MicroRNA inhibitors for use in the treatment of metabolic diseases

Abstract

The present invention provides compositions for use in treating or preventing metabolic diseases, diseases related to metabolic disorders, and / or cancer. [Solution] A composition is provided comprising: an inhibitor of miR-379 or a part or fragment thereof and an inhibitor of miR-541 or a part or fragment thereof; and / or an inhibitor of a target site of miR-379 or a part or fragment thereof and an inhibitor of a target site of miR-541 or a part or fragment thereof; and / or a combination of an inhibitor of miR-379 or a part or fragment thereof and an inhibitor of a target site of miR-541 or a part or fragment thereof, or a combination of an inhibitor of a target site of miR-379 or a part or fragment thereof and an inhibitor of miR-541 or a part or fragment thereof.

Description

[Technical Field]

[0001] The present invention relates to compositions comprising inhibitors of miR-379 or part or fragment thereof and inhibitors of miR-541 or part or fragment thereof, and / or inhibitors of target sites of miR-379 or part or fragment thereof and inhibitors of target sites of miR-541 or part or fragment thereof, and / or combinations of inhibitors of miR-379 or part or fragment thereof and inhibitors of target sites of miR-541 or part or fragment thereof, or combinations of inhibitors of miR-379 or part or fragment thereof and inhibitors of target sites of miR-541 or part or fragment thereof. The present invention further relates to such compositions for use in treating or preventing metabolic diseases, diseases associated with metabolic disorders, and / or cancer. [Background technology]

[0002] Metabolic dysfunction and metabolic diseases are often associated with unbalanced levels of glucocorticoid hormones and conditions such as fasting, cancer cachexia, aging, Cushing's syndrome, GC therapy, obesity, insulin resistance, type 1 and type 2 diabetes, hyperglycemia, lipid metabolism disorders, and HCC (hepatocellular carcinoma). For example, metabolic syndrome (a set of metabolic disorders, all resulting from or associated with primary diseases of insulin resistance) is characterized by a group of metabolic risk factors, including abdominal obesity, elevated triglyceride levels, decreased high-density lipoprotein (HDL) cholesterol levels, hypertension, and poor fasting blood glucose (a measure of decreased insulin sensitivity and increased risk of developing diabetes). Patients suffering from such conditions and diseases have an increased risk of coronary heart disease and other atherosclerotic conditions, such as stroke and peripheral vascular disease and type 2 diabetes.

[0003] The hypothalamic-pituitary-adrenal (HPA) endocrine axis is a crucial physiological stress circuit for maintaining bodily homeostasis in diverse situations such as trauma, exercise, or nutritional depletion. In metabolic regulation, glucocorticoid (GC) signaling functions as a major counter-regulatory system to insulin action, and abnormally elevated GC activity is closely associated with major components of metabolic syndrome, including obesity, insulin resistance, hyperglycemia, and systemic lipid metabolism disorders. Indeed, elevated GC levels have been observed in patients with insulin resistance and are strongly associated with hyperglycemia and fatty liver phenotypes mediated through the glucocorticoid receptor (GR), a member of the nuclear receptor transcription factor family. Similarly, obesity is characterized by enhanced local GC action, and any state of endogenous or exogenous GC deficiency or excess, such as Addison's disease, Cushing's syndrome, or GC therapy, is characterized by severe perturbations of systemic energy metabolism that closely mimic aspects of metabolic syndrome.

[0004] Certain classes of small non-coding RNAs (microRNAs, also known herein as miRNAs or miRs) have emerged as a layer of significant metabolic regulatory importance. MicroRNAs (miRNAs, miRs) are a class of small (e.g., 18-24 nucleotides) non-coding RNAs found in a variety of organisms, including mammals, and are conserved in evolution. miRNAs are processed from a hairpin-shaped precursor of approximately 70 nucleotides and arise from primary transcripts through sequential cleavage by RNAse III enzymes. Many microRNAs can be encoded in intergeneric regions, either within introns of pre-mRNA or within non-coding RNA genes. Many miRNAs also tend to form clusters and be transcribed as polycistrons, often exhibiting similar spatiotemporal expression patterns. miRs have been shown to play roles in a variety of biological processes, including development, differentiation, apoptosis, cell proliferation, organogenesis, and metabolism. Indeed, individual miRNAs have been shown to regulate diverse aspects of energy homeostasis, including pancreatic β-cell insulin secretion, adipose tissue lipid storage, and hepatic cholesterol and lipid processing. Furthermore, some miRNAs, such as miR-379, have been identified as being involved in glucocorticoid (GC) signaling (WO 2015 / 063081 (Patent Document 1)). For example, inhibition of miR-379 activity resulted in a decrease in circulating triglyceride (TG) levels (de Guia et al., EMBO J (2015), 34(3): 344-360 (Non-Patent Document 1)). However, metabolic dysfunction and metabolic diseases are still not adequately treatable, and appropriate therapies for such diseases, including type 1 and type 2 diabetes, are desired. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] WO 2015 / 063081 [Non-patent literature]

[0006] [Non-Patent Document 1] de Guia et al., EMBO J (2015), 34(3): 344-360 [Overview of the Initiative]

[0007] The present invention addresses these needs and objectives by providing solutions as described herein and defined in the claims.

[0008] As previously demonstrated in animal studies, inhibition of miR-379 activity results in a decrease in circulating triglyceride (TG) levels (de Guia et al., EMBO J (2015), 34(3): 344-360). Further recognized in the context of this invention, another miRNA, namely miRNA-541 (miR-541), was found to be overexpressed in obese patients, and a correlation between miR-541 and insulin sensitivity was identified (see Table 1). However, inhibition or knockdown of miR-541 did not result in a substantial metabolic phenotype (data not shown). Furthermore, surprisingly recognized in the context of this invention and as shown herein, simultaneous inhibition of both miR-379 and miR-541 improves both glucose and lipid metabolism by lowering TG and glucose levels. This remarkable finding has led to the present invention, which inhibits both miR-379 and miR-541, enabling the treatment of disorders and diseases related to glucose and lipid metabolism, including, but not limited to, glucocorticoid hormone-induced metabolic dysfunction, obesity, diabetes (including type 1 and type 2), diabetic obesity (diabetes), metabolic syndrome, insulin resistance, hyperglycemia, (systemic) lipid metabolism disorders, Cushing's syndrome, adverse effects or side effects associated with or caused by glucocorticoid (GC) treatment or excessive GC, atherosclerosis, heart disease, stroke, (cancerous) cachexia, and growth retardation, fatty liver, NASH, and hepatic fibrosis, particularly type 1 and type 2 diabetes, and these treatments include personalized medicine for type 1 and type 2 diabetes.

