Methods and compositions relating to miR-10 mimetics and their targets

Abstract

The present invention relates to methods and compositions for treating gastrointestinal motility disorders, obesity, and diabetes, comprising miR-10a-5p or miR-10b-5p mimics.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 837,988, filed April 24, 2019, and U.S. Provisional Patent Application No. 62 / 964,382, filed January 22, 2020, each of which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with United States Government support under Grant No. DK091725 awarded by the National Institutes of Health (NIH). The United States Government has certain rights in this invention. [Background technology]

[0003] 2. Background of the Invention According to data from the World Health Organization, there are over 425 million people with diabetes in the world, and as the prevalence of obesity increases, the number of diabetic patients is also increasing rapidly. Obesity and diabetes are closely related, as 90% of diabetic patients are also obese. Type 2 diabetes (T2D), a complex, heterogeneous, polygenic disease, occurs mainly in adults over the age of 40, and is therefore known as adult diabetes. T2D accounts for up to 95% of all diagnosed cases of diabetes. Unfortunately, so far, it is difficult to develop a drug that can fundamentally cure T2D, as the causes and induction mechanisms of T2D remain elusive. Interestingly, about half of diabetic patients also have gastrointestinal (GI) complications, including gastroparesis. It is known that abnormally high blood glucose levels (hyperglycemia), a characteristic sign of diabetes, lead to gastroparesis.

[0004] MicroRNAs (miRNAs) are important regulators of molecular and cellular processes. Several studies have shown that miRNAs are involved in the regulation of various cellular processes, including cell differentiation, proliferation, and apoptosis.

[0005] There remains a need for methods and compositions for treating and preventing T2D, obesity, diabetic fatty liver disease, and GI complications. The present invention addresses this need. Summary of the Invention

[0006] The present invention is based on the unexpected discovery that miR-10a-5p mimics and miR-10b-5p mimics can reduce or reverse various conditions and pathologies associated with diabetes mellitus, including insulin-resistant diabetes, obesity, fatty liver disease, cardiac function and inflammation. The present invention is also based on the unexpected discovery that treatment with miR-10a-5p mimics and miR-10b-5p mimics can restore normal function in intramuscular interstitial cells of Cajal within the muscular layer of the gastrointestinal tract and improve gastrointestinal motility disorders.

[0007] Thus, in certain aspects, the present invention provides a method of treating diabetes in a subject in need thereof, comprising administering to the subject an effective amount of a miR-10a-5p mimic, thereby treating the diabetic condition.

[0008] In various embodiments, the miR-10a-5p mimic is a miR-10a-5p duplex, a chemically modified double-stranded miR-10a-5p, an unmodified double-stranded miR-10a-5p, a single-stranded chemically modified miR-10a-5p, or a single-stranded unmodified miR-10a-5p.

[0009] In various embodiments, the diabetes is type 2 diabetes. In one embodiment, the miR-10a-5p mimic is mammalian.

[0010] In various embodiments, the miR-10a-5p mimic is human.

[0011] In various embodiments, the miR-10a-5p mimic is engineered.

[0012] In various embodiments, the miR-10a-5p mimic further comprises a pharma- ceutically acceptable carrier or adjuvant.

[0013] In another aspect, the present invention provides a method for reducing body weight in a subject comprising administering an effective amount of a miR-10a-5p mimic, thereby reducing the body weight of the subject.

[0014] In another aspect, the present invention provides a method for lowering blood glucose in a subject, comprising administering an effective amount of a miR-10a-5p mimic, thereby lowering blood glucose in the subject.

[0015] In another aspect, the present invention provides a method for increasing insulin sensitivity, comprising administering to a subject in need thereof an effective amount of a miR-10a-5p mimic, thereby increasing insulin sensitivity in the subject.

[0016] In another aspect, the present invention provides a method of treating diabetes-associated fatty liver disease in a subject in need thereof, comprising administering to the subject an effective amount of a miR-10a-5p mimic, thereby treating diabetes-associated fatty liver disease.

[0017] In another aspect, the present invention provides a method of reducing diabetes-associated inflammation in a subject in need thereof, comprising administering to the subject an effective amount of a miR-10a-5p mimic, thereby reducing diabetes-associated inflammation.

[0018] In certain exemplary embodiments, the miR-10a-5p mimic is a miR-10a-5p duplex, a chemically modified double-stranded miR-10a-5p, an unmodified double-stranded miR-10a-5p, a single-stranded chemically modified miR-10a-5p, or a single-stranded unmodified miR-10a-5p.

[0019] In various embodiments, the miR-10a-5p mimic is mammalian.

[0020] In various embodiments, the miR-10a-5p mimic is human.

[0021] In various embodiments, the miR-10a-5p mimic is engineered.

[0022] In various embodiments, the miR-10a-5p mimic further comprises a pharma- ceutically acceptable carrier or adjuvant.

[0023] In another aspect, the present invention provides a method for treating a gastrointestinal disorder comprising administering to a subject in need thereof an effective amount of a miR-10a-5p mimic, thereby treating the gastrointestinal disorder in the subject.

[0024] In various embodiments, the miR-10a-5p mimic is a miR-10a-5p duplex, a chemically modified double-stranded miR-10a-5p, an unmodified double-stranded miR-10a-5p, a single-stranded chemically modified miR-10a-5p, or a single-stranded unmodified miR-10a-5p.

[0025] In various embodiments, the miR-10a-5p mimic is mammalian.

[0026] In various embodiments, the miR-10a-5p mimic is human.

[0027] In various embodiments, the gastrointestinal disorder is selected from the group consisting of gastroparesis, functional gastrointestinal disorder, functional gastrointestinal motility disorder, and intestinal pseudo-obstruction.

[0028] In various embodiments, the functional gastrointestinal disorder is selected from the group consisting of irritable bowel syndrome, functional constipation, and unspecified functional bowel disorder.

[0029] In another aspect, the invention provides a composition comprising a miR-10a-5p mimic and a pharma- ceutically acceptable carrier or adjuvant.

[0030] In various embodiments, the miR-10a-5p mimic is a miR-10a-5p duplex, a chemically modified double-stranded miR-10a-5p, an unmodified double-stranded miR-10a-5p, a single-stranded chemically modified miR-10a-5p, or a single-stranded unmodified miR-10a-5p.

[0031] In various embodiments, the miR-10a-5p mimic is engineered.

[0032] In certain exemplary embodiments, the miR-10a-5p mimic comprises a nucleic acid sequence comprising SEQ ID NO: 1, 2, 3, 4, 5, or a combination thereof.

[0033] In another aspect, the present invention provides a method for increasing interstitial cells of Cajal (ICC) proliferation, comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby increasing ICC proliferation in the subject.

[0034] In various embodiments, the miR-10b-5p mimic is a miR-10b-5p duplex, a chemically modified double-stranded miR-10b-5p, an unmodified double-stranded miR-10b-5p, a single-stranded chemically modified miR-10b-5p, or a single-stranded unmodified miR-10b-5p.

[0035] In various embodiments, the miR-10b-5p mimic is mammalian.

[0036] In various embodiments, the miR-10b-5p mimic is human.

[0037] In various embodiments, increasing ICC proliferation in a subject restores function of ICCs in the subject.

[0038] In various embodiments, the ICC is located within the smooth muscle of the gastrointestinal tract of the subject.

[0039] In various embodiments, the smooth muscle is located within the stomach, small intestine, or colon of a subject.

[0040] In certain exemplary embodiments, the ICC comprises an ICC precursor, an ICC-MY, an ICC-IM, an ICC-DMP, an ICC-SM, or an ICC-SMP.

[0041] In various embodiments, the subject is a human.

[0042] In another aspect, the present invention provides a method for increasing KIT expression in ICC, comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby increasing KIT expression in the subject.

[0043] In various embodiments, the miR-10b-5p mimic is a miR-10b-5p duplex, a chemically modified double-stranded miR-10b-5p, an unmodified double-stranded miR-10b-5p, a single-stranded chemically modified miR-10b-5p, or a single-stranded unmodified miR-10b-5p.

[0044] In various embodiments, the miR-10b-5p mimic is mammalian.

[0045] In various embodiments, the miR-10b-5p mimic is human.

[0046] In various embodiments, increasing KIT expression in a subject restores function of ICC in the subject.

[0047] In another aspect, the present invention provides a method for treating a gastrointestinal disorder comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby treating the gastrointestinal disorder in the subject.

[0048] In various embodiments, the miR-10b-5p mimic is a miR-10b-5p duplex, a chemically modified double-stranded miR-10b-5p, an unmodified double-stranded miR-10b-5p, a single-stranded chemically modified miR-10b-5p, or a single-stranded unmodified miR-10b-5p.

[0049] In various embodiments, the miR-10b-5p mimic is mammalian.

[0050] In various embodiments, the miR-10b-5p mimic is human.

[0051] In various embodiments, the gastrointestinal disorder is selected from the group consisting of gastroparesis, functional gastrointestinal disorder, functional gastrointestinal motility disorder, and intestinal pseudo-obstruction.

[0052] In various embodiments, the functional gastrointestinal disorder is selected from the group consisting of irritable bowel syndrome, functional constipation, and unspecified functional bowel disorder.

[0053] In another aspect, the present invention provides a method for reducing body weight comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby reducing the subject's body weight.

[0054] In another aspect, the present invention provides a method for lowering blood glucose, comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby lowering blood glucose in the subject.

[0055] In another aspect, the present invention provides a method for treating pancreatic cancer, comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby increasing pancreatic stem cell (PSC) proliferation or pancreatic progenitor cell (PPC) proliferation in the subject. + PSC expansion or KIT + Methods for increasing PPC proliferation are provided.

[0056] In another aspect, the present invention provides a method for increasing insulin sensitivity, comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby increasing insulin sensitivity in the subject.

[0057] In another aspect, the present invention provides a method for treating diabetes comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby treating diabetes in the subject.

[0058] In various embodiments, the diabetes is type 1 diabetes or type 2 diabetes.

[0059] In another aspect, the present invention provides a method for increasing KIT expression in PSCs or PPCs by comprising the step of administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby increasing KIT expression.

[0060] In another aspect, the present invention provides a method for increasing expression of INSR, IRS2, and IRS1 in skeletal muscle cells (SkMCs), comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby increasing expression of INSR, IRS2, and IRS1.

[0061] In another aspect, the present invention provides a method for reducing diabetes-associated inflammation in a subject in need thereof, comprising administering an effective amount of a miR-10b-5p mimic, thereby reducing diabetes-associated inflammation.

[0062] In certain exemplary embodiments, the miR-10b-5p mimic is a miR-10b-5p duplex, a chemically modified double-stranded miR-10b-5p, an unmodified double-stranded miR-10b-5p, a single-stranded chemically modified miR-10b-5p, or a single-stranded unmodified miR-10b-5p.

[0063] In various embodiments, the miR-10b-5p mimic is mammalian.

[0064] In various embodiments, the miR-10b-5p mimic is human.

[0065] In various embodiments, the miR-10b-5p mimic is engineered.

[0066] In various embodiments, the miR-10b-5p mimic further comprises a pharma- ceutically acceptable carrier or adjuvant.

[0067] In another aspect, the invention provides a composition comprising a miR-10b-5p mimic and a pharma- ceutically acceptable carrier or adjuvant.

[0068] In various embodiments, the miR-10b-5p mimic is a miR-10b-5p duplex, a chemically modified double-stranded miR-10b-5p, an unmodified double-stranded miR-10b-5p, a single-stranded chemically modified miR-10b-5p, or a single-stranded unmodified miR-10b-5p.

[0069] In various embodiments, the miR-10b-5p mimic is engineered.

