% (weight by volume) of the formulation. In some embodiments, the casein phosphoproteins (e.g., a mixture of an a-sl casein subunit, an a-s2 casein subunit, a 0 casein subunit, and a K casein subunit) are present in the composition collectively at 1.2-1.6% (weight by volume) of the formulation. In some embodiments, the casein phosphoproteins (e.g., a mixture of an a-sl casein subunit, an a-s2 casein subunit, a 0 casein subunit, and a K casein subunit) are present in the composition collectively at or at about 1.4% (weight by volume) of the formulation. In some embodiments, the casein phosphoproteins (e.g., a mixture of an a-sl casein subunit, an a-s2 casein subunit, a 0 casein subunit, and a K casein subunit) are present in the composition at 2-3% (weight by volume) of the formulation. In some embodiments, the casein phosphoproteins (e g., a mixture of an a-sl casein subunit, an a-s2 casein subunit, a 0 casein subunit, and a K casein subunit) are present in the composition at 2.2-2.8% (weight by volume) of the formulation. In some embodiments, the casein phosphoproteins (e.g., a mixture of an a-sl casein subunit, an a-s2 casein subunit, a 0 casein subunit, and a K casein subunit) are present in the composition at 3-5% (weight by volume) of the formulation. In some embodiments, the casein phosphoproteins are present in the composition at about 5-10 % (weight by volume). Unless otherwise indicated, the use of the term weight per volume or weight by volume assumes that 1 g / lOOmL = 1% w / v. In some embodiments, the casein phosphoproteins are collectively present in the composition at about 8 % (weight by volume).

[0073] In some embodiments, the formulation includes a ratio of the percent w / v of nucleic acid in the formulation to percent w / v of casein phosphoproteins (e.g., a mixture of an a-sl casein subunit, an a-s2 casein subunit, a 0 casein subunit, and a K casein subunit) inthe formulation of, of about, or of at least 1 : 100, 1 :200, 1 :300, 1 :400, 1 :500, 1 : 1000, 1 :2000, 1 :5000, 1: 10,000, 1 : 12,000, 1:15,000, or optionally the ratio of the percent w / v of nucleic acid in the formulation to percent w / v of casein phosphoproteins (e.g., a mixture of an a-sl casein subunit, an a-s2 casein subunit, a 0 casein subunit, and a K casein subunit) in the formulation that is in a range defined by any two of the preceding values (e.g., 1 : 100-1 : 15,000, 1 :200- 1 : 12,000, 1 : 100-1 :10,000, etc.). In some embodiments, the formulation includes a ratio of the percent w / v of nucleic acid in the formulation to percent w / v of casein phosphoproteins (e.g., a mixture of an a-sl casein subunit, an a-s2 casein subunit, a 0 casein subunit, and a K casein subunit) in the formulation of 1 :100-1 : 15,000. In some embodiments, the formulation includes a ratio of the percent w / v of nucleic acid in the formulation to percent w / v of casein phosphoproteins (e.g., a mixture of an a-sl casein subunit, an a-s2 casein subunit, a 0 casein subunit, and a K casein subunit) in the formulation of at least 1 : 15,000. In some embodiments, the formulation includes a ratio of the percent w / v of nucleic acid in the formulation to percent w / v of casein phosphoproteins (e.g., a mixture of an a-sl casein subunit, an a-s2 casein subunit, a 0 casein subunit, and a K casein subunit) in the formulation of at least 1 :2000. In some embodiments, the formulation includes a ratio of the percent w / v of nucleic acid in the formulation to percent w / v of casein phosphoproteins (e.g., a mixture of an a-sl casein subunit, an a-s2 casein subunit, a 0 casein subunit, and a K casein subunit) in the formulation of at least 1 :200.

[0074] 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 (e.g., with or without 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, the formulation or composition does not contain any other bovine milk protein other than the casein phosphoproteins. In some embodiments, a composition or formulation, e.g., pharmaceutical composition or therapeutic formulation, of the present disclosure formulated with casein, as provided herein, is suitable for oral administration to the subject. Without being bound by theory, the casein phosphoproteins in the composition are thought to increase thebioavailability of orally administered nucleic acids of the present disclosure (e.g., with or without EV and / or liposomes and their nucleic acid cargo).

[0075] In some embodiments, alpha si casein is a phosphoprotein associated with the gene name CSN1S1. The alpha si casein can be a CSN1S1 phosphoprotein from any suitable mammal. In some embodiments, the alpha si casein is bovine (Gene ID: 282208), porcine (Gene ID: 445514), equine (Gene ID: 100033982), ovine (Gene ID: 443382), caprine (Gene ID: 100750242), cameline (Gene ID: 105090954), or human (Gene ID: 1446). In some embodiments, the alpha si casein is a non-human alpha si casein. In some embodiments, the alpha si casein is a polypeptide having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or about 100% identical to the sequence set forth in SEQ ID NO: 17.

[0076] In some embodiments, the composition includes any suitable amount of alpha si casein. In some embodiments, the composition includes the alpha si casein in an amount, by weight, between about 0% to about 50%, e.g., between about 5% to about 50%, between about 10% to about 50%, between about 15% to about 45%, between about 20% to about 45%, including between about 25% to about 40%, of the phosphoprotein mass in the composition. In some embodiments, the composition includes the alpha si casein in an amount, by weight, of about 0%, 5%, 10%, 15%, 20%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or an amount within a range defined by any two of the preceding values of the phosphoprotein mass in the composition.

[0077] In some embodiments, the alpha s2 casein is a phosphoprotein associated with the gene name CSN1 S2. The alpha s2 casein can be a CSN1 S2 phosphoprotein from any suitable mammal. In some embodiments, the alpha s2 casein is bovine (Gene ID: 282209), porcine (Gene ID: 445515), equine (Gene ID: 100327035), ovine (Gene ID: 443383), caprine (Gene ID: 100861229), or cameline (Gene ID: 105090951). In some embodiments, the alpha s2 casein is a polypeptide having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or about 100% identical to the sequence set forth in SEQ ID NO: 18.

[0078] The composition can include any suitable amount of alpha s2 casein. In some embodiments, the composition includes the alpha s2 casein in an amount, by weight, between about 0% to about 20%, e.g., between about 2% to about 18%, between about 3% toabout 18%, between about 4% to about 17%, between about 5% to about 16%, including between about 5% to about 15%, of the phosphoprotein mass in the composition. In some embodiments, the composition includes the alpha s2 casein in an amount, by weight, of about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 18%, 20%, or an amount within a range defined by any two of the preceding values of the phosphoprotein mass in the composition.

[0079] In some embodiments, the beta casein is a phosphoprotein associated with the gene name CSN2. The beta casein can be a CSN2 phosphoprotein from any suitable mammal. In some embodiments, the beta casein is bovine (Gene ID: 281099), porcine (Gene ID: 404088), equine (Gene ID: 100033903), ovine (Gene ID: 443391), caprine (Gene ID: 100860784), cameline (Gene ID: 105080412), or human (Gene ID: 1447). In some embodiments, the beta casein is a non-human beta casein. In some embodiments, the beta casein is a polypeptide having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or about 100% identical to the sequence set forth in SEQ ID NO: 19 or 20.

[0080] The composition can include any suitable amount of beta casein. In some embodiments, the composition includes the beta casein in an amount, by weight, between about 0% to about 50%, e.g., between about 5% to about 50%, between about 10% to about 50%, between about 15% to about 45%, between about 20% to about 45%, including between about 25% to about 40%, of the phosphoprotein mass in the composition. In some embodiments, the composition includes the beta casein in an amount, by weight, of about 0%, 5%, 10%, 15%, 20%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or an amount within a range defined by any two of the preceding values of the phosphoprotein mass in the composition.

[0081] In some embodiments, the kappa casein is a phosphoprotein associated with the gene name CSN3. The beta casein can be a CSN3 phosphoprotein from any suitable mammal. In some embodiments, the kappa casein is bovine (Gene ID: 281728), porcine (Gene ID: 445511), equine (Gene ID: 100033983), ovine (Gene ID: 443394), caprine (Gene ID: 100861231), cameline (Gene IDs: 105080408 or 105090949), or human (Gene ID: 1448). In some embodiments, the kappa casein is a non-human kappa casein. In some embodiments, the kappa casein is a polypeptide having an amino acid sequence at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or about 100% identical to the sequence set forth in SEQ ID NO: 21.

[0082] The composition can include any suitable amount of kappa casein. In some embodiments, the composition includes the kappa casein in an amount, by weight, between about 0% to about 20%, e.g., between about 2% to about 18%, between about 3% to about 18%, between about 4% to about 17%, between about 5% to about 16%, including between about 5% to about 15%, of the phosphoprotein mass in the composition. In some embodiments, the composition includes the kappa casein in an amount, by weight, of about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 18%, 20%, or an amount within a range defined by any two of the preceding values of the phosphoprotein mass in the composition.

[0083] Combinations of caseins from different species are used, in some embodiments. For example, in several embodiments, one or more human casein is used in combination with one or more bovine casein. Ratios of caseins are used in some embodiments, for example a 3: 1 :3: 1 ratio of alpha SI casein:alpha s2 caseimbeta casein:kappa casein. Different ratios may be used in some embodiments, for example 4: 1:4: 1, 2: 1 :2: 1, or 1 : 1 : 1 : 1. Ratios may also be used between any two given caseins in a composition, ranging from 1 :1, 2:1, 3: 1, 4: 1, 5: 1, 10: 1, 1 :5, 1 :4, 1 :3, 1 :2, etc.

[0084] Any suitable total amount of the phosphoproteins may be present in the composition. In some embodiments, the phosphoproteins are present in an amount between 5% to about 10%, e.g., about 6% to about 10%, about 6% to about 9%, including about 6% to about 8%, (weight by volume) of the composition. In some embodiments, the phosphoproteins are present in an amount of about 5%, 6%, 7%, 8%, 9%, 10%, or an amount within a range defined by any two of the preceding values, (weight by volume) of the composition.

[0085] In some embodiments, one or more of the casein phosphoproteins are nonhuman casein phosphoproteins. In some embodiments, the exosomes and at least one of the casein phosphoproteins are from different species. In some embodiments, the exosomes are human exosomes, and one or more of the casein phosphoproteins are non-human casein phosphoproteins. In some embodiments, the exosomes are human exosomes, and one or more of the casein phosphoproteins are bovine (or ovine, porcine, caprine, cameline, or equine) casein phosphoproteins.

[0086] In some embodiments, the composition includes micellar structures formed by at least a portion of the casein phosphoproteins. In some embodiments, the casein micelles are substantially spherical. In some embodiments, a casein micelle in the composition has an average diameter (as measured per micelle) of about 40 nm, about 50 nm, about 60 nm, about 70 nm, about80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm or more, or an average diameter within a range defined by any two of the preceding values. In some embodiments, a casein micelle in the composition has an average diameter (as measured per micelle) in a range from about 40 nm to about 500 nm, e.g., from about 40 nm to about 400 nm, from about 50 nm to about 300 nm, from about 60 nm to about 250 nm, from about 70 nm to about 250 nm, from about 80 nm to about 200 nm, including from about 90 nm to about 150 nm. The casein micelles of the present composition are generally not precipitated or in gel form.

[0087] In some embodiments, the composition includes one or more colloidal minerals (e.g., minerals in suspension). In several embodiments, a complex (e.g., two or more) minerals are used as a colloidal mineral complex. The colloidal mineral complex can include any suitable mineral compounds and / or their salts. In some embodiments, the colloidal mineral complex includes, without limitation, one or more of calcium, magnesium, inorganic phosphate, citrate, sodium, potassium, and chloride, or their respective salts. In some embodiments, the colloidal mineral complex is present in an amount between about 2% and about 15%, e.g., about 2% to about 12%, about 5% to about 10%, about 5% to about 9%, including about 6% to about 9% (by weight) of the phosphoprotein mass in the composition. In some embodiments, the colloidal mineral complex is present in an amount of about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or an amount within a range defined by any two of the preceding percentages.

[0088] In some embodiments, the composition includes two, three, or all four phosphoproteins selected from alpha si casein, alpha s2 casein, beta casein, and kappa casein. In some embodiments, the amount of a phosphoprotein in the composition depends on the amount of one or more other phosphoprotein present in the composition.

[0089] In several embodiments, a formulation or composition for enhancing the oral bioavailability of a therapeutic nucleic acid of the present disclosure comprises at leasttwo phosphoproteins selected from alpha si casein, alpha s2 casein, beta casein, and kappa casein, where the phosphoproteins are present in an amount between 0.25%-7%, 0.5%-5%, or about 5% to about 10% (weight by volume) of the composition, in a physiologically compatible excipient. In some embodiments, the formulation or composition contains alpha si casein and beta casein in about equal amounts, and alpha s2 casein and kappa casein in lesser amounts than the alpha si casein and beta casein. In some embodiments, the formulation or composition contains alpha si casein, alpha s2 casein, beta casein, and kappa casein in proportion to each other as they are found typically in bovine milk. In some embodiments, the formulation or composition does not contain any other bovine milk protein other than the casein phosphoproteins. In several embodiments, the formulation or composition includes the alpha si casein in an amount between about 0% to about 50% (e.g., about 10% to about 45%, about 20% to about 40%, about 25% to about 40%, about including 30% to about 40%) (by weight), the alpha s2 casein in an amount between about 0% to about 20% (e.g., about 5% to about 15%, about 7% to about 12%, including about 8% to about 12%) (by weight), the beta casein in an amount between about 0% to about 50% (e.g., about 10% to about 45%, about 20% to about 40%, about 25% to about 40%, about including 30% to about 40%) (by weight), and the kappa casein in an amount between about 0% to about 20% (e.g., about 5% to about 18%, about 8% to about 18%, including about 10% to about 15%) (by weight) of the phosphoprotein mass in the composition. In some embodiments, the formulation or composition contains (by weight per volume): 30-40% (e.g., about 36%) alpha si casein, 5-15% (e.g., about 10%) alpha s2 casein, 30-40% (e.g., about 34%) beta casein, and 5-15% (e.g., about 12%) kappa casein. In some embodiments, the formulation or composition contains (by weight per volume): 30- 40% (e.g., about 36%) alpha si casein, 5-15% (e.g., about 10%) alpha s2 casein, 30-40% (e.g., about 34%) beta casein, and 5-15% (e.g., about 12%) kappa casein, where the total amount of casein phosphoproteins is in a range of 0.5% to 5% of the formulation or composition, weight by volume. The present compositions can provide for enhanced oral bioavailability of therapeutic nucleic acids, such as non-coding RNA (such as and without limitation, TY1, miR- 1246, uREXl, TT1, TT8, and yREX3, etc.). In several embodiments, the therapeutic nucleic acid comprises RNA, such as, but not limited to mRNA and non-coding RNA (e.g., miRNA, IncRNA). In some embodiments, the payload is a synthetic molecule, e.g., a small molecule or drug.

[0090] In some embodiments, the formulation or composition includes chitosan. In some embodiments, the formulation or composition includes casein and chitosan. In some embodiments, the formulation or composition includes a casein-chitosan complex. In some embodiments, the isolated nucleic acid in the formulation or composition is encapsulated in a casein-chitosan complex. In some embodiments, the chitosan is medium molecular weight chitosan. In some embodiments, the chitosan is or includes deacetylated chitin, poly(D- glucosamine). In some embodiments, the medium molecular weight chitosan has a molecular weight in the range of 150,000-350,000 Da. In some embodiments, the medium molecular weight chitosan has a molecular weight of, of about, or of at least 150,000 Da, 175,000 Da, 200,000 Da, 225,000 Da, 250,000 Da, 275,000 Da, 300,000 Da, 325,000 Da, 350,000 Da, or more, and optionally, the medium molecular weight chitosan has a molecular weight in a range defined by any two of the preceding values (e g., 150,000-350,000 Da, 200,000-300,000 Da, 175,000-325,000 Da, etc.). In some embodiments, the chitosan is present in the formulation at an amount in a range of 0.001% to 1% of the formulation by weight per volume. In some embodiments, the chitosan is present in the formulation (by weight per volume) at, at about, or at no less than 0.001%, 0.002%, 0.005%, 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or more, and optionally the chitosan is present in the formulation at a percentage (by weight per volume) in a range defined by any two of the preceding values (e.g., 0.001-1%, 0.01-1%, 0.05-0.5%, 0.005-0.3%, etc.). In some embodiments, the chitosan is present in the formulation at an amount in a range of 0.01% to 0.1% of the formulation by weight per volume. In some embodiments, the chitosan is present in the formulation at an amount in a range of 0.06% to 0.09% of the formulation by weight per volume. In some embodiments, the chitosan is present in the formulation at or at about 0.07% of the formulation by weight per volume.

[0091] In some embodiments, the formulation includes a ratio of the percent w / v of nucleic acid in the formulation to percent w / v of chitosan (e.g., medium molecular weight chitosan) in the formulation of, of about, or of at least 1 :5, 1 : 10, 1:20, 1:50, 1: 100, 1 :200, 1 :500, 1 : 1000, or optionally the ratio of the percent w / v of nucleic acid in the formulation to percent w / v of chitosan (e.g., medium molecular weight chitosan) in the formulation that is in a range defined by any two of the preceding values (e.g., 1 :5-1 : 1000, 1: 10-1: 1000, 1 :5-1 :500, etc.). In some embodiments, the formulation includes a ratio of the percent w / v of nucleic acid in theformulation to percent w / v of chitosan (e.g., medium molecular weight chitosan) in the formulation of 1 :5-1 : 1000. In some embodiments, the formulation includes a ratio of the percent w / v of nucleic acid in the formulation to percent w / v of chitosan (e.g., medium molecular weight chitosan) in the formulation of at least 1 : 1000. In some embodiments, the formulation includes a ratio of the percent w / v of nucleic acid in the formulation to percent w / v of chitosan (e.g., medium molecular weight chitosan) in the formulation of at least 1 : 100. In some embodiments, the formulation includes a ratio of the percent w / v of nucleic acid in the formulation to percent w / v of chitosan (e.g., medium molecular weight chitosan) in the formulation of at least 1 : 10.

[0092] In several embodiments, the composition or formulation comprises caseinchitosan coated lipid micelles, where the casein phosphoproteins are present in the composition in suitable amounts (e.g., suitable total amount of phosphoprotein mass in the composition, suitable proportions of phosphoproteins relative to each other), as described herein.