[0009] Therefore, the present invention is (a) inhibitors of miR-379 or part or fragment thereof, and inhibitors of miR-541 or part or fragment thereof, and / or (b) inhibitors of the target site of miR-379 or part or fragment thereof, and inhibitors of the target site of miR-541 or part or fragment thereof, and / or (c) A combination of an inhibitor of miR-379 or a part or fragment thereof and an inhibitor of the target site of miR-541 or a part or fragment thereof, or a combination of an inhibitor of the target site of miR-379 or a part or fragment thereof and an inhibitor of miR-541 or a part or fragment thereof This relates to a composition containing the following:

[0010] As used herein, the term “target site” means a site within a cell’s mRNA that is typically targeted by a miRNA to suppress or inhibit the translation of such mRNA, enable the cleavage of such mRNA, or destabilize such mRNA and promote its degradation (also known to those skilled in the art as “silencing” mRNA). Accordingly, the term “target site inhibitor” in the context of this invention means an inhibitor of a “target site” as defined above. Thus, compositions described and provided in the context of this invention may include inhibitors of miR-379 or part or fragment thereof, and inhibitors of miR-541 or part or fragment thereof, and / or inhibitors of each target site of such miR, thereby preventing the miR from docking to the target site and silencing the respective mRNA. As will be readily apparent to those skilled in the art, such inhibitors on the target site of miR-379 or part or fragment thereof, or on the target site of miR-541 or part or fragment thereof, do not interfere with the proper translation of each mRNA, nor do they cleave or destabilize the mRNA, or at least do so to a lower degree compared to each miR-379 or part or fragment thereof, or miR-541 or part or fragment thereof itself.

[0011] Furthermore, as used herein, the terms “microRNA,” “miRNA,” and “miR” are interchangeable and typically include non-coding RNA of 18 to 26 nucleic acid bases in length, which may be the product of pre-miRNA cleavage by an enzyme dicer. Examples of mature miRNAs can be found in miRNA databases known in the art, such as miRBase (http: / / microma.sanger.ac.uk / ).

[0012] Furthermore, as used herein, the terms “inhibit” or “inhibit” miR-379 or miR-541 (or their respective target sites) or part or fragment thereof include, respectively, that the binding or docking between miR-379 or part or fragment thereof or miR-541 or part or fragment thereof and their respective target sites is inhibited or suppressed, for example, by directly binding to the respective miR or its target site, or by assisting or inducing the cleavage or degradation of miR-379 or miR-541, or by otherwise impairing the function and / or expression of miR-379 or part or fragment thereof or miR-541 or part or fragment thereof. For example, the inhibitor may be or may contain a nucleic acid molecule. In one aspect of the present invention, the inhibitor of miR-379 or part or fragment thereof and / or the inhibitor of miR-541 or part or fragment thereof may function as an antisense molecule against the respective miR.

[0013] In the context of the present invention, inhibitors of miR-379 or part or fragment thereof and inhibitors of miR-541 or part or fragment thereof, as included in the compositions described and provided herein, may be located within the same molecule or on different molecules. For example, the compositions of the present invention may include a nucleic acid molecule containing both a sequence that functions as an inhibitor of miR-379 or part or fragment thereof and a sequence that functions as an inhibitor of miR-541 or part or fragment thereof, or they may include two different nucleic acid molecules, one containing a sequence that functions as an inhibitor of miR-379 or part or fragment thereof and the other containing a sequence that functions as an inhibitor of miR-541 or part or fragment thereof. In one embodiment of the present invention, a composition comprises an inhibitor of miR-379 or a part or fragment thereof and an inhibitor of miR-541 or a part or fragment thereof on the same molecule, wherein the molecule comprises both a nucleotide sequence that is complementary to or hybridizes with miR-379 or a part or fragment thereof (e.g., under stringent conditions) as described herein, and a nucleotide sequence that is complementary to or hybridizes with miR-541 or a part or fragment thereof (e.g., under stringent conditions) as described herein.

[0014] In general, as used herein, the terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” should be interpreted as synonymous. Generally, a nucleic acid molecule may include, in particular, DNA molecules, RNA molecules, oligonucleotide thiophosphates, substituted ribooligonucleotides, or PNA molecules. Furthermore, the term “nucleic acid molecule” may refer to DNA or RNA or hybrids thereof or any modification thereof known in the art (see, for example, US 5525711, US 471 1955, US 5792608, or EP 302175 as examples of modifications). Polynucleotide sequences may be single-stranded or double-stranded, linear or cyclic, natural or synthetic, and of no particular size. For example, the polynucleotide sequence may be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribosomal RNA, or DNA encoding such RNA, or a chimeroplast (Gamper, Nucleic Acids Research, 2000, 28, 4332-4339). The polynucleotide sequence may take the form of a vector, a plasmid, or viral DNA or RNA. Nucleic acid molecules complementary to the nucleic acid molecules described above, and nucleic acid molecules that can hybridize to the nucleic acid molecules described herein, are also described herein. The nucleic acid molecules described herein may also be fragments of nucleic acid molecules in the context of the present invention. In particular, such fragments are functional fragments. An example of such a functional fragment is a nucleic acid molecule that can function as a primer.

[0015] As used herein, nucleic acid molecules may include various types of nucleotides, including naturally occurring nucleotides, modified nucleotides, and synthetic nucleotides. Nucleotides as used herein generally include nucleosides, naturally occurring nucleosides, modified nucleosides, and synthetic nucleosides. Naturally occurring nucleosides known in the art include purine bases or pyrimidine bases. Examples of naturally occurring nucleosides include (deoxy)adenosine, (deoxy)guanosine, (deoxy)uridine, thymidine, and (deoxy)cytidine. Nucleosides as part of nucleotides (and therefore nucleic acids) as described herein generally encompass structures containing any purine or pyrimidine nucleoside, and their derivatives or analogues. That is, as used in the context of the present invention, "purine nucleoside" or "pyrimidine nucleoside" generally encompass any type of purine or pyrimidine as described herein, and their derivatives or analogues, as well as sugars, such as pentoses. In one aspect of the present invention, the purine nucleoside may be selected from the group consisting of (deoxy)adenosine, inosine, and (deoxy)guanosine, and their derivatives or analogs. The derivative may be a nucleoside having a purine selected from the group consisting of deazapurines, such as 7-deazapurine, azidoprine, alkylpurine, thiopurine, bromopurine, O-alkylpurine, and isoprine. That is, in one aspect of the present invention, the purine nucleoside may be a nucleoside having a purine selected from the group consisting of deazapurines, such as 7-deazapurine, azidoprine, alkylpurine, thiopurine, bromopurine, O-alkylpurine, and isoprine. In another aspect of the present invention, the purine nucleoside may be 1-methyl-(deoxy)adenosine, 2-methyl-(deoxy)adenosine, N 6 -methyl-(deoxy)adenosine, N 6 ,N 6-Dimethyl-(deoxy)adenosine, 7-deaza-(deoxy)adenosine, 7-deaza-8-aza(deoxy)adenosine, 7-deaza-7-bromo(deoxy)adenosine, 7-deaza-7-iodo(deoxy)adenosine, 8-azido(deoxy)adenosine, 8-bromo(deoxy)adenosine, 8-iodo(deoxy)adenosine, 8-bromo-2'-deoxy(deoxy)adenosine, 2'-O-methyladenosine, inosine, 1-methylinosine, 2'-O-methylinosine, 1-methyl(deoxy)guanosine, 7-methyl(deoxy)guanosine, N 2 -Methyl(deoxy)guanosine, N 2 ,N 2 -Dimethyl-guanosine, isoguanosine, 7-deaza(deoxy)guanosine, 7-deaza-8-aza(deoxy)guanosine, 7-deaza-7-bromo(deoxy)guanosine, 7-deaza-7-iodo(deoxy)guanosine, 6-thio(deoxy)guanosine, O 6 -methyl(deoxy)guanosine, 8-azido(deoxy)guanosine, 8-bromo(deoxy)guanosine, 8-iodo(deoxy)guanosine, 2'-O-methylguanosine, 8-azidoinosine, 7-azainosine, 8-bromoinosine, 8-iodoinosine, 1-methylinosine, and 4-methylinosine may be selected from the group consisting of -methyl(deoxy)guanosine, 8-azidoinosine, 7-azainosine, 8-bromoinosine, 8-iodoinosine, 1-methylinosine, and 4-methylinosine. In a further aspect of the present invention, the purine nucleoside may be keuosin, alkaeosin, uiosin, and N 6- It may be selected from the group consisting of thienylcarbamoyladenosine. In one aspect of the present invention, the pyrimidine nucleoside may be selected from the group consisting of (deoxy)cytidine, (deoxy)thymidine, (deoxy)ribothymidine, (deoxy)uridine, and their derivatives. The derivatives may be, for example, nucleosides having a pyrimidine selected from the group consisting of alkylpyrimidine, thiopyrimidine, bromopyrimidine, O-alkylpyrimidine, isopyrimidine, acetylpyrimidine, hydropyrimidine, and pseudopyrimidine. That is, in one aspect of the present invention, the pyrimidine nucleoside may be a nucleoside having a pyrimidine selected from the group consisting of alkylpyrimidine, thiopyrimidine, bromopyrimidine, O-alkylpyrimidine, isopyrimidine, acetylpyrimidine, hydropyrimidine, and pseudopyrimidine. In another aspect of the present invention, the pyrimidine nucleoside is 3-methyl-(deoxy)cytidine, N 4 -methyl (deoxy)cytidine, N 4 ,N 4 -dimethyl (deoxy)cytidine, iso(deoxy)cytidine, pseudo(deoxy)cytidine, pseudoiso(deoxy)cytidine, 2-thio(deoxy)cytidine, N 4 -acetyl (deoxy)cytidine, 3-methyl (deoxy)uridine, pseudo(deoxy)uridine, 1-methyl-pseudo(deoxy)uridine, 5,6-dihydro(deoxy)uridine, 2-thio(deoxy)uridine, 4-thio(deoxy)uridine, 5-bromodeoxy(deoxy)uridine, 2'-deoxyuridine, 4-thio(deoxy)thymidine, 5,6-dihydro(deoxy)thymidine, O 4-The group consisting of methylthymidine, difluoltoluene, and other nucleic acid base substitutes may be selected. As described above, the nucleosides described and provided herein generally include purines or pyrimidines or their derivatives or analogues, as well as sugar moieties, such as pentoses. Generally, the pentose as part of the purine or pyrimidine nucleosides or their derivatives or analogues described herein may be ribose, deoxyribose, arabinose, or methylribose (2-O-methylribose), for example, ribose or deoxyribose. That is, the nucleoside may be, for example, (ribosyl)nucleoside, deoxy(ribosyl)nucleoside, arabinosylnucleoside, or (methylribosyl)nucleoside, for example, (ribosyl)nucleoside or deoxy(ribosyl)nucleoside.