[0070] In certain exemplary embodiments of the above or any other aspect of the invention, the miR-10b-5p mimic comprises a nucleic acid sequence comprising SEQ ID NO: 6, 7, 8, 9, 10, 12, 13, or a combination thereof. [The present invention 1001] A method of treating diabetes in a subject in need thereof, comprising administering to the subject an effective amount of a miR-10a-5p mimic, thereby treating the diabetic condition. [The present invention 1002] 1001. The method of claim 1001, wherein said miR-10a-5p mimic is a miR-10a-5p double-stranded, a chemically-modified double-stranded miR-10a-5p, an unmodified double-stranded miR-10a-5p, a single-stranded chemically-modified miR-10a-5p, or a single-stranded unmodified miR-10a-5p. [The present invention 1003] 1001. The method of claim 1001, wherein said diabetes is type 2 diabetes. [The present invention 1004] 1001. The method of claim 1001, wherein said miR-10a-5p mimic is mammalian. [The present invention 1005] 1001. The method of claim 1001, wherein said miR-10a-5p mimic is human. [The present invention 1006] The method of any one of claims 1 to 10, wherein said miR-10a-5p mimic is engineered. [The present invention 1007] 1001. The method of claim 1001, wherein said miR-10a-5p mimic further comprises a pharma- ceutically acceptable carrier or adjuvant. [The present invention 1008] 23. A method of reducing body weight in a subject comprising administering an effective amount of a miR-10a-5p mimic, thereby reducing the body weight of the subject. [The present invention 1009] A method of lowering blood glucose in a subject comprising administering an effective amount of a miR-10a-5p mimic, thereby lowering blood glucose in the subject. [The present invention 1010] A method for increasing insulin sensitivity comprising administering to a subject in need thereof an effective amount of a miR-10a-5p mimic, thereby increasing insulin sensitivity in the subject. [The present invention 1011] A method of treating diabetes-associated fatty liver disease in a subject in need thereof, comprising administering to the subject an effective amount of a miR-10a-5p mimic, thereby treating the diabetes-associated fatty liver disease. [The present invention 1012] A method of reducing diabetes-associated inflammation in a subject in need thereof, comprising administering to the subject an effective amount of a miR-10a-5p mimic, thereby reducing said diabetes-associated inflammation. [The present invention 1013] The method of any of claims 1008 to 1012, wherein the miR-10a-5p mimic is a miR-10a-5p double-stranded, a chemically modified double-stranded miR-10a-5p, an unmodified double-stranded miR-10a-5p, a single-stranded chemically modified miR-10a-5p, or a single-stranded unmodified miR-10a-5p. [The present invention 1014] The method of any one of claims 1 to 3, wherein said miR-10a-5p mimic is mammalian. [The present invention 1015] The method of any one of claims 1 to 4, wherein said miR-10a-5p mimic is human. [The present invention 1016] The method of any one of claims 1 to 5, wherein said miR-10a-5p mimic is engineered. [The present invention 1017] The method of claim 1013, wherein said miR-10a-5p mimic further comprises a pharma- ceutically acceptable carrier or adjuvant. [The present invention 1018] 23. A method of treating a gastrointestinal disorder comprising administering to a subject in need thereof an effective amount of a miR-10a-5p mimic, thereby treating the gastrointestinal disorder in the subject. [The present invention 1019] The method of claim 1018, wherein the miR-10a-5p mimic is a miR-10a-5p duplex, a chemically modified double-stranded miR-10a-5p, an unmodified double-stranded miR-10a-5p, a single-stranded chemically modified miR-10a-5p, or a single-stranded unmodified miR-10a-5p. [The present invention 1020] The method of any one of claims 10 to 19, wherein said miR-10a-5p mimic is mammalian. [The present invention 1021] The method of claim 1020, wherein said miR-10a-5p mimic is human. [The present invention 1022] The method of claim 1018, wherein said gastrointestinal disorder is selected from the group consisting of gastroparesis, functional gastrointestinal disorder, functional gastrointestinal motility disorder, and intestinal pseudo-obstruction. [The present invention 1023] The method of claim 1022, wherein said functional gastrointestinal disorder is selected from the group consisting of irritable bowel syndrome, functional constipation, and unspecified functional bowel disorder. [The present invention 1024] A composition comprising a miR-10a-5p mimic and a pharma- ceutically acceptable carrier or adjuvant. [The present invention 1025] The composition of the present invention 1024, wherein the miR-10a-5p mimic is a miR-10a-5p duplex, a chemically modified double-stranded miR-10a-5p, an unmodified double-stranded miR-10a-5p, a single-stranded chemically modified miR-10a-5p, or a single-stranded unmodified miR-10a-5p. [The present invention 1026] The composition of any one of claims 10 to 24, wherein said miR-10a-5p mimic is engineered. [The present invention 1027] The method of any one of claims 1001 to 1023 or the composition of any one of claims 1024 to 1026, wherein the miR-10a-5p mimic comprises a nucleic acid sequence comprising SEQ ID NO:1, 2, 4, 5, or a combination thereof. [The present invention 1028] A method for increasing interstitial cells of Cajal (ICC) proliferation, comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby increasing ICC proliferation in the subject. [The present invention 1029] The method of claim 1028, wherein said miR-10b-5p mimic is a miR-10b-5p duplex, a chemically-modified double-stranded miR-10b-5p, an unmodified double-stranded miR-10b-5p, a single-stranded chemically-modified miR-10b-5p, or a single-stranded unmodified miR-10b-5p. [The present invention 1030] The method of claim 1029, wherein said miR-10b-5p mimic is mammalian. [The present invention 1031] The method of claim 1030, wherein said miR-10b-5p mimic is human. [The present invention 1032] The method of claim 1028, wherein increasing ICC proliferation in said subject restores function of ICC in said subject. [The present invention 1033] The method of claim 1028, wherein said ICC is located within the smooth muscle of the gastrointestinal tract of said subject. [The present invention 1034] The method of claim 1033, wherein said smooth muscle is located within the stomach, small intestine, or colon of said subject. [The present invention 1035] The method of claim 1034, wherein the ICC comprises an ICC precursor, an ICC-MY, an ICC-IM, an ICC-DMP, an ICC-SM, or an ICC-SMP. [The present invention 1036] The method of claim 1028, wherein the subject is a human. [The present invention 1037] A method for increasing KIT expression in ICC, comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby increasing KIT expression in the subject. [The present invention 1038] The method of the present invention 1037, wherein said miR-10b-5p mimic is a miR-10b-5p duplex, a chemically-modified double-stranded miR-10b-5p, an unmodified double-stranded miR-10b-5p, a single-stranded chemically-modified miR-10b-5p, or a single-stranded unmodified miR-10b-5p. [The present invention 1039] The method of claim 1038, wherein said miR-10b-5p mimic is mammalian. [The present invention 1040] The method of claim 1039, wherein said miR-10b-5p mimic is human. [The present invention 1041] The method of claim 1037, wherein increasing KIT expression in said subject restores the function of said ICC in said subject. [The present invention 1042] A method for treating a gastrointestinal disorder comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby treating the gastrointestinal disorder in the subject. [The present invention 1043] The method of claim 1042, wherein said miR-10b-5p mimic is a miR-10b-5p duplex, a chemically-modified double-stranded miR-10b-5p, an unmodified double-stranded miR-10b-5p, a single-stranded chemically-modified miR-10b-5p, or a single-stranded unmodified miR-10b-5p. [The present invention 1044] The method of claim 1043, wherein said miR-10b-5p mimic is mammalian. [The present invention 1045] The method of claim 1044, wherein said miR-10b-5p mimic is human. [The present invention 1046] The method of claim 1043, wherein said gastrointestinal disorder is selected from the group consisting of gastroparesis, functional gastrointestinal disorder, functional gastrointestinal motility disorder, and intestinal pseudo-obstruction. [The present invention 1047] The method of claim 1046, wherein said functional gastrointestinal disorder is selected from the group consisting of irritable bowel syndrome, functional constipation, and unspecified functional bowel disorder. [The present invention 1048] 23. A method for reducing body weight comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby reducing the body weight of the subject. [The present invention 1049] A method for lowering blood glucose comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby lowering blood glucose in the subject. [The present invention 1050] administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby increasing pancreatic stem cell (PSC) proliferation or pancreatic progenitor cell (PPC) proliferation in the subject. + PSC proliferation or KIT + • Methods for increasing PPC proliferation. [The present invention 1051] A method for increasing insulin sensitivity comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby increasing insulin sensitivity in the subject. [The present invention 1052] 23. A method for treating diabetes comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby treating diabetes in the subject. [The present invention 1053] The method of claim 1051, wherein said diabetes is type 1 or type 2 diabetes. [The present invention 1054] A method for increasing KIT expression in PSCs or PPCs, comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby increasing KIT expression. [The present invention 1055] A method for increasing expression of INSR, IRS2, and IRS1 in skeletal muscle cells (SkMCs), comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby increasing expression of INSR, IRS2, and IRS1. [The present invention 1056] 23. A method for reducing diabetes-associated inflammation in a subject in need thereof, comprising administering an effective amount of a miR-10b-5p mimic, thereby reducing said diabetes-associated inflammation. [The present invention 1057] The method of any of claims 1048 to 1056, wherein the miR-10b-5p mimic is a miR-10b-5p double-stranded, a chemically modified double-stranded miR-10b-5p, an unmodified double-stranded miR-10b-5p, a single-stranded chemically modified miR-10b-5p, or a single-stranded unmodified miR-10b-5p. [The present invention 1058] The method of claim 1057, wherein said miR-10b-5p mimic is mammalian. [The present invention 1059] The method of claim 1058, wherein said miR-10b-5p mimic is human. [The present invention 1060] The method of claim 1057, wherein said miR-10b-5p mimic is engineered. [The present invention 1061] The method of claim 1057, wherein said miR-10b-5p mimic further comprises a pharma- ceutically acceptable carrier or adjuvant. [The present invention 1062] A composition comprising a miR-10b-5p mimic and a pharma- ceutically acceptable carrier or adjuvant. [The present invention 1063] The composition of the present invention 1062, wherein the miR-10b-5p mimic is a miR-10b-5p duplex, a chemically-modified double-stranded miR-10b-5p, an unmodified double-stranded miR-10b-5p, a single-stranded chemically-modified miR-10b-5p, or a single-stranded unmodified miR-10b-5p. [The present invention 1064] The composition of claim 1062, wherein the miR-10b-5p mimic is engineered. [The present invention 1065] The method of any one of claims 1028 to 1061 or the composition of any one of claims 1062 to 1064, wherein the miR-10b-5p mimic comprises a nucleic acid sequence comprising SEQ ID NO:6, 7, 8, 9, 10, 12, 13, or a combination thereof. [Brief description of the drawings]