[0093] In some embodiments, the formulation or composition includes a therapeutically effective amount of the nucleic acid. In some embodiments, the formulation or composition includes the nucleic acid in an amount in a range of 0.0001 to 0.01% of the formulation by weight per volume. In some embodiments, the formulation or composition includes the nucleic acid in an amount in a range of 0.0001 to 0.01% of the formulation by weight per volume, wherein the at least one casein protein comprises at least an a-sl casein subunit and wherein the at least one casein protein is present in an amount in a range of 0.5 to 5% of the formulation by weight per volume, and wherein the chitosan is present in an amount in a range of 0.001 to 1% of the formulation by weight per volume. In some embodiments, the at least one casein protein includes at least one of alpha si casein, alpha s2 casein, beta casein, and kappa casein. In some embodiments, the at least one casein protein includes alpha si casein, alpha s2 casein, beta casein, and kappa casein. In some embodiments, the nucleic acid is present at, at about, or at no less than 0.0001%, 0.0002%, 0.0005%, 0.001%, 0.002%, 0.005%, 0.01%, 0.02%, 0.05%, 0.1% or greater of the formulation by weight per volume, or optionally the RNA or the chemically modified variant thereof is present in an amount in a range defined by any two of the preceding values (e.g., 0.0001-0.1%, 0.0001-0.01%, 0.0005- 0.05%, 0.001-0.01%, etc.). In some embodiments, the nucleic acid comprises a ribonucleic acid (RNA) (or a chemically modified variant thereof) and wherein the RNA (or the chemicallymodified variant thereof) is present in an amount ranging between about 0.0001 and 0.01% (or in a range of 0.0001 % to 0.01%) of the formulation by weight per volume, wherein the at least one casein protein comprises at least an a-sl casein subunit and wherein the at least one casein protein is present in an amount ranging between about 0.5 and 5% of the formulation by weight per volume, and wherein the chitosan is present in an amount ranging between about 0.001 and 1% of the formulation by weight per volume. In some embodiments, the at least one casein protein includes at least one of alpha si casein, alpha s2 casein, beta casein, and kappa casein. In some embodiments, the at least one casein protein includes alpha si casein, alpha s2 casein, beta casein, and kappa casein. In some embodiments, the RNA or the chemically modified variant thereof is present in an amount in a range of 0.0001% to 0.01% of the formulation by weight per volume. In some embodiments, the RNA or the chemically modified variant thereof is present at, at about, or at no less than 0.0001%, 0.0002%, 0.0005%, 0.001%, 0.002%, 0.005%, 0.01%, 0.02%, 0.05%, 0.1% or greater of the formulation by weight per volume, or optionally the RNA or the chemically modified variant thereof is present in an amount in a range defined by any two of the preceding values (e.g., 0.0001-0.1%, 0.0001-0.01%, 0.0005- 0.05%, 0.001-0.01%, etc.). In some embodiments, the formulation includes a therapeutically effective amount of the RNA or the chemically modified variant thereof.

[0094] In some embodiments, the formulation has an acidic pH. In some embodiments, the formulation has a pH of, of about, or of at most 6. In some embodiments, the formulation has a pH of 5, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, or optionally the formulation has a pH in a range defined by any two of the preceding values (e.g., 5-6.5, 5.5- 6.3, 5.5-6.5, 5.9-6.1, etc.). In some embodiments, the formulation includes an acid, e.g., acetic acid. In some embodiments, the acid is selected from acetic acid, phosphoric acid and citric acid. In some embodiments, the liposome-free formulation includes an acid, e.g., acetic acid. In some embodiments, the acid is present in an amount in a range of 0.001 to 1% of the formulation by weight per volume. In some embodiments, the acid is present at, at about, or at no less than 0.001%, 0.002%, 0.005%, 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% of the formulation by weight per volume, or optionally the acid is present in an amount in a range defined by any two of the preceding amounts (e.g., 0.001-1%, 0.005-0.5%, 0.002-0.8%, 0.01-1%, etc.). In some embodiments, the acid is present in an amount ranging between about 0.001 and 1% of the formulation by volume and wherethe acid is selected from acetic acid, phosphoric acid and citric acid. In some embodiments, the liposome-free formulation includes acetic acid, wherein the acetic acid is present in an amount ranging between about 0.01 and 1% of the formulation by weight per volume. In some embodiments, the liposome-free formulation includes acetic acid, wherein the acetic acid is present at or at about 0.04% of the formulation by weight per volume.

[0095] In some embodiments, the nucleic acid comprises a ribonucleic acid (RNA), or a chemically modified variant thereof. In some embodiments, the chemically modified variant includes one or more chemical modification to a backbone sugar and / or phosphate relative to a naturally occurring structure (e.g., nucleosides linked by phosphate groups). In some embodiments, the nucleic acid comprises a deoxyribonucleic acid (DNA). In some embodiments, the nucleic acid comprises a nucleotide sequence of 20-40 nucleotides in length. In some embodiments, the nucleic acid comprises a nucleotide sequence of 19-35 nucleotides in length. In some embodiments, the nucleic acid comprises a nucleotide sequence of, of about, of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50 nucleotides or more, or optionally the nucleotide sequence is in a range defined by any two of the preceding values (e.g., 15-50 nucleotides long, 19-35 nucleotides long, 20-40 nucleotides long, 20-45 nucleotides long, etc.). In some embodiments, the nucleic acid comprises a nucleotide sequence at most 35 or 36 nucleotides long. In some embodiments, the nucleic acid is at least partially single-stranded. In some embodiments, the nucleic acid is substantially single-stranded. In some embodiments, the nucleic acid is single-stranded. In some embodiments, the nucleic acid sequence is “single- stranded” when the formulation or composition does not include another nucleic acid molecule that is at least partially or fully complementary to the single-stranded nucleic acid. In some embodiments, the nucleic acid sequence is “single-stranded” even when a portion of the nucleic acid can anneal to itself at another portion (e.g., can form a stem-loop structure). In some embodiments, the formulation does not comprise a second nucleic acid that is at least partially complementary to the nucleic acid. In some embodiments, the formulation does not comprise a second nucleic acid that is complementary to the nucleic acid.

[0096] The formulation includes any suitable nucleic acid, such as any suitable therapeutic nucleic acid. In some embodiments, the thenucleic acid is a synthetic or recombinantly produced nucleic acid. In some embodiments, the nucleic acid acts on or targetsa macrophage. In some embodiments, the nucleic acid acts on or targets a macrophage to modulate an anti-inflammatory activity of the macrophage. In some embodiments, the nucleic acid acts on or targets a macrophage to modulate an anti-inflammatory activity of the macrophage at a site of inflammation (e.g., cardiac and / or pulmonary inflammation). In some embodiments, the nucleic acid increases expression of one or more anti-inflammatory cytokines (e.g., IL-10). In some embodiments, the nucleic acid reduces expression of one or more inflammatory cytokines (e.g., IL-6 and / or IL-ip). In some embodiments, the nucleic acid modulates expression of one or more of IL-10, IL-la, IL-ip, IL-6, IL-12, TGFp, NFKB, TNF- a, CCL3, IFN-gamma, CD40, and / or VGEF-A. In some embodiments, the nucleic acid is DNA (with or without one or more chemically modified residues). In some embodiments, the nucleic acid is RNA (with or without one or more chemically modified residues). Non-limiting examples of suitable nucleic acids for use in formulations of the present disclosure are listed in Table 0.4. In some embodiments, the nucleic acid comprises a nucleotide sequence of any one of the nucleotide sequences provided in Table 0.4 or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA. In some embodiments, the nucleic acid comprises a nucleotide sequence at least 80, 85, 90, 95, 96, 97, 89, 99, or 100 % identical to any one of the nucleotide sequences provided in Table 0.4. In some embodiments, the nucleic acid includes a nucleotide sequence having no more than 1, 2, 3, 4, or 5 mutations in any one of the nucleotide sequences provided in Table 0.4. In some embodiments, the nucleic acid includes a nucleotide sequence having no more than 1, 2, 3, 4, or 5 substitutions in any one of the nucleotide sequences provided in Table 0.4. In some embodiments, the nucleic acid consists essentially of or consists of a nucleotide sequence of any one of the nucleotide sequences provided in Table 0.4 or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA. In some embodiments, the nucleic acid consists essentially of or consists of a nucleotide sequence at least 80, 85, 90, 95, 96, 97, 89, 99, or 100 % identical to any one of the nucleotide sequences provided in Table 0.4. In some embodiments, the nucleic acid consists essentially of or consists of a nucleotide sequence having no more than 1, 2, 3, 4, or 5 mutations in any one of the nucleotide sequences provided in Table 0.4. In some embodiments, the nucleic acid consists essentially of or consists of a nucleotide sequence having no more than 1, 2, 3, 4, or 5 substitutions in any one of the nucleotide sequences provided in Table 0.4.Table 0.4LNA residues are indicated with underlining.

[0097] The nucleic acid can be any of the nucleic acids described herein (e.g., TY1). In some embodiments, the nucleic acid includes a nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12) or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA, and wherein the nucleic acid is at most 30 nt long. In some embodiments, the nucleic acid is TY1 , as described herein. Suitable, nonlimiting examples of nucleic acids, such as TY1, are described in PCT publication No. WO 2023 / 278799, which is incorporated herein by reference in its entirety.

[0098] In some embodiments, the nucleic acid includes a nucleotide sequence at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 98%, 99% identical to CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, the nucleic acid includes a nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12) with a sequence variation at up to 1, 2, 3, 4, or 5 positions in the nucleotide sequence. As used herein, a “position” within a nucleotide sequence or nucleic acid is defined relative to the 5’ end of the nucleotide sequence or nucleic acid. In some embodiments, the nucleotide sequence of the nucleic acid is CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12), or a sequence variant thereof. The nucleic acid can be any suitable length. In some embodiments, the nucleic acid is 24 nucleotides (nt) long. In some embodiments, the nucleicacid is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nt long, or longer. In some embodiments, the nucleic acid is at most 30 nt long. In some embodiments, the nucleic acid is 16-30 nt long, or 24-30 nt long. In some embodiments, the nucleic acid is 24 nucleotides long.

[0099] A nucleic acid of the present disclosure can be single stranded or double stranded (e.g., RNA / DNA hybrid). In some embodiments, the nucleic acid is single stranded.

[0100] An isolated nucleic acid of the present disclosure in some embodiments includes one or more chemically modified nucleotides, e g., nucleotides with a modified backbone. In general, the chemical modification(s) is one that substantially preserves or enhances the therapeutic potency of the nucleic acid. Any suitable number of nucleotides of the nucleic acid can be chemically modified. In some embodiments, the nucleic acid includes 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more chemically modified nucleotides in the nucleotide sequence. In some embodiments, the nucleic acid includes 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-25, or 1-30 chemically modified nucleotides in the nucleotide sequence. In some embodiments, the nucleic acid includes 1-10 chemically modified nucleotides in the nucleotide sequence. In some embodiments, the nucleic acid includes 8 chemically modified nucleotides in the nucleotide sequence. In some embodiments, the nucleic acid includes 6 chemically modified nucleotides in the nucleotide sequence.

[0101] The isolated nucleic acid, in some embodiments, can include any suitable chemical modification. In some embodiments, the chemical modification is a backbone modification, e.g., modification of the sugar / phosphate backbone. In some embodiments, the chemical modification is a backbone sugar modification. In some embodiments, the chemically modified nucleotide includes a LNA. In some embodiments, the chemical modification includes the introduction of a phosphorothioate group as linker between nucleotides. Suitable backbone modifications of the chemically modified nucleotides include, without limitation, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. In some embodiments, the chemical modification is a base modification.

[0102] The chemically modified nucleotides can be distributed along the isolated nucleic acid in any suitable manner. In some embodiments, the chemically modified nucleotides are within the nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12), or sequence variant thereof. In some embodiments, the nucleic acid includes at least one chemically modified nucleotide within the first half of the nucleic acid comprising the nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12), e g., the 5’ half of the nucleic acid. In some embodiments, the nucleic acid includes at least one chemically modified nucleotide within the second half of the nucleic acid comprising the nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12), e g., the 3’ half of the nucleic acid. In some embodiments, the nucleic acid includes at least one chemically modified nucleotide within the first half of the nucleic acid comprising the nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12), e g., the 5’ half of the nucleic acid, and at least one chemically modified nucleotide within the second half of the nucleic acid, e.g., the 3’ half of the nucleic acid. In some embodiments, the nucleic acid includes one or more chemically modified nucleotides within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides from the 5’ end of the nucleic acid comprising the nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, the nucleic acid includes one or more chemically modified nucleotides within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides from the 3’ end of the nucleic acid comprising the nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, no two chemically modified nucleotides are adjacent each other in the nucleic acid. In some embodiments, the nucleic acid includes 1, 1, 2, 2, 3, 3, 4, 4, 5, 5 chemically modified nucleotides within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides, respectively, from the 5’ end of the nucleic acid comprising the nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, the nucleic acid includes 1, 1, 2, 2, 3, 3, 4, 4, 5, 5 chemically modified nucleotides within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides, respectively, from the 3’ end of the nucleic acid comprising the nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, the nucleic acid includes the same number of chemically modified nucleotides in the 5’ half and 3’half of the nucleic acid comprising the nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12) In some embodiments, thenucleic acid includes 3 chemically modified nucleotides within 5 nucleotides from the 5’ end of the nucleic acid and / or 3 chemically modified nucleotides within 5 nucleotides from the 3’ end of the nucleic acid comprising the nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12).

[0103] In some embodiments, the chemically modified nucleotides are within the nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12), or sequence variant thereof. In some embodiments, the nucleic acid includes at least one chemically modified nucleotide within the first half of the nucleotide sequence comprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12), e g., the 5’ half of the nucleotide sequence. In some embodiments, the nucleic acid includes at least one chemically modified nucleotide within positions 1-12 of the nucleotide sequence comprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, the nucleic acid includes at least one chemically modified nucleotide within the second half of the nucleotide sequence comprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12), e.g., the 3’ half of the nucleotide sequence. In some embodiments, the nucleic acid includes at least one chemically modified nucleotide within positions 13-24 of the nucleotide sequence comprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, the nucleic acid includes at least one chemically modified nucleotide within positions 1-12 of the nucleotide sequence comprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12), and at least one chemically modified nucleotide within positions 13-24 of the nucleotide sequence. In some embodiments, the nucleic acid includes one or more chemically modified nucleotides within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides from the 5’ end of the nucleotide sequence comprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, the nucleic acid includes one or more chemically modified nucleotides within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides from the 3’ end of the nucleotide sequence comprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, no two chemically modified nucleotides are adjacent each other in the nucleotide sequence comprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, the nucleic acid includes 1, 1, 2, 2, 3, 3, 4, 4, 5, 5 chemically modified nucleotides within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides, respectively, from the 5’ end of the nucleotide sequencecomprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, the nucleic acid includes 1, 1, 2, 2, 3, 3, 4, 4, 5, 5 chemically modified nucleotides within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides, respectively, from the 3’ end of the nucleotide sequence comprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, the nucleic acid includes the same number of chemically modified nucleotides in the 5’ half and 3 ’half of the nucleotide sequence comprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, the nucleic acid includes 3 chemically modified nucleotides within 5 nucleotides from the 5’ end of the nucleotide sequence comprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12) and / or 3 chemically modified nucleotides within 5 nucleotides from the 3’ end of the nucleotide sequence. In some embodiments, the nucleic acid includes a different number of chemically modified nucleotides in the 5’ half and 3’half of the nucleotide sequence comprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, the nucleic acid includes a greater number of chemically modified nucleotides in the 3’ half than in the 5 ’half of the nucleotide sequence comprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, the nucleic acid includes 3 chemically modified nucleotides within 5 nucleotides from the 5’ end of the nucleotide sequence comprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12) and / or 3, 4, or 5 chemically modified nucleotides within 5 nucleotides from the 3’ end of the nucleotide sequence.

[0104] In some embodiments, the isolated nucleic acid includes a chemically modified nucleotide at one or more of positions 1, 3, 5, 20, 22 and 24 of the nucleotide sequence comprising CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12). In some embodiments, the isolated nucleic acid includes a chemically modified nucleotide at positions 1, 3, 5, 20, 22 and 24 of the nucleotide sequence. In some embodiments, the isolated nucleic acid has the nucleotide sequence CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12), or a sequence variant thereof, where one or more of positions 1, 3, 5, 20, 22, and 24 are chemically modified. In some embodiments, the chemically modified nucleotide(s) increases in vitro and / or in vivo stability of the nucleic acid. In some embodiments, the chemically modified nucleotide(s) increases therapeutic potency of the nucleic acid, e.g., for treating an inflammatory condition, cardiac injury, or muscular dystrophy.

[0105] The isolated nucleic acid in some embodiments includes one type, or two or more different types of chemically modified nucleotides. In some embodiments, the chemically modified nucleotide has a methylene bridge connecting the 2’-0 atom and the 4’- C atom of the nucleotide sugar ring to lock the conformation (Locked Nucleic Acid (LNA)). In some embodiments, the isolated nucleic acid includes the nucleotide sequence CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12), or a sequence variant thereof, where one or more of positions 1, 3, 5, 20, 22, and 24 are LNA. In some embodiments, the isolated nucleic acid has the nucleotide sequence CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12), or a sequence variant thereof, where one or more of positions 1, 3, 5, 20, 22, and 24 are LNA. In some embodiments, the isolated nucleic acid includes the nucleotide sequence CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 2), or a sequence variant thereof, where positions 1, 3, 5, 20, 22, and 24 are LNA. In some embodiments, the isolated nucleic acid has the nucleotide sequence CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 2), where positions 1, 3, 5, 20, 22, and 24 are LNA. In some embodiments, the nucleic acid is TY1, e.g., as set forth in SEQ ID NO:2.

[0106] In some embodiments, the nucleic acid includes a nucleotide sequence of AAUGGAUUUUUGGAGCAGG (SEQ ID NO: 26) or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA, and wherein the nucleic acid is 19-25 nt long. In some embodiments, the nucleic acid consists essentially of, or consists of the nucleotide sequence of AAUGGAUUUUUGGAGCAGG (SEQ ID NO: 26), where the nucleic acid is RNA. In some embodiments, the nucleic acid is miR-1246.

[0107] In some embodiments, the nucleic acid includes a nucleotide sequence of GUGGUCUAGUGGUUAGGAUUCGG (SEQ ID NO: 27) or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA, and wherein the nucleic acid is 23-30 nt long. In some embodiments, the nucleic acid consists essentially of, or consists of the nucleotide sequence of GUGGUCUAGUGGUUAGGAUUCGG (SEQ ID NO: 27), where the nucleic acid is RNA. In some embodiments, the nucleic acid is uREXl.

[0108] In some embodiments, the nucleic acid includes a nucleotide sequence of UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC (SEQ ID NO: 28) or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA, and wherein the nucleic acid is 30-35 nt long. In some embodiments, the nucleic acid consists essentially of, or consists ofthe nucleotide sequence of UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC (SEQ ID NO: 28), where the nucleic acid is RNA. In some embodiments, the nucleic acid is tREXl . In some embodiments, the nucleic acid includes a nucleotide sequence of UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC (SEQ ID NO: 33), where there is a phosphorothioate bond between each pair of consecutive nucleic acid residues in the nucleotide sequence, wherein the nucleic acid is RNA, and wherein the nucleic acid is 30-35 nt long. In some embodiments, the nucleic acid consists essentially of, or consists of the nucleotide sequence of UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC (SEQ ID NO: 33), where the nucleic acid is RNA. In some embodiments, the nucleic acid is TT1. In some embodiments, the nucleic acid includes a nucleotide sequence of UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC (SEQ ID NO: 33), where the nucleic acid includes 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, wherein the nucleic acid is RNA, and wherein the nucleic acid is 30-35 nt long. In some embodiments, the nucleic acid consists essentially of, or consists of the nucleotide sequence of UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGC (SEQ ID NO: 33), where the nucleic acid includes 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, and where the nucleic acid is RNA. In some embodiments, the nucleic acid is TT8 (or SEQ ID NO:35). Suitable, non-limiting examples of nucleic acids, such as TT1 and TT8, are described in PCT App. No. PCT / US2025 / 022136, fded March 28, 2025, which is incorporated herein by reference in its entirety.

[0109] In some embodiments, the nucleic acid includes a nucleotide sequence of CCCCCCACUGCUAAAUUUGACUGGUU (SEQ ID NO: 34) or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA, and wherein the nucleic acid is 20-30 nt long. In some embodiments, the nucleic acid consists essentially of, or consists of the nucleotide sequence of CCCCCCACUGCUAAAUUUGACUGGUU (SEQ ID NO: 34), wherein the nucleic acid is RNA. In some embodiments, the nucleic acid is yREX3. In some embodiments, the nucleic acid includes a nucleotide sequence of CCCCCCACUGCUAAAUUUGACUGGUA (SEQ ID NO: 32), wherein the nucleic acid is RNA, where positions 1, 3, 5, 22, 24, and 26 of the nucleotide sequence are LNA, and whereinthe nucleic acid is 20-30 nt long. In some embodiments, the nucleic acid consists essentially of, or consists of the nucleotide sequence of CCCCCCACUGCUAAAUUUGACUGGUA (SEQ ID NO: 32), wherein the nucleic acid is RNA, and where positions 1, 3, 5, 22, 24, and 26 of the nucleotide sequence are LNA. In some embodiments, the nucleic acid is TY2. Suitable, non-limiting examples of nucleic acids, such as yREX3, are described in PCT Publication No. WO 2021 / 237238, which is incorporated herein by reference in its entirety.