[0016] As used herein, the terms “desoxy” and “deoxy” as molecular prefixes are used synonymously to indicate, for example, the absence of oxygen atoms and hydroxyl groups in a given pentose such as ribose.

[0017] In one aspect of the present invention, inhibitors of miR-379 and / or miR-541 or part or fragment thereof may each function as an antisense molecule to the respective miR. In one aspect of the present invention, at least one (or all) inhibitors of miR-379 or part or fragment thereof comprises a nucleotide sequence that is complementary to or hybridizes with miR-379 or part or fragment thereof (e.g., under stringent conditions), and at least one (or all) inhibitors of miR-541 or part or fragment thereof comprises a nucleotide sequence that is complementary to or hybridizes with miR-541 or part or fragment thereof (e.g., under stringent conditions). In this context, the inhibitors of miR-379 or part or fragment thereof and miR-541 or part or fragment thereof can be located on different molecules, or they may be located on a single molecule, i.e., one molecule contains both the inhibitor of miR-379 or part or fragment thereof and the inhibitor of miR-541 or part or fragment thereof. In one embodiment of the present invention, the inhibitors of miR-379 or part or fragment thereof and miR-541 or part or fragment thereof are located on a single molecule.

[0018] In the context of inhibitors and nucleic acid molecules / DNA sequences containing miR or parts or fragments thereof, the terms “hybridization” or “to hybridize” as used herein may relate to hybridization under stringent conditions, low-stringent conditions, or non-stringent conditions. In one embodiment, the conditions are preferably stringent. The hybridization conditions can be established according to conventional protocols, such as those described in Sambrook, Russell, "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, NY (2001); Current Protocols in Molecular Biology, Update May 9, 2012, Print ISSN: 1934-3639, Online ISSN: 1934-3647; Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, NY (1989); or Higgins and Hames (Eds.), "Nucleic acid hybridization, a practical approach", IRL Press Oxford, Washington DC, (1985). Setting the conditions is well within the skill of those skilled in the art and can be determined according to protocols described in the art. Therefore, the detection of only sequences that specifically hybridize typically requires stringent hybridization and washing conditions, e.g., 0.1×SSC, 0.1%SDS at 65°C (as used herein, “stringent conditions”). Non-stringent hybridization conditions for the detection of homologous or not strictly complementary sequences may be set at 6×SSC, 1%SDS at 65°C (as used herein, “non-stringent conditions”).As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Changes in the above conditions may be made by the inclusion and / or substitution of alternative blocking reagents used to suppress background in hybridization tests. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of a particular blocking reagent may require a change in the hybridization conditions described above due to compatibility issues. According to the invention described herein, low stringency hybridization conditions for the detection of homologous or not strictly complementary sequences may be set, for example, at 65° C. with 6×SSC, 0.5% SDS (the "low stringency conditions" used herein). As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions.

[0019] The nucleic acid molecules that hybridize also include fragments of the molecules described above. Such fragments may be nucleic acid molecules or functional fragments thereof that function as inhibitors, as described herein. Furthermore, nucleic acid molecules that hybridize with any of the nucleic acid molecules described above also include complementary fragments, derivatives, and variants of these molecules. In addition, a hybridization complex refers to a complex between two nucleic acid sequences resulting from the formation of hydrogen bonds between complementary G bases and C bases and between complementary A bases and T bases (or U in RNA, as known to those skilled in the art); these hydrogen bonds may be further stabilized by stacking interactions of the bases. The hydrogen bonds may take an antiparallel configuration. Hybridization complexes may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., a membrane, filter, tip, pin, or glass slide on which cells are fixed). The terms “complementary” or “complementary” refer to the innate bonding of polynucleotides by base pairing under acceptable salt and temperature conditions. For example, the sequence "AGU" binds to the complementary sequence "UCA". The complementarity between two single-stranded molecules may be "partial," where only a portion of the nucleic acid is bound, or it may be complete, as complete complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions that depend on binding between nucleic acid strands.The term “hybridizes sequence” preferably refers to a sequence that exhibits sequence identity of at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and most preferably at least 100% with a nucleic acid sequence described herein that functions as an inhibitor as described herein and provided herein.