[0071] The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings, in which: For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred; it is to be understood, however, that the invention is not limited to the precise apparatus and instrumentality of the embodiments shown in the drawings. [Figure 1]Figure 1A-1I illustrate the absence of miR-10a-5p and miR-10b-5p in jejunum and colon KIT+ ICCs of diabetic KitcopGFP / +;Lep / ob male mice. Figure 1A shows gross anatomical changes in KitcopGFP / +;Lep+ / + (wild type: WT) male mice at 4, 8 and 12 weeks. Figure 1B shows the progressive increase in body weight in diabetic Lep mutant mice compared to WT mice. Figure 1C shows increased blood glucose levels in diabetic mice. Diabetic mice become significantly hyperglycemic between 11 and 13 weeks. Figure 1D is an automated Western blot of KIT in the jejunum and colon of diabetic Lep mutant and WT mice. Figure 1E is a Pearson correlation analysis between miRNA-seq data from pooled colonic and jejunal ICCs (CICCs and JICCs, respectively) isolated from diabetic KitcopGFP / +;Lep / ob mice (n=30), non-diabetic KitcopGFP / +;Lep / + mice (n=20), and KitcopGFP / +;Lep+ / + mice (n=20). Figure 1F and Figure 1G are heat maps of the most dynamically regulated miRNAs in diabetic and non-diabetic jejunal and colonic ICCs. Both jejunal and colonic KIT+ICCs were isolated by FACS and miRNA expression profiles were obtained by miRNA-seq. miR-10a-5p and miR-10b-5p are abundantly expressed in both WT JICCs and WT CICCs, but are depleted in both diabetic ICCs. Figures 1H and 1I show the expression levels of the 10 significantly expressed miRNAs in Figures 1F (boxed in black) and 1G obtained by miRNA-seq. [Diagram 2]Figures 2A-F illustrate the generation of KIT+ (ICC-specific) mir-10b knockout mice. Figure 2A shows the genomic maps of tamoxifen-inducible KitCreERT2 / +;mir-10bLacZ / LacZ and KitCreERT2 / +;mir-10b- / - mice. KitCreERT2 / +;mir-10bLacZ / LacZ (resulting in mir-10b KO with tamoxifen and mir-10b WT with oil) and ROSA26FLP / +;mir-10blox / lox mice were generated by crossing mir-10bLacZ-FLP-lox / + mice with KitCreERT2 / + or ROSA26FLP / + mice. ROSA26FLP / +;mir-10blox / lox mice were further crossed with KitCreERT2 / + mice to generate KitCreERT2 / +;10blox / lox (mir-10b KO with tamoxifen and mir-10b WT with oil) mice. Figure 2B shows gross dissections of male mir-10b KO mice (injected with tamoxifen) and mir-10b WT mice (injected with oil). Figure 2C shows validation of miR-10b depletion in mir-10b KO mice. Expression levels of miR-10b-5p were measured by qPCR in jejunal and colonic smooth muscle of mir-10b KO and mir-10b WT. Figure 2D shows western blots of proteins regulated by or associated with Mir10b. KIT was decreased in the jejunum and colon of mir-10b KO, while KLF11 was increased in the KO tissues. There were no significant changes in HOXD3 and HOXD4. GAPDH was used as a loading control. Protein ladder (L) is shown with molecular weight (kDa). Figure 2E shows the reduction of ICCs (CD117+CD45-) in stomach, jejunum and colon from mir-10b KO compared to mir-10b WT mice.Figures 2F-G show cross-sections, whole mount optical stacks and separated images of jejunal and / or colonic ICCs (KIT+ANO1+) in mir-10b KO mice (ICC-MY, ICC-DMP and ICC-SMP) compared to mir-10b WT mice. [Diagram 3] Figures 3A-F illustrate that male mir-10b knockout mice develop diabetes. Figure 3A shows that the body weight of mir-10b KO male mice increases progressively from 16 weeks onwards compared to control mir-10b WT male mice. Figure 3B shows that the fasting blood glucose levels of mir-10b KO male mice also increase progressively, leading to hyperglycemia after 24 weeks. Figure 3C shows intraperitoneal glucose tolerance test (GTT) in mir-10b KO and WT mice. The glucose clearance of mir-10b KO mice decreases progressively with age from 1 month to 7 months. Figure 3D shows the area under the curve (AUC) plot of the GTT. Glucose intolerance is significantly increased in 6 and 7 month old mir-10b KO mice. Figure 3E shows intraperitoneal insulin tolerance test (ITT) in mir-10b KO and WT mice. These ITTs show that mir-10b KO mice become increasingly insensitive to insulin as they age from 1 to 7 months of age. Figure 3F shows the AUC plot of the ITTs. Insulin resistance is significantly increased in mir-10b KO mice at 4 months of age compared to mir-10b WT mice, which become excessive over time. [Figure 4]Figures 4A-4F illustrate that male mir-10b knockout mice exhibit delayed gastrointestinal (GI) transit. Figure 4A shows total GI transit time measured using Evans Blue in mir-10b KO and WT mice from 1 to 7 months of age. Figure 4B shows gastric emptying profiles of 7-month-old mir-10b KO and WT mice using a GastroSense IVIS in vivo imaging system. Figure 4C shows quantification of the percentage of gastric emptying at 30 minutes. Figure 4D shows colonic transit time measured using the bead emptying method. Figures 4E-4F show the mean frequency and output of fecal pellets within 24 hours. [Diagram 5]Figure 5A-G illustrate the pathway analysis and target validation of miR-10b-5p in diabetes. Figure 5A shows the pathway analysis of miR-10b-5p and its target genes related to diabetes by Ingenuity Pathway Analysis (IPA). Figure 5B shows the Western blot analysis for screening of synthetic miR-10b-5p mimics (10b duplex mimics 1-3), single-stranded miR-10b-3p (10b 3p), single-stranded miR-10b-5p (10b 5p), annealed miR-10b-3p and miR-10b-5p (10b 3p+5p), and mouse precursor miR-10b (mPre-10b), which can regulate KLF11 and KIT in NIH3T3 and HEK293 cells. Non-targeting RNA (scrambled) and non-transfected control (NTC) were used as negative controls. Protein ladder (L) is shown with molecular weight (kDa). Figure 5C shows the validation of miR-10b-5p target proteins in NIH3T3 cells, HEK293 cells. Figure 5D shows NIT-1 cells (mβC) and Panc10.05 cells (hβC) transfected with 10b mimic 1, miR-10b-5p antisense (10b inhibitor), two siRNAs targeting mouse Klf11 (siKlf11-1 and siKlf11-2) and human KLF11 (siKLF11-1 and siKLF11-2), scrambled, and NTC. Figure 5E shows the line diagram of the luciferase reporter plasmid and the miR-10b-5p target sites (10b mimic binding sites) of human and mouse KLF11 (hKLF11 10b TS and mKlf11 10b TS) and mutants (mKlf11 10b TSM). Figure 5F shows the targeting effect of 10b mimics and 10b inhibitors in stable HEK293 cells transfected with luciferase reporter plasmids over a period of 6 to 48 hours post-transfection.FIG. 5G shows target validation of KLF11 by 10b mimics and 10b inhibitors in four cell lines transfected with luciferase reporter plasmids and the dose-dependent targeting effects of 10b mimics and 10b inhibitors in each cell after 12 hours. [Figure 6]Figures 6A-J illustrate the rescue of diabetic conditions and gastroparesis in male mir-10b knockout mice by miR-10b mimic injection. Figure 6A shows the body weight comparison in male diabetic mir-10b knockout (KO) mice injected with miR-10b-5p mimic (263 ng / g body weight), mir-10b KO mice without injection, and WT mice. Figure 6B shows the restoration of normal blood glucose levels in mir-10b KO male mice immediately after injection with miR-10b-5p mimic. Mice that did not receive miR-10b mimic injection (WT and "no injection" mice) did not show a decrease in glucose levels. Normal and stable glucose levels were maintained for 4 weeks after injection. Figure 6C shows GTT showing dramatically improved glucose tolerance in miR-10b-5p mimic treated mice. Figure 6D shows ITT indicating significantly improved insulin sensitivity in miR-10b-5p mimic-treated mice. Figure 6E shows gradual recovery of total GI transit time in miR-10b-5p mimic-treated mice. Figure 6F shows increased mean fecal output in miR-10b-5p mimic-treated mice. Figure 6G shows gastric emptying study in miR-10b KO mice injected with miR-10b-5p mimic, as well as uninjected mir-10b KO and WT mice using GastroSense IVIS in vivo imaging system. Figure 6H shows ICC in jejunum and colon is restored in mir-10 KO mice after miR-10b-5p mimic injection. Figure 6I shows KLF11 and KIT expression in pancreas and colon is reversed in mir-10 KO mice after miR-10b-5p mimic injection. FIG. 6J shows that miR-10b-5p expression was increased in blood, pancreas, jejunum, and colon following miR-10b-5p mimic injection. [Figure 7]Figures 7A-G illustrate that miR-10b-5p mimic rescues the diabetic state and slows GI transit time in high fat high sucrose diet (HFHSD) fed mice. Figure 7A shows the weight comparison up to 15 weeks in HFHSD-induced diabetic male mice and normal diet (ND) male mice injected twice with miR-10b-5p mimic [Thermo Fisher Scientific, Assay ID MC11108 (Cat. No. 4464070)] (500 ng / g body weight) or miRNA mimic negative control or no injection. Figure 7B shows that normal blood glucose levels are restored in HFHSD-induced diabetic male mice immediately after injection with miR-10b-5p mimic but not in either the negative control or no injection group. Normal fasting glucose levels similar to those of untreated normal diet male mice were maintained for 5 weeks after the first injection, and for an additional 10 weeks after the second injection. In addition, normal diet fed mice injected with miR-10b-5p mimics showed normal glucose levels similar to those of mice injected with negative control and mice not injected with anything. Figure 7C features GTT results showing significantly improved glucose tolerance in miR-10b-5p mimic-treated HFHSD mice 2 and 4 weeks (1-2W and 1-4W) after the first injection and 2 and 4 weeks (2-2W and 2-4W) after the second injection. Figure 7D illustrates the ITT results showing significantly improved insulin sensitivity in miR-10b-5p mimic-treated HFHSD mice. Figure 7E shows improvement in total GI transit time in miR-10b-5p mimic-treated HFHSD-fed mice 2 and 4 weeks after the first and second injections, and Figures 7F-G show that fecal pellet output returned to normal in the miR-10b-5p mimic-treated HFHSD-fed group after the first and second injections for 5 weeks after injection. [Figure 8]Figures 8A-8G illustrate the recovery of miR-10b, its regulated proteins, ICC and β-cells in diabetic mice treated with miR-10b-5p mimics. Figure 8A is a graph showing the amount of miR-10b-5p in blood in normal diet (ND) healthy mice, HFHSD diabetic mice, and miR-10b-5p mimic HFHSD mice at 1-4 weeks after injection. Figure 8B is a graph showing the change in insulin levels after 6 hours of fasting and after glucose injection in the three groups of mice. Figure 8C is a graph showing hemoglobin A1C levels. Figure 8D shows the change in KLF11 protein expression at 2 weeks in HFHSD diabetic mice and ND healthy mice treated with 10b mimics or negative control RNA or without injection. Figures 8E-8F show a comparison of KLF11 and KIT expression levels in blood, pancreas, stomach, colon and skeletal muscle of ND, HFHSD and miR-10b-5p mimic-injected HFHSD mice at 1 and 3 weeks. Figure 8G illustrates cross-section and whole mount images showing recovery of ICC(KIT+) in stomach, jejunum, colon and pancreas of the three groups at 3 weeks. [Figure 9] Figures 9A-G illustrate the lack of adverse effects of miR-10b-5p mimic injection in healthy C57 mice. Figure 9A shows body weight comparisons in healthy C57 male mice injected twice with a range of concentrations of miR-10b-5p mimic (263ng / g, 132ng / g, 66ng / g, 33ng / g body weight) and observed over 10 weeks. Figure 9B shows blood glucose level comparisons. Figure 9C shows GTTs showing dose-dependent improvement in glucose tolerance in mice injected with miR-10b-5p mimic 2 and 4 weeks (1-2 and 1-4 weeks) after the first injection and 2 and 4 weeks (2-2 and 2-4 weeks) after the second injection. Figure 9D shows ITTs showing dose-dependent improvement in insulin sensitivity. Figures 9E-9F show fecal pellet frequency and output. Figure 9G shows total GI transit time. [Figure 10]FIG. 10A-I illustrate that conditional ablation of KIT+ cells in male KitCre-ERT2 / +;ROSA26DTA / + mice leads to a diabetic state and slow GI transit. Kit-tdTom (KitCre-ERT2 / +;ROSA26tdTom / +) and Kit-DTA (KitCre-ERT2 / +;ROSA26DTA / +) mice were treated with tamoxifen at 2 months of age. FIG. 10A is a graph showing that the weight of Kit-DTA male mice increases progressively from 10 weeks onwards compared to control male mice. FIG. 10B is a graph showing that the blood glucose levels of Kit-DTA male mice also increase progressively, with the mice becoming hyperglycemic after 24 months. FIG. 10C is a graph showing the results of an intraperitoneal glucose tolerance test (GTT) in Kit-DTA and WT mice. Kit-DTA male mice show a gradual decline in glucose clearance at 2 and 5 months of age. Figure 10D is a graph showing intraperitoneal insulin tolerance test (ITT) in Kit-DTA and WT mice. Kit-DTA KO mice show progressive insulin insensitivity at 2 and 5 months of age. Figure 10E shows degeneration of β cells in Kit-DTA mice compared to Kit-tdTom mice. At 7 days and 5 months after tamoxifen injection, KIT+ cell-derived (tdTom+) cells are found among β (INS+) cells in the islets of Kit-tdTom, but are depleted in Kit-DTA. Figure 10F is a graph showing expression of miR-10b-5p measured by qPCR. Figure 10G is a graph showing the amount of insulin and C-peptide in blood by ELISA. Figure 10H shows a comparison of KLF11 and KIT expression levels in the pancreas, colon, and blood of Kit-tdTom and Kit-DTA mice after 7 days and 5 months, and Figure 10I is a graph showing total GI transit time in Kit-DTA mice after 2 and 5 months. [Figure 11]Figures 11A-H illustrate that human patients with gastroparesis and / or T2D have aberrant miR-10b-5p, KLF11 and KIT expression patterns similar to the mouse data obtained above. Figure 11A shows the expression of miR-10b-5p in blood samples collected from idiopathic gastroparesis patients (IG, n=13) and diabetic gastroparesis patients (DG, n=2) compared to healthy controls (HC, n=15). The IG group was divided into two groups according to high (IG-H, n=7) and low (IG-L, n=6) miR-10b. **p<0.05, ***p<0.01. Figures 11B and 11C show insulin and C-peptide in blood of the above four groups. Figure 11D shows hemoglobin A1C levels. Figure 11E shows the expression of KLF11 and KIT in blood and stomach samples. KLF11 protein levels in serum and antrum were also higher in the DG group than in the HC group, while KIT levels were lower in the DG group. Figure 11F shows fasting blood glucose levels. Figure 11G shows Pearson correlation analysis between miRNA-seq data obtained from blood in HC, IG-H, IG-L and DG (n=2 for each group). Figure 11H shows a heat map of the 70 most dynamically regulated miRNAs in HC, IG-H, IG-L and DG (n=2 for each group). The levels of miR-10a-5p and miR-10b-5p were highest in HC, followed by IG-H, IG-L and DG, respectively. [Figure 12]Illustrating the regulation of diabetic conditions by miR-10b. miR-10b-5p regulates key metabolic genes (LEP, ADIPOQ, GLP1R, KIT, INS, INSR, SLC2A2, SLC2A4, IRS1 and IRS2) through two metabolism-related transcription factors KLF7 and KLF11, which orchestrate cell proliferation, differentiation, insulin production and insulin sensitivity in adipocytes, ICCs, pancreatic progenitor cells, β-cells and skeletal muscle cells. Under normal glucose conditions, miR-10b is abundantly expressed to inhibit KLF7 and KLF11, whereas under hyperglycemic conditions this miRNA is suppressed allowing overexpression of these transcriptional genes, which leads to diabetes, GI dysmotility and obesity. [Figure 13] Figures 13A-B illustrate the sequence comparison of miR-10a-5p and miR-10b-5p mimics and inhibitors used in this study. Figure 13A