[0110] The nucleic acids 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 nucleic acids are prepare using chemical synthesis. Any suitable option for chemical synthesis of nucleic acids can be used. Suitable options include, without limitation, phosphodiester, phosphotriester, phosphoramidite, phosphite-triester, and solid phase synthesis approaches. In some embodiments, preparing the nucleic acids includes in vitro transcription. In some embodiments, the nucleic acids are prepared using recombinant DNA technology. In some embodiments, the nucleic acids are prepared by chemically modifying an unmodified nucleic acid having a nucleotide sequence of interest.[0U1] In some embodiments, the formulation includes the nucleic acid that includes a RNA (e.g., a non-coding RNA), wherein the at least one casein protein comprises at least an a-sl casein subunit and wherein the at least one casein protein is present in an amount in a range of 0.5 to 5% of the formulation by weight per volume, and wherein the chitosan is present in an amount in a range of 0.001 to 1% of the formulation by weight per volume. In some embodiments, the formulation includes the nucleic acid that includes a RNA (e.g., a non-coding RNA) and wherein the RNA is present in an amount in a range of 0.0001 to 0.01% of the formulation by weight per volume, wherein the at least one casein protein comprises at least an a-sl casein subunit and wherein the at least one casein protein is present in an amount in a range of 0.5 to 5% of the formulation by weight per volume, and wherein the chitosan is present in an amount in a range of 0.001 to 1% of the formulation by weight per volume. In some embodiments, the formulation includes the nucleic acid that includes a RNA (e.g., a non-coding RNA), wherein the at least one casein protein comprises at least an a-sl casein subunit and wherein the at least one casein protein is present in an amount in a range of 0.5 to 5% of the formulation by weight per volume, wherein the chitosan is present in anamount in a range of 0.001 to 1% of the formulation by weight per volume; and wherein the acid is acetic acid and is present in an amount in a range of 0.01 to 1% of the formulation by weight per volume. In some embodiments, the formulation includes the nucleic acid that includes a RNA (e.g., a non-coding RNA), wherein the at least one casein protein comprises a mixture of an a-sl casein subunit, an a-s2 casein subunit, a P casein subunit, and a K casein subunit, wherein the casein subunits are present in a range of 1 and 3% of the formulation by weight per volume; and wherein the chitosan is present in a range of 0.01 to 0.1% of the formulation by weight per volume. In some embodiments, the formulation includes the nucleic acid that includes a RNA (e.g., a non-coding RNA) and wherein the RNA is present in an amount in a range of 0.0001 to 0.01% of the formulation by weight per volume, wherein the at least one casein protein comprises a mixture of an a-sl casein subunit, an a-s2 casein subunit, a casein subunit, and a K casein subunit, wherein the casein subunits are present in a range of 1 and 3% of the formulation by weight per volume; and wherein the chitosan is present in a range of 0.01 to 0.1% of the formulation by weight per volume. In some embodiments, the formulation includes the nucleic acid that includes a RNA (e.g., a non-coding RNA), wherein the at least one casein protein comprises a mixture of an a-sl casein subunit, an a-s2 casein subunit, a P casein subunit, and a K casein subunit, wherein the casein subunits are present in a range of 1 and 3% of the formulation by weight per volume; wherein the chitosan is present in a range of 0.01 to 0.1% of the formulation by weight per volume; and wherein the acid is acetic acid and is present in an amount in a range of 0.01 to 1% of the formulation by weight per volume. In some embodiments, the formulation includes the nucleic acid that includes a RNA (e.g., a non-coding RNA), wherein the casein subunits are present in an amount in a range of 1 to 2% of the formulation by weight per volume; wherein the chitosan is present in an amount in a range of 0.05 to 0.1% of the formulation by weight per volume. In some embodiments, the formulation includes the nucleic acid that includes a RNA (e.g., a noncoding RNA) and wherein the RNA is present in an amount in a range of 0.0001 to 0.01% of the formulation by volume, wherein the casein subunits are present in an amount in a range of 1 to 2% of the formulation by weight per volume; wherein the chitosan is present in an amount in a range of 0.05 to 0.1% of the formulation by weight per volume. In some embodiments, the formulation includes the nucleic acid that includes a RNA (e.g., a non-coding RNA), wherein the casein subunits are present in an amount in a range of 1 to 2% of the formulationby weight per volume; wherein the chitosan is present in an amount in a range of 0.05 to 0.1% of the formulation by weight per volume; and wherein the acid is acetic acid and is present in an amount in a range of 0.01 to 1% of the formulation by weight per volume. In some embodiments, the formulation includes the nucleic acid that includes a RNA (e.g., a noncoding RNA), wherein the casein subunits are present in an amount in a range of 1 to 2% of the formulation by weight per volume; wherein the chitosan is present in an amount in a range of 0.06 to 0.09% of the formulation by weight per volume. In some embodiments, the formulation includes the nucleic acid that includes a RNA (e.g., a non-coding RNA), wherein the casein subunits are present in an amount in a range of 1 to 2% of the formulation by weight per volume; wherein the chitosan is present in an amount in a range of 0.06 to 0.09% of the formulation by weight per volume; and wherein the acid is acetic acid and is present in an amount in a range of 0.01 to 0.05% of the formulation by weight per volume. In some embodiments, the formulation includes the nucleic acid that includes a RNA (e.g., a noncoding RNA) and wherein the RNA is present in an amount in a range of 0.0015 and about 0.0035% of the formulation by volume, wherein the casein subunits are present in an amount ranging between about 2.2 and 2.8% of the formulation by weight per volume; wherein the chitosan is present in an amount ranging between about 0.06 and 0.09% of the formulation by weight per volume.

[0112] In some embodiments, the formulation consists essentially of the nucleic acid; the at least one casein protein; the chitosan; and the acid. In some embodiments, the formulation consists of the nucleic acid; the at least one casein protein; the chitosan; and the acid. In some embodiments, the formulation consists essentially of or consists of the nucleic acid; the at least one casein protein, wherein the at least one casein protein comprises a mixture of an a-sl casein subunit, an a-s2 casein subunit, a P casein subunit, and a K casein subunit, wherein the casein subunits are present in a range of 1 and 3% of the formulation by weight per volume; the chitosan, wherein the chitosan is present in an amount in a range of 0.05 to 0.1% of the formulation by weight per volume; and the acid, wherein the acid is acetic acid and is present in an amount in a range of 0.01 to 1% of the formulation by weight per volume.

[0113] Also provided is a therapeutic formulation for oral delivery of a nucleic acid, consisting essentially of: a nucleic acid comprising RNA or a chemically modified variant thereof, wherein the RNA or the chemically modified variant thereof is present in an amountin a range of 0.0001 to 0.01% of the formulation by weight per volume; at least one casein protein, wherein the at least one casein protein comprises a mixture of an a-sl casein subunit, an a-s2 casein subunit, a casein subunit, and a K casein subunit, wherein the casein subunits are collectively present in an amount in a range of 0.5% to 5% of the formulation by weight per volume; a chitosan present in an amount in a range of 0.001 to 1% of the formulation by weight per volume; and acetic acid present in an amount in a range of 0.01 and 1% of the formulation by weight per volume.

[0114] In some embodiments, the liposome-free formulation for oral delivery of a nucleic acid, comprising: an RNA molecule (e.g., TY1, miR-1246, uREXl, TT1, TT8, or yREX3) at 0.0001 and 0.01% of the formulation by weight per volume; alpha si casein, alpha s2 casein, beta casein, and kappa casein; and a chitosan that includes deacetylated chitin, poly(D-glucosamine) and has a molecular weight in the range of 150,000-350,000 Da.

[0115] In some embodiments, any of the liposome-free formulations of the present disclosure find use in delivering a nucleic acid (e.g., a therapeutic nucleic acid) to a subject in need thereof by orally administering the liposome-free formulation containing a therapeutically effective amount of the nucleic acid to the subject. The nucleic acid can be any suitable nucleic acid, for example, those described herein, including TY 1.

[0116] Also provided is a composition that includes the isolated nucleic acid (e.g., TY1) and second therapeutic (e.g., semaglutide), for use in a method of treating a metabolic disorder or disease, as described herein. Provided herein is a composition that includes an isolated nucleic acid comprising a nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12) or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA, and wherein the nucleic acid is at most 30 nt long; and a second therapeutic that is an incretin or functional analogue thereof, and / or is an activator of glucagon-like peptide 1 (GLP-1) receptor signaling; and a pharmaceutically acceptable excipient. The nucleic acid of the composition can be any of the nucleic acids (e.g., TY1 and other ncRNA derivatives) as described herein. The second therapeutic can be any incretin or functional analogue thereof, or an activator of glucagon-like peptide 1 (GLP-1) receptor signaling (e.g., GLP-1 receptor agonist, GIP receptor agonist, as described herein). In some embodiments, the nucleic acid is TY1. In some embodiments, the second therapeutic is semaglutide.

[0117] 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.

[0118] In some embodiments, the composition does not include a transfection reagent, e.g., to promote delivery of the nucleic acid to a target cellular target. In some embodiments, the composition includes a transfection reagent, e.g., to promote delivery of the nucleic acid to a target 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 a polyethylene glycol (PEG)-cationic lipid complex (PCLC). In some embodiments, the transfection reagent includes a lipid (e.g., a liposomeforming 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 nucleic acid of the present disclosure is formulated with the transfection reagent in the composition so as to promote cellular uptake and / or pharmacokinetics of the nucleic acid.

[0119] 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.

[0120] 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.).

[0121] In some embodiments, the formulation or composition does not include a cationic lipid. In some embodiments, the formulation or composition is substantially free of a cationic lipid. In some embodiments, the formulation or composition comprises less than 0.1 microliters of a cationic lipid for each microgram of the nucleic acid in the formulation, or comprises no cationic lipid. 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 cationic lipid 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. A nucleic acid of the present disclosure can be mixed with the PCLC to generate a complex of the nucleic acid and the PCLC.

[0122] 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. etal. 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.

[0123] 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, 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 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.

[0124] The composition containing the EV and nucleic acid of the present disclosure can be prepared using any suitable option. In some embodiments, loading the nucleic acid into the EV includes: formulating the nucleic acid with liposomes and / or PCLC, e.g., as provided above, to generate a nucleic acid-liposome mixture; combining the nucleic acid-liposome mixture with the EV; and enriching for EV associated with exosome markers to generate a population of EV enriched for the nucleic acid. Combining the nucleic acidliposome mixture with the EV can be done using any suitable option. In some embodiments, the nucleic acid-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 nucleic acid 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 nucleic acid 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 nucleic acid 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.

[0125] In some embodiments, the formulations provided for herein do not comprise lipid-bound vesicles, e.g., micelles or liposomes. In some embodiments, the formulations provided for herein are substantially free of lipid-bound vesicles, e.g., micelles or liposomes. In several embodiments, the formulations provided for herein are in the form of lipid-bound vesicles, e.g., micelles or liposomes, and can therefore include any suitable number of particles. In some embodiments, the amount of micelles (e.g., casein-chitosan coated micelles) is in a range of about 106to about 1010particles, e.g., about 2 x 106to about 1010particles, about 5 xlO6to about 1010particles, about 107to about 5 x 109particles, about 2 xlO7to about 5 x 109particles, about 5 xlO7to about 5 x 109particles, including about 1 xlO8to about 2 x 109particles. In some embodiments, the amount of micelles (e.g., casein-chitin coated micelles) in the population is about 106, about 2 x 106, about 5 x 106, about 107, about 2 x 107, about 5 x107, about 108, about 2 x 108, about 5 x 108, about 109, about 2 x 109, about 5 x 109, or about IO10particles, or an amount in between any two of the preceding values.

[0126] In some embodiments, the composition is in a parenteral dose 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 nucleic acid 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.

[0127] In some embodiments, the formulation is effective for treatment of a condition associated with inflammation and / or fibrosis when the formulation is administered orally. In some embodiments, the condition associated with inflammation and / or fibrosis is a cardiac condition.METHODS

[0128] Also provided are methods of manufacturing a liposome-free therapeutic formulation for oral delivery of a nucleic acid (also referred to herein as “manufacturing methods”). In some embodiments, a manufacturing method of the present disclosure is for preparing any one of the formulations, e.g., liposome-free therapeutic formulations, of the present disclosure. The method can include: contacting a nucleic acid with a solution comprising 2% to 10% casein proteins by weight per volume, to generate a first mixture; and contacting the first mixture with an acid and chitosan polymers, to generate a formulation for oral delivery of the nucleic acid, wherein the method does not comprise contacting the nucleic acid with a solution comprising cationic lipids as transfection reagents, or does not includeformation of lipid nanoparticles. In some embodiments, the method does not include contacting the nucleic acid with a solution comprising cationic lipids as transfection reagents. In some embodiments, the method does not include formation of lipid nanoparticles. In some embodiments, the method includes encapsulating a nucleic acid in an assembly of casein and chitosan by contacting the nucleic acid with an acidic solution, thereby generating a casein and chitosan micelle comprising the nucleic acid, wherein the mixture of the acid and the chitosan polymers allows intercalation of the chitosan with the casein proteins and precipitation of casein-chitosan micelles comprising the nucleic acid. In some embodiments, the casein proteins are within a solution of 5% (w / v) bovine casein solution and are added to the nucleic acid at a volume ratio of about 1: 10. In some embodiments, the casein proteins are within a solution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30% (w / v) or more bovine casein solution and are added to the nucleic acid at a volume ratio of about 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1: 10, 1: 15, 1 :20, 1 :25, or 1:30 or more.

[0129] In some embodiments, contacting the first mixture with an acid and chitosan polymers comprises contacting the first mixture with a second mixture of the acid and the chitosan polymers. In some embodiments, the acid is selected from acetic acid, phosphoric acid and citric acid. In some embodiments, the acid is acetic acid. In some embodiments, the second mixture comprises: an acetic acid solution of 0.05 to 2%, weight per volume; and a chitosan solution of 0.1% to 2%, weight per volume. In some embodiments, the second mixture comprises: an acetic acid solution of 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.2%, 1.5%, or 2%, weight per volume; and a chitosan solution of 0.1%, 0.2%, 0.5%, 1%, 1.2%, 1.5%, or 2%, weight per volume.

[0130] Provided herein are methods of immunomodulation that includes orally administering to a subject an effective amount of the formulation, wherein the nucleic acid modulates an anti-inflammatory activity of a macrophage in the subject. Any suitable nucleic acid as described herein can be used in the formulation for use in the method of immunomodulation. In some embodiments, the method is effective to promote an antiinflammatory activity of macrophages in the subject, e.g., at a site of inflammation and / or fibrosis, to treat a condition or disease associated with inflammation and / or fibrosis.

[0131] Also provided herein are methods of treating a condition associated with inflammation and / or fibrosis, or a cardiometabolic disorder, comprising orally administeringto a subject having or suspected of having a condition associated with inflammation and / or fibrosis, or a cardiometabolic disorder, a therapeutically effective amount of any one of the formulations (e.g., liposome-free therapeutic formulation) of the present disclosure. In some embodiments, the condition associated with inflammation and / or fibrosis or the cardiometabolic disorder comprises a heart condition, optionally wherein the heart condition is heart failure with preserved ejection fraction, myocardial infarction, muscular dystrophy, scleroderma, viral infection, diabetes, and / or hypertrophic cardiomyopathy. In some embodiments, any one of the formulations of the present disclosure can be used for the treatment of a condition associated with inflammation and / or fibrosis, or a cardiometabolic disorder by a method comprising orally administering a therapeutically effective amount of the formulation to a subject having or suspected of having a condition associated with inflammation and / or fibrosis, or a cardiometabolic disorder.

[0132] Provided herein are methods of treating a metabolic disorder or disease, e.g., a cardiometabolic disorder or diseases (also referred to herein as “treatment methods”). A “metabolic” disorder or disease has its customary and ordinary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and denotes at least one condition associated with metabolic dysregulation, such as obesity, insulin resistance, hypertension, chronic kidney disease (CKD), atrial fibrillation (AF), obstructive sleep apnea, and dyslipidemia, that can increase the risk of developing diabetes, heart disease, and / or stroke. The method can include identifying a subject in need of treating metabolic disorder or disease. The method can further include administering to the subject therapeutically effective amounts of: an anti-inflammatory, cardioprotective nucleic acid therapeutic, where the nucleic acid therapeutic is RNA; and a second therapeutic that is an incretin or functional analogue thereof, and / or is an activator of glucagon-like peptide 1 (GLP-1) receptor signaling, thereby treating the metabolic disorder or disease. In some embodiments, the second therapeutic is semaglutide.

[0133] In some embodiments, the anti-inflammatory, cardioprotective nucleic acid therapeutic is or includes a nucleotide sequence of 15-50 nt in length and at least 90% identical to a non-coding RNA derived from cardiosphere-derived cell (CDC) derived extracellular vesicles (EV) (e.g., EV-YF1 having the sequence set forth in SEQ ID NO: 1). In some embodiments, the nucleotide sequence is, is about, or is at least 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 45, 50 nt in length, or optionally the nucleotide sequence has a length in a range defined by any two of the preceding values (e.g., 15-40 nt, 20-30 nt, 18-28 nt, 20-25 nt, etc.). In some embodiments, the nucleotide sequence is, is about, or is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical or 100% identical to the noncoding RNA derived from CDC-EV (e.g., EV-YF1 having the sequence set forth in SEQ ID NO: 1), or optionally the nucleotide sequence has sequence identity to the non-coding RNA in a range defined by any two of the preceding values (e.g., 90-99%, 95-98%, 92-97%, 95-99%, etc.). In some embodiments, the non-coding RNA is a Y-RNA or a fragment thereof (e.g., EV- YF1 having the sequence set forth in SEQ ID NO: 1), as described herein. In some embodiments, the nucleic acid therapeutic is TY1 (e.g., having the sequence set forth in SEQ ID N0:2).

[0134] Also provided is a method that includes identifying a subject in need of treating metabolic disorder or disease; and co-administering to the subject therapeutically effective amounts of: an isolated nucleic acid comprising a nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12) or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA, and wherein the nucleic acid is at most 30 nt long; and a second therapeutic that is an incretin or functional analogue thereof, and / or is an activator of glucagon-like peptide 1 (GLP-1) receptor signaling, thereby treating the metabolic disorder or disease.

[0135] As used herein, “co-administering” denotes administering at least two different therapeutics to the same subject, where each therapeutic is administered to the subject within the duration of the therapeutic effect of at least one of the other therapeutics administered to the subject. In some embodiments, the co-administering includes administering the nucleic acid and the second therapeutic simultaneously (including, without limitation, in the same composition, in the same session or on the same day). In some embodiments, the co-administering includes administering the nucleic acid to the subject within or within about 5, 10, 15, 20, 30, 40, 50, 60, 120, 180, 240, 300, 360, 500, 600 minutes or more of administering the second therapeutic to the subject, optionally, within a time period defined by any two of the preceding values (e.g., 5-600 minutes, 5-360 minutes, 5-240 minutes, 10-60 minutes, 30-180 minutes, etc.). In some embodiments, the co-administering includes administering the nucleic acid to the subject on the same day as administering the secondtherapeutic to the subject. In some embodiments, the co-administering includes administering the nucleic acid to the subject during the same visit to the doctor as administering the second therapeutic to the subject. In some embodiments, the co-administering includes administering the nucleic acid and the second therapeutic at different times (including, without limitation, on different days). In some embodiments, the co-administering includes administering the nucleic acid and the second therapeutic under the same dosing frequency. In some embodiments, the co-administering includes administering the nucleic acid and the second therapeutic under different dosing frequencies.