[0020] As used herein, a “portion” or “fragment” of a given microRNA (miRNA) may be any part of the microRNA, and may specifically include a part of the microRNA or its precursor (e.g., pri-microRNA or pre-microRNA) that contains or consists of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides of each microRNA or its precursor. In one aspect, a portion or fragment of a given microRNA is a portion or fragment found in a cell after (intranuclear and / or cytoplasmic) processing of the microRNA (e.g., pri-microRNA or pre-microRNA), such as the 5p arm (also called the 5p strand) or 3p arm (also called the 3p strand) of each miR. In one aspect of the invention, a portion or fragment of a microRNA is the 5p arm of the microRNA or its precursor. For example, according to the invention, a portion or fragment of miR-379 may be, in particular, miR-379-5p, and / or a portion or fragment of miR-541 may be, in particular, miR-541-5p. In one aspect of the invention, a portion or fragment of a microRNA is the 3p arm of the microRNA or its precursor. For example, according to the invention, a portion or fragment of miR-379 may be, in particular, miR-379-3p, and / or a portion or fragment of miR-541 may be, in particular, miR-541-3p.

[0021] In this context, as will be readily understood by those skilled in the art, according to the invention, inhibition of a portion or fragment of a given microRNA as defined herein also inhibits the function of the entire microRNA as defined herein, and thus an inhibitor of a portion or fragment of a given microRNA is also an inhibitor of the microRNA itself or its precursor.

[0022] Therefore, as used herein, inhibitors of microRNAs (e.g., miR-379 or miR-541) or their precursors may also each include inhibitors of a portion or fragment of such microRNA (e.g., miR-379 or miR-541) if such inhibitors bind to, are complementary to, or hybridize to (e.g., under stringent conditions). Similarly, according to the present invention, inhibitors of a portion or fragment of such microRNAs (e.g., miR-379 or miR-541) also include inhibitors of each of such microRNAs (e.g., miR-379 or miR-541) or their precursors.

[0023] In one embodiment of the present invention, a portion or fragment of miR-379 is miR-379-5p, having a nucleotide sequence according to SEQ ID NO: 1, with substitutions of 6, 5, 4, 3, 2, or one or fewer nucleotides. For example, the nucleotides substituted compared to SEQ ID NO: 1 may be any other nucleotides that enable hybridization of each inhibitor described herein. In one embodiment of the present invention, the substitution may be located within the last 6, 5, 4, 3, 2, or 1 nucleotide at the 3' end of SEQ ID NO: 1 and / or the first nucleotide at the 5' end of SEQ ID NO: 1. In a particular embodiment of the present invention, a portion or fragment of miR-379 is miR-379-5p, having a nucleotide sequence according to SEQ ID NO: 1.

[0024] In a further embodiment of the present invention, a portion or fragment of miR-541 is miR-541-5p, having a nucleotide sequence according to SEQ ID NO: 2, with substitutions of 8, 7, 6, 5, 4, 3, 2, or one or fewer nucleotides. For example, the nucleotide substituted for SEQ ID NO: 2 may be any other nucleotide that enables hybridization of each inhibitor described herein. In one embodiment of the present invention, the substitution may be located within the last 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide at the 3' end of SEQ ID NO: 2 and / or the first nucleotide at the 5' end of SEQ ID NO: 2. In a particular embodiment of the present invention, a portion or fragment of miR-541 is miR-541-5p, having a nucleotide sequence according to SEQ ID NO: 2.

[0025] In one embodiment of the composition of the present invention, a portion of miR-379 has a nucleotide sequence according to SEQ ID NO: 1 in which six or fewer nucleotides are substituted, and a portion of miR-541 has a nucleotide sequence according to SEQ ID NO: 2 in which eight or fewer nucleotides are substituted.

[0026] Each inhibitor contained in the compositions described and provided by the present invention may be any inhibitor, preferably one that can inhibit or suppress the binding or docking between miR-379 (or a part or fragment thereof) or miR-541 (or a part or fragment thereof) and its respective target site by directly binding to the respective miR or its target site, assisting or inducing the cleavage or degradation of miR-379 (or a part or fragment thereof) or miR-541 (or a part or fragment thereof), or by otherwise impairing the function and / or expression of miR-379 (or a part or fragment thereof) or miR-541 (or a part or fragment thereof). For example, the inhibitor may be a nucleic acid molecule or may contain one.

[0027] In one embodiment of the present invention, at least one inhibitor comprises a nucleic acid sequence containing at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, for example, at least 15 or 16 nucleotides. In a further embodiment of the present invention, the inhibitor does not exceed a length of 250, 200, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, or 25 nucleotides.

[0028] In the context of the present invention, inhibitors of miR-379 (or a part or fragment thereof) and / or miR-541 (or a part or fragment thereof) may directly target each of the miRs or parts or fragments thereof described herein. Common inhibitory molecules capable of inhibiting miRs are known in the art. In one aspect of the present invention, inhibitors against miR-379 (or a part or fragment thereof) and / or miR-541 (or a part or fragment thereof) can inhibit or suppress the function of each miR described herein (for example, by hybridizing to at least a part of the target site of each miR), including Tough Decoy (TuD) (e.g., Tough Decoy RNA), Decoy, Antisense Oligonucleotide (Antisense RNA or DNA, Chimeric Antisense Molecules), Anti-miR (Anti-miR), Block-miR (Block-miR), Ribozyme, External Guide Sequence (EGS), Oligonucleotide, Small Interfering RNA (siRNA), Small Temporal RNA (stRNA), Small Hairpin RNA (shRNA), Small RNA-Induced Gene Activation (RNAa), Small Activating RNA (SRNA) The inhibitor may be selected from the group consisting of RNA (saRNA), locked nucleic acid (LNA), antagonist mir, peptide nucleic acid (PNA), and other oligomeric nucleic acid molecules. In certain embodiments of the present invention, at least one inhibitor is tough decoy RNA (TuD). Tough decoys are generally known in the art and are available, for example, from SignaGen® Laboratories (USA).In the context of the present invention, the composition of the present invention may include, for example, a TuD that contains both a sequence that inhibits miR-379 (or a part or fragment thereof) and another sequence that inhibits miR-541 (or a part or fragment thereof) on the same TuD molecule, or the composition of the present invention may include two different TuDs, one of which contains a sequence that inhibits miR-379 (or a part or fragment thereof) and the other contains a sequence that inhibits miR-541 (or a part or fragment thereof). For example, the composition of the present invention includes a TuD that contains both a sequence that inhibits miR-379 (or a part or fragment thereof) and another sequence that inhibits miR-541 (or a part or fragment thereof) on the same TuD molecule. In a particular embodiment of the present invention, the composition includes a nucleotide sequence of SEQ ID NO: 5 in which 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotides are substituted for SEQ ID NO: 5. In a further particular embodiment of the present invention, the inhibitor is a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 5.

[0029] Furthermore, inhibitors contained in the compositions described and provided herein may also include chemical modifications, for example, to improve stability or to enable appropriate administration to subjects that need it. In one embodiment of the present invention, at least one inhibitor includes a chemical modification of a nucleic acid sequence selected from the group consisting of nucleic acid analogs including N-acetylgalactosamine (GalNAc), phosphorothioate DNA (PS), 2'-O-methyl RNA (OMe), 2'-O-methoxy-ethyl RNA (MOE), peptide nucleic acid (PNA), N3'-P5'-phosphoramidate (NP), 2'-fluoroarabino nucleic acid (FANA), morpholinophosphoramidate (MF), cyclohexene nucleic acid (CeNA), and tricycle DNA (tc-DNA).