shows the sequence and structure of mouse miR-10a precursor encoding miR-10a-5p and miR-10a-3p, synthetic miR-10a-5p molecule (miR-10a-5p mimic) and synthetic miR-10a-5p antisense molecule (miR-10a-5p inhibitor). Figure 13B shows the sequence and structure of mouse miR-10b precursor encoding miR-10b-5p and miR-10b-3p, synthetic miR-10b-5p molecule (miR-10b-5p mimic) and synthetic miR-10b-5p antisense molecule (miR-10b-5p inhibitor). Note that single nucleotide base differences between the miR-10a-5p mimic (U) and the miR-10b-5p mimic (A) and between the miR-10a-5p inhibitor (A) and the miR-10b-5p inhibitor (U) are shown in bold. [Figure 14]Figure 14A-B illustrates that miR-10a-5p mimic rescues obesity and diabetes phenotypes in mice fed a high-fat, high-sucrose diet (HFHSD) or normal diet (ND). (Figure 14A and Figure 14B) Comparison of body weight and fasting blood glucose. Male C57 mice were fed a HFHSD throughout the experiment (4-43 weeks) and injected twice with miR-10a-5p mimic or negative control (scrambled RNA) (first injection at 22 weeks and second injection at 31 weeks), or no injection at 22 weeks, and then monitored for 21 weeks post-injection (PI). n=3-6 per group. *p<0.05 and **p<0.01 (miR-10a-5p mimic vs no injection on HFHSD). [Figure 15] Figures 15A-B illustrate that miR-10a-5p mimic injection results in significant weight loss (40%) in HFHSD-induced obese diabetic mice. (Figure 15A) Gross anatomical changes in HFHSD-induced obese diabetic mice injected with miR-10a-5p mimic or negative control (scrambled RNA) or no injection compared to age-matched ND mice. (Figure 15B) Gross anatomical changes before injection and at 4 weeks PI in the same HFHSD-induced obese diabetic mice injected with miR-10a-5p mimic at 22 weeks. [Figure 16] Figure 16A-B illustrates that miR-10a-5p mimic injection reduces body mass index (BMI) and waist circumference in HFHSD-induced obese diabetic mice. Changes in mean BMI and mean waist circumference in HFHSD-induced obese diabetic mice injected with miR-10a-5p mimic or negative control (scrambled RNA) or without injection compared to healthy mice fed ND in Figure 14 (measured at 8 weeks PI after the first injection at 23 weeks, and 1 week PI after the second injection at 31 weeks). n=6-12 per group. *p<0.05 and **p<0.01. [Figure 17]We demonstrate that insulin regulation by miR-10a-5p and miR-10b-5p mimics is opposite. miR-10a-5p mimic injection slightly reduces insulin levels in HFHSD-induced diabetic mice, followed by a slow recovery, whereas miR-10b-5p mimic injection transiently increases insulin levels, followed by a gradual decrease. Changes in insulin levels after 6 hours of fasting over 4 weeks in male mice fed HFHSD or ND and injected with either miR-10a-5p mimic or miR-10b-5p mimic or no injection. n=6-12 per condition per experiment. **p<0.01 (miR-10a-5p mimic vs. no injection in HFHSD);##p<0.01 (miR-10b-5p mimic vs. no injection in HFHSD). [Figure 18] Figures 18A-B illustrate that miR-10a-5p mimic injection improves glucose tolerance in HFHSD-induced diabetic mice. (Figure 18A) Intraperitoneal (IP) glucose tolerance test (GTT) 4 weeks PI after the first injection. (Figure 18B) Area under the curve (AUC) plot of GTT. n=6-12 per group. **p<0.01. [Figure 19] Figures 19A-B illustrate that miR-10a-5p mimic injection improves insulin resistance in HFHSD-induced diabetic mice. (Figure 19A) IP insulin tolerance test (ITT) 4W PI after 1st injection. (Figure 19B) Area under the curve (AUC) plot of ITT. n=6-12 per group. **p<0.01. [Figure 20] Figures 20A-B illustrate that miR-10a-5p mimic injection increases (Figure 20A) GLP-1 and (Figure 20B) leptin in HFHSD-induced diabetic mice. miR-10a-5p mimic injection increases and restores (Figure 20A) GLP-1 and (Figure 20B) leptin in HFHSD-induced diabetic mice to hormone levels close to those of ND-fed healthy mice over 4 weeks. n=4 per group per experiment. **p<0.01 (miR-10a-5p mimic vs. no injection in HFHSD). [Figure 21] Figures 21A-F illustrate that miR-10a-5p mimic injection improves GI function. (Figure 21A) Total GI transit time, (Figure 21B) fecal pellet output, and (Figure 21C) colonic transit time in HFHSD-fed male diabetic mice or ND-fed male healthy mice with or without miR-10a-5p mimic injection at 4 weeks PI. (Figures 21D-F) Changes in total GI transit time, fecal pellet output, and colonic transit time in individual mice from Figures 21A-C. n=6-18 per group. **p<0.01. [Figure 22] A series of fluorescent images demonstrating that miR-10a-5p mimic injection improves gastric emptying. Male diabetic mice fed HFHSD or healthy mice fed ND were injected with or without miR-10a-5p mimic and given fluorescent GastroSense 750 (GS) by oral gavage, and gastric emptying was measured over 60 min with an IVIS Lumina III system. [Figure 23] Figures 23A-D illustrate that both miR-10a-5p and miR-10b-5p mimics confer protection from obesity and diabetes phenotypes in HFHSD-fed mice. Male C57 mice were injected twice (first and second injections) with miR-10a-5p mimic, miR-10b-5p mimic, negative control (scrambled RNA), or no injection, then fed HFHSD or ND. (A and B) Body weight and fasting blood glucose in ND-fed mice. (C and D) Body weight and fasting blood glucose in HFHSD-fed mice. n=3 per group. *p<0.05 and **p<0.01 (miR-10a-5p mimic vs. no injection); #p<0.05 and ##p<0.01 (miR-10b-5p mimic vs. no injection). [Figure 24]Figures 24A-D are a series of graphs showing that both miR-10a-5p and miR-10b-5p inhibitors trigger and exacerbate the obese and diabetic phenotype in ND-fed and HFHSD-fed mice. Male C57 mice were injected twice, once at 4 weeks and then once at 9 weeks (first and second injections), with either miR-10a-5p inhibitor, miR-10b-5p inhibitor, both miR-10a-5p and miR-10b-5p inhibitors (miR-10a / b-5p inhibitor), negative control (scrambled RNA), or no injection, then fed HFHSD or ND. (Figures 24A and 24B) Body weight and fasting blood glucose in ND-fed mice. (FIG. 24C and FIG. 24D) Body weight and fasting blood glucose in mice fed HFHSD. n=3 per group. *p<0.05 and **p<0.01 (miR-10a-5p inhibitor vs. no injection), #p<0.05 and ##p<0.01 (miR-10b-5p inhibitor vs. no injection), ^p<0.05 and ^^p<0.01 (miR-10a / b-5p inhibitor vs. no injection). [Diagram 25] Figures 25A-B illustrate that miR-10a-5p inhibitors increase fasting insulin levels, while miR-10b-5p inhibitors decrease fasting insulin levels at 2 weeks PI after the second injection at 9 weeks in ND-fed male healthy mice and HFHSD-fed male diabetic mice. **p<0.01. [Figure 26] Illustrates gastric emptying profiles in ND-fed mice after the second injection of either miR-10a-5p inhibitor, miR-10b-5p inhibitor or negative control (scrambled RNA) at 4 weeks PI or without injection at 10 weeks. GS stands for GastroSense 750. [Figure 27]A series of western blots demonstrating that miR-10a-5 and miR-10b-5p differentially target key proteins (LGR5, REG4, PDX1, NEROG3 and PDGFRA) in regulating stem or progenitor cells in colonic mucosa, pancreas and white adipocytes of ND-fed mice 4 weeks PI after the first injection. GAPDH was used as an endogenous control in this western blot. [Figure 28] Figure 28A-C illustrates that miR-10a-5p and miR-10b-5p target the same obesity- and diabetes-related proteins in different ways. Comparison of the targeting effect of miR-10a-5p mimic, miR-10b-5p mimic, miR-10a-5p inhibitor and miR-10b-5p inhibitor on obesity- and diabetes-related proteins. Mouse pancreatic β cells (NIT-1 cells) or mouse adipocytes (LM cells) were transfected with miR-10a-5p mimic, miR-10b-5p mimic, miR-10a-5p inhibitor, miR-10b-5p inhibitor or negative control (scrambled RNA), or were not transfected. (FIG. 28A) Western blot of KLF11, KIT, LEP, GLU4, ADIPOQ, IRS-1, IRS-2 and GAPDH control in NIT-1 cells. Protein markers (M) are shown with the corresponding molecular weight (kDa). (FIG. 28B) Quantification of proteins in A, normalized to GAPDH. (C) Western blot of LEP, LEPR and GAPDH control in LM cells. n=3 for each group. *p<0.05 and **p<0.01. [Figure 29]A series of images and micrographs illustrating that miR-10a-5p mimic rescues fatty, inflammatory and fibrotic liver phenotypes in diabetic mice fed a HFHSD. Male diabetic C57 mice fed a HFHSD for 18 weeks (4-22 weeks) or male healthy mice fed a ND diet were injected once at week 22 with miR-10a-5p mimic or miR-10a-5p inhibitor or continued on the same diet without injection. Macroscopic and stained images of the livers of those mice are shown. Liver tissues were dissected from HFHSD and / or ND mice at 2 weeks PI and stained with H&E, Oil Red O (lipids), Picrosirius Red (fibrosis) and CD64 (macrophages). [Diagram 30] Figure 30A-B illustrates that miR-10a-5p mimic rescues liver injury in HFHSD-fed diabetic mice. (Figure 30A and Figure 30B) Blood tests for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in HFHSD-fed male diabetic mice or ND-fed male healthy mice for 18 weeks, injected with miR-10a-5p mimic once at 22 weeks or not, and continued to feed the same diet. AST and ALT were measured in HFHSD and / or ND mice at 2 weeks PI. n=3 for each group. *p<0.05 and **p<0.01. [Diagram 31] Figures 31A-B illustrate that miR-10a-5p mimic rescues high cholesterol in HFHSD-fed diabetic mice. (Figures 31A and 31B) Blood tests for high-density lipoprotein (HDL) and low-density lipoprotein (LDL) / very low-density lipoprotein (VLDL) cholesterol in HFHSD-fed male diabetic mice or ND-fed male healthy mice for 18 weeks, injected with miR-10a-5p mimic once at 22 weeks or not, and continued to feed the same diet. HDL and LDL / VLDL were measured in HFHSD and / or ND mice at 2 weeks PI. n=6 per group. **p<0.01. [Diagram 32] Figures 32A-B illustrate that miR-10a-5p mimics reduce inflammation in HFHSD-fed diabetic mice. (Figures 32A and 32B) Blood tests for the pro-inflammatory cytokine IL-6 and the anti-inflammatory cytokine IL-10 in HFHSD-fed male diabetic mice or ND-fed male healthy mice for 18 weeks, injected with miR-10a-5p mimics once at 22 weeks or not, and continued to feed the same diet. IL-6 and IL-10 were measured in HFHSD and / or ND mice at 2 weeks PI. n=6-8 per group. **p<0.01. [Diagram 33] FIG. 33A-D are a series of graphs illustrating that miR-10b-5p mimics improve cardiac function in HFHSD-induced diabetic mice. Cardiac function in HFHSD-fed male diabetic mice and ND-fed male healthy mice was assessed using echocardiography with or without injection of miR-10b-5p mimics or miR-10b-5p inhibitors. (FIG. 33A) Ejection fraction (EF). (FIG. 33B) Fractional shortening (FS). (FIG. 33C) Stroke volume (SV). (FIG. 33D) Heart rate (HR). HFHSD-fed diabetic mice (no injection) showed reduced EF, FS and SV compared to ND-fed mice (no injection). Injection of miR-10b-5p mimics in ND-fed mice did not change EF and FS but improved SV. Injection of miR-10b-5p inhibitor in ND-fed mice reduced EF, FS, and SV. Injection of miR-10b-5p mimic or miR-10b-5p inhibitor in HFHSD-fed mice did not significantly change EF, FS, and SV. Heart rates of 400-500 beats / min were maintained in all mice during echocardiography. n=3 per group. *p<0.05 and **p<0.01. [Diagram 34]Figure 34A-B illustrates the comparison of drug effects of miR-10a-5p, miR-10b-5p, miR-103 / 107 inhibitor (RG-125) on body weight and blood glucose. miR-10a-5p mimic rescues obesity and diabetes phenotypes in HFHSD-fed mice. (Figure 34A and Figure 34B) Comparison of body weight and fasting blood glucose. Male C57 mice were fed HFHSD or ND throughout the experiment and were injected with either miR-10a-5p mimic, miR-10b-5p mimic, or miR-103 / 107 inhibitor or no injection at 22 weeks and then monitored for 9 weeks PI. AstraZeneca, in collaboration with Regulus Therapeutics, has completed phase 1 and 2 clinical trials with a miR-103 / 107 inhibitor (RG-125) in patients with type 2 diabetes and nonalcoholic fatty liver disease. miR-10a-5p and miR-10b-5p have significantly improved and durable effects on lowering blood glucose and body weight compared to miR-103 / 107 inhibitors. n=3-6 per group. *p<0.05 and **p<0.01 (miR-10a-5p mimic vs. miR-103 / 7 inhibitor), #p<0.05 and ##p<0.01 (miR-10b-5p mimic vs. miR-103 / 7 inhibitor). [Diagram 35]Figure 1 illustrates the study design of drug effects of miR-10a-5p mimic, miR-10b-5p mimic, popular antidiabetic drug treatments and FDA-approved GI motility enhancers on blood glucose, body weight and GI function in diabetic mice fed HFHSD for 8 weeks after treatment or, as a control, healthy mice fed ND without injection. miR-10a-5p (500ng / g body weight), miR-10b-5p (500ng / g body weight) or scrambled RNA (500ng / g body weight) were injected twice, once at week 1 and once at week 2, by IP injection. Metformin (250mg / kg body weight) or sitagliptin (DPP4 inhibitor) (10mg / kg body weight) were administered orally (PO) daily for 4 weeks. Liraglutide (GLP-1 receptor agonist) (0.2 mg / kg body weight) was injected twice daily by subcutaneous (SC) injection for 2 weeks. Insulin (0.75 U / kg body weight) was injected once daily by IP injection for 4 weeks. Prucalopride (5-HT4 receptor agonist) (2 mg / kg body weight) was administered PO daily for 4 weeks. Drug effects are shown in Figures 36-38. [Diagram 36] Figures 36A-B illustrate that miR-10a-5p mimics and miR-10b-5p mimics have better and longer-lasting effects on lowering (Figure 36A) blood glucose and (Figure 36B) body weight than the antidiabetic drug treatment and gastrointestinal motility enhancer described in Figure 35. n=5 for each group. *p<0.05 and **p<0.01 (miR-10a-5p mimic vs. liraglutide), #p<0.05 and ##p<0.01 (scrambled vs. liraglutide), ^p<0.05 and ^^p<0.01 (miR-10a-5p mimic vs. liraglutide). [Figure 37] FIG. 35 illustrates that miR-10a-5p mimics and miR-10b-5p mimics have better and longer-lasting effects on improving GI motility than antidiabetic medications used to treat chronic constipation. Total GI transit time was measured in each group at 2-8 weeks after treatment. n=5 per group. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. The frequency of drug treatment is shown in FIG. 35. [Figure 38] Figure 38A-B illustrates that miR-10a-5p mimics and miR-10b-5p mimics have better and longer-lasting effects on improving gastric emptying than anti-diabetic medications used to treat chronic constipation. Figure 38A is a series of images demonstrating that delayed gastric emptying in control scrambled RNA HFHSD-fed diabetic mice is improved by miR-10a-5p mimics, miR-10b-5p mimics and prucalopride at 4 weeks, but liraglutide does not improve delayed gastric emptying. Figure 38B shows that gastric emptying is still normal at 8 weeks in mice injected with miR-10a-5p mimics or miR-10b-5p mimics, but is delayed again in prucalopride-treated mice. The frequency of drug treatments is shown in Figure 35. [Figure 39] Figures 39A-H illustrate that miR-10b-5p mimics prevent diabetes and slow GI transit in HFHSD-fed mice. Figure 39A shows macroscopic images of mice injected with 10b mimics or 10b inhibitors or without injections, fed HFHSD or ND. Figures 39B and 39C show changes in body weight and fasting blood glucose in mice 4 weeks PI. Figures 39D and 39E show GTT and ITT. Figures 39F-H show A1C (Figure 9F), miR-10b-5p (Figure 39G) and insulin (Figure 39H) levels in mice. Figure 39I shows total GI transit time and Figure 39J shows total fecal pellet output. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] Detailed Description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. In carrying out the testing of the present invention, any methods and materials similar or equivalent to those described herein can be used, but the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology is used:

[0073] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0074] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0075] As used herein, "about" when referring to a measurable value, such as an amount, duration, etc., is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the stated value, as such variations are appropriate for carrying out the disclosed methods.

[0076] As used herein, "activation" refers to the state of T cells that have been stimulated sufficiently to induce detectable cell proliferation. Activation may also be accompanied by induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells that are undergoing cell division.

[0077] The term "autoimmune disease" as used herein is defined as a disorder caused by autoimmune response. Autoimmune disease is the result of inappropriate and excessive response to self-antigens. Examples of autoimmune disease include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, etc.

[0078] As used herein, the term "autologous" is intended to refer to any material derived from the same individual into whom the material is subsequently reintroduced.

[0079] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0080] "Xenogeneic" refers to a graft derived from an animal of a different species.

[0081] The term "cancer" as used herein is defined as a disease characterized by rapid and uncontrolled growth of abnormal cells.Cancer cells can spread locally or to other parts of the body via bloodstream and lymphatic system.Various examples of cancer include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.In certain embodiments, the cancer is medullary thyroid cancer.

[0082] The term "cleavage" refers to the breaking of a covalent bond, for example, the breaking of a covalent bond in the backbone of a nucleic acid molecule. Cleavage can be initiated in a variety of ways, including but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand breaks are possible. Double-strand breaks can occur as a result of two distinct single-strand break events. DNA cleavage can result in the generation of either blunt ends or overhanging ends. In certain embodiments, fusion polypeptides can be used to target double-stranded DNA to be cleaved.

[0083] The term "conservative sequence modification" as used herein refers to an amino acid modification that does not significantly affect or significantly change the binding characteristics of the antibody containing that amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of an antibody can be replaced with another amino acid residue from the same side chain family, and the altered antibody can be tested for antigen binding ability using the functional assays described herein.

[0084] A "disease" is a condition in an animal's health in which the animal is unable to maintain homeostasis and, unless the condition is ameliorated, the animal's health will continue to deteriorate. In contrast, a "disorder" in an animal is a condition in which the animal is able to maintain homeostasis, but the animal's health is less favorable than it would be in the absence of the disorder. If left untreated, a disorder does not necessarily cause a further deterioration in the animal's health.

[0085] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result or to provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.

[0086] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template in a biological process for the synthesis of other polymers and macromolecules having a given nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or having a given amino acid sequence. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, i.e., the nucleotide sequence which is identical to the mRNA sequence and which is usually listed in the sequence listing, and the non-coding strand which is used as a template for transcription of the gene or cDNA, can be said to code for the protein or other product of the gene or cDNA.

[0087] As used herein, the terms "engineered," "genetically engineered," "recombinant," "non-naturally occurring," and "non-natural" are used interchangeably to refer to synthetic polynucleotides and polypeptides that have been intentionally manipulated by man.

[0088] As used herein, "endogenous" refers to any substance that originates or is produced within an organism, cell, tissue or system.

[0089] As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside an organism, cell, tissue or system.

[0090] As used herein, the term "RNA interference" or RNAi refers to the process by which synthetic small interfering RNA (siRNA) or the expression of RNA molecules such as microRNA (miRNA), short interfering RNA (siRNA) or short hairpin RNA (shRNA) causes sequence-specific degradation or translational suppression of complementary mRNA molecules.Thus, RNAi is a form of post-transcriptional gene silencing.

[0091] The term "miRNA mimic" as used herein refers to a double-stranded synthetic version of endogenous miRNA that can be similar to endogenous miRNA or mimic its function.Synthetic miRNA mimics can be modified (e.g., chemically) to have greater or less activity than endogenous counterparts (e.g., by increasing resistance to degradation).In contrast, "miRNA inhibitor" or "anti-miR" refers to a synthetic single-stranded RNA molecule that binds to endogenous target miRNAs and prevents them from regulating their respective mRNA targets.

[0092] The term "expand" as used herein refers to an increase in number, such as when the number of T cells is increased. In one embodiment, the T cells expanded ex vivo are increased in number compared to the number originally present in the culture. In another embodiment, the T cells expanded ex vivo are increased in number compared to other cell types in the culture. As used herein, the term "ex vivo" refers to cells that are removed from a living organism (e.g., a human) and expanded outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0093] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0094] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids incorporating a recombinant polynucleotide, plasmids (e.g., naked plasmids or plasmids contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0095] As used herein, "homologous" refers to the subunit sequence identity between two polymer molecules, e.g., two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or two polypeptide molecules. If a subunit position is occupied by the same monomer subunit in both molecules, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions. For example, if half of the positions in two sequences (e.g., 5 positions in a polymer of length 10 subunits) are homologous, then the two sequences are 50% homologous, and if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

[0096] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, chimeric immunoglobulin chains or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, "humanized antibodies" can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, with all or substantially all of the CDR regions corresponding to a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin sequence. A humanized antibody will optimally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

[0097] "Fully human" refers to an immunoglobulin, such as an antibody, whose entire molecule is of human origin or consists of an amino acid sequence identical to that of a human antibody.

[0098] "Identity" as used herein refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, e.g., between two polypeptide molecules. If two amino acid sequences have the same residue at the same position, e.g., if a position in each of the two polypeptide molecules is occupied by arginine, then they are identical at that position. Identity, or the degree to which two amino acid sequences have the same residue at the same position in an alignment, is often expressed as a percentage. Identity between two amino acid sequences is a direct function of the number of matching or identical positions. For example, if half of the positions in two sequences (e.g., 5 positions in a polymer of 10 amino acids in length) are identical, then the two sequences are 50% identical, and if 90% of the positions (e.g., 9 out of 10) are matched or identical, then the two amino acid sequences are 90% identical.

[0099] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can be present in a substantially purified form or can exist in a non-native environment, such as, for example, a host cell.

[0100] The term "modified" as used herein refers to an altered state or structure of a molecule or cell of the invention. Molecules can be modified in a variety of ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of a nucleic acid.

[0101] The term "modulate" as used herein means to mediate a detectable increase or decrease in the level of response in a control compared to the level of response in a control in the absence of a treatment or compound and / or compared to the level of response in an otherwise identical subject without treatment. This term encompasses perturbing and / or affecting a native signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0102] In the present invention, the following abbreviations are used for the commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0103] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerates of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that a nucleotide sequence encoding a protein may contain introns in some versions.

[0104] The term "operably linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence if the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0105] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection, or infusion techniques.

[0106] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide and can be hydrolyzed into monomeric "nucleotides". The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR™, and synthetic means.

[0107] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up a protein's or peptide's sequence. A polypeptide encompasses any peptide or protein that contains two or more amino acids joined together by peptide bonds. As used herein, the term encompasses both short chains, often referred to in the art as peptides, oligopeptides and oligomers, and the like, and longer chains, commonly referred to in the art as proteins, of which there are many types. A "polypeptide" encompasses, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide encompasses natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0108] The term "promoter" as used herein is defined as a DNA sequence that is recognized by the synthetic machinery of a cell or introduced synthetic machinery and is necessary to initiate the specific transcription of a polynucleotide sequence.

[0109] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that allows tissue-specific expression of the gene product.

[0110] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes production of that gene product in a cell under most or all physiological conditions of the cell.

[0111] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes production of that gene product in a cell substantially only if an inducer corresponding to that promoter is present in that cell.

[0112] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes production of that gene product in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0113] "Signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and complexes of molecules that have the ability to receive and transmit a signal across the plasma membrane of a cell.

[0114] The term "specifically binds" as used herein with respect to an antibody means an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind that antigen from one or more species. However, such cross-species reactivity, in and of itself, does not change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity, in and of itself, does not change the classification of the antibody as specific. In some cases, the terms "specifically bind" or "specifically bind" can be used with respect to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species. For example, an antibody recognizes and binds to a specific protein structure, rather than proteins in general. If an antibody is specific for epitope "A", then in a reaction involving labeled "A", the presence of a molecule containing epitope A (or free unlabeled A) would reduce the amount of labeled A bound to the antibody.

[0115] The term "subject" is intended to include any living organism in which an immune response can be elicited (e.g., a mammal). As used herein, a "subject" or "patient" can be a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is a human.

[0116] "Target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0117] As used herein, the term "therapeutic" refers to treatment and / or prophylaxis. The therapeutic effect is achieved by suppression, amelioration, or eradication of the disease state.

[0118] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell refers to a cell that has been transfected or transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0119] The term "transgene" refers to genetic material that has been or is being artificially introduced into the genome of an animal, particularly a mammal, and more particularly a mammalian cell of a living animal.

[0120] The term "transgenic animal" refers to a non-human animal, usually a mammal, in which a non-endogenous (i.e., heterologous) nucleic acid sequence is present in a portion of its cells as an extrachromosomal element or stably integrated into its germline DNA (i.e., into most or all of the genomic sequences of the animal's cells), e.g., a transgenic mouse. The heterologous nucleic acid is introduced into the germline of such a transgenic animal, e.g., by gene manipulation of the host animal's embryo or embryonic stem cells.

[0121] The term "knockout mouse" refers to a mouse in which an existing gene has been inactivated (i.e., "knocked out"). In some embodiments, the gene is inactivated by homologous recombination. In some embodiments, the gene is inactivated by replacement or disruption with an artificial nucleic acid sequence.

[0122] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0123] As used herein, the phrases "under transcriptional control" or "operably linked" mean that the promoter is in the correct location and orientation relative to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0124] A "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0125] Ranges: Throughout this disclosure, various aspects of the invention may be expressed in a range format. It should be understood that the description in range format is merely for convenience and brevity of expression and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed the possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed the subranges 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as the individual numerical values ​​within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0126] explanation To investigate the effect of mir-10b deficiency in ICCs, we generated an ICC-specific miR-10b knockout (KO) mouse line. Surprisingly, conditional deletion of the miRNA gene in ICCs of healthy adult mice caused the mice to develop gastroparesis and become progressively overweight and hyperglycemic, both hallmarks of type 2 diabetes (T2D). Here, we discovered that impaired peristaltic activity in the GI tract leads to both obesity and T2D in a novel mouse model. The most common pathological GI abnormality in diabetic patients is depletion of ICCs, the GI pacemaker cells that regulate peristaltic activity.