[0136] Non-coding RNA (ncRNA) in CDC-EVs are implicated in diseasemodifying bioactivity of CDC-EV. Among ncRNA, Y RNAs are of interest as they are abundant in CDC-EVs (18% of small RNAs). EV-YF1 is a ncRNA found in CDC-EV and encoded by the human Y-RNA4 gene. EV-YF1 increases secretion of interleukin 10 (IL-10), an anti-inflammatory cytokine, by macrophages and is cardioprotective against myocardial infarction (MI). EV-YF1 is also antifibrotic and anti-hypertrophic in a model of hypertension and hypertrophy induced by angiotensin II infusion. The nucleotide sequence of EV-YF1 is: GGCUGGUCCG AUGGUAGUGG GUUAUCAGAA CUUAUUAACA UUAGUGUCAC UAAAGU (SEQ ID NO: 1). In some embodiments, nucleic acids, e.g., TY1, of the present disclosure that are co-administered with the second therapeutic are bioinspired by EV-YF1, for example, to improve stability and potency as a therapeutic agent. In some embodiments, the anti-inflammatory, cardioprotective nucleic acid therapeutic is or includes a nucleotide sequence that is, is about, or is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the non-coding RNA derived from CDC-EV (e.g., EV-YF1), optionally where the nucleotide sequence has a percent identity to the non-coding RNA derived from CDC-EV (e.g., EV-YF1) in a range defined by any two of the preceding values (e.g., 90-100%, 94-98%, 95- 97%, 92-98%, etc.). TY1 (SEQ ID NO:2) is a chemically modified variant of EV-YF1 and has strong disease-modifying bioactivity in a number of diseases associated with inflammation and / or fibrosis, such as heart failure, hypertrophic cardiomyopathy, heart failure with preserved ejection fraction (HFpEF), Duchenne muscular dystrophy, or scleroderma.

[0137] In some embodiments, the anti-inflammatory, cardioprotective nucleic acid therapeutic includes a sequence of 15, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 nucleotides (nt) in length, optionally, the sequence is a length in a range defined by any two ofthe preceding values (e.g., 15-50 nt, 20-30 nt, 15-30 nt, 20-26 nt, 23-25 nt, etc.). In some embodiments, the anti-inflammatory, cardioprotective nucleic acid therapeutic is at most 30 nt long.

[0138] In some embodiments, the isolated nucleic acid includes a nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12) or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA, and wherein the nucleic acid is at most 30 nt long.

[0139] The second therapeutic can be any suitable compound that is an incretin or functional analogue thereof, and / or is an activator of glucagon-like peptide 1 (GLP-1) receptor signaling. In some embodiments, the incretin is glucagon-like peptide 1 (GLP-1) or gastric inhibitory peptide (GIP). In some embodiments, the second therapeutic is GLP-1 or a functional analogue thereof. The second therapeutic can be GLP-1 or any suitable analogue thereof. In some embodiments, the second therapeutic includes the sequence of HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO:23) (e g., as shown in FIG. 20B), or a sequence that is, is about, or is at least 80, 85, 90, 95, 97, 98, 99% or about 100% identical to SEQ ID NO:23. In some embodiments, the second therapeutic includes the sequence of HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO:23) without the last glycine in SEQ ID NO:23, or a sequence that is, is about, or is at least 80, 85, 90, 95, 97, 98, 99% or about 100% identical thereto. In some embodiments, the second therapeutic includes the sequence of SEQ ID NO:23 with 1, 2, 3, 4, or 5 amino acid substitutions thereto (e.g., conservative substitution). In some embodiments, the second therapeutic includes the sequence of SEQ ID NO:23 without the last glycine in SEQ ID NO:23, with 1, 2, 3, 4, or 5 amino acid substitutions thereto (e.g., conservative substitution). In some embodiments, the second therapeutic includes the sequence of SEQ ID NO:23 with a substitution of the alanine at A2 (position 2 within SEQ ID NO:23) with 2-aminoisobutyric acid. In some embodiments, the second therapeutic includes the sequence of SEQ ID NO:23 with a substitution of K28 (position 28 within SEQ ID NO:23) with an arginine (K28R). In some embodiments, the second therapeutic includes the sequence of SEQ ID NO:23 with a substitution of A2 with 2- aminoisobutyric acid and a substitution of K28 with an arginine (K28R). In some embodiments, the second therapeutic includes the sequence of SEQ ID NO:23, which is acylated at at least one position (e.g., at a lysine). In some embodiments, the second therapeuticincludes the sequence of HXEGTFTSDVSSYLEGQAAKEFIAWLVRGRG, wherein X is 2- aminoisobutyric acid (SEQ ID NO:22) (e.g., as shown in FIG. 20A).

[0140] In some embodiments, the second therapeutic includes the sequence of SEQ ID NO:22 or 23, which is acylated at K20 (lysine at position 20 within SEQ ID NO:22 or 23). In some embodiments, the second therapeutic includes the sequence of SEQ ID NO:22 or 23, which is acylated with a C12-C20 fatty acid. In some embodiments, the second therapeutic includes the sequence of SEQ ID NO:22 or 23, which is acylated with a C12-C20 fatty di-acid. In some embodiments, the second therapeutic includes the sequence of SEQ ID NO:23, which is acylated with a C18 fatty di-acid. In some embodiments, the second therapeutic is acylated via a linker (e.g., a hydrophilic linker). In some embodiments, the second therapeutic is or includes the compound shown in FIG. 19. In some embodiments, the second therapeutic is semaglutide.

[0141] In some embodiments, the second therapeutic is selected from: semaglutide, dulaglutide, tirzepatide, liraglutide, exenatide, albiglutide, benaglutide [INN], lixisenatide, pegloxenatide [INN], Diabegone, dulaglutide biosimilar, mazdutide, ecnoglutide [INN], EFPEGLENATIDE, GMA-102, GXG-6, HR-17031, HRS-9531, ORFORGLIPRON CALCIUM, PB-119, RETATRUTIDE, SAL-015, survodutide, Uni-E4, AZD-9550, BGM- 0504, CT-388, CT-868, DANUGLIPRON TROMETHAMINE, dapiglutide [INN], DD-01, E- 2HSA, EFINOPEGDUTIDE, EFOCIPEGTRUTIDE [INN], FRONIGLUTIDE [INN], GL- 0034, GMA-105, GSBR-1290, GZR-18, HEC-88473, HRS-7535, HS-20004, HS-20094, IY- 09, MARIDEBART CAFRAGLUTIDE [INN], MDR-001, NNC0519-0130, PB-718, PEGAPAMODUTIDE, PEGSEBRENATIDE [INN], PEMVIDUTIDE, RGT-075, TTP-273, VK-2735, YN-012, AP-026, CT-996, DA-1726, DR-10624, DR-10627, ECC-5004, GLP-06, GMA-106, HB-1085, HDM-1002, HL-08, HZ-010, ID-110521156, KN-056, MWN-101, NN- 6177, NN-9542, NN-9650, NN-9904, NNC-04870111, peptides 1 to agonize glp-1 and gcgr for diabetes and obesity, peptides 2 to agonize glp-1 and gcgr for, diabetes and obesity, PF- 06954522, PG-102, SAL-0112, SCO-094, TERN-601, THDBH-110, THDBH-120, UBT-251, XW-014, YH-25724, YN-015, ZT-002, APH-01727, DB-081, GLP-l / GIP / GCG FUSION PROTEIN, GW-002, HDM-1005, HM-15275, HZCX-012, THDB-0211, THDBH-121, ZX- 2010, ZX-2021, ACT-1003, AER-601, AGM-212, BEBT-808, BZ-043B, C-2816, CIN-209, CIN-210, DD-02S, DD-15, DR- 10625, DR- 10628, DS-004, DS-005, DS-006, DS-012,EXTENDTN-FC, fusion protein to agonize glplr for type 1 diabetes, G-49, GLP-1 incretin triagonist, GLP-1 oral preparation, GLP-1 oral small molecule, GLP-l-GIP combinations, GLPIR-agonist miniprotein, GLUCAGON [INN], INSULIN HUMAN, GPCR-targeted project 012, GPCR-targeted project 013, GT-01123, HFG-1, HLB-1006, HPG-5119, HRS- 4729, HSP-001, HSP-004, HSP-005, HSP012-C, I2O-105S, KP-405, LA-EX, MBX-4291, MK-1462, MLX-7000, MWN-105, MWN-109, NA-931, NLY-12, obesity, OGB-21502, oral GLP-1 agonist, OXM, P-11, PB-2301, PB-2309, peptides to agonize glplr for diabetes, recombinant peptide- 1 to activate glp-1 for type 2 diabetes, recombinant protein to agonize glplr for metabolic diseases, RGT-028, RGT-274, RPC-8844, SHX-022, SL-209, synthetic peptides to agonize GLP1 and GLUCAGON receptor for diabetes and obesity, synthetic peptides to agonize GLP1R for type 2 diabetes, TB-592, TE-8105, UDS-003, ultra long acting GLP-1, VTCG-15, XL-310, XW-003 + XW-015, XW-003 + XW-017, YGX-1, ZT-003, ZT- 006, ZT-007, APB-GLP-1, ATBB-22, BEM-012, CLMOB-01, DD-03, DD-14, dual amylin / cal citonin receptor agonist, GLP-1 receptor agonist, HLB-1015, incretin program 2 oral, INV-610, LXM-2, MD-02, MET-06, MRANK-111, next gen GSBR, next generation TERN-600 series, next-generation therapeutics, obesity (GLP1R agonist), OGB-21501, oral GLP-1 nonpeptide, oral incretin, P-01, PAT-201, Peptide to Agonize GLP-1 R for Metabolic Disorders, peptides to agonize GLP-1 and GCGR for diabetes and obesity, peptides to agonize GLP-1 for diabetes and obesity, PF-1807, PT-3, and / or synthetic peptide to agonize GLP-1R for type 2 diabetes. In some embodiments, the second therapeutic is selected from Table 0.1.Table 0.1

[0142] In some embodiments, the second therapeutic is GIP or a functional analogue thereof. The second therapeutic can be GIP or any suitable analogue thereof. In some embodiments, the second therapeutic includes the sequence of YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ (SEQ ID NO:25) (e g., as shown in FIG. 20D). or a sequence that is, is about, or is at least 80, 85, 90, 95, 97, 98, 99% or about 100% identical to SEQ ID NO:25. In some embodiments, the second therapeutic includes the sequence of SEQ ID NO:25 with 1, 2, 3, 4, or 5 amino acid substitutions thereto (e.g., conservative substitution).

[0143] In some embodiments, the second therapeutic is selected from: tirzepatide, HRS-9531, retatrutide, BGM-0504, CT-388, CT-868, efocipegtrutide [INN], HS-20094, NNC0519-0130, VK-2735, DR-10624, DR-10627, HZ-010, LY-3532226, LY-3537021, MWN-101, NN-9542, NN-9650, THDBH-120, UBT-251, GLP-l / GIP / GCG FUSION PROTEIN, HDM-1005, HM- 15275, HZCX-012, THDBH-121, ZX-2010, ZX-2021, DD-15, DR-10625, DR-10628, GLP-1 incretin triagonist, GLP-l-GIP combinations, GLP- 1 / GIP / FGF21 fusion protein, KP-405, MBX-4291, MWN-105, MWN-109, NA-931, PB-2309, TERN-800 SERIES, ZP-6590, DD-03, DD-14, GIPR, incretin program 2 oral, OGB-21501, oral GIP nonpeptide, oral incretin. In some embodiments, the second therapeutic is selected from Table 0.2.Table 0.2

[0144] In some embodiments, the second therapeutic is glucagon or a functional analogue thereof (e.g, that is an activator of glucagon-like peptide 1 (GLP-1) receptor signaling). In some embodiments, the second therapeutic includes the sequence of HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO:24) (e.g, as shown in FIG. 20C), or a sequence that is, is about, or is at least 80, 85, 90, 95, 97, 98, 99% or about 100% identical to SEQ ID NO:24. In some embodiments, the second therapeutic includes the sequence of SEQ ID NO:24 with 1, 2, 3, 4, or 5 amino acid substitutions thereto (e.g, conservative substitution).

[0145] In some embodiments, the second therapeutic is selected from: glucagon [INN], dasiglucagon, mazdutide, retatrutide, survodutide, AZD-9550, DD-01, efmopegdutide, efocipegtrutide [INN], efpegerglucagon [INN], PB-718, pegapamodutide, pemvidutide, DA- 1726, MWN-101, NN-6177, Peptides 1 to Agonize GLP-1 and GCGR for Diabetes and Obesity, Peptides 2 to Agonize GLP-1 and GCGR for Diabetes and Obesity, UBT-251, GLP- 1 / GIP / GCG fusion protein, HM-15275, PB-722, ZX-2021, ABG-023, DD-15, G-49, GLP-1 incretin triagonist, MK-1462, MWN-109, OGB-21502, OXM, synthetic peptides to agonizeglpl and glucagon receptor for diabetes and obesity, DD-03, GCGR, OGB-21 01 , peptides to agonize GLP-1 and GCGR for diabetes and obesity. In some embodiments, the second therapeutic is selected from Table 0.3.Table 0.3

[0146] The method can include any suitable option for administering the nucleic acid (e.g., anti-inflammatory, cardioprotective nucleic acid therapeutic) and the second therapeutic to the subject. In some embodiments, the co-administering includes administering the nucleic acid orally or intravenously. In some embodiments, the co-administering includes administering the nucleic acid orally. In some embodiments, the co-administering includes administering the second therapeutic orally, subcutaneously, or intravenously. In some embodiments, the co-administering includes administering the second therapeutic orally. In some embodiments, the co-administering includes administering the nucleic acid orally and administering the second therapeutic subcutaneously. In some embodiments, the coadministering includes administering the nucleic acid orally and administering the second therapeutic orally. Any suitable option for formulating the nucleic acid and / or the second therapeutic (e g., semaglutide) for oral administration can be used. Non-limiting options for oral formulation of a nucleic acid, including TY1, is described in e.g., PCT publication numbers WO 2023 / 278799 and WO 2023 / 278802, each of which are incorporated herein by reference in its entirety.

[0147] The nucleic acid (e.g., anti-inflammatory, cardioprotective nucleic acid therapeutic) and the second therapeutic can be administered to the subject using any suitable dosing regimen. In some embodiments, the co-administering comprises administering the nucleic acid at a frequency in the range of once every day to once every month. In some embodiments, the co-administering comprises administering the second therapeutic at a frequency in the range of once every day to once every month. In some embodiments, the coadministering includes administering the nucleic acid at a frequency of, of about, or no more frequently than once a day, e.g., at a frequency of once every 1, 2, 3, 4, 5, 6, or 7 days, or more days. In some embodiments, the co-administering includes administering the second therapeutic at a frequency of, of about, or no more frequently than once a day, e.g., at a frequency of once every 1, 2, 3, 4, 5, 6, or 7 days, or more days. In some embodiments, the co-administering includes administering the nucleic acid at a frequency of, or of about once a day, and administering the second therapeutic about once a day. In some embodiments, the co-administering includes administering the nucleic acid at a frequency of, of about, or no more frequently than twice a week (or administering biweekly). In some embodiments, the co-administering includes administering the second therapeutic at a frequency of, of about, orno more frequently than twice a week (or administering biweekly). In some embodiments, the co-administering includes administering the nucleic acid twice a week (or administering biweekly), and administering the second therapeutic twice a week (or administering biweekly). In some embodiments, the co-administering includes administering the nucleic acid at a frequency of, of about, or no more frequently than once every week, e.g., at a frequency of once every 1, 2, 3, 4, 5, 6, 7, 8 weeks. In some embodiments, the co-administering includes administering the second therapeutic at a frequency of, of about, or no more frequently than once every week, e g., at a frequency of once every 1, 2, 3, 4, 5, 6, 7, 8 weeks. In some embodiments, the nucleic acid and the second therapeutic are administered to the subject at the same frequency. In some embodiments, the nucleic acid and the second therapeutic are administered to the subject at the same frequency, and at the same time (e.g., in the same composition, on the same day, in the same session, etc ). In some embodiments, the nucleic acid and the second therapeutic are administered to the subject at different frequencies. In some embodiments, the nucleic acid and the second therapeutic are each administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more times, optionally, they are each administered to the subject a number of times in a range defined by any two of the preceding values (e.g., 1-30 times, 1-25 times, 4-15 times, 2-6 times, etc.). In some embodiments, the nucleic acid and the second therapeutic are administered to the subject at regular intervals.

[0148] In some embodiments, the subject is a human subject. In some embodiments, the subject is a non-human subject, e.g., a non-human mammal.

[0149] Metabolic conditions and diseases that may be treated by the present treatment methods include, without limitation, obesity, fatty liver disease, and / or diabetes. “Obesity” has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and denotes a condition of having excessive body fat. In some embodiments, a subject having a body mass index (BMI) of 30 or higher is considered to be obese. BMI can be calculated using any suitable option. In some embodiments, BMI is determined by multiplying a subject’s weight in pounds by 703, dividing by height in inches and then dividing again by height in inches. In some embodiments, BMI is determined by dividing weight in kilograms by height in meters squared. In some embodiments, the method includes identifying a subject having a BMI of, of about, or of at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or optionally a subject having a BMI in a range definedby any two of the preceding values (e.g., 25-40, 26-38, 30-40, 28-40, etc.). In some embodiments, the method includes identifying a subject in need of treating obesity and / or one or more symptoms thereof.

[0150] ‘Diabetes” has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and denotes a condition characterized by excessive blood glucose levels. In some embodiments, the diabetes is type 2 diabetes. In some embodiments, the method includes identifying a subject having diabetes (e.g., type 2 diabetes). In some embodiments, the method includes identifying a subject in need of treating diabetes (e.g., type 2 diabetes) and / or one or more symptoms thereof.

[0151] In some embodiments, the method includes identifying a subject in need of treating fatty liver disease. In some embodiments, the fatty liver disease is nonalcoholic steatohepatitis (NASH) (or MASH (metabolic dysfunction associated steatohepatitis)) or nonalcoholic fatty liver disease (NAFLD) (or MASLD (metabolic dysfunction associated steatotic liver disease)). NAFLD (or MASLD) has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and denotes a condition characterized by excess fat in the liver. NASH (or MASH) has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and denotes a condition characterized by inflammation of the liver and liver damage, and excess fat in the liver.

[0152] In some embodiments, the metabolic disorder or disease includes a cardiovascular disorder or disease (e.g., a cardiometabolic disorder or disease). In some embodiments, the cardiovascular disorder includes heart failure or a symptom and / or sequelae thereof (e.g., myocardial inflammation, myocardial fibrosis, damage to the heart, etc.). In some embodiments the cardiovascular disorder includes: heart failure with preserved ejection fraction (HFpEF) or a symptom and / or sequelae thereof; or heart failure with reduced ejection fraction (HFrEF) or a symptom and / or sequelae thereof. In some embodiments, the method includes identifying a subject in need of treating a metabolic disorder or disease that includes a cardiovascular disorder (e.g., HFpEF, HFrEF) or a symptom and / or sequelae thereof. In some embodiments, the method includes identifying a subject in need of treating heart failure with preserved ejection fraction (HFpEF) or a symptom and / or sequelae thereof. In someembodiments, the subject has or suffers from one or more of hypertension, diabetes, chronic kidney disease (CKD), atrial fibrillation (AF), obesity, and obstructive sleep apnea.