[0030] In another embodiment of the present invention, the inhibitor may also be contained by a suitable medium or carrier. Thus, the present invention also relates to mediums and carriers containing compositions comprising inhibitors described and provided herein. For example, in the context of the present invention, at least one inhibitor may be contained by a delivery medium selected from the group consisting of adeno-associated virus (AAV), lentiviral vector, polyethyleneimine (PEI), cationic liposomes (e.g., lipid nanoparticles), silica nanoparticles, PEGylated PLGA, and neutral lipids. The medium of the present invention can serve the purpose of ensuring the uptake of the composition of the present invention at a target site of interest in an object requiring treatment with such a composition. In the context of the present invention, further exemplary delivery mediums for the inhibitors described herein include lipid (e.g., cationic lipids)-containing mediums (e.g., liposomes), virus-containing mediums (e.g., vectors), polymer-containing mediums (e.g., biodegradable polymers or dendrimers), and peptide-containing mediums (e.g., penetrating peptides), exosomes, and intact mini-cells derived from bacteria. In a particular embodiment of the present invention, the delivery medium comprises two or more compounds. For example, this may include one or more lipid components, one or more peptides, one or more polymers, one or more viral vectors, or combinations thereof. Certain embodiments relate to a delivery medium, which is an associated complex such as a liposome. Liposomes generally contain one or more components, such as cationic lipids (e.g., aminolipids), a targeting component, a fusion lipid, and a PEGylated lipid. In some embodiments, the PEG-lipid may be a targeted PEG-lipid. For example, the liposome may contain nucleic acids and amine-lipids and PEGylated lipids. In some embodiments of the present invention, the PEG-lipid is a targeted PEG-lipid. In further embodiments, the preparation may also include structural components such as cholesterol. In the context of the present invention, a viral delivery medium is most preferred. The viral vector may be a retrovirus such as a lentivirus, or an adenovirus, preferably an adeno-associated virus (e.g., AAV).Accordingly, the present invention also provides viral vectors comprising inhibitors of miR-379 (or a portion or fragment thereof) and miR-541 (or a portion or fragment thereof) as described herein, and / or compositions comprising viral vectors comprising inhibitors of miR-379 (or a portion or fragment thereof) as described herein and viral vectors comprising inhibitors of miR-541 (or a portion or fragment thereof) as described herein. If the inhibitors are nucleic acid molecules, their sequences can be inserted, for example, into an untranslated region of a gene that is part of a construct or cassette and then delivered by the vector. Upon transduction, host cells can express the sequences of the present invention and thus silence or express any miR of the present invention. The vectors may be viral capsids and may not contain any viral polynucleotides other than the constructs or other polynucleotides.

[0031] As previously stated, inhibitors of compositions described and provided herein may be nucleic acid molecules. For example, at least one (or all) inhibitors of miR-379 (or a part or fragment thereof) comprises a nucleotide sequence that is complementary to or hybridizes with miR-379 (or a part or fragment thereof) (e.g., under stringent conditions), and at least one (or all) inhibitors of miR-541 (or a part or fragment thereof) comprises a nucleotide sequence that is complementary to or hybridizes with miR-541 (or a part or fragment thereof) (e.g., under stringent conditions). In one embodiment of the present invention, the inhibitor of miR-379 comprises a nucleotide sequence of SEQ ID NO: 3 in which 5, 4, 3, 2, or 1 or fewer nucleotides are substituted. In a further embodiment, 30, 25, 20, 15, 10, 6, or 5 or fewer nucleotides are added to SEQ ID NO: 3. For example, the nucleotide added to or substituted for SEQ ID NO: 3 may be any other nucleotide that enables the hybridization of the inhibitor to miR-379 as described herein. In one embodiment, when a nucleotide is added to SEQ ID NO: 3, such added nucleotide is added in such a manner that it further increases the hybridization to the nucleic acid sequence by SEQ ID NO: 1. For example, up to 5, 4, 3, 2, or 1 nucleotide, e.g., 5'-CCUTC-3' or any subset thereof, may be added to the 5' end of SEQ ID NO: 3. In a particular embodiment of the present invention, the inhibitor of miR-379 is a nucleic acid molecule having the sequence of SEQ ID NO: 3.

[0032] In a further embodiment of the present invention, the inhibitor of miR-541 comprises a nucleotide sequence of SEQ ID NO: 4, with 5, 4, 3, 2, or 1 or fewer nucleotides substituted. In a further embodiment, 30, 25, 20, 15, 10, 9, or 8 or fewer nucleotides are added to SEQ ID NO: 4. For example, the nucleotides added to or substituted on SEQ ID NO: 4 may be any other nucleotides that enable the hybridization of the inhibitor to miR-541 as described herein. In one embodiment, when nucleotides are added to SEQ ID NO: 4, such added nucleotides are added in such a manner that hybridization to the nucleic acid sequence by SEQ ID NO: 2 is further increased. For example, up to 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide, e.g., 5'-TGUGUGTC-3' or any subset thereof, may be added to the 5' end of SEQ ID NO: 4. In a particular aspect of the present invention, the inhibitor of miR-541 is a nucleic acid molecule having the sequence SEQ ID NO: 4.

[0033] In a further embodiment of the composition of the present invention, the inhibitor of miR-379 or part or fragment thereof comprises a nucleotide sequence with SEQ ID NO: 3 in which five or fewer nucleotides are substituted, and / or the inhibitor of miR-541 or part or fragment thereof comprises a nucleotide sequence with SEQ ID NO: 4 in which five or fewer nucleotides are substituted.

[0034] The present invention also relates to pharmaceutical compositions comprising compositions described and provided herein and / or media or carriers containing such compositions.

[0035] The present invention also relates to compositions, media or carriers and / or pharmaceutical compositions described and provided herein for use in treating or preventing metabolic dysfunction, metabolic diseases, or metabolic disorders related to lipid (e.g., triglycerides) and sugar (e.g., glucose) metabolism and / or cancer. In one aspect of the present invention, such metabolic dysfunction, metabolic diseases, or metabolic disorders may include glucocorticoid hormone-induced metabolic dysfunction, obesity, diabetes mellitus (type 1 and type 2), diabetic obesity, metabolic syndrome, insulin resistance, hyperglycemia, (systemic) lipid metabolism disorders, Cushing's syndrome, adverse effects or side effects associated with or caused by glucocorticoid (GC) treatment or excessive GC, atherosclerosis, heart disease, stroke, (cancerous) cachexia, growth retardation, fatty liver, NASH, and hepatic fibrosis. In the context of the present invention, the compositions, media or carriers and / or pharmaceutical compositions described and provided herein may also be for use in (personalized) type 1 and / or type 2 diabetes therapy. In further embodiments of the present invention, such cancers treated as described herein may include hepatocellular carcinoma (HCC), as well as tumors that have been shown to be associated with metabolic dysfunction, such as obesity, including pancreatic cancer, colon cancer, endometrial cancer, breast cancer, esophageal cancer, and gastric cancer.

[0036] The embodiments that characterize the present invention are described herein, shown in the drawings, illustrated in the examples, and reflected in the claims.

[0037] It should be noted that, as used herein, the singular forms “a,” “an,” and “the” also include references to the plural unless otherwise explicitly indicated in the context. For example, a reference to “a reagent” includes one or more of such reagents, and a reference to “the method” includes references to equivalent steps and methods known to those skilled in the art that can be modified or replaced by the methods described herein.

[0038] Unless otherwise indicated, the term “at least” preceding a set of components should be understood to refer to all components within that set. Those skilled in the art will recognize, or can verify, many equivalents to specific aspects of the invention described herein using routine experimental methods. Such equivalents are intended to be encompassed by the present invention.