[0127] This novel finding of gastroparesis-induced diabetes is unexpected and contradicts the currently widespread view that the diabetic state causes ICC degeneration and gastroparesis. Without wishing to be bound by theory, we suggest that miR-10b deficiency-mediated ICC degeneration is not merely a symptom, but may actually contribute to obesity and T2D-like symptoms.

[0128] The present invention is further based on the unexpected discovery that miR-10a-5p mimics and the genes they regulate, such as Krüppel-like factor 11 (KLF11), leptin (LEP), adiponectin (ADIPOQ), insulin-dependent glucose transporter type 4 (GLU4 / GLUT4) and tyrosine-protein kinase (KIT), can result in reduction of body fat and body weight, restoration of glucose homeostasis and insulin sensitivity, and improved gastrointestinal (GI) function.

[0129] Recent studies have confirmed that miR-10b-5p mimics and their targets can be used in the treatment of diabetes and gastrointestinal motility disorders. Subsequent studies have identified another miRNA, miR-10a-5p mimic, that has similar effects on restoring glucose homeostasis and insulin sensitivity and improving GI function in diabetic mice. The only difference between miR-10a-5p and miR-10b-5p is a single nucleotide base in the middle of the sequence. However, injection of miR-10a-5p mimics into obese diabetic mice dramatically reduces body fat and weight in addition to lowering blood glucose and improving GI function. Furthermore, although both miRNAs lower blood glucose, they contrast in how they regulate insulin levels. While miR-10a-5p mimics reduce insulin levels, miR-10b-5p mimics increase insulin production. Consistent with the miRNA mimic data, miR-10a-5p inhibitors and miR-10b-5p inhibitors have opposing and opposite effects on insulin levels. These observations suggest that unlike miR-10b-5p mimics, which lower blood glucose by increasing insulin production, miR-10a-5p mimics have the ability to lower blood glucose by improving insulin sensitivity without increasing insulin. The improvement of insulin sensitivity by miR-10a-5p mimics can be achieved by upregulating the insulin-dependent glucose transporter GLUT4. Treatment with miR-10a-5p also increases the expression of leptin, a hormone produced primarily by adipocytes, which regulates energy balance by inhibiting hunger and reduces fat storage in adipocytes. Without wishing to be bound by theory, miR-10a-5p mimics are ideal for treating patients with obese type 2 diabetes and GI motility disorders, and miR-10b-5p mimics are ideal for treating patients with type 1 diabetes, non-obese type 2 diabetes and GI motility disorders.

[0130] Diabetes and Related Conditions In one aspect, the disclosed invention provides a method of treating diabetes in a subject in need thereof, comprising administering to the subject an effective amount of a miR-10a-5p mimic or miR-10b-5p mimic. Diabetes or diabetes mellitus refers to a group of diseases broadly associated with failure to properly regulate glucose or sugar use as a result of defects in the production, secretion, or function of the hormone insulin. Abnormal insulin function leads to abnormal carbohydrate, lipid, and protein metabolism. Diabetes is broadly divided into two types. Type 1, or insulin-dependent diabetes mellitus, is due to an autoimmune reaction that leads to the destruction of insulin-producing beta cells in the pancreas, which results in a systemic deficiency of insulin. Treatment of the disease mainly consists of regular monitoring of blood glucose levels and injections of insulin several times a day. Failure to manage insulin dosage can result in severe hypoglycemia and life-threatening damage to the brain and other functions.

[0131] Type 2, or non-insulin-dependent diabetes mellitus (T2DM), typically occurs in adults and is a more complex disease involving glucose-responsive tissues such as adipose fat tissue, muscle, and liver that develop resistance to the action of insulin. In the early stages of T2DM, pancreatic islet cells compensate by secreting excessive amounts of insulin. Without intervention, beta islet cell dysfunction occurs, which leads to decompensation and chronic hyperglycemia. In addition, T2DM may be accompanied by peripheral insulin resistance, in which cells that are normally insulin-sensitive fail to respond normally. Whereas type 1 diabetes is often an acute disease that appears in early life, type 2 diabetes can develop gradually from middle age onwards as a result of a number of factors, including genetics and lifestyle. There are several types of drug treatments that are commonly used to treat T2DM: 1) insulin-releasing agents, which directly stimulate insulin secretion but carry the risk of causing hypoglycemia; 2) diet insulin releasers, which enhance glucose-induced insulin secretion but must be taken before each meal; 3) biguanides, including metformin, which reduce the production of glucose from digestion; 4) insulin sensitizers, such as the thiazolidinedione derivatives rosiglitazone and pioglitazone, which improve the peripheral response to insulin by modulating the expression of glucose metabolism genes but have side effects such as weight gain, edema and liver toxicity; and 5) insulin injections, which are often required in end-stage T2DM.

[0132] The metabolic nature of type 2 diabetes and the abnormally high blood glucose that results from this condition often leads to the development of symptoms and disorders that affect a wide range of body systems. Diabetes is associated with an increased incidence of obesity, fatty liver disease, hyperlipidemia, fatty liver disease, and GI motility disorders, including gastroparesis and constipation.

[0133] T2DM-associated insulin resistance is commonly associated with atherosclerosis, obesity, hyperlipidemia, and essential hypertension. This group of abnormal conditions constitutes "metabolic" or insulin resistance. In addition, insulin resistance is associated with fatty liver disease, which can lead to chronic inflammation or nonalcoholic hepatitis, fibrosis, and cirrhosis. Nonalcoholic fatty liver disease begins with the accumulation of triacylglycerol in the liver and is defined as the presence of cytoplasmic lipid droplets in more than 5% of hepatocytes or TAG levels above the 95th percentile of healthy individuals. Both T2DM and fatty liver disease are associated with each other's deleterious outcomes. Type 2 diabetes is a risk factor for progressive liver disease and liver-related death in patients with fatty liver disease, whereas fatty liver disease can be a marker of cardiovascular risk and mortality in individuals with type 2 diabetes. Nonalcoholic steatohepatitis, a histologic subtype of NAFLD characterized by hepatocellular injury and inflammation, is present in approximately 10% of patients with T2DM and is associated with an increased risk of developing cirrhosis and liver-related death.

[0134] Diabetic gastroparesis, a common but serious chronic disorder of the upper gastrointestinal tract, is defined by the presence of delayed gastric emptying in the absence of physical obstruction and is associated with symptoms such as nausea, vomiting, early satiety, abdominal distension and abdominal pain. Currently, the only FDA-approved drug for diabetic gastroparesis is metoclopramide, a dopamine D2 receptor antagonist and 5-HT3 receptor antagonist with weak 5-HT4 agonist activity, indicated for relief of symptoms associated with acute and recurrent diabetic gastric stasis for treatment periods of 12 weeks or less. However, metoclopramide treatment is associated with significant side effects such as sudden muscle spasms and depression / mood swings.

[0135] Regulation of gene expression by miRNAs In one aspect, the invention provides methods of using miRNA mimics of miR-10a-5p and miR-10b-5p to treat diabetes and diabetes-related conditions.

[0136] miRNAs are small non-coding RNA molecules, typically 20-22 nucleotides in length. miRNAs act as key regulators of gene expression and function, which act to modify gene expression by interacting with post-transcriptional RNAs to regulate their stability and subsequent translation. Our understanding of the biological roles of ncRNAs, including miRNAs, is rapidly evolving. Numerous evolutionary studies have revealed that non-coding RNAs can be expressed nearly four times more than protein-coding RNAs.

[0137] Endogenous miRNAs are transcribed by RNA polymerase II as 100-1000 nucleotides (nt) primary miRNAs (pri-RNAs). miRNAs can be modified by 5' capping and 3' poly(A) tailing. The miRNA-coding portion of the pri-miRNA forms a hairpin that is cleaved by the dsRNA-specific ribonuclease Drosha and its cofactor DiGeorge syndrome critical region 8 (DGCR8) to form the approximately 60-70 nt long pre-miRNA. The pre-miRNA is further processed by Dicer and the transactivator RNA-binding protein TRBP to give a miRNA duplex containing two mature miRNAs (5' and 3' strand miRNAs). Each mature miRNA is approximately 22-23 nt long.

[0138] Depending on the degree of complementarity between the mature miRNA and its target, several mechanisms of mRNA silencing can occur. The first bases of the mature miRNA, from position 2 to position 7, are called the "seed" sequence and provide most of the pairing specificity with the target mRNA. In some cases, perfect pairing between the seed sequence and its cognate target is sufficient to mediate cleavage and degradation of the cognate mRNA. However, for mammalian and viral mRNA targets, cleavage is more typically impaired by pairing containing mismatches in the seed and other regions, and translation inhibition occurs due to physical interference with the binding of the translation machinery. Because the complementary length of the seed sequence required for miRNAs to target their cognate mRNAs is short, each miRNA has the potential to target and regulate hundreds of transcripts. Furthermore, conversely, an mRNA molecule can be acted upon by many different miRNAs. Most miRNAs only reduce target protein levels by a factor of two at most, but this is often sufficient to exert a significant physiological effect. Thus, the endogenous miRNA pathway is a highly efficient system for regulating specific functional pathways and fine-tuning the expression of numerous genes simultaneously. miRNAs are predicted to control the activity of approximately 30% of all protein-coding genes in mammals and play important roles in normal physiological processes ranging from embryonic development to hematopoietic cell development, as well as in diseases such as cardiovascular disease, cancer, and immune disorders. Recent studies have demonstrated that miR-10a-5p mimics and miR-10b-5p mimics can successfully treat diabetes and other conditions such as gastroparesis, fatty liver disease, and obesity.

[0139] miRNA mimics In certain embodiments of the present disclosure, the present invention provides some miRNA mimics that target human genes regulated by endogenous miR-10b-5p miRNA and miR-10a-5p miRNA.miRNA mimics are a strategy of gene silencing that utilizes non-natural double-stranded miRNA-like RNA fragments. The 5' end of these fragments has a sequence that is partially complementary to the cognate sequence in the 3'UTR that is unique to the target gene. When this RNA fragment is expressed or introduced into cells, it specifically binds to the target gene, mimicking the function of endogenous miRNA, which leads to post-transcriptional suppression of the gene, typically by inhibiting transcription.Therefore, miRNA mimics can be precisely targeted to affect specific genes. In various embodiments of the invention, miR-10a-5p mimics and miR-10b-5p mimics can affect the expression of several target genes, including, but not limited to, KLF11, KIT, leupeptin, GLU / GLUT4, adiponectin, LGR5, REG4, PDX1, NEUROG3, PDGFA, LEP, LEPR, and IRS21 (miR-10a-5p), as well as KLF7, KLF11, KIT, INSR, IRS2, and IRS1 (miR-10b-5p).

[0140] miR-10a-5p mimics In some embodiments of the invention, the miR-10a-5p mimic is a miR-10a-5p duplex, a chemically modified double-stranded miR-10a-5p, an unmodified double-stranded miR-10a-5p, a single-stranded chemically modified miR-10a-5p, or a single-stranded unmodified miR-10a-5p.

[0141] The sequences and / or sources of several miR-10a-5p mimics and miR-10a-5p mimic inhibitors are listed below. 10a-5p mimic 1: 10a double-stranded RNA Sense: TIFF0007660521000001.tif4128 Antisense: TIFF0007660521000002.tif4128miR-10b-5p:10a-5p single-stranded RNA (miR-10a-5p) TIFF0007660521000003.tif412810a inhibitor: 10a-3p single-stranded RNA (miR-10a-5p inhibitor) TIFF0007660521000004.tif4128 Human mir-10a gene with mature sequence underlined (stem-loop) Accession: MI0000266 ID:hsa-mir-10a TIFF0007660521000005.tif9162 Mature sequence mmu-miR-10a-5p (miR-10a mimic) Accession:MIMAT0000253 ID:hsa-miR-10a TIFF0007660521000006.tif5128 Mouse mir-10a gene with mature sequence underlined (stem-loop) Accession: MI0000685 ID:mmu-mir-10a TIFF0007660521000007.tif12162 Mature sequence mmu-miR-10a-5p (miR-10a mimic) Accession:MIMAT0000648 ID:mmu-miR-10a TIFF0007660521000008.tif4128

[0142] miR-10b-5p mimics In some embodiments, the miR-10b-5p mimic is a miR-10b-5p duplex, a chemically modified double-stranded miR-10b-5p, an unmodified double-stranded miR-10b-5p, a single-stranded chemically modified miR-10b-5p, or a single-stranded unmodified miR-10b-5p.

[0143] The sequences and / or sources of several miR-10b-5p mimics and miR-10b-5p mimic inhibitors are listed below. 10b mimic 1 [Figure 5B-Figure 5C] (MC11108, Thermo Fisher Scientific): 10b double-stranded RNA Sense: TIFF0007660521000009.tif4128 Antisense: TIFF0007660521000010.tif412810b Mimic 2 [Figure 5C] (C-310431-07-0005, Dharmacon): 10b double-stranded RNA 10b mimic 3 [Figure 5C] (219600, QIAGEN): 10b RNA duplex miR-10b-5p(IDT):10b-5p single-stranded RNA(miR-10b-5p) TIFF0007660521000011.tif4128 Annealed miR-10b-3p and miR-10b-5p (IDT): 10b double-stranded RNA by miR-10b-5p / 3p Sense: TIFF0007660521000012.tif4128 Antisense: TIFF0007660521000013.tif412810b mimic 7 (IDT):10b precursor RNA TIFF0007660521000014.tif11168

[0144] miRNA mimics are commercially available. Human miR-10b-5p mimics (has-miR-10b-5p) are commercially available from Thermo Fisher Scientific (product name mirVana® miRNA mimics; product ID MC11108).