[0153] The subject treated by the present methods can exhibit one or more symptoms and / or sequelae of the metabolic disease or disorder. In some embodiments, the subject exhibits before the co-administering, at least one of the following: hypertension, elevated E / e’ ratio, cardiac hypertrophy, myocardial fibrosis, obesity, reduced endurance, and elevated systemic inflammatory markers.

[0154] In some embodiments, the subject is at risk of developing the cardiovascular disorder or disease. In some embodiments, the subject is at risk of developing the cardiovascular disorder or disease based on one or more of the subject’s family history, genetic predisposition, lifestyle, and medical history. In some embodiments, the subject has one or more comorbidities for the cardiovascular disorder or disease. In some embodiments, the one or more comorbidities includes obesity and hypertension. In some embodiments, the subject has, or is diagnosed with, the cardiovascular disorder or disease.

[0155] In some embodiment, co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic reduces or restores a pathological cardiovascular function (e.g., elevated E / e’ ratio, elevated left ventricular end-diastolic pressures were measured (LVEDP)) after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiment, the subject’s pathological cardiovascular function is mitigated by , by at least, or by about 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more, or by a percentage in a range defined by any two of the preceding values (e.g., 5-75%, 10-50%, 7.5-40%, 15-60%, etc.) after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiment, the subject’s pathological cardiovascular function is mitigated at least to a level that is deemed no longer to be clinically relevant after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiments, the reduction is relative to a pre-treatment value. In some embodiments, the reduction is relative to a suitable control population (e.g., an average value for a cohort of subjects to which the nucleic acid and the second therapeutic were not co-administered). In some embodiments, the subject exhibits a mitigation in the pathological cardiovascular function after co-administering the therapeutically effectiveamounts of the nucleic acid and the second therapeutic that is greater than the mitigation expected from the therapeutic effect of administering either the nucleic acid alone or the second therapeutic alone.

[0156] In some embodiment, the subject has an elevated E / e’ ratio (before the coadministering), and co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic reduces the E / e’ ratio. In some embodiment, the subject’s E / e’ ratio is reduced by, by at least, or by about 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more, or by a percentage in a range defined by any two of the preceding values (e.g., 5-75%, 10-50%, 7.5-40%, 15-60%, etc.) after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiment, the subject’s E / e’ ratio is reduced by 10-50% after coadministering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiment, the subject’s E / e’ ratio is reduced at least to a level that is deemed no longer to be clinically relevant after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiments, the reduction is relative to a pre-treatment value. In some embodiments, the reduction is relative to a suitable control population (e.g., an average value for a cohort of subjects to which the nucleic acid and the second therapeutic were not co-administered). In some embodiments, the subject exhibits a reduction in E / e’ ratio after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic that is greater than the reduction expected from the therapeutic effect of administering either the nucleic acid alone or the second therapeutic alone. The E / e’ ratio can be measured using any suitable option, e.g., by echocardiography.

[0157] In some embodiment, the subject has an elevated left ventricular end- diastolic pressure (LVEDP) (before the co-administering), and co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic reduces the (LVEDP). In some embodiment, the subject’s LVEDP is reduced by, by at least, or by about 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more, or by a percentage in a range defined by any two of the preceding values (e.g., 5-75%, 10-50%, 7.5-40%, 15-60%, etc.) after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiment, the subject’s LVEDP is reduced by 10-70% after co-administering the therapeutically effectiveamounts of the nucleic acid and the second therapeutic. In some embodiment, the subject’s LVEDP is reduced at least to a level that is deemed no longer to be clinically relevant after coadministering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiments, the reduction is relative to a pre-treatment value. In some embodiments, the reduction is relative to a suitable control population (e.g., an average value for a cohort of subjects to which the nucleic acid and the second therapeutic were not coadministered). In some embodiments, the subject exhibits a reduction in LVEDP after coadministering the therapeutically effective amounts of the nucleic acid and the second therapeutic that is greater than the reduction expected from the therapeutic effect of administering either the nucleic acid alone or the second therapeutic alone. The LVEDP can be measured using any suitable option, e.g., by a pressure catheter in the left ventricle or at the pulmonary artery.

[0158] In some embodiment, the subject exhibits reduced exercise capacity, e.g., exercise endurance, and co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic at least slows the decline in exercise capacity. In some embodiment, the subject exhibits an increase in exercise capacity of, of at least, or of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500% or more, or optionally a percentage increase in exercise capacity in a range defined by any two of the preceding values (e.g., 10-500%, 20-400%, 50-200%, 100-400%, etc.), after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiment, the subject’s exercise capacity recovers to, or is maintained at substantially the pre-treatment level after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiments, the improvement in exercise capacity after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic is sustained over the duration of treatment. In some embodiments, the improvement in exercise capacity after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic is sustained across multiple doses of administration. In some embodiments, the subject exhibits an increase in exercise capacity after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic that is greater than the increase expected from the therapeutic effect ofadministering either the nucleic acid alone or the second therapeutic alone. The exercise capacity can be measured using any suitable option, e.g., using a treadmill.

[0159] In some embodiments, the subject has an elevated non-fasting blood glucose level (hyperglycemia), and co-admini st ering the therapeutically effective amounts of the nucleic acid and the second therapeutic reduces the subject’s non-fasting blood glucose level. In some embodiment, the subject’s non-fasting blood glucose level is reduced by, by at least, or by about 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more, or by a percentage in a range defined by any two of the preceding values (e.g., 5-75%, 10-50%, 7.5-40%, 15-60%, etc.) after co-admini stering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiment, the subject’s non-fasting blood glucose level is reduced by 10-60% after coadministering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiment, the subject’s non-fasting blood glucose level is reduced at least to a level that is deemed no longer to be clinically relevant after co-admini stering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiments, the reduction is relative to a pre-treatment value. In some embodiments, the reduction is relative to a suitable control population (e.g., an average value for a cohort of subjects to which the nucleic acid and the second therapeutic were not co-administered).

[0160] In some embodiment, the subject has hypertension, and co-admini stering the therapeutically effective amounts of the nucleic acid and the second therapeutic reduces the subject’s blood pressure. In some embodiments, a subject having hypertension has a resting blood pressure of over 130 / 90 mmHg. In some embodiments, a subject having hypertension has a resting blood pressure of over 140 / 90 mmHg. In some embodiment, coadministering the therapeutically effective amounts of the nucleic and the second therapeutic reduces the subject’s systolic blood pressure and / or diastolic blood pressure. In some embodiment, the subject’s blood pressure (systolic or diastolic blood pressure) is reduced by, by about, or by at least 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 25%, 30%, 35%, 40% or more, or by a percentage in a range defined by any two of the preceding values (e.g., 5-40%, 10-35%, 15-40%, etc.) after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiments, the subject’s blood pressure (systolic or diastolic blood pressure) is reduced by at least 10% (e.g., 10-30%) after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiment, the subject’s blood pressure (systolic or diastolic blood pressure) is reduced at least to a level that is deemed no longer to be hypertensive after coadministering the therapeutically effective amounts of the nucleic acid and the second therapeutic.

[0161] In some embodiments, the subject exhibits elevated levels of systemic inflammatory markers, e.g., in the peripheral blood. In some embodiments, the systemic inflammatory marker includes one or more of IL-6 and brain natriuretic peptide (BNP). In some embodiments, the level of the systemic inflammatory marker is reduced by at least about 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more, or by a percentage in a range defined by any two of the preceding values, after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic. In some embodiments, the subject’s systemic inflammatory marker is reduced at least to a level that is deemed no longer to be elevated after co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic.

[0162] In some embodiments, any of the therapeutic effects of co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic herein is sustained over the duration of treatment. In some embodiments, any of the therapeutic effects of co-administering the therapeutically effective amounts of the nucleic acid and the second therapeutic herein is sustained across multiple doses of administration is sustained across multiple doses of administration. In some embodiments, any of the therapeutic effects of coadministering the therapeutically effective amounts of the nucleic acid and the second therapeutic herein is not transient over the duration of treatment.

[0163] The nucleic acid can be administered to the subject at any suitable amount. In some embodiments, the therapeutically effective amount of the nucleic acid includes, includes about, or includes at least 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 optionally includes 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, l Omg-lOOmg). In some embodiments, the therapeutically effective amount of the nucleic acid includes, includes about, or include at least 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 nucleic acid is, is about, or is at least 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 optionally 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 nucleic acid includes from 1 pg / kg to 1 mg / kg of body weight.

[0164] The second therapeutic can be administered to the subject at any suitable amount. In some embodiments, the therapeutically effective amount of the second therapeutic includes, includes about, or includes at least 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000 nmol or more, or optionally, includes an amount in a range defined by any two of the preceding values (e.g., 0.1-10,000 nmol, 0.1-10 nmol, 10-100 nmol, 100- 500 nmol, 500-5,000 nmol, etc ). In some embodiments, the therapeutically effective amount of the second therapeutic includes, includes about, or include at least 0.01 nmol / kg, 0.1 nmol / kg, 1 nmol / kg, 2 nmol / kg, 5 nmol / kg, 10 nmol / kg, 15 nmol / kg, 20 nmol / kg, 25 nmol / kg, 30 nmol / kg, 35 nmol / kg, 40 nmol / kg, 45 nmol / kg, 50 nmol / kg, 60 nmol / kg, 70 nmol / kg, 80 nmol / kg, 90 nmol / kg, 100 nmol / kg, 200 nmol / kg, 500 nmol / kg, 1,000 nmol / kg of body weight, or more, or an amount in a range defined by any two of the preceding values (e.g., 0.01 nmol / kg-0.1 nmol / kg, 0.1 nmol / kg- 1 nmol / kg, 1 nmol / kg- 10 nmol / kg, 10 nmol / kg- 100 nmol / kg, 100 nmol / kg-1,000 nmol / kg, etc.). In some embodiments, the therapeutically effective amount of the second therapeutic is, is about, or is at least 0.1 nmol / kg, 1 nmol / kg, 10 nmol / kg, 30 nmol / kg, 50 nmol / kg, or 100 nmol / kg of body weight, or more, or optionallyit is an amount in a range defined by any two of the preceding values (e.g., 0.1 nmol / kg-1 nmol / kg, 1 nmol / kg-30 nmol / kg, or 30 nmol / kg-100 nmol / kg, etc.).

[0165] The nucleic acid 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 nucleic acid or composition is administered intravenously. In some embodiments, the nucleic acid or composition is administered by infusion.KITS

[0166] Provided herein is a kit for oral delivery of a nucleic acid. A kit can include at least one casein protein; a chitosan; and an acid. The kit can include any suitable collection of the at least one casein protein, or a mixture thereof, as described herein. The kit can include any suitable chitosan (e.g., medium molecular weight chitosan), as described herein. The kit can include any suitable acid (e.g., acetic acid), as described herein. In some embodiments, the kit includes instructions for using the kit to prepare a therapeutic formulation of a nucleic acid (e.g., a liposome-free therapeutic formulation of a therapeutic RNA). The information and instructions may be in the form of words, pictures, or both, and the like. In some embodiments, the kit includes a nucleic acid (e.g., a therapeutic nucleic acid or RNA, as described herein). In some embodiments, the kit does not include a transfection reagent for a nucleic acid, such as a cationic lipid.

[0167] Also provided herein are kits that include the nucleic acid and second therapeutic (or a composition thereof) of the present disclosure. The present kit in some embodiments finds use in treating a metabolic disorder or disease (e.g., a cardiometabolic disorder or disease), as provided herein. The kit can include the nucleic acid of the present disclosure and a transfection reagent. The transfections 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.

[0168] In some embodiments, the kit includes a pharmaceutically acceptable excipient, as provided herein. In some embodiments, the kit includes casein and / or chitosan.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., obesity, diabetes, HFpEF). The information and instructions may be in the form of words, pictures, or both, and the like.Additional Embodiments

[0169] The incidence and prevalence of HFpEF is rising relative to HF with reduced ejection fraction (HFrEF); 5-year mortality is 75.3% and similar to that observed in HFrEF2. Furthermore, 30-day hospital readmission rate is 21%2. The increasing risk of HFpEF is driven by an aging population and an increasing burden of associated cardiometabolic risk factors including hypertension, diabetes, chronic kidney disease (CKD), atrial fibrillation (AF), obesity, and obstructive sleep apnea. The projected overall lifetime risk is approximately 20% at age 40 years1'3.

[0170] Unlike HFrEF, where pharmacological agents and devices are effective, few treatments have proven effective for HFpEF, and none has been shown to reduce mortality. Thus far two classes of drugs have demonstrated benefit in human clinical trials: glucagon-like peptide- 1 receptor agonists (GLP-lRAs) and sodium-glucose co-transporter-2 inhibitors (SGLT2i). These drugs have demonstrated efficacy in HFpEF patients including improved exercise tolerance, greater KCCQ survey scores, and reduced hospitalizations, but have failed to reduce mortality10'12.

[0171] Provided herein, in some embodiments, is a combination therapy consisting of a novel non-coding RNA drug, TY1, and the FDA approved GLP-1 RA, semaglutide. The latter has been shown to be effective in HFpEF patients with obesity and diabetes. Semaglutide is a potent weight loss drug that exerts effects on the gastrointestinal tract and the brain to suppress appetite and augment satiety. In addition, semaglutide also exerts direct cardioprotective effects independent of weight loss. The novel non-coding RNA drug, TY1, targets cell stress pathways that drive tissue inflammation and fibrosis which are critical elements contributing to HFpEF pathobiology. TY1 has been extensively characterized in cardiovascular diseases including acute myocardial infarction, HF, and other inflammatory disorders13'14. Because TY1S (TY1 + semaglutide) targets the root causes, this combinationcan revolutionize the treatment of HFpEF and transcend the narrow limits of the currently available HFpEF therapeutics.

[0172] The data provided herein shows that TY1S was effective in reversing the key disease manifestations of HFpEF in two preclinical model systems. In well-accepted mouse and rat models of cardiometabolic HFpEF, TY1S improved primary disease indicators of HFpEF without apparent toxicity.

[0173] In well-validated preclinical models, TY1S induced consistent and substantial reductions of the following disease conditions: left ventricular (LV) diastolic dysfunction, exercise intolerance, obesity, hyperglycemia, and vascular dysfunction. Translated to the clinic, these effects can revolutionize the treatment of HFpEF, not only improving signs and symptoms but also decreasing morbidity and mortality.

[0174] In some embodiments, TY1S can be applied to additional cardiometabolic diseases including obesity, diabetes, chronic kidney disease, and HFrEF.

[0175] References:1. Tsao, C.W. et al. Heart disease and stroke statistics-2023 Update: A report from the American Heart Association. Circulation 2023;147:e93-e621, DOI: 10.1161.2. Borlaug, B.A. et al. Heart failure with preserved ejection fraction: JACC Scientific Statement. J. Am. Coll. Cardiol. 2023; 81 : 1810-1834.3. Jeffries, A. and W.P.A. Chan. Heart failure with preserved ejection fraction: Advances in management. Medicine Today 2023; 24(10):21-27.10. Butler, J., et al. Empagliflozin, health status, and quality of life in patients with heart failure and preserved ejection fraction: The EMPEROR-preserved trial. 2022; Circulation, 145, 184-193.11. Cimino, G. et al. Obesity, heart failure with preserved ejection fraction, and the role of glucagon-like peptide-1 receptor agonists. ESC Heart Failure 2024; 11 :649-661 DOI: 10: 1002 / ehf2.14560.12. Withaar, C. et al. The cardioprotective effects of semaglutide exceed those of dietary weight loss in mice with HFpEF. 2023; JACC Basic Trans. Sci. 8(10): 1298-1314.13. Ibrahim A. et al. Augmentation of DNA exonuclease TREX1 in macrophages as a therapy for cardiac ischemic injury. 2024; bioRXiv doi : 10.1101 / 2024.02.20.581294.14. Yamaguchi, S. et al. Oral bioavailability of a noncoding RNA drug, TY1, that acts on macrophages. 2024; bioRxiv doi: 10.1101 / 2024.04.27.59147437. Doiron, J.E. Adjunctive therapy with an oral H2S donor provides additional therapeutic benefit beyond SGLT2 inhibition in cardiometabolic heart failure with preserved ejection fraction. 2024; Br. J. Pharmacol. DOI: 10.1111 / bph.16493.38. Schauer, A. et al. ZSF1 rat as animal model for HFpEF: Development of reduced diastolic function and skeletal muscle dysfunction. 2020; ESC Heart Failure 7:2123- 2134.39. Nguyen, I.T.N. et al. Both male and female obese zsfl rats develop cardiac dysfunction in obesity -induced heart failure with preserved ejection fraction. PLOS ONE 15(5):e0232399 doi.org / 10.1371.40. LaPenna, K.B. et al., Combination sodium nitrite and hydralazine therapy attenuates heart failure with preserved ejection fraction severity in a “2-hit” murine model. 2023; JAHA 12:e028480.DOI: 10.1161 / J AHA.122.028480.41. Sharp, T.E. Novel gottingen miniswine model of heart failure with preserved ejection fraction integrating multiple comorbidities. 2020; JACC BTS doi . org / 10.1016 / j . j acbts .2020.11.012.