[0039] As used herein, the terms “and / or” include the meanings of “and,” “or,” and “all or any other combination of the components linked by the terms.”

[0040] As used herein, the terms “about” or “approximately” mean within 20%, preferably within 10%, more preferably within 5%, and most preferably within 3% of a given value or range.

[0041] Throughout this specification and the accompanying claims, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” are understood to mean the inclusion of any integer or process or set of integers or processes described, but not the exclusion of any other integer or process or set of integers or processes. Where used herein, the term “comprising” may be replaced by the terms “containing,” “including,” or, as sometimes used herein, “having.”

[0042] As used herein, “consisting of” excludes any component, process, or ingredient not specified in the components of the invention. As used herein, “essentially consisting of” does not exclude any material or process that does not substantially affect the basic and novel features of the claim.

[0043] Unless otherwise specified, in each case herein, any of the terms “contains,” “essentially consists of,” and “consistes of” may be substituted for any of the other two terms. For example, where a given feature, compound, or range is indicated as “contains” its respective broader term, such broader term may also consist of such feature, compound, or range.

[0044] The present invention is not limited to the specific methodologies, protocols, reagents, etc., described herein, and should therefore be understood to be subject to change. The technical terms used herein are for illustrative purposes only and are not intended to limit the scope of the invention, which is defined solely by the claims.

[0045] All publications and patents (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) referenced throughout this specification, whether above or below, are incorporated herein by reference in their entirety. Nothing herein should be construed as an acknowledgment that the present invention does not have prior rights to such disclosures on the grounds that they are prior inventions. To the extent that any material incorporated by reference is inconsistent with or contradicts this specification, this specification takes precedence over any such material. [Brief explanation of the drawing]

[0046] [Figure 1] Experimental protocol for liver-specific inhibition of miR-541 and miR-379 activity by rAAV-mediated delivery of TuD inhibitor in mice. [Figure 2] Reduced HOMA-IR (a surrogate measure of insulin resistance) observed in animals with liver-specific inhibition of miR-541 and miR-379 activity (AAV-TuD, white bars) compared to negative controls (AAV-NC, black bars). [Figure 3] Reduced circulating triglyceride levels in animals with liver-specific inhibition of miR-541 and miR-379 activity (AAV-TuD, white bars) compared to negative controls (AAV-NC, black bars). [Figure 4A] Improved glucose clearance after intraperitoneal glucose loading (2 g / kg) in mice with liver-specific inhibition of miR-541 and miR-379 activity (AAV-TuD, ○) compared to negative controls (AAV-NC, ■). Glucose profile (A). [Figure 4B] Improvement in glucose clearance after intraperitoneal glucose loading (2 g / kg) in mice with liver-specific inhibition of miR-541 and miR-379 activity (AAV-TuD, white bars) compared to negative controls (AAV-NC, black bars), and area under the curve (B). [Figure 5A] Improvement in glucose tolerance after intraperitoneal glucose loading (ipGTT, 2 g glucose / kg) at week 4 of treatment in wild-type mice (AAV-TuD, ○ in the upper graph and white bars in the lower graph) with combined liver-specific inhibition of miR-541 and miR-379 (same profile as shown in Figure 4), compared to negative controls (AAV-NC, ■ in the upper part of the panel and black bars in the lower part). The glucose profile over time is shown in the upper graph of the panel, and the area under the corresponding curve is shown in the lower graph. A synergistic improvement in glucose clearance was observed in response to combined inhibition of miR-541 and miR-379 activity. [Figure 5B]Improvement in glucose tolerance after intraperitoneal glucose loading (ipGTT, 2 g glucose / kg) at week 4 of treatment in wild-type mice with liver-specific inhibition of miR-379 activity (AAV-TuD, ○ in the upper graph and white bars in the lower graph) compared to negative controls (AAV-NC, ■ in the upper part of the panel and black bars in the lower part). The glucose profile over time is shown in the upper graph of the panel, and the area under the corresponding curve is shown in the lower graph. A synergistic improvement in glucose clearance was observed in response to combined inhibition of miR-541 and miR-379 activity. [Figure 5C] Improvement in glucose tolerance after intraperitoneal glucose loading (ipGTT, 2 g glucose / kg) at week 4 of treatment in wild-type mice with liver-specific inhibition of miR-541 activity (AAV-TuD, ○ in the upper graph and white bars in the lower graph) compared to negative controls (AAV-NC, ■ in the upper part of the panel and black bars in the lower part). The glucose profile over time is shown in the upper graph of the panel, and the area under the corresponding curve is shown in the lower graph. A synergistic improvement in glucose clearance was observed in response to combined inhibition of miR-541 and miR-379 activity. [Figure 6A] Serum glucose levels in response to an intraperitoneal exogenous insulin bolus (0.7 IU insulin / kg) at week 3 of treatment in wild-type mice (AAV-TuD, ○) with combined liver-specific inhibition of miR-541 and miR-379 (Figure 6A), compared to a negative control (AAV-NC, ■). A significant decrease in glucose levels compared to the negative control was observed at all time points tested, only in animals possessing liver-specific inhibition of both miR-541 and miR-379 activity. [Figure 6B]Serum glucose levels in response to an intraperitoneal exogenous insulin bolus (0.7 IU insulin / kg) at week 3 of treatment in wild-type mice (AAV-TuD, ○) with hepatic-specific inhibition of miR-379 (Figure 6B) compared to negative controls (AAV-NC, ■). A significant decrease in glucose levels compared to negative controls was observed at all time points tested, only in animals possessing hepatic-specific inhibition of both miR-541 and miR-379 activity. [Figure 6C] Serum glucose levels in response to an intraperitoneal exogenous insulin bolus (0.7 IU insulin / kg) at week 3 of treatment in wild-type mice (AAV-TuD, ○) with liver-specific inhibition of miR-541 activity (Figure 6C), compared to negative controls (AAV-NC, ■). A significant decrease in glucose levels compared to negative controls was observed at all time points tested, only in animals possessing liver-specific inhibition of both miR-541 and miR-379 activity. [Figure 7A] Plasma triglyceride levels (5-6 hour fasting) 2, 3, and 4 weeks after treatment in wild-type mice (AAV-TuD, white bars) with combined liver-specific inhibition of miR-541 and miR-379 (Figure 7A) compared to negative controls (AAV-NC, black bars). A robust reduction in circulating triglycerides was observed in response to combined liver-specific inhibition of miR-541 and miR-379 activity. [Figure 7B] Plasma triglyceride levels (5-6 hour fasting) 2, 3, and 4 weeks after treatment in wild-type mice (AAV-TuD, white bars) with liver-specific inhibition of miR-379 (Figure 7B) compared to negative controls (AAV-NC, black bars). A robust reduction in circulating triglycerides was observed in response to combined liver-specific inhibition of miR-541 and miR-379 activity. [Figure 7C]Plasma triglyceride levels (5-6 hour fasting) 2, 3, and 4 weeks after treatment in wild-type mice (AAV-TuD, white bars) with liver-specific inhibition of miR-541 activity (Figure 7C) compared to negative controls (AAV-NC, black bars). A robust reduction in circulating triglycerides was observed in response to combined liver-specific inhibition of miR-541 and miR-379 activity. [Modes for carrying out the invention]