[0145] The mature miR-10b sequence is The file is TIFF0007660521000015.tif4128. Human mir-10b gene with mature sequence underlined (stem-loop) Accession number: MI0000267 ID:hsa-mir-10b >hsa-mir-10b MI0000267 TIFF0007660521000016.tif14164 Mature sequence hsa-miR-10b-5p (miR-10b mimic) Accession:MIMAT0000254 ID:hsa-miR-10b >hsa-miR-10b-5p MIMAT0000254 TIFF0007660521000017.tif4128 Mouse mir-10b gene with mature sequence underlined (stem-loop) Accession: MI0000221 ID:mmu-mir-10b >mmu-mir-10b MI0000221 TIFF0007660521000018.tif9163 Mature sequence mmu-miR-10b-5p (miR-10b mimic) Accession:MIMAT0000208 ID:mmu-miR-10b >mmu-miR-10b-5p MIMAT0000208 TIFF0007660521000019.tif4128Negative control or inhibitor: Negative control #1 (4464058, Thermo Fisher Scientific): non-targeting double-stranded RNA 10b inhibitor (Thermo Fisher Scientific): 10b-5p inhibitor RNA (antisense to miR-10b-5p) TIFF0007660521000020.tif4128miR-10b-3p(IDT):10b-3p single-stranded RNA (miR-10b-5p inhibitor) TIFF0007660521000021.tif4128

[0146] Treatment Method Provided is a method for treating diabetes in a subject in need thereof, comprising administering an effective amount of a miR-10a-5p mimic to the subject, thereby treating the diabetic condition.In some embodiments, the miR-10a-5p mimic is a miR-10a-5p double-stranded, a chemically modified double-stranded miR-10a-5p, an unmodified double-stranded miR-10a-5p, a single-stranded chemically modified miR-10a-5p, or a single-stranded unmodified miR-10a-5p.

[0147] In some embodiments, the diabetes is type 2 diabetes. In some embodiments, the miR-10a-5p mimic is mammalian. In some embodiments, the miR-10a-5p mimic is human. In some embodiments, the miR-10a-5p mimic is engineered. In some embodiments, the miR-10a-5p mimic further comprises a pharma- ceutically acceptable carrier or adjuvant.

[0148] Also provided is a method for reducing subject's body weight, comprising administering an effective amount of miR-10a-5p mimic, thereby reducing subject's body weight.In some embodiments, miR-10a-5p treatment leads to upregulation of leptin, thereby reducing hunger and reducing fat storage, thereby leading to weight loss.

[0149] Also provided is a method for lowering blood glucose in a subject comprising administering an effective amount of a miR-10a-5p mimic, thereby lowering blood glucose in the subject.

[0150] Also provided is a method for increasing insulin sensitivity comprising administering to a subject in need thereof an effective amount of a miR-10a-5p mimic, thereby increasing insulin sensitivity in the subject.

[0151] In some embodiments of any one of the above methods, the miR-10a-5p mimic is a miR-10a-5p duplex, a chemically modified double-stranded miR-10a-5p, an unmodified double-stranded miR-10a-5p, a single-stranded chemically modified miR-10a-5p, or a single-stranded unmodified miR-10a-5p. In further embodiments, the miR-10a-5p mimic is mammalian. In further embodiments, the miR-10a-5p mimic is human. In further embodiments, the miR-10a-5p mimic is engineered. In further embodiments, the miR-10a-5p mimic further comprises a pharma- ceutically acceptable carrier or adjuvant.

[0152] Also provided is a method for treating gastrointestinal disease, comprising administering an effective amount of miR-10a-5p mimic to a subject in need thereof, thereby treating gastrointestinal disease in the subject.In some embodiments, the miR-10a-5p mimic is miR-10a-5p double-stranded, chemically modified double-stranded miR-10a-5p, unmodified double-stranded miR-10a-5p, single-stranded chemically modified miR-10a-5p, or single-stranded unmodified miR-10a-5p.In some embodiments, the miR-10b-5p mimic is mammalian.In some embodiments, the miR-10b-5p mimic is human.

[0153] In some embodiments, the gastrointestinal disorder is selected from the group consisting of gastroparesis, functional gastrointestinal disorder, functional gastrointestinal motility disorder, and pseudo-obstruction. In further embodiments, the functional gastrointestinal disorder is selected from the group consisting of irritable bowel syndrome, functional constipation, and unspecified functional bowel disorder.

[0154] Also provided is a method for increasing insulin sensitivity, comprising administering an effective amount of miR-10a-5p mimic to a subject in need thereof, thereby increasing insulin sensitivity in the subject.In some embodiments, administration of miR-10a-5p mimic results in increased expression of insulin-dependent glucose transporter 4 (GLU4 / GLUT4).In further embodiments, administration of miR-10a-5p mimic results in decreased blood glucose levels in the subject.

[0155] In some embodiments of any one of the aforementioned methods, the miR-10a-5p mimic targets a gene selected from the group consisting of KLF11, KIT, leupeptin, GLU4 / GLUT4, adiponectin, and IRS2, and combinations thereof.

[0156] Also provided is a composition comprising miR-10a-5p mimic and a pharmaceutically acceptable carrier or adjuvant.In some embodiments, a composition comprising miR-10a-5p mimic and a pharmaceutically acceptable carrier or adjuvant is provided.In some embodiments, the miR-10a-5p mimic is miR-10a-5p double-stranded, chemically modified double-stranded miR-10a-5p, unmodified double-stranded miR-10a-5p, single-stranded chemically modified miR-10a-5p, or single-stranded unmodified miR-10a-5p.In some embodiments, the miR-10a-5p mimic is engineered.

[0157] A method for increasing interstitial cells of Cajal (ICC) proliferation is provided, comprising administering an effective amount of miR-10b-5p mimic to a subject in need thereof, thereby increasing ICC proliferation in the subject. In some embodiments, the miR-10b-5p mimic is a miR-10b-5p double-stranded, a chemically modified double-stranded miR-10b-5p, an unmodified double-stranded miR-10b-5p, a single-stranded chemically modified miR-10b-5p, or a single-stranded unmodified miR-10b-5p. In further embodiments, the miR-10b-5p mimic is mammalian. In further embodiments, the miR-10b-5p mimic is human.

[0158] In some embodiments, increasing ICC proliferation in a subject restores the function of ICC in the subject. In further embodiments, the ICC is located within the smooth muscle of the gastrointestinal tract of the subject. In further embodiments, the smooth muscle is located within the stomach, small intestine, or colon of the subject.

[0159] In some embodiments, the ICC comprises an ICC precursor, an ICC-MY, an ICC-IM, an ICC-DMP, an ICC-SM, or an ICC-SMP.

[0160] In some embodiments, the subject is a human.

[0161] Provided is a method for increasing KIT expression in ICC, comprising administering an effective amount of miR-10b-5p mimic to a subject in need thereof, thereby increasing KIT expression in the subject.In some embodiments, the miR-10b-5p mimic is miR-10b-5p double-stranded, chemically modified double-stranded miR-10b-5p, unmodified double-stranded miR-10b-5p, single-stranded chemically modified miR-10b-5p, or single-stranded unmodified miR-10b-5p.In further embodiments, the miR-10b-5p mimic is mammalian.In further embodiments, the miR-10b-5p mimic is human.

[0162] In some embodiments, increasing KIT expression in a subject restores function of ICC in the subject.

[0163] In some embodiments, administration of a miR-10-b-5p mimic to a subject results in decreased expression of KLF7 and KLF11.

[0164] In some embodiments, the gastrointestinal disease results in phenotypically inactivated and non-functional ICC.

[0165] Also provided is a method for treating gastrointestinal disease, comprising administering an effective amount of miR-10b-5p mimic to a subject in need thereof, thereby treating the gastrointestinal disease in the subject.In some embodiments, the miR-10b-5p mimic is a miR-10b-5p double-stranded, a chemically modified double-stranded miR-10b-5p, an unmodified double-stranded miR-10b-5p, a single-stranded chemically modified miR-10b-5p, or a single-stranded unmodified miR-10b-5p.In further embodiments, the miR-10b-5p mimic is mammalian.In further embodiments, the miR-10b-5p mimic is human.

[0166] In some embodiments, the gastrointestinal disorder is selected from the group consisting of gastroparesis, functional gastrointestinal disorder, functional gastrointestinal motility disorder, and pseudo-obstruction. In further embodiments, the functional gastrointestinal disorder is selected from the group consisting of irritable bowel syndrome, functional constipation, and unspecified functional bowel disorder.

[0167] A method for reducing body weight is provided comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby reducing the body weight of the subject.

[0168] Also provided is a method for lowering blood glucose comprising administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby lowering blood glucose in the subject.

[0169] administering to a subject in need thereof an effective amount of a miR-10b-5p mimic, thereby increasing pancreatic stem cell (PSC) proliferation or pancreatic progenitor cell (PPC) proliferation in the subject. + PSC expansion or KIT + Methods for increasing PPC proliferation are also provided. In some embodiments, PSC proliferation or PPC proliferation is increased in a subject compared to a control.

[0170] Also provided is a method for increasing insulin sensitivity, comprising administering an effective amount of miR-10b-5p mimic to a subject in need thereof, thereby increasing insulin sensitivity in the subject.In some embodiments, administering miR-10b-5p mimic results in increased glucose uptake into skeletal muscle cells (SkMCs) in the subject.In further embodiments, administering miR-10b-5p mimic results in reduced blood glucose levels in the subject.In some diabetic subjects, the SkMCs of the subject may exhibit insulin resistance.

[0171] Also provided is a method for increasing insulin gene expression, comprising administering an effective amount of miR-10b-5p mimic to a subject in need thereof, thereby increasing insulin gene expression in the subject.In some embodiments, administering miR-10b-5p mimic results in restoration of beta cell function, thereby increasing the amount of insulin produced from beta cells compared to control.In some embodiments, administering miR-10-b-5p mimic to a subject results in decreased expression of KLF7 and KLF11.In some embodiments, decreased expression of KLF11 results in increased expression of NEUROG3 and INS in beta cells.

[0172] In some untreated diabetic subjects, insulin is not produced or is produced at low levels in the subject's beta cells. Untreated diabetic subjects have reduced beta cells located in pancreatic islet cells. Pancreatic islet cells are located in the subject's pancreas.

[0173] Also provided is a method for treating diabetes, comprising administering an effective amount of a miR-10b-5p mimic to a subject in need thereof, thereby treating diabetes in the subject. In some embodiments, the diabetes is type 1 diabetes or type 2 diabetes. In some embodiments, administration of a miR-10b-5p mimic results in increased glucose uptake into skeletal muscle cells (SkMCs) in the subject. In further embodiments, administration of a miR-10b-5p mimic results in decreased blood glucose levels in the subject.

[0174] Provided is a method for increasing KIT expression in PSC or PPC, comprising administering an effective amount of miR-10b-5p mimic to a subject in need thereof, thereby increasing KIT expression.In some embodiments, administering miR-10-b-5p mimic to a subject results in the reduction of KLF11 expression.

[0175] Also provided is a method for increasing the expression of INSR, IRS2, and IRS1 in skeletal muscle cells (SkMCs), comprising administering an effective amount of miR-10b-5p mimic to a subject in need thereof, thereby increasing the expression of INSR, IRS2, and IRS1. In some embodiments, administering miR-10-b-5p mimic to a subject results in a decrease in the expression of KLF11. In some embodiments, the decrease in the expression of KLF11 results in an increase in the expression of INSR, IRS2, and IRS1.

[0176] In some embodiments of any one of the above methods, the miR-10b-5p mimic is a miR-10b-5p duplex, a chemically modified double-stranded miR-10b-5p, an unmodified double-stranded miR-10b-5p, a single-stranded chemically modified miR-10b-5p, or a single-stranded unmodified miR-10b-5p. In further embodiments, the miR-10b-5p mimic is mammalian. In further embodiments, the miR-10b-5p mimic is human.

[0177] In some embodiments of any one of the aforementioned methods, the miR-10b-5p mimic is engineered.

[0178] In some embodiments of any one of the above methods, the miR-10b-5p mimic is administered by injection. In some embodiments, the miR-10b-5p mimic can be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by nasal injection, by implantation, by intracavitary or intravesical injection, intraocularly, intraarterially, intralesionally, transdermally, or by application to a mucosa.

[0179] In some embodiments of any one of the aforementioned methods, the miR-10b-5p mimic further comprises a pharma- ceutically acceptable carrier or adjuvant.