[0176] Additional, non-limiting embodiments of the present disclosure are provided by the following numbered embodiments.1. A method of treating a metabolic disorder or disease, comprising: identifying a subject in need of treating metabolic disorder or disease; and co-administering to the subject therapeutically effective amounts of: an isolated nucleic acid comprising a nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12) or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA, and wherein the nucleic acid is at most 30 nt long; and a second therapeutic that is an incretin or functional analogue thereof, and / or is an activator of glucagon-like peptide 1 (GLP-1) receptor signaling, thereby treating the metabolic disorder or disease.2. The embodiment of embodiment 1, wherein the second therapeutic comprises: a GLP-1 receptor agonist and / or a gastric inhibitory peptide (GIP) receptor agonist.3. The method of embodiment 1 or 2, wherein the second therapeutic comprises:GLP-1 or a functional analogue thereof; glucagon or a functional analogue thereof; and / orGIP or a functional analogue thereof.4. The method of any one of the preceding embodiments, wherein the second therapeutic is selected from: Tables 0.1, 0.2 or 0.3, optionally wherein the second therapeutic is semaglutide.5. The method of any one of the preceding embodiments, wherein the nucleic acid comprises at least one chemically modified nucleotide in the nucleotide sequence, optionally wherein the nucleic acid comprises between 1-10 chemically modified nucleotides in thenucleotide sequence, optionally wherein the nucleic acid comprises the at least one chemically modified nucleotide at one or more of positions 1, 3, 5, 20, 22 and 24 of the nucleotide sequence.6. The method of any one of the preceding embodiments, wherein the chemically modified nucleotide comprises a backbone modification, optionally wherein the backbone modification comprises a backbone sugar modification, optionally wherein the chemically- modified nucleotide is a locked nucleic acid (LNA).7. The method of any one of the preceding embodiments, wherein the nucleic acid is 24 nucleotides long.8. A method of treating metabolic disorder or disease, comprising: identifying a subject in need of treating metabolic disorder or disease; and co-administering to the subject therapeutically effective amounts of: an anti-inflammatory, cardioprotective nucleic acid therapeutic, wherein the nucleic acid therapeutic is RNA; and a second therapeutic that is an incretin or functional analogue thereof, and / or an activator of glucagon-like peptide 1 (GLP-1) receptor signaling, thereby treating the metabolic disorder or disease.9. The method of embodiment 8, wherein the second therapeutic comprises: a GLP-1 receptor agonist and / or a gastric inhibitory peptide (GIP) receptor agonist.10. The method of embodiment 8 or 9, wherein the second therapeutic comprises:GLP-1 or a functional analogue thereof; glucagon or a functional analogue thereof; and / orGIP or a functional analogue thereof.11. The method of any one of embodiments 8-10, wherein the second therapeutic is selected from: Tables 0.1, 0.2 or 0.3, optionally wherein the second therapeutic is semaglutide.12. The method of any one of embodiments 8-11, wherein the nucleic acid therapeutic comprises a nucleotide sequence of 15-50 nt in length and at least 90% identical to a non-coding RNA derived from cardiosphere-derived cell (CDC) derived extracellular vesicles, optionally wherein the non-coding RNA is a Y-RNA or a fragment thereof.13. The method of any one of the preceding embodiments, wherein the nucleic acid is TYl.14. The method of any one of the preceding embodiments, wherein the coadministering comprises administering the nucleic acid orally or intravenously, optionally administering the nucleic acid orally.15. The method of any one of the preceding embodiments, wherein the coadministering comprises administering the second therapeutic orally, subcutaneously, or intravenously, optionally administering the second therapeutic orally.16. The method of any one of the preceding embodiments, wherein the coadministering comprises administering the nucleic acid and the second therapeutic simultaneously.17. The method of any one of the preceding embodiments, wherein the coadministering comprises administering the nucleic acid no more frequently than once a day, and / or administering the second therapeutic no more frequently than about once a day.18. The method of any one of the preceding embodiments, wherein the coadministering comprises administering the nucleic acid at frequency in the range of once every day to once every month, and / or administering the second therapeutic at frequency in the range of once every day to once every month.19. The method of any one of the preceding embodiments, wherein the metabolic disorder or disease comprises obesity, fatty liver disease, and / or diabetes, optionally the diabetes is type 2 diabetes.20. The method of any one of the preceding embodiments, wherein the metabolic disorder or disease comprises a cardiovascular disorder, optionally wherein the cardiovascular disorder comprises heart failure or a symptom and / or sequelae thereof, optionally wherein the cardiovascular disorder comprises: heart failure with preserved ejection fraction (HFpEF) or a symptom and / or sequelae thereof; or heart failure with reduced ejection fraction (HFrEF) or a symptom and / or sequelae thereof.21. The method of any one of the preceding embodiments, wherein the metabolic disorder or disease comprises heart failure with preserved ejection fraction (HFpEF) or a symptom and / or sequelae thereof.22. The method of any one of the preceding embodiments, wherein the subject exhibits, before the co-administering, at least one of the following: hypertension, elevated E / e’ratio, cardiac hypertrophy, myocardial fibrosis, obesity, reduced endurance, and elevated systemic inflammatory markers.23. An isolated nucleic acid for co-administering with a second therapeutic to treat a metabolic disorder or disease, the isolated nucleic acid comprising a nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12) or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA, and wherein the nucleic acid is at most 30 nt long, wherein the second therapeutic is an incretin or functional analogue thereof, and / or is an activator of glucagon-like peptide 1 (GLP-1) receptor signaling.24. An anti-inflammatory, cardioprotective nucleic acid therapeutic for coadministering with a second therapeutic to treat a metabolic disorder or disease, wherein the nucleic acid therapeutic is RNA, and wherein the nucleic acid is at most 30 nt long, wherein the second therapeutic is an incretin or functional analogue thereof, and / or is an activator of glucagon-like peptide 1 (GLP-1) receptor signaling.25. A composition comprising: an isolated nucleic acid comprising a nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12) or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA, and wherein the nucleic acid is at most 30 nt long; a second therapeutic that is an incretin or functional analogue thereof, and / or is an activator of glucagon-like peptide 1 (GLP-1) receptor signaling; and a pharmaceutically acceptable excipient.26a. A liposome-free formulation for oral delivery of a nucleic acid, comprising: a nucleic acid; at least one casein protein; and a chitosan.26b. A therapeutic formulation for oral delivery of a nucleic acid, comprising: a nucleic acid; at least one casein protein; and a chitosan, wherein the formulation is substantially free of cationic lipids.26c. A therapeutic formulation for oral delivery of a nucleic acid, comprising:a nucleic acid; at least one casein protein; and a chitosan, wherein the formulation does not comprise a cationic lipid as a transfection reagent.27. The formulation of any one of embodiments 26a-26c, wherein the nucleic acid comprises a ribonucleic acid (RNA) and wherein the RNA is present in an amount ranging between about 0.0001 and 0.01% of the formulation by weight per volume, wherein the at least one casein protein comprises at least an a-sl casein subunit and wherein the at least one casein protein is present in an amount ranging between about 0.5 and 5% of the formulation by weight per volume, and wherein the chitosan is present in an amount ranging between about 0.001 and 1% of the formulation by weight per volume.28. The formulation of any one of embodiments 26a-27, comprising an acid, optionally wherein the acid is present in an amount ranging between about 0.001 and 1% of the formulation by volume and where the acid is selected from acetic acid, phosphoric acid and citric acid.29. The formulation of any one of embodiments 26a-27, comprising acetic acid, wherein the acetic acid is present in an amount ranging between about 0.01 and 1% of the formulation by weight per volume.30. The formulation of any one of embodiments 26a-29, wherein the chitosan is medium molecular weight chitosan, optionally wherein the chitosan is or comprises deacetylated chitin, Poly(D-glucosamine).31. The formulation of embodiment 30, wherein the medium molecular weight chitosan has a molecular weight in the range of 150,000-350,000 Da.32. The formulation of any one of embodiments 26a-31, wherein the nucleic acid comprises a nucleotide sequence of CGUCCGAUGGUAGUGGGUUAUCAG (SEQ ID NO: 12) or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA, and wherein the nucleic acid is at most 30 nt long, optionally wherein the nucleic acid is TY1.33. The method of any one of embodiments 1-22, the co-administering comprises administering the isolated nucleic acid to the subject by administering the formulation of any one of embodiments to the subject.34. The formulation of any one of embodiments 26a-32, wherein the nucleic acid comprises a ribonucleic acid (RNA) or a chemically modified variant thereof, or wherein the nucleic acid comprises a deoxyribonucleic acid (DNA) or a chemically modified variant thereof.35. The formulation of embodiment 34, wherein the nucleic acid is present in an amount in a range of 0.0001% to 0.01% of the formulation by weight per volume, optionally wherein the RNA or the chemically modified variant thereof is present in an amount in a range of 0.0001% to 0.01% of the formulation by weight per volume.36. The formulation of any one of embodiments 26a-32, 34, and 35, wherein the at least one casein protein is present in an amount in a range of 0.5% to 5% of the formulation by weight per volume, optionally wherein the at least one casein protein comprises at least an a- sl casein subunit.37. The formulation of any one of embodiments 26a-32 and 34-36, wherein the at least one casein protein comprises a mixture of an a-sl casein subunit, an ot-s2 casein subunit, a P casein subunit, and a K casein subunit, wherein the casein subunits are collectively present in an amount in a range of 0.5% to 5% of the formulation by weight per volume.38. The formulation of any one of embodiments 26a-32 and 34-37, wherein the chitosan is present in an amount in a range of 0.001% to 1% of the formulation by weight per volume.39. The formulation of any one of embodiments 26a-32 and 34-38, wherein the chitosan is or comprises medium molecular weight chitosan, optionally wherein the chitosan is or comprises deacetylated chitin, Poly(D-glucosamine).40. The formulation of embodiment 39, wherein the medium molecular weight chitosan has a molecular weight in the range of 150,000-350,000 Da.41. The formulation of any one of embodiments 26a-32 and 34-40, wherein the nucleic acid comprises a nucleotide sequence of 20-40 nucleotides in length.42. The formulation of any one of embodiments 26a-32 and 34-41 , wherein the nucleic acid is at least partially single-stranded, or wherein the formulation does not comprise a second nucleic acid that is at least partially complementary to the nucleic acid.43. The formulation of any one of embodiments 26a-32 and 34-42, wherein the nucleic acid modulates an anti-inflammatory activity of a macrophage in a subject when the formulation is orally administered to the subject.44. The formulation of any one of embodiments 26a-32 and 34-43, wherein the nucleic acid comprises a nucleotide sequence of any one of the nucleotide sequences provided in Table 0.4 or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA.45. The formulation of any one of embodiments 26a-32 and 34-44, comprising an acid, optionally wherein the acid is selected from acetic acid, phosphoric acid and citric acid.46. The formulation of embodiment 45, wherein the acid is present in an amount in a range of 0.001 to 1% of the formulation by weight per volume, optionally wherein the acid is acetic acid.47. The formulation of any one of embodiments 26a-32 and 34-46, consisting essentially of the nucleic acid; the at least one casein protein; the chitosan; and the acid.48. The formulation of any one of embodiments 26a-32 and 34-47, wherein the formulation comprises less than 0.1 microliters of a cationic lipid for each microgram of the nucleic acid in the formulation, or comprises no cationic lipid.49. A therapeutic formulation for oral delivery of a nucleic acid, consisting essentially of: a nucleic acid comprising RNA or a chemically modified variant thereof; at least one casein protein, wherein the at least one casein protein comprises a mixture of an a-sl casein subunit, an a-s2 casein subunit, a P casein subunit, and a K casein subunit, wherein the casein subunits are collectively present in an amount in a range of 0.5% to 5% of the formulation by weight per volume; a chitosan present in an amount in a range of 0.001 to 1% of the formulation by weight per volume; and acetic acid present in an amount in a range of 0.01 and 1% of the formulation by weight per volume, optionally wherein the RNA or the chemically modified variant thereof is present in an amount in a range of 0.001 to 0.005% of the formulation by weight per volume.50. The formulation of any one of embodiments 26a-32 and 34-49, wherein the formulation is effective for treatment of a condition associated with inflammation and / or fibrosis when the formulation is administered orally, optionally wherein the condition associated with inflammation and / or fibrosis is a cardiac condition.51. A method for manufacturing a liposome-free therapeutic formulation for oral delivery of a nucleic acid, comprising: contacting a nucleic acid with a solution comprising 2% to 10% casein proteins by weight per volume, to generate a first mixture; and contacting the first mixture with an acid and chitosan polymers, to generate a formulation for oral delivery of the nucleic acid, wherein the method does not comprise contacting the nucleic acid with a solution comprising cationic lipids as transfection reagents, or formation of lipid nanoparticles.52. The method of embodiment 51 , wherein the casein proteins are within a solution of 5% bovine casein solution and are added to the nucleic acid at a volume ratio of about 1 : 10.53. The method of embodiment 51 or 52, wherein contacting the first mixture with an acid and chitosan polymers comprises contacting the first mixture with a second mixture of the acid and the chitosan polymers, optionally wherein the acid is acetic acid.54. The method of embodiment 53, wherein the second mixture comprises: an acetic acid solution of 0.05 to 2%, weight per volume; and a chitosan solution of 0.1% to 2%, weight per volume.55. The method of any one of embodiments 50-54, wherein the formulation is the formulation of any one of embodiments 26a-32 and 34-50.56. A method for treating a condition associated with inflammation and / or fibrosis, or a cardiometabolic disorder, comprising orally administering to a subject having or suspected of having a condition associated with inflammation and / or fibrosis, or a cardiometabolic disorder, a therapeutically effective amount of any one of the formulation of embodiments 26a- 32 and 34-50.57. The method of embodiment 56, wherein the condition associated with inflammation and / or fibrosis or the cardiometabolic disorder comprises a heart condition, optionally wherein the heart condition is heart failure with preserved ejection fraction,myocardial infarction, muscular dystrophy, scleroderma, viral infection, diabetes, and / or hypertrophic cardiomyopathy.58. The formulation of any one of embodiments 26a-32 and 34-50 for the treatment of a condition associated with inflammation and / or fibrosis, or a cardiometabolic disorder by a method comprising orally administering a therapeutically effective amount of the formulation to a subject having or suspected of having a condition associated with inflammation and / or fibrosis, or a cardiometabolic disorder.59. A method of immunomodulation, comprising administering to a subject an effective amount of the formulation of any one of embodiments 26a-32 and 34-50, wherein the nucleic acid modulates an anti-inflammatory activity of a macrophage in the subject.60. A kit comprising: at least one casein protein; a chitosan; and an acid.61. The kit of embodiment 60, further comprising a nucleic acid, wherein the kit does not comprise a cationic lipid as a transfection reagent, optionally wherein the nucleic acid is RNA.62. The kit of embodiment 60 or 61, where the acid is selected from acetic acid, phosphoric acid and citric acid, optionally wherein the acid is acetic acids.63. The kit of any one of embodiments 60-62, wherein the chitosan is medium molecular weight chitosan, optionally wherein the medium molecular weight chitosan has a molecular weight in the range of 150,000-350,000 Da.64. The kit of any one of embodiments 60-63, wherein the at least one casein protein comprises a mixture of an a-sl casein subunit, an a-s2 casein subunit, a 0 casein subunit, and a K casein subunit.

[0177] 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 disclosurecan 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 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.

[0178] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain 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.

[0179] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLESExample 1

[0180] This non-limiting example shows that combination therapy of semaglutide plus novel noncoding RNA drug TY 1 exerts synergistic benefits in heart failure with preserved ejection fraction (HFpEF).

[0181] Background: The International Diabetes Federation (IDF) indicates that approximately 537 million adults worldwide had diabetes in 2021, accounting for around 10.5% of the global population. Projections suggest that by 2030 and 2045, these numbers are expected to soar to 643 million and 783 million, respectively.

[0182] Obesity is a complex, chronic, multifactorial disease that the American Medical Association (AMA) has recognized since 2013. It affects over 1 billion people worldwide, according to recently updated figures from The Lancet, and, in the US precisely, 42.4% of adults and 19.4% of the pediatric population per the latest National Health and Nutrition Examination Survey.

[0183] Obesity and diabetes are key drivers of cardiometabolic heart failure with preserved ejection fraction (HFpEF). This large population of obese and diabetic children and adults is increasing.

[0184] Heart failure with preserved ejection fraction (HFpEF) is a major unmet medical need, comprising over 50% of all heart failure cases. This multi-organ cardiometabolic disorder, characterized by diastolic dysfunction and exercise intolerance, often involves hypertension, obesity and diabetes.

[0185] Currently available therapies only manage symptoms and do not improve mortality rates. New approaches are urgently needed to combat HFpEF.

[0186] Hypothesis: Glucagon-like peptide-1 (GLP-1) receptor agonists show promise in HFpEF clinical trials and are expected to become standard-of-care treatment for this condition. Semaglutide is a GLP-1 agonist that has been shown to improve quality of life, exercise performance, and weight loss in HFpEF patients with obesity and diabetes.

[0187] It was hypothesized that combining semaglutide with TY1, a novel synthetic bioinspired non-coding RNA, which attenuates innate immunity pathways in myocardial infarction, would provide synergistic benefits in cardiometabolic HFpEF.

[0188] Methods: Male ZSF1 obese rats were divided into four groups, n = 6 each; HFpEF Control, HFpEF + TY 1 (0.15 mg / kg P.O. twice per week), HFpEF + semaglutide group (30 nmol / kg S.Q. twice per week) and combination therapy (TY1 + Semaglutide). Treatments were initiated at 10 weeks of age (baseline). Body weight, food intake, blood glucose, echocardiography (including LVEF and E / e’ ratio), and treadmill exercise performance (distance and work) were measured at baseline, after 4 weeks and after 8 weeks.

[0189] Results: While all groups gained weight, those treated with TY1, semaglutide, or the combination showed a trend towards reduced weight gain compared to control. Semaglutide and combination therapy significantly lowered blood glucose compared to control and TY1 groups. TY1 alone, semaglutide alone, and the combination therapy, significantly improved E / e' ratio, a marker of diastolic dysfunction. Exercise distance and work were also significantly improved in TY1, semaglutide, and combination groups compared to control. Notably, combination therapy showed greater improvements in E / e' ratio and exercise work compared to either single agent alone.

[0190] Conclusions: Monotherapy with either TY1 or semaglutide improves signs of HFpEF, but the pair together is superior to either agent alone. The combination of semaglutide’ s metabolic benefits, plus TYl’s attenuation of the DNA damage response,harnesses complementary mechanisms to more effectively combat this complex multisystem disease.Example 2

[0191] This non-limiting example shows synergistic effect of co-administration of TY1 and semaglutide (“TY1S”) in a “two-hit” mouse model of cardiometabolic HFpEF.

[0192] To assess TY1S in the target indication, HFpEF, the well-characterized “two-hit” mouse model was utilized. This model involves the co-administration of L-NAME and a high-fat diet (HFD). Male C57BL6 / N mice received L-NAME + HFD for 5 wks to induce HFpEF (FIG. 1). The effects of TY 1 S following the onset of HFpEF were investigated. Mice received oral TY1 (0.15 mg / kg) alone or in combination with semaglutide (30 nmol / kg, s.c. injection) biweekly for 10 wks. A group of animals that received semaglutide alone were also investigated.

[0193] Food intake and body weight were monitored weekly (FIG. 2). Animals that were administered semaglutide alone or semaglutide and TY1 reduced food intake upon administration compared to control animals and animals that were administered TY1 alone (FIG. 2, left panel). Administration of semaglutide alone or semaglutide and TY1 also prevented the gain in body weight observed in control animals and animals that were administered TY1 alone (FIG. 2, right panel). Non-fasting glucose levels were measured at 0, 5, 10, and 15 weeks. Animals that were administered semaglutide alone or semaglutide and TY1 had reduced non-fasting glucose levels compared to control animals and animals that were administered TY1 alone (FIG. 3; from left to right for each time point: Control; TY1; Serna; Sema+TYl; ).

[0194] Echocardiography and treadmill exercise performance were both measured (FIGs. 4A and 4B). At 5 weeks following L-NAME + HFD, the HFpEF phenotype was established, with increased left ventricular E / e’ (a measure of diastolic function) (FIG. 4A) and reduced exercise capacity (FIGs. 4B and 5; from left to right for each time point: Control; TY 1 ; Serna; Sema+TYl). Administration of TY1 or semaglutide alone resulted in significant reductions in E / e’ (FIG. 4A). Interestingly, TY1S (here labeled as Sema + TY1) restored left ventricular E / e’ to a significantly greater extent than either TY1 or semaglutide alone; indeed, E / e’ values were comparable to baseline in the TY1S group. At 15 weeks of HFpEF leftventricular end-diastolic pressures were measured (LVEDP; FIG. 4C). LVEDP was reduced to a comparable extent with TY 1 or semaglutide alone, but TY 1 S resulted in a significantly great reduction in LVEDP to normal levels. This demonstrated complete reversal of the HFpEF phenotype, which was unprecedented.

[0195] FIGs. 4A-4C. TY1S reduces diastolic dysfunction and improves exercise capacity in a “two-hit” mouse model of cardiometabolic HFpEF. (FIG. 4A) LV diastolic dysfunction (E / e’) was significantly reduced with TY1 alone and returned to baseline levels following TY1S. From left to right for each time point: Control; TY1; Sema; Sema+TYl; (FIG. 4B) Exercise performance was increased with TY1S. From left to right for each time point: Control; TY1; Sema; Sema+TYl; (FIG. 4C) LV end-diastolic pressure. n=12 mice per group. *p < 0.05 vs. Control.