[0047] The following sequences are provided herein: SEQ ID NO: 1 RNA Homo sapiens (H. sapiens) miR-379-5p Bold: Inhibitor SEQ ID NO: Sequence complementary to 3 TIFF2026091891000001.tif3128SEQ ID NO: 6 RNA Homo sapiens miR-541-5p Bold: Inhibitor SEQ ID NO: Sequence complementary to 7 TIFF2026091891000002.tif4128SEQ ID NO: 3 dna artificial miR-379 inhibitor TIFF2026091891000003.tif3128SEQ ID NO: 7 dna artificial miR-541 inhibitor TIFF2026091891000004.tif3128SEQ ID NO: 8 RNA artificial Inhibitory sequences against miR-379 and miR-541 contained in TuD TIFF2026091891000005.tif17162

[0048] The present invention is further illustrated by the following embodiments, but is not limited to these embodiments or any particular aspect thereof. [Examples]

[0049] The expression levels of various microRNAs belonging to the Dlk1-Dio3 locus were determined by semi-quantitative real-time PCR using the TaqMan microRNA assay in liver biopsies from healthy volunteers (n=10) not undergoing diabetes treatment and obese subjects (n=37). Consistent upregulation of the tested microRNAs (miR-127, miR-337, miR-379, miR-382, miR-134, miR-541, miR-409) was observed in liver samples from obese subjects. The two microRNAs showing the highest increase in expression were miR-379 and miR-541. Significant correlations between the expression levels of these transcripts and various metabolic indicators were detected, as shown for miR-541 in Table 1.

[0050] The effects of inhibiting hepatic miR-541 and miR-379 activity on metabolism were investigated in vivo by rAAV delivery of tough decoy (TuD) inhibitors under the control of a liver-specific LP1 promoter. The construction of the AAV-delivered constructs was carried out according to Rose AJ et al. Cell Metab 2011, 14(1): 123-30. Briefly, to clone these inhibitors into the rAAV-delivered constructs, the negative control sequence of the original vector was replaced with the tough decoy sequence using Bglll and Sall restriction enzymes. These inhibitor types have been previously demonstrated to potently inhibit the activity of their target microRNAs in vitro (unpublished observations). In three separate tests conducted by the inventors of the present invention, C57BL / 6J mice (12 mice per group) were given AAV (5 × 10¹⁶ mice per group) expressing a negative control sequence or a tough decoy inhibitor against both miR-541 and miR-379 (Test 1, sequence by SEQ ID NO: 5), a negative control sequence or a tough decoy inhibitor against miR-379 (Test 2), and a tough decoy inhibitor against a negative control sequence or miR-541 (Test 3). 11The viral genome was administered. Body weight, as well as diet and water intake, were monitored regularly. An ipGTT (2 g glucose / kg) was performed 2 and 4 weeks after viral administration, and an ITT (0.7 IU / kg) was performed 3 weeks after the start of the experiment. In both cases, the animals were fasted 6 hours prior to the experiment, and the tests were started at 14:00-15:00 h (a schematic diagram of the experimental protocol is shown in Figure 1). In addition, postprandial blood samples were collected at 23:00 h at weeks 2.5 and 4.5. The experiment was concluded 5 weeks after viral vector administration, with half of the animals (n=6 mice per group) killed at 14:00 h after 5-6 hours of fasting, and the other half killed at 23:00 h after feeding. In Study 1, no differences were detected in body weight, diet, or water intake between animals receiving the negative control sequence and animals possessing combined miR-541 and miR-379 liver-specific inhibition (AAV-TuD). Fasting glucose levels were significantly lower in the AAV-TuD group from week 2 to the end of the study, fasting insulin concentrations were significantly lower from week 3, and hepatic insulin resistance, calculated from fasting plasma insulin (FPI) and fasting plasma glucose (FPG) concentrations [FPI(mU / l) × FPG(mmol / l) / 22.5] as estimated by the Homeostasis Model-IR (HOMA-IR) index, was significantly lower in the AAV-TuD group from week 2 to the end (Figure 2). In addition, plasma triglyceride levels were also significantly lower in this group, regardless of the animals' feeding status (Figures 3 and 7A). Glucose clearance was also significantly superior in the AAV-TuD group (60% improvement at week 4, p<0.001; Figures 4A, 4B, and 5A). Significant improvements in glucose clearance were also observed in animals treated with the other two tough decoy inhibitors tested, but the effects were less pronounced (14% and 36% improvements in response to a single tough decoy against miR-379, Figure 5B, and miR-541 activity, Figure 5C, respectively), suggesting a synergistic effect in response to simultaneous inhibition of both microRNAs.Furthermore, combined inhibition of both microRNAs also induced a marked enhancement of the glucose-lowering effect in response to exogenous bolus insulin administration, resulting in a significant (p<0.001) decrease in glucose levels over 2 hours compared to negative controls (Figure 6A). Here again, this effect was not consistent with the single inhibition of either of the two microRNAs (Figures 6B and 6C). Blood glucose levels were determined by a glucose meter (Accu-Check). Triglyceride levels were measured by an enzyme assay (Sigma-Aldrich), and insulin levels were quantified by ELISA (Alpco). The area under the curve of the glucose profile in response to intraperitoneal glucose loading (2 g / kg) was used to calculate the improvement in glucose clearance.

[0051] (Table 1) Correlation between hepatic levels of miR-541 expression and various metabolic parameters in healthy volunteers not undergoing diabetes treatment and obese subjects. TIFF2026091891000006.tif59164