[0196] The time constant of isovolumic left ventricular pressure decay did not show significant difference across different treatment groups (FIG. 9). Left ventricular ejection fraction (LVEF) was also measured, and no difference was observed in LVEF among the different treatment groups (FIG. 6; from left to right for each time point: Control; TY1; Sema; Sema+TYl). Hang wire test of animals showed administration of semaglutide alone or TY1S improved performance compared to control or TY 1 only (FIG. 7). Systolic and diastolic blood pressure was also reduced with administration of semaglutide alone or TY1S compared to control (FIG. 8).Example 3

[0197] This non-limiting example shows synergistic effect of co-administration of TY1 and semaglutide (“TY1S”) in a model of HFpEF in rat that involves genetically driven hypertension and obesity-metabolic syndrome. This example relates to Example 1.

[0198] TY1S in the target indication was assessed in a severe genetic model, the ZSF1 Ob rats, of cardiometabolic HFpEF as shown in FIG. 10. For these studies male, ZSF1 obese (ob) rats were used.

[0199] The ZSF1 ob rat is a model of HFpEF that involves genetically driven hypertension and obesity -metabolic syndrome37'39. The ZSF1 ob rat is a hybrid rat strain between the spontaneously hypertensive heart failure (SHHF) and Zucker diabetic fatty rat37'39. These animals exhibit profound obesity, metabolic syndrome, and hypertension thatworsens with age38,39. Studies were initiated in 10-wks of age which is consistent with a robust HFpEF phenotype. TY1 (0.15 mg / kg, oral) and semaglutide (30 nmol / kg, s.c. injection) were administered biweekly for 16 weeks.

[0200] Food intake and body weight were monitored weekly (FIG. 11). Animals that were administered semaglutide alone or semaglutide and TY1 reduced food intake upon administration compared to control animals and animals that were administered TY1 alone (FIG. 11, left panel). Administration of semaglutide alone or semaglutide and TY 1 also slowed the gain in body weight compared to control animals (FIG. 11, right panel).

[0201] Non-fasting blood glucose, LV E / e’, and LVEDP were evaluated. Hyperglycemia was reduced in a highly significant manner with semaglutide alone and with TY1 S (here labeled as Serna + TY1) consistent with the anti-diabetic actions of GLP-1 receptor agonists (RAs) (FIG. 12A). Similar to the “two-hit” mouse HFpEF studies, reductions in LV E / e’ and LVEDP were observed with TY1 or semaglutide monotherapy (FIGs. 12B and 12C). TY1S provided significantly greater benefits when compared to either agent alone.

[0202] FIGs. 12A-12C. TY 1 S reduces diastolic dysfunction and improves exercise capacity in the genetic rat model of severe cardiometabolic HFpEF model. (FIG. 12A) Blood glucose was significantly reduced by semaglutide and TY1S to baseline levels at all experimental timepoints; (FIG. 12B) LV diastolic dysfunction as measured by echocardiography (E / e’) was significantly reduced by TY1 and further reduced by TY1S at 8- and 16- weeks timepoints; (FIG. 12C) LV end-diastolic pressure (LVEDP) at 16 weeks of HFpEF as measured by invasive hemodynamic was reduced by TY1 single agent treatment, and was further reduced by TY1S. n=5-6 per group. * p < 0.05 vs. WKY Control rats.

[0203] Treadmill exercise performance was measured and showed that treatment with TY1 only, semaglutide only, or TY1S improved exercise capacity compared to control (FIG. 13; from left to right for each time point: Control; TY1; Sema; Sema+TYl). Left ventricular ejection fraction (LVEF) was also measured, and no difference was observed in LVEF among the different treatment groups (FIG. 14; from left to right for each time point: Control; TY1; Sema; Sema+TYl). Levels of 8-isoprostane, a marker of oxidative stress, were measured, and reduction was observed in animals treated with semglutide only or TY1S compared to control or TY1 only-treated animals (FIG. 16).Example 4

[0204] This non-limiting example shows ex vivo aortic vascular reactivity in a model of HFpEF in rat that involves genetically driven hypertension and obesity-metabolic syndrome.

[0205] HFpEF is associated with abnormalities in endothelium that contribute to cardiovascular dysfunction. Thus, ex vivo endothelium-dependent and independent vascular responses were evaluated in the ZSF1 ob rat (FIGs. 15A-15D). At 16 wks of HFpEF thoracic aortae from ZSF1 ob rats that had been treated with TY1, semaglutide, or TY1S were isolated. Vasorelaxation responses to increasing concentrations of acetylcholine (ACh) and sodium nitroprusside (SNP) were studied. ACh releases endothelial nitric oxide (NO), a marker of endothelial function while SNP elicits smooth muscle cell relaxation as an index of endothelium-independent function. TY1 or semaglutide resulted in improvements in both endothelial-dependent and independent vascular reactivity. Remarkably, TY1S (here labeled as Serna + TY1) resulted in synergistic improvements in vascular responses that were greater than the responses to either drug alone. These data support a direct vascular effect of TY 1 S in HFpEF and provide mechanistic insights underlying the beneficial effects.

[0206] FIGs. 15A-15D. Effects of TY1S on vasorelaxation of aortic vascular rings. (FIG. 15A) Relaxation to acetylcholine (Ach); (FIG. 15B) Relaxation responses to Sodium nitroprusside (SNP); (FIG. 15C) ECso for ACh relaxation; (FIG. 15D) ECso for SNP relaxation. n=6 animals per group. *p < 0.05 vs. HFpEF.

[0207] Summary of Examples 1-4: The mechanistically-motivated hypothesis that TY1S is therapeutically superior, in HFpEF, to either drug alone was tested. Such a hypothesis is based on the premise that the two drugs have entirely complementary mechanisms of action (MoA). The data provided herein are consistent with this hypothesis: in “two-hit” HFpEF mice, TY1S is significantly better than either agent alone in improving diastolic heart function and in enhancing exercise capacity. In the complementary ZSF1 ob rat model of HFpEF, the superiority of TY1S in improving diastolic cardiac function and in reducing LV filling pressure, both key hallmarks of HFpEF, were confirmed. These benefits occurred without apparent toxicity at the doses tested. In vascular rings from ZSF1 ob rats, TY1S restored vascular function, and did so more effectively than either agent alone.Example 5

[0208] This non-limiting examples shows a simplified oral TY 1 formulation.

[0209] A simplified oral formulation was developed by omitting the lipid transfection reagent Dharmafect® from previous formulations. This removes one excipient, with its potential CMC complications. In the new formulation, TY1 is the ncRNA active pharmaceutical ingredient (API), which is admixed with casein+chitosan (C2) to create the oral formulation, TY1-C2. TY1 is synthesized using standard options. The remaining two constituents are the excipients: casein and chitosan. The casein used in this formulation is a mixture of alpha-, beta- and kappa-caseins. The chitosan used is medium molecular weight deacetylated chitin, Poly(D-glucosamine). TY1-C2 was first formulated in casein protein followed by admixture with chitosan under acidic conditions to form casein chitosan micelles that encapsulate TY1 (FIG. 21). TY1-C2 formed micelles that were absorbed orally and protect the ncRNA from degradation, consistently among various batches (FIGs. 22A-22C), TY1-C2 was straightforward to produce, casein and chitosan are common, natural food entities generally regarded as safe. All the data shown in Examples 1-4 and 6 were obtained using the TY1-C2 oral formulation.Example 6

[0210] This non-limiting examples shows repeated administration of TY1 is well- tolerated in healthy mice.

[0211] In a safety study, healthy animals fed TY1 (0.15 mg / kg / dose) twice weekly for four weeks (FIG. 17A) showed a favorable safety profile. After four weeks of chronic exposure, animals receiving TY1 orally did not exhibit weight loss compared to vehicle-fed animals (FIG. 17B). Furthermore, exercise endurance, as measured by maximal distance (on a treadmill) was preserved (FIG. 17C). Four weeks post-exposure (eight total oral doses), blood and tissue samples were isolated for signs of tissue damage or inflammation. Sections of heart, lung, liver, and kidney tissue from TYl-fed animals showed no increases in fibrosis (Masson’s trichrome staining). Furthermore, blood chemistry showed no remarkable electrolyte disturbance, liver or kidney function abnormalities, or acute phase reactants. Finally, complete blood cell counts (CBC) showed no changes in total white blood cell counts includinglymphocytes or neutrophils. These findings suggest that repeated oral administration of TY1 is well tolerated.

[0212] Semaglutide is FDA-approved with an extensive record of clinical safety and efficacy as monotherapy. Semaglutide is also FDA-approved in an oral formulation (Rybelsus®).Example 7

[0213] This non-limiting examples shows dose optimization of TY1S in HFpEF (FIG. 18A).Methods

[0214] “Two-Hit” cardiometabolic HFpEF mouse model. Mice (C57BL6 / N, Charles River) are fed with L-NAME implemented (1,500 mg / Kg diet) high fat diet (HFD, 60 kcal% fat, Research Diets DI 2492) for a total duration of 15 weeks to induce cardiometabolic HFpEF as described previously37’40. Mice are fed L-NAME+HFD for 5 wks to induce HFpEF at which time oral treatment with either vehicle, TY1 (0.02, 0.04, 0.08 mg / kg / day), or semaglutide (4.5, 9, 18 nmol / kg / day) is initiated. Once the optimal doses of oral TY1 and semaglutide are determined, TY1S therapy is investigated. Mice receive TY1, semaglutide, or TY1S for 10 weeks. Experiments are performed in both male and female mice with n=12.

[0215] Echocardiographic assessment of LV function. Baseline echocardiography is performed in all animals at baseline and at weeks 5, 10, and 15. In vivo transthoracic echocardiography is performed using the VEVO 3100 echocardiography system (VisualSonics, Canada)37,40. Isoflurane (3%) supplemented with 100% oxygen in an induction chamber is used for sedation. The mice receive 0.5 -1.5 % of isoflurane via nose cone during image acquisition.

[0216] Exercise capacity assessment. Mice are subjected to exercise capacity assessment using a rodent-specific treadmill (IITC Life Science, Model 805 treadmill, Woodland Hills, CA)37,40. Before the test, animals are acclimated in the treadmill chamber. Next, animals undergo a short walk / run protocol to help train the animals for treadmill running. The treadmill is set to start at 0 m / min speed, and increased Im / min every minute for 10 minutes. During the test phase in which the treadmill is set to 10-degree incline. The startingspeed is 10 m / min and is programed to increase by 2.67 m / min every minute for 3 min., at which time the treadmill reaches and is maintained at its top speed of 18 m / s.

[0217] LV hemodynamic assessment. At study termination animals are anesthetized with isoflurane in oxygen (3% isoflurane for induction, 1-1.5% isoflurane for maintenance)37'40. The right common carotid artery is dissected and a pre-calibrated 1.0 Fr pressure catheter SPR-1000, (Millar, USA) is inserted and arterial blood pressures is recorded. The catheter is then advanced into the LV to record end-diastolic pressure (LVEDP) and relaxation constant (Tau).

[0218] Aortic Vascular Reactivity. At euthanasia the thoracic aorta is carefully dissected and excised for ex vivo vascular reactivity testing as described previously37'40. Aortic ring segments are equilibrated in Krebs-Henseleit solution with a tension of 0.5g for a period of 60-90 minutes. Rings are precontracted with phenylephrine (1 pmol / L) and then treated with acetylcholine ( 109to 105M) followed by sodium nitroprusside ( 1010to 107M). These data are reported as the % relaxation as compared with the maximal contraction induced by phenylephrine.

[0219] Biomarker Assays for Heart Failure, Oxidative Stress, and Nitrosative Stress. Biomarkers of oxidative stress, nitrosative stress, inflammation are measured in mouse plasma samples isolated from fresh blood collected from the inferior vena cava at the time of sacrifice.

[0220] Tissue Fibrosis Determination. Fibrosis is measured in the heart, liver, and kidney using Masson’s tri chrome and Picrosirius red staining at 15 weeks of HFpEF in all mice as previously described37,40. Tissue processing and staining is performed at the Cedars-Sinai Biobank and Research Pathology Core Lab.

[0221] Statistical Analysis. All data are expressed as the mean ± SEM. Data are statistically analyzed using Prim 6 (GraphPad Software, San Diego, California) with Student unpaired, 2-tailed, T test when only comparing at a single time point and a repeated 2-way analysis of variance (ANOVA) with a Bonferroni post-test when performing multiple comparisons between two groups, or within group, at multiple time points. All p values of < 0.05 is considered statistically significant.

[0222] Results. When administered individually TY1 and Semaglutide attenuate the severity of HFpEF pathophysiology in a dose-dependent manner. Therapy with TY 1 Ssignificantly improve HF outcomes compared to monotherapy. Reductions in LV E / e’ and LVEDP in conjunction with improved treadmill exercise performance and vascular reactivity is observed. TY1S attenuates cardiac fibrosis and circulating biomarkers of heart failure severity and oxidative-nitrosative stress. The beneficial effects of TY1S is observed in both sexes.Example 8

[0223] This non-limiting example shows efficacy studies in a translational model of cardiometabolic HFpEF (FIG. 18B).Methods

[0224] Multiple HFpEF Comorbidities in Gottingen miniswine. Gottingen minipigs (adult, females) receive mineralocorticoid-excess (DOCA salt) and a Western high fat diet to induce HFpEF as described previously41. Pigs are fed a diet 50 / 50 (wt / wt) mix of standard diet and custom diet containing high levels of fat, fructose, cholesterol and salt (9GZC TestDiet, St. Louis, MO: Ossabaw atherosclerotic diet type 5B4L w / 2% total salt). Animals are fed once per day and water provided. Minipigs also receive a subcutaneous deoxycorticosterone acetate (DOCA) depot (50 mg / kg, 200 mg pellets, 60-day release, Innovative Research of America, Sarasota, FL).

[0225] Conscious Arterial Blood Pressure Monitoring. Prior to the onset of HFpEF, high fidelity pressure catheters (Data Sciences International) are advanced into the descending aortic via the external carotid artery. Arterial pressures (systolic, diastolic, mean) as well as heart rate are continuously collected during the 20 wk protocol.

[0226] Blood Collection. Venous blood samples are obtained from anesthetized animals at baseline and at 4, 8, 10, 12, 14, 16, 18, and 20 wks, processed for plasma and serum, snap frozen and stored at -80°C.

[0227] Biomarker Assays for Heart Failure, Oxidative Stress, and Nitrosative Stress. Biomarkers of oxidative stress, nitrosative stress, and inflammation re measured in swine plasma samples isolated from fresh blood collected the jugular vein at the time of sacrifice. Samples are placed on ice and centrifuged at 4,000g for 20 minutes. Plasma are assayed using commercially available ELISA kits for 8-isoprostane (Cayman Chemical), 3- nitrotyrosine (Novus Biologicals), hsCRP (My BioSource).

[0228] Transthoracic B-mode, Two-dimensional Speckle Tracking and Doppler Echocardiography. At baseline, 4, 10, 16, and 20 wks, transthoracic echocardiography is performed in miniswine under ketamine / xyl azine sedation and isoflurane anesthesia. LVEF is measured using 2D speckle tracking with LV volumes, dimensions and wall thickness at systole and diastole measured from subcostal 2D B-mode images acquired at the level of the mitral valve leaflets. Left atrial area and fractional area of change is measured from subcostal 2D B-mode views. PW tissue doppler imaging, with a maximal corrected angle of 40°, is used to calculate tissue velocities during early ventricular fdling (e’) at the mitral valve medial and lateral annulus with medial and lateral ratios of early transmitral inflow and tissue velocity (E / e’) calculated.

[0229] MRI Imaging for Body Composition. Myocardial lipid content and body composition are measured at baseline and at 20 wks of HFpEF in all animals. These measurements are performed using MRI in the Cedars-Sinai Research Imaging Core Laboratory.

[0230] Treadmill Testing. Minipigs are acclimated and then subjected to treadmill testing at baseline, prior to treatment at the 10-week timepoint, and at the 20 wk timepoint. Total time and distance are recorded.

[0231] Vascular Reactivity. At 20 wks the left anterior descending (LAD) coronary is cut into 5-mm rings and mounted in organ baths for isometric tension experiments as described previously41. Artery contractile responsiveness is assessed by potassium chloride. Vascular rings pre-contracted with prostaglandin PGF2 alpha is tested for endotheliumdependent relaxation responses to bradykinin and substance P and endothelial-independent relaxation responses to sodium nitroprusside. Maximum relaxation responses and half maximal effective concentrations (ECso) are calculated.

[0232] Capillary Density. LV tissue samples are fixed in neutral buffered formalin, paraffin-embedded, 5pm cross-sections mounted onto glass slides and immunostained with anti-CD31 antibody to identify blood vessels. For each tissue section, 20 images at 40x magnification are acquired, tissue area measured and number of CD31 positive blood vessels counted. The total number of blood vessels per total area is calculated for each sample as previously described.

[0233] Tissue Fibrosis Determination. Fibrosis is measured in the heart, liver, and kidney using Masson’s trichrome and Picrosirius red staining at 20 weeks of the HFpEF study protocol in all pigs. Tissue processing and staining will be performed at the Cedars-Sinai Biobank and Research Pathology Core Lab.

[0234] Natriuretic Peptide measurements. Plasma samples are collected at baseline and at 4-wk intervals at the same time the echocardiography studies are performed41. WA., USA). NT proBNP and NT proANP are quantified using ELISA technique according to the manufacturer’s instructions (My Biosource). LV tissue is obtained at 20 wks and rtPCR analysis of BNP and ANP is performed.

[0235] Statistical Analysis. All data are expressed as the mean ± SEM. Data are statistically analyzed using Prim 6 (GraphPad Software, San Diego, California) with Student unpaired, 2-tailed, T test when only comparing at a single time point and a repeated 2-way analysis of variance (ANOVA) with a Bonferroni post-test when performing multiple comparisons between two groups, or within group, at multiple time points. All p values of < 0.05 is considered statistically significant.

[0236] Results. Administration of TY 1 S results in significant improvements in LV diastolic function, decrease LV and pulmonary pressures, decrease circulating NT-proBNP and oxidative-nitrosative stress biomarker levels, improve vascular endothelial function, increase myocardial vascularity, decrease tissue fibrosis, and improve exercise performance in Gottingen miniswine with HFpEF. TY1S provides superior benefit as compared to semaglutide alone and TY 1 S is well-tolerated in this swine model of HFpEF. Given the potent anti-obesity effects of semaglutide, TY1S therapy decreases myocardial lipid content and improves overall body composition in the setting of HFpEF. The high-fat Western diet and DOCA administration is titrated to appropriately modulate the severity of HFpEF. If significant beneficial effects are not observed, dosing is escalated in an additional cohort of animals.Example 9

[0237] This non-limiting examples shows dose-ranging studies for toxicology in healthy mice and swine (FIG. 18C).Methods

[0238] Healthy C57BL6 mice (n=10 per group) are subjected to either control, oral TY1S -2x therapeutic dosing, and oral TY1S -5x therapeutic dosing for a period of 8 weeks. Both male and female mice are studied. In addition, healthy Gottingen miniswine (n=3 per group) are subjected to either control, oral TY1S -2x therapeutic dosing, and oral TY1S -5x therapeutic dosing for a period of 4 weeks. Both male and female miniswine are investigated.

[0239] Observations of Test Animals. Routine cage-side observations are made on all animals at least once or twice a day throughout the study for general signs of pharmacologic and toxicologic effects, morbidity and mortality. The usual interval between observations is at least 6 hours. Individual records are maintained for each animal and the time of onset and the characteristics and progression of any effects are recorded, preferably using a scoring system. Animal body weights are collected on a weekly basis.

[0240] Hematology. Hematology (i.e., CBC) measurements are performed at baseline and following dosing with TY 1 S in all animals. Hematological measurements include: hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte counts, mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration, and platelet count.

[0241] Clinical Chemistry. Clinical chemistry measurements are performed at baseline and following dosing with TY1S in all animals. Animals undergo fasting overnight prior to sample collection. Blood samples are analyzed individually, and not pooled. Blood samples are drawn at approximately the same time each sampling day. The following are evaluated: alanine aminotransferase (ALT, SGPT), aspartase aminotransferase (AST, SGOT), sorbitol dehydrogenase, alkaline phosphatase, bilirubin (total), gamma-glutamyl transpeptidase (GG transferase), albumin, calcium, chloride, total cholesterol, cholinesterase, globulin (calculated), glucose, phosphorus, potassium, total protein, sodium, triglycerides, urea nitrogen.

[0242] Urinalyses. Urine samples are collected in all animals at baseline and following TY 1 S dosing. Specific gravity, pH, glucose, and protein are measured. Microscopic evaluation for sediment, and presence of blood / blood cells are evaluated.

[0243] Gross Necropsy. All test animals in the toxicology study are subjected to complete gross necropsy, including examination of external surfaces, orifices, cranial, thoracic and abdominal cavities, carcass, and all organs.

[0244] Organ Weights. Adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thyroid / parathyroid, thymus, ovaries and uterus are carefully dissected and trimmed to remove fat and other contiguous tissue and then weighed immediately to minimize the effects of drying on organ weight.

[0245] Tissue Preparation and Evaluation. For these studies adrenal glands, aorta, bone (femur), bone marrow (sternum), brain, cecum, colon, duodenum, esophagus, eyes, heart, ileum, jejunum, kidneys, liver, lung, mammary glands, ovaries, skeletal muscle, skin, spleen, spinal cord, stomach, testes, and trachea are collected. Tissues are fixed in 10% buffered formalin and sections prepared and stained with hematoxylin and eosin (or another appropriate stain) in preparation for microscopic examination. All tissues from the animals in the control and high dose groups are examined by random tissue sampling (in the absence of gross lesions).

[0246] Results. No unusual toxicity or adverse reactions in these mouse and swine toxicity studies are observed. As shown above, no sign of toxicity or adverse events were observed in mice exposed to TY1S for 10 weeks in the setting of severe HFpEF. Similarly, ZSF1 obese rats suffering from obesity, hypertension, hyperglycemia and severe HFpEF were treated with TY1S for 16 weeks without any evidence of toxicity. In rodent preclinical studies of TY1S, this drug combination was highly efficacious and very well tolerated.Example 10

[0247] This non-limiting example shows an oral formulation for a therapeutic RNA.

[0248] As provided for herein, several embodiments relate to the generation of compositions comprising a therapeutic nucleic acid, such as a coding or non-coding RNA, the compositions being formulated for oral administration. In several embodiments, these compositions are formulated, by way of example, according to the general schematic of Figure 23, which depicts a non-limiting embodiment in which a nucleic acid (such as RNA, in particular a non-coding RNA with therapeutic effects upon administration (e.g., including oraladministration) is encapsulated in an assembly of casein and chitosan through exposure to an acidic solution. The oral RNA formulation is prepared without the use of a lipid transfection reagent. The casein-chitosan formulation allows for oral delivery of the nucleic acid with increased bioavailability of the nucleic acid due to the casein-chitosan coated micelle imparting acid resistance to the composition, allowing it to pass through the acidic environment of the stomach with limited degradation. For the formulation, casein (e.g., 5-10% w / v casein, or about 8% w / v casein) and chitosan (e.g., medium molecular weight chitosan, 0.01-0.1% w / v), were used without a lipid transfection reagent, and encapsulation was achieved under acidic conditions (e.g., acetic acid, 0.01-1% w / v). (FIG. 23).Example 11

[0249] This non-limiting example shows the effectiveness of the oral formulation (without the lipid transfection reagent) of a therapeutic RNA as a treatment in the 48-hour cardiac ischemia / reperfusion injury model.

[0250] To study the therapeutic efficacy of orally delivered RNA formulated without lipid transfection reagent, the 48-hour cardiac ischemia / reperfusion injury model was used (FIG. 24). Rats were given cardiac ischemia using temporary ligation of the left descending coronary artery for 45 minutes. After the ischemic period, the suture was removed to start reperfusion injury. Twenty minutes after reperfusion, animals were given oral administration of a therapeutic RNA (e.g., TY1) formulated as shown in Table 11.1, which lists the components used to prepare the oral formulation.Table 11.1: Orally-formulated TY1TY1 : 32 pg for a 160 g animal (0.2 mg / kg)Casein: 100 pl of 5% solution (w / v)Chitosan: 125 pl of 0.2% solution (w / v)Acetic Acid: 125 pl of 0.1% solution (w / v)

[0251] An intravenous group (given retroorbitally: RO; Tx - IV) was also included as a positive control. 48 hours after injury, the heart was excised and sectioned to assess infarct size (using TTC staining) and blood was collected to assess circulating levels of cardiac troponin (cTnl), which correlates with amount of cardiac damage.

[0252] Oral administration of TY1 was tested using a previously optimized dose (FIGs. 25A-25C). The orally administered TY1 (TYl_Oral, 200 ng / g body weight) significantly reduced both scar size (FIGs. 25A-25B) and cTnl (FIG. 25C) compared to vehicle.

[0253] The results show that the oral formulation of a therapeutic RNA (e.g., TY1) without the lipid transfections reagent is at least as effective as the parenterally administered formulation in treating cardiac ischemia / reperfusion injury.Example 12

[0254] This non-limiting example shows the effectiveness of the oral formulation (without the lipid transfection reagent) of a therapeutic RNA as a treatment in the 48-hour cardiac ischemia / reperfusion injury model.

[0255] Oral administration of miR-1246 was tested using previously optimized doses, in a cardiac ischemia / reperfusion injury model according to the protocol described in Example 11 (FIGs. 26A-26C). miR-1246 was formulated as described herein (e.g., see Example 11). Orally administered miR-1246 (20 ng / g body weight) reduced infarct size (FIG. 26A and FIG. 26B) and cardiac troponin levels (cTnl) (FIG. 26C) compared to retroorbitally (RO) administered miR-1246.

[0256] The results show that the oral formulation of a therapeutic RNA (e.g., miR- 1246) without the lipid transfections reagent is at least as effective as the parenterally administered formulation in treating cardiac ischemia / reperfusion injury.Example 13

[0257] This non-limiting example shows the effectiveness of the oral formulation (without the lipid transfection reagent) of a therapeutic RNA as a treatment in the 48-hour cardiac ischemia / reperfusion injury model.

[0258] Oral administration of uREXl was tested using previously optimized doses, according to the protocol described in Example 11 (FIGs. 26A-26C). uREXl was formulated as described herein (e.g., see Example 10). The orally administered uREXl (20 ng / g body weight) significantly reduced infarct size (FIG. 26A and FIG. 26B) and cardiac troponin levels (cTnl) (FIG. 26C) compared to retroorbitally (RO) administered uREXl .

[0259] The results show that the oral formulation of a therapeutic RNA (e.g., uREXl) without the lipid transfections reagent is at least as effective as the parenterally administered formulation in treating cardiac ischemia / reperfusion injury.Example 14

[0260] This non-limiting example shows the effectiveness of the oral formulation (without the lipid transfection reagent) of a therapeutic RNA as a treatment in the 48-hour cardiac ischemia / reperfusion injury model.

[0261] To study the therapeutic efficacy of orally delivered RNA formulated without lipid transfection reagent, the 48-hour cardiac ischemia / reperfusion injury model was used (see Example 11). Rats were given cardiac ischemia using temporary ligation of the left descending coronary artery for 45 minutes. After the ischemic period, the suture was removed to start reperfusion injury. Twenty minutes after reperfusion, animals were given oral administration of a therapeutic RNA (e.g., TT1) formulated as shown in Table 14.1, which lists the components used to prepare the oral formulation.Table 14.1; Orally-formulated TT1TT1 : 3.2 pg for a 160 g animal (0.02 mg / kg)Casein: 100 pl of 5% solution (w / v)Chitosan: 125 pl of 0.2% solution (w / v)Acetic Acid: 125 pl of 0.1% solution (w / v)

[0262] A control group was administered the therapeutic RNA with the lipid transfection reagent (e.g., Dharmafect® (DF)). 48 hours after injury, the heart was excised and sectioned to assess infarct size (using TTC staining).

[0263] Oral administration of TT1 (20 ng / g body weight) was tested using previously optimized doses (FIG. 27A and FIG. 27B). The oral formulation of TT1 (without a lipid transfection reagent) significantly reduced infarct size and at least as effectively as the oral formulation that included the lipid transfection reagent (FIG. 27A and FIG. 27B).

[0264] The results show that the oral formulation of a therapeutic RNA (e.g., TT1) without the lipid transfections reagent is at least as effective as the oral formulation with the lipid transfections reagent in treating cardiac ischemia / reperfusion injury.Example 15

[0265] This non-limiting example shows the effectiveness of the oral formulation (without the lipid transfection reagent) of a therapeutic RNA as a treatment in the 48-hour cardiac ischemia / reperfusion injury model.

[0266] Oral administration of yREX3 was tested using previously optimized doses, according to the protocol described in Example 14 (FIG. 28). yREX3 was formulated as shown in Table 15.1, which lists the components used to prepare the oral formulation.Table 15.1; Orally-formulated yREX3 yREX3: 0.4 pg for a 160 g animal (2.5 pg / kg)Casein: 100 pl of 5% solution (w / v)Chitosan: 125 pl of 0.2% solution (w / v)Acetic Acid: 125 pl of 0.1% solution (w / v)

[0267] The oral formulation of yREX3 (without a lipid transfection reagent) significantly reduced infarct size and at least as effectively as an oral formulation that included the lipid transfection reagent (FIG. 28).

[0268] The results show that the oral formulation of a therapeutic RNA (e.g., yREX3) without the lipid transfections reagent is at least as effective as the oral formulation with the lipid transfections reagent in treating cardiac ischemia / reperfusion injury.

[0269] Although the foregoing has been described in some detail by way of illustrations and examples for purposes of clarity and understanding, it will be understood by those of skill in the art that modifications can be made without departing from the spirit of the present disclosure. Therefore, it should be understood that the forms disclosed herein are illustrative only and are not intended to limit the scope of the present disclosure, but rather to also cover all modification and alternatives coming with the true scope and spirit of the embodiments of the present disclosure.

[0270] It is contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments disclosed above may be made. Further, the disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, or the like in connection with an embodiment can be used in all other embodiments set forth herein. Accordingly, it should be understood that various features and aspects of thedisclosed embodiments can be combined with or substituted for one another in order to form varying modes of the disclosed subject matter. Thus, it is intended that the scope of the present disclosure should not be limited by the particular disclosed embodiments described above. Moreover, while the disclosed subject matter is susceptible to various modifications, and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. It should be understood, however, that the present disclosure is not to be limited to the particular forms or methods disclosed, but is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the various embodiments described and the appended claims.

[0271] Any methods disclosed herein need not be performed in the order recited. The methods disclosed herein include certain actions taken by a practitioner; however, they can also include any third-party instruction of those actions, either expressly or by implication. For example, actions such as “administering to a subject in need of treating a heart condition or symptom thereof a therapeutically effective amount of the nucleic acid” include “instructing the administration of an effective amount of the nucleic acid to a subject.” In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0272] The ranges disclosed herein also encompass any and all overlap, sub-ranges, and combinations thereof. Language such as “up to,” “at least,” “greater than,” “less than,” “between,” and the like includes the number recited. Numbers preceded by a term such as “about” or “approximately” include the recited numbers. For example, “about 90%” includes “90% .” In some embodiments, at least 95% homologous includes 96%, 97%, 98%, 99%, and 100% homologous to the reference sequence. In addition, when a sequence is disclosed as “comprising” a nucleotide or amino acid sequence, such a reference shall also include, unless otherwise indicated, that the sequence “comprises”, “consists of’ or “consists essentially of’ the recited sequence.

[0273] Terms and phrases used in this application, and variations thereof, especially in the appended claims, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing, the term ‘including’ should be read to mean ‘including, without limitation,’ ‘including but not limited to,’ or the like.

[0274] 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 applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.

Claims

WHAT IS CLAIMED IS:

1. A liposome-free therapeutic formulation for oral delivery of a nucleic acid, comprising: a nucleic acid; at least one casein protein; and a chitosan.

2. A therapeutic formulation for oral delivery of a nucleic acid, comprising: a nucleic acid; at least one casein protein; and a chitosan, wherein the formulation is substantially free of cationic lipids.

3. A therapeutic formulation for oral delivery of a nucleic acid, comprising: a nucleic acid; at least one casein protein; and a chitosan, wherein the formulation does not comprise a cationic lipid as a transfection reagent.

4. The formulation of any one of claims 1-3, wherein the nucleic acid comprises a ribonucleic acid (RNA) or a chemically modified variant thereof, or wherein the nucleic acid comprises a deoxyribonucleic acid (DNA) or a chemically modified variant thereof.

5. The formulation of claim 4, wherein the nucleic acid is present in an amount in a range of 0.0001% to 0.01% of the formulation by weight per volume, optionally wherein the RNA or the chemically modified variant thereof is present in an amount in a range of 0.0001% to 0.01% of the formulation by weight per volume.

6. The formulation of any one of the preceding claims, wherein the at least one casein protein is present in an amount in a range of 0.5% to 5% of the formulation by weight per volume, optionally wherein the at least one casein protein comprises at least an a-sl casein subunit.

7. The formulation of any one of the preceding claims, wherein the at least one casein protein comprises a mixture of an a-sl casein subunit, an a-s2 casein subunit, a caseinsubunit, and a K casein subunit, wherein the casein subunits are collectively present in an amount in a range of 0.5% to 5% of the formulation by weight per volume.

8. The formulation of any one of the preceding claims, wherein the chitosan is present in an amount in a range of 0.001% to 1% of the formulation by weight per volume.

9. The formulation of any one of the preceding claims, wherein the chitosan is or comprises medium molecular weight chitosan.

10. The formulation of claim 9, wherein the medium molecular weight chitosan has a molecular weight in the range of 150,000-350,000 Da.

11. The formulation of claim 1, wherein the nucleic acid comprises a ribonucleic acid (RNA) and wherein the RNA is present in an amount in a range of 0.0001 to 0.01% of the formulation by weight per volume, wherein the at least one casein protein comprises at least an a-sl casein subunit and wherein the at least one casein protein is present in an amount in a range of 0.5 to 5% of the formulation by weight per volume, and wherein the chitosan is present in an amount in a range of 0.001 to 1% of the formulation by weight per volume.

12. The formulation of any one of the preceding claims, wherein the nucleic acid comprises a nucleotide sequence of 20-40 nucleotides in length.

13. The formulation of any one of the preceding claims, wherein the nucleic acid is at least partially single-stranded, or wherein the formulation does not comprise a second nucleic acid that is at least partially complementary to the nucleic acid.

14. The formulation of any one of the preceding claims, wherein the nucleic acid modulates an anti-inflammatory activity of a macrophage in a subject when the formulation is orally administered to the subject.

15. The formulation of any one of the preceding claims, wherein the nucleic acid comprises a nucleotide sequence of any one of the nucleotide sequences provided in Table 0.4 or a sequence at least 95% identical thereto, wherein the nucleic acid is RNA.

16. The formulation of any one of the preceding claims, comprising an acid, optionally wherein the acid is selected from acetic acid, phosphoric acid and citric acid.

17. The formulation of claim 16, wherein the acid is present in an amount in a range of 0.001 to 1% of the formulation by weight per volume, optionally wherein the acid is acetic acid.

18. The formulation of any one of claims 1-15, comprising an acid in an amount in a range of 0.001 to 1% of the formulation by weight per volume, and wherein the acid is selected from acetic acid, phosphoric acid and citric acid.

19. The formulation of any one of claims 1-15, comprising acetic acid, wherein the acetic acid is present in an amount in a range of 0.01 and 1% of the formulation by weight per volume.

20. The formulation of any one of claims 16-19, consisting essentially of the nucleic acid; the at least one casein protein; the chitosan; and the acid.

21. The formulation of any one of the preceding claims, wherein the formulation comprises less than 0.1 microliters of a cationic lipid for each microgram of the nucleic acid in the formulation, or comprises no cationic lipid.

22. A therapeutic formulation for oral delivery of a nucleic acid, consisting essentially of: a nucleic acid comprising RNA or a chemically modified variant thereof; at least one casein protein, wherein the at least one casein protein comprises a mixture of an a-sl casein subunit, an a-s2 casein subunit, a casein subunit, and a K casein subunit, wherein the casein subunits are collectively present in an amount in a range of 0.5% to 5% of the formulation by weight per volume; a chitosan present in an amount in a range of 0.001 to 1% of the formulation by weight per volume; and acetic acid present in an amount in a range of 0.01 and 1% of the formulation by weight per volume, optionally wherein the RNA or the chemically modified variant thereof is present in an amount in a range of 0.001 to 0.005% of the formulation by weight per volume.

23. The formulation of any one of the preceding claims, wherein the formulation is effective for treatment of a condition associated with inflammation and / or fibrosis when the formulation is administered orally, optionally wherein the condition associated with inflammation and / or fibrosis is a cardiac condition.

24. A method for manufacturing a liposome-free therapeutic formulation for oral delivery of a nucleic acid, comprising: contacting a nucleic acid with a solution comprising 2% to 10% casein proteins by weight per volume, to generate a first mixture; and contacting the first mixture with an acid and chitosan polymers, to generate a formulation for oral delivery of the nucleic acid, wherein the method does not comprise contacting the nucleic acid with a solution comprising cationic lipids as transfection reagents or formation of lipid nanoparticles.

25. The method of claim 24, wherein the casein proteins are within a solution of 5% bovine casein solution and are added to the nucleic acid at a volume ratio of about 1: 10.

26. The method of claim 24 or 25, wherein contacting the first mixture with an acid and chitosan polymers comprises contacting the first mixture with a second mixture of the acid and the chitosan polymers, optionally wherein the acid is acetic acid.

27. The method of claim 26, wherein the second mixture comprises: an acetic acid solution of 0.05 to 2%, weight per volume; and a chitosan solution of 0.1% to 2%, weight per volume.

28. The method of any one of claims 24-27, wherein the formulation is the formulation of any one of claims 1-23.

29. The formulation of any one of claims 1-23 for the treatment of a condition associated with inflammation and / or fibrosis, or a cardiometabolic disorder by a method comprising orally administering a therapeutically effective amount of the formulation to a subject having or suspected of having a condition associated with inflammation and / or fibrosis, or a cardiometabolic disorder.

30. The formulation of any one of claims 1-23, for use in a method of immunomodulation, the method comprising orally administering to a subject an effective amount of the formulation, wherein the nucleic acid modulates an anti-inflammatory activity of a macrophage in the subject.

31. A kit comprising: at least one casein protein; a chitosan; and an acid.

32. The kit of claim 31, further comprising a nucleic acid, wherein the kit does not comprise a cationic lipid as a transfection reagent, optionally wherein the nucleic acid is RNA.

33. The kit of claim 31 or 32, where the acid is selected from acetic acid, phosphoric acid and citric acid.

34. The kit of any one of claims 31-33, wherein the chitosan is medium molecular weight chitosan, optionally wherein the medium molecular weight chitosan has a molecular weight in the range of 150,000-350,000 Da.

35. The kit of any one of claims 31-34, wherein the at least one casein protein comprises a mixture of an a-sl casein subunit, an a-s2 casein subunit, a P casein subunit, and a K casein subunit.

36. The formulation, method, or kit of any one of the preceding claims, wherein the formulation is free or substantially free of exosomes and / or extracellular vesicles.-MO-

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