[0052] The present invention is further characterized by the following: 1. (a) inhibitors of miR-379 or part or fragment thereof, and inhibitors of miR-541 or part or fragment thereof; and / or (b) inhibitors of the target site of miR-379 or part or fragment thereof, and inhibitors of the target site of miR-541 or part or fragment thereof; and / or (c) A combination of an inhibitor of miR-379 or a part or fragment thereof and an inhibitor of the target site of miR-541 or a part or fragment thereof, or a combination of an inhibitor of the target site of miR-379 or a part or fragment thereof and an inhibitor of miR-541 or a part or fragment thereof A composition containing the following: 2. The composition of Item 1, wherein at least one inhibitor of miR-379 or part or fragment thereof comprises a nucleotide sequence that is complementary to or hybridizes with miR-379 or part or fragment thereof, and at least one inhibitor of miR-541 or part or fragment thereof comprises a nucleotide sequence that is complementary to or hybridizes with miR-541 or part or fragment thereof. 3. A portion of miR-379 has a nucleotide sequence shown in SEQ ID NO: 1, in which six or fewer nucleotides are substituted; and A portion of miR-541 has a nucleotide sequence shown in SEQ ID NO: 2, in which eight or fewer nucleotides are substituted. A composition of item 1 or 2. 4. One of the compositions from items 1 to 3, wherein at least one inhibitor comprises a nucleic acid sequence containing at least 10 nucleotides. 5. One composition of any one of items 1 to 4, wherein at least one inhibitor is selected from the group consisting of Tough Decoy (TuD), decoy, antisense oligonucleotide, anti-miR, block miR, ribozyme, external guide sequence (EGS), oligonucleotide, small interfering RNA (siRNA), small temporal RNA (stRNA), small hairpin RNA (shRNA), small RNA-induced gene activation (RNAa), small activating RNA (saRNA), locked nucleic acid (LNA), antagonist miR, and peptide nucleic acid (PNA). 6. A composition of item 5, wherein at least one inhibitor is tough decoy RNA (TuD). 7. At least one inhibitor, N-acetylgalactosamine (GalNAc), phosphorothioate DNA (PS), 2'-O-methyl RNA (OMe), 2'-O-methoxyethyl RNA (MOE), peptide nucleic acid (PNA), N3'-P5'-phosphoramidate (NP), 2'-fluoroarabino nucleic acid (FANA), morpholinophosphoramidate (MF), cyclohexene nucleic acid (CeNA), and tricycled DNA (tc-DNA) A composition comprising any one of items 1 to 6, comprising a chemical modification of a nucleic acid sequence selected from the group consisting of nucleic acid analogs including [specific component]. 8. Any one of items 1 to 7, wherein at least one inhibitor is contained in a delivery medium selected from the group consisting of adeno-associated virus (AAV), lentiviral vector, polyethyleneimine (PEI), cationic liposomes, silica nanoparticles, PEGylated PLGA, and neutral lipids. 9. One composition of any of items 1-8, wherein at least one inhibitor is found in adeno-associated virus (AAV). 10. The inhibitor of miR-379 or part or fragment thereof contains a nucleotide sequence of SEQ ID NO: 3 in which five or fewer nucleotides are substituted; and / or The inhibitor of miR-541 or part or fragment thereof contains a nucleotide sequence with SEQ ID NO: 4 in which five or fewer nucleotides are substituted. One of the compositions from items 1 to 9. 11. A composition comprising an inhibitor of miR-379 or a part or fragment thereof and an inhibitor of miR-541 or a part or fragment thereof on the same molecule, wherein the molecule comprises a nucleotide sequence that is complementary to or hybridizes with miR-379 or a part or fragment thereof, and a nucleotide sequence that is complementary to or hybridizes with miR-541 or a part or fragment thereof, any one of items 1 to 10. 12. The composition of item 11, wherein the inhibitor comprises a nucleotide sequence of SEQ ID NO: 5 in which 10 or fewer nucleotides are substituted. 13. A pharmaceutical composition, one of items 1 through 12. 14. Any one of items 1-13 for use in the treatment or prevention of metabolic diseases, diseases related to metabolic disorders, and / or cancer. 15. A composition of Item 14, wherein the disease associated with metabolic disease or metabolic disorder is selected from the group consisting of glucocorticoid hormone-induced metabolic dysfunction, obesity, diabetes, diabetic obesity, metabolic syndrome, insulin resistance, hyperglycemia, (systemic) lipid metabolism disorders, Cushing's syndrome, adverse effects or side effects associated with or caused by glucocorticoid (GC) treatment or excessive GC, atherosclerosis, heart disease, stroke, (cancerous) cachexia, growth retardation, fatty liver, NASH, and hepatic fibrosis.

Claims

1. (a) inhibitors of miR-379 or part or fragment thereof and inhibitors of miR-541 or part or fragment thereof; and / or (b) inhibitors of the target site of miR-379 or part or fragment thereof, and inhibitors of the target site of miR-541 or part or fragment thereof; and / or (c) A combination of an inhibitor of miR-379 or a part or fragment thereof and an inhibitor of the target site of miR-541 or a part or fragment thereof, or a combination of an inhibitor of the target site of miR-379 or a part or fragment thereof and an inhibitor of miR-541 or a part or fragment thereof A composition containing the following:

2. The composition according to claim 1, wherein at least one inhibitor of miR-379 or part or fragment thereof comprises a nucleotide sequence that is complementary to or hybridizes with miR-379 or part or fragment thereof, and at least one inhibitor of miR-541 or part or fragment thereof comprises a nucleotide sequence that is complementary to or hybridizes with miR-541 or part or fragment thereof.

3. A portion of miR-379 has a nucleotide sequence shown in SEQ ID NO: 1, in which six or fewer nucleotides are substituted; and A portion of miR-541 has a nucleotide sequence shown in SEQ ID NO: 2, in which eight or fewer nucleotides are substituted. The composition according to claim 1 or 2.

4. The composition according to any one of claims 1 to 3, wherein at least one inhibitor comprises a nucleic acid sequence comprising at least 10 nucleotides.

5. The composition according to any one of claims 1 to 4, wherein at least one inhibitor is selected from the group consisting of Tough Decoy (TuD), decoy, antisense oligonucleotide, anti-miR, block miR, ribozyme, external guide sequence (EGS), oligonucleotide, small interfering RNA (siRNA), small temporal RNA (stRNA), small hairpin RNA (shRNA), small RNA-induced gene activation (RNAa), small activating RNA (saRNA), locked nucleic acid (LNA), antagonist mir, and peptide nucleic acid (PNA).

6. The composition according to claim 5, wherein at least one inhibitor is tough decoy RNA (TuD).

7. At least one inhibitor, N-acetylgalactosamine (GalNAc), phosphorothioate DNA (PS), 2'-O-methyl RNA (OMe), 2'-O-methoxyethyl RNA (MOE), peptide nucleic acid (PNA), N3'-P5'-phosphoramidate (NP), 2'-fluoroarabino nucleic acid (FANA), morpholinophosphoramidate (MF), cyclohexene nucleic acid (CeNA), and tricycled DNA (tc-DNA) The composition according to any one of claims 1 to 6, comprising chemical modification of a nucleic acid sequence selected from the group consisting of nucleic acid analogs including the above.

8. The composition according to any one of claims 1 to 7, wherein at least one inhibitor is contained in a delivery medium selected from the group consisting of adeno-associated virus (AAV), lentiviral vector, polyethyleneimine (PEI), cationic liposomes, silica nanoparticles, PEGylated PLGA, and neutral lipids.

9. The composition according to any one of claims 1 to 8, wherein at least one inhibitor is contained in adeno-associated virus (AAV).

10. The inhibitor of miR-379 or part or fragment thereof contains a nucleotide sequence of SEQ ID NO: 3 in which five or fewer nucleotides are substituted; and / or The inhibitor of miR-541 or part or fragment thereof contains a nucleotide sequence with SEQ ID NO: 4 in which five or fewer nucleotides are substituted. The composition according to any one of claims 1 to 9.

11. A composition comprising an inhibitor of miR-379 or a part or fragment thereof and an inhibitor of miR-541 or a part or fragment thereof on the same molecule, wherein the molecule comprises a nucleotide sequence that is complementary to or hybridizes with miR-379 or a part or fragment thereof, and a nucleotide sequence that is complementary to or hybridizes with miR-541 or a part or fragment thereof, according to any one of claims 1 to 10.

12. The composition according to claim 11, wherein the inhibitor comprises a nucleotide sequence of SEQ ID NO: 5 in which 10 or fewer nucleotides are substituted.

13. A pharmaceutical composition according to any one of claims 1 to 12.

14. A composition according to any one of claims 1 to 13, for use in the treatment or prevention of metabolic diseases, diseases related to metabolic disorders, and / or cancer.

15. The composition according to claim 14, wherein the disease associated with metabolic disease or metabolic disorder is selected from the group consisting of glucocorticoid hormone-induced metabolic dysfunction, obesity, diabetes, diabetic obesity, metabolic syndrome, insulin resistance, hyperglycemia, (systemic) lipid metabolism disorder, Cushing's syndrome, adverse effects or side effects associated with or caused by glucocorticoid (GC) treatment or excessive GC, atherosclerosis, heart disease, stroke, (cancerous) cachexia, growth retardation, fatty liver, NASH, and hepatic fibrosis.

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