[0180] It is envisioned that miR-10b-5p mimics may be administered to a subject in combination with other appropriate treatments for diabetes (type 1 or type 2). In some embodiments, the other treatments are insulin, insulin sensitizers (thiazolidinediones), metformin, glucagon-like peptide-1 (GLP-1) receptor agonists (exenatide, albiglutide, dulaglutide, liraglutide, lixisenatide), dipeptidyl peptidase-4 (DPP4) inhibitors (sitagliptin, vildagliptin, alogliptin, linagliptin), sodium-glucose transporter-2 (SGLT2) inhibitors (dapagliflozin, empagliflozin) and sulfonylureas (glimepiride).

[0181] It is envisaged that miR-10b-5p mimics can be administered to subjects in combination with other suitable treatments for gastrointestinal disorders.In some embodiments, these other treatments are intestinal motility enhancers, 5-HT4 receptor agonists (prcalopride, tegaserod and velsetrag), ghrelin agonists (reramorelin), dopamine receptor antagonists and 5-HT4 agonists (metoclopramide and domperidone), motilin receptor agonists (macrolide antibiotics: erythromycin and azithromycin)], antiemetics (aprepitant, promethazine, prochlorperazine and ondansetron) and drugs acting on secretion (lubiprostone and tenapanol).

[0182] In some embodiments, the miR-10b-5p mimic comprises a nucleic acid sequence comprising SEQ ID NO:6, 7, 8, 9, 10, 12, 13, or a combination thereof.

[0183] In any one of the above aspects or embodiments, the miR-10b-5p mimic may be chemically modified or unmodified.

[0184] Pharmaceutical Compositions Provided is a composition comprising miR-10a-5p mimic or miR-10b-5p mimic and pharmaceutically acceptable carrier or adjuvant.In some embodiments, miR-10b-5p mimic or miR-10a-5p mimic is miR-10a-5p double-strand or miR-10b-5p double-strand, chemically modified double-strand miR-10a-5p or chemically modified double-strand miR-10b-5p, unmodified double-strand miR-10a-5p or unmodified double-strand miR-10b-5p, single-strand chemically modified miR-10a-5p or single-strand chemically modified miR-10b-5p, or single-strand unmodified miR-10a-5p or single-strand unmodified miR-10b-5p.In further embodiments, miR-10a-5p mimic or miR-10b-5p mimic is mammalian. In further embodiments, the miR-10a-5p mimic or the miR-10b-5p mimic is human.

[0185] In some embodiments of any one of the aforementioned compositions, the miR-10a-5p mimic or miR-10b-5p mimic is engineered.

[0186] In some embodiments, the miR-10a-5p mimic comprises a nucleic acid sequence comprising SEQ ID NO:1, 2, 4, 5, or a combination thereof.

[0187] In some embodiments, the miR-10b-5p mimic comprises a nucleic acid sequence comprising SEQ ID NO:6, 7, 8, 9, 10, 12, 13, or a combination thereof.

[0188] In some embodiments, the miR-10a-5p mimic or miR-10b-5p mimic may be chemically modified or unmodified.

[0189] The pharmaceutical composition of the present invention may comprise the miR-10a-5p mimic or miR-10b-5p mimic described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, adjuvants, or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The composition of the present invention is preferably formulated for intravenous administration.

[0190] The pharmaceutical composition of the present invention can be administered in a manner appropriate for the disease to be treated (or prevented). The appropriate dosage can be determined by clinical trials, but the amount and frequency of administration will be determined by factors such as the condition of the patient, the type and severity of the patient's disease, etc.

[0191] The pharmaceutical compositions of the present invention may be administered in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, emulsions, etc. The pharmaceutical compositions of the present invention may be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by nasal drops, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes. In some embodiments, the compositions may be applied to the nose, throat, or bronchi, for example, by inhalation.

[0192] The dosage of the above treatments to be administered to a patient will vary depending on the exact nature of the condition being treated and the recipient of the treatment, and may be determined by physical and physiological factors such as body weight, severity of the condition, previous or current therapeutic interventions, etc., as well as the route of administration. Scaling of dosages for human administration can be performed according to accepted practices in the art. For example, doses of miR mimics will generally range from 1 to about 100 mg for adult patients, and will usually be administered monthly for a period of 1 to 12 months. A preferred monthly dose is 1 to 10 mg per month, although larger doses of more than 10 mg per month may be used in some cases.

[0193] Human dosage can be determined by first extrapolating from the amount of compound used in mice. As those skilled in the art will appreciate, it is a routine procedure in the art to adjust dosage for humans by comparing with animal models. In certain embodiments, it is contemplated that dosage can include effective amounts of about 1 microgram / kg / body weight, 5 microgram / kg / body weight, 10 microgram / kg / body weight, 50 microgram / kg / body weight, 100 microgram / kg / body weight, 200 microgram / kg / body weight, 350 microgram / kg / body weight, 500 microgram / kg / body weight, 1 milligram / kg / body weight, 5 milligram / kg / body weight, 10 milligram / kg / body weight, 50 milligram / kg / body weight, 100 milligram / kg / body weight, 200 milligram / kg / body weight, 350 milligram / kg / body weight, or 500 milligram / kg / body weight to 1000 mg / kg / body weight or more per administration, and any range derivable therein. In another embodiment, the effective amount can be about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100 mg / Kg body weight. In another embodiment, it is contemplated that the effective amount may range from about 1 microgram of compound to about 100 mg of compound. In another embodiment, the effective amount may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg per administration. In another embodiment, the effective amount includes less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 mg per day. In an exemplary embodiment, the effective amount includes less than about 50 mg per day.Of course, the dosage amount or daily dosage amount may be adjusted upwards or downwards, as is routine in such treatment protocols, depending on the results of initial clinical trials and the needs of the individual subject. Conditions and circumstances that warrant changes in dosage will be known to those skilled in the art.

[0194] The exact determination of what will be considered an effective amount will depend on factors individual to each subject, such as the subject's size, age, sex, weight, and condition of the individual subject. Dosage amounts can be readily ascertained by one of ordinary skill in the art from this disclosure and knowledge in the art.

[0195] Optionally, the methods of the invention provide for administration of the compositions of the invention to appropriate animal models to identify the dosage of the composition, the concentration of components therein, and the timing of administration of the composition that induces tissue repair, reduces cell death, or induces other desired biological responses. Such determinations do not require undue experimentation and are routine and can be ascertained without undue experimentation.

[0196] Biologically active agents can be conveniently provided to a subject as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which can be buffered to a selected pH. The cells and agents of the present invention can be provided as a liquid or viscous formulation. For some applications, a liquid formulation is desirable because it is convenient for administration, particularly administration by injection. If prolonged contact with tissue is desired, a viscous composition may be preferred. Such compositions are formulated within an appropriate viscosity range. A liquid or viscous composition can include a carrier, which can be a solvent or dispersion medium, including, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0197] Sterile injectable solutions are prepared by suspending talampanel and / or perampanel in the required amount of a suitable solvent with various amounts of other ingredients as desired. Such compositions may be mixed with a suitable carrier, diluent or excipient, such as sterile water, saline, glucose, dextrose, and the like. The compositions may also be lyophilized. Depending on the route of administration and the desired formulation, the compositions may contain auxiliary substances such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or thickening additives, preservatives, flavoring agents, coloring agents, and the like. Suitable preparations may be prepared without undue experimentation by consulting standard textbooks such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th Edition, 1985, which is incorporated herein by reference.

[0198] Various additives can be added to improve the stability and sterility of the composition, such as antimicrobial preservatives, antioxidants, chelating agents and buffers. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of injectable pharmaceutical forms can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin. However, according to the present invention, it is considered that any vehicle, diluent or additive used must be compatible with the agent present in the cell or its conditioned medium.

[0199] The composition can be isotonic, i.e., the composition can have the same osmotic pressure as blood and tears. The desired isotonicity of the compositions of the present invention can be achieved using sodium chloride or other pharma- ceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride is particularly preferred for buffers containing sodium ions.

[0200] If desired, methylcellulose and other pharma- ceutically acceptable thickening agents can be used to maintain the viscosity of the composition at a selected level.Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like.The selection of suitable carriers and other additives will depend on the exact route of administration and the nature of a particular dosage form, for example, a liquid dosage form (for example, whether the composition is formulated into a solution, suspension, gel, or other liquid form, for example, a sustained release dosage form or a liquid fill dosage form).Those skilled in the art will understand that the components of the composition should be selected to be chemically inert.

[0201] It should be understood that the methods and compositions that may be useful in the present invention are not limited to the specific formulations described in the examples. The following examples are provided to fully disclose and describe to those of skill in the art the methods of making and using the cells, the methods of expansion and culture, and the methods of treatment of the present invention, but are not intended to limit the scope of what the inventors regard as their invention.

[0202] The practice of the present invention uses conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology that are well within the scope of those skilled in the art, unless otherwise indicated. Such techniques are described in detail in such publications as "Molecular Cloning: A Laboratory Manual" 4th Edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology" and "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting" (Babar, 2011); "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention and therefore may be considered in making and practicing the invention. Techniques that are particularly useful for particular embodiments are discussed in the sections below. EXAMPLES

[0203] Experimental Example The present invention will be described in more detail below with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any and all variations that become obvious as a result of the teachings provided herein.

[0204] Without further description, it is believed that one of ordinary skill in the art can, using the above description and the following specific examples, make and utilize the compounds of the present invention and practice the claimed methods. Therefore, the following examples specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0205] Example 1 - Hyperglycemia Kit copGFP / + ;Lep ob / ob Jejunal and colonic KIT from male mice + Absence of highly expressed miR-10b-5p in ICC Obese female spontaneous leptin gene mutant mouse Lep ob / ob (Type 2 diabetes model) (The Jackson Laboratory) ICC-copGFP male mouse kit copGFP / + (Ro, 2010) copGFP / + ;Lep ob / ob We generated diabetic male Kit mice at 4, 8, and 12 weeks of age (Ro, 2010). copGFP / + ;Lep ob / ob Mice and healthy control males copGFP / + ;Lep + / + Mice were sacrificed and compared (Figure 1A). Leptin mutant males had much more body fat than healthy controls. Leptin mutant mice gained weight faster than their wild-type (WT) siblings at 4 weeks of age and continued to weigh twice as much as controls at 18 weeks of age (Figure 1B). Fasting blood glucose levels were much higher in mutants compared to healthy controls, reaching clearly hyperglycemic levels (>250 mg / dL) after 11 weeks (Figure 1C). However, glucose declined to normal levels after 14 weeks. During the transient hyperglycemic (diabetic) state, copGFP in the small intestine and colon was downregulated due to loss of KIT expression. + It has been previously reported that the number of ICCs is reduced (Ro, 2010). + ICCs were isolated by FACS from the colon and jejunum of three male diabetic mice, respectively, at 12–13 weeks of age, along with age- and sex-matched healthy non-diabetic controls. +ICC (JICC) and colon copGFP + ICCs (CICCs) were pooled into one sample each, which was used for small RNA isolation and subsequent miRNA sequencing. Global miRNA expression patterns within colonic and jejunal ICCs of diabetic homozygous Lep mutants and non-diabetic WT Lep are highly distinct (Pearson correlation coefficients: 0.55 for CICCs and 0.68 for JICCs), whereas Lep heterozygous ICCs show similarity to homozygous mutants (Pearson correlation coefficients: 0.75 for CICCs and 0.81 for JICCs) and to WT mice (Pearson correlation coefficients: 0.63 for CICCs and 0.61 for JICCs) (Figure 1E). Surprisingly, colonic and jejunal ICCs show remarkably similar miRNA expression profiles within Lep heterozygous mutant and homozygous mutant genotypes (Pearson correlation coefficients: 0.98 and 0.99, respectively) (Figure 1E). miR-10a-5p, miR-143-3p and miR-10b-5p were the most highly expressed miRNAs in WT ICCs, whereas their expression was significantly reduced in Lep heterozygous mutant and homozygous mutant ICCs. Among these three miRNAs, miR-10b-5p exhibited a diabetes-dependent reduction, as a further reduction was observed in diabetic Lep homozygous mutant ICCs (Figure 1F and Figure 1G). This facilitated the determination that miR-10b-5p was selectively depleted in diabetic ICCs. Sequencing and annotation of miRNAs showed that miR-10a-5p and miR-10b-5p were the most highly expressed in healthy WT JICCs and CICCs, but were dramatically depleted in diabetic JICCs and CICCs (Figure 1F-G). These results are shown in Figure 1A-G.

[0206] Example 2 - KIT + Generation of ICC-specific mir-10b knockout mice The Mir10b gene is located within the fifth intron of Hoxd4, which also overlaps with intron 1 of Hoxd3 on chromosome 2 (Figure 2A).LacZ-FLP-lox / + ) was retrieved from the cryo-archive (The Jackson Laboratory). LacZ-FLP-lox / + Mouse Kit CreERT2 / + Mouse or ROSA26 FLP / + By mating with mice, Kit CreERT2 / + ;mir-10b LacZ / LacZ Mice and ROSA26 FLP / + ;mir-10b lox / lox We generated ROSA26 mice (Figure 2A). FLP / + ;mir-10b lox / lox More Mouse Kit CreERT2 / + By mating with mice, Kit CreERT2 / + ;mir-10b lox / lox The mice were generated. CreERT2 / + ;mir-10b LacZ / LacZ Mouse and Kit CreERT2 / + ;mir-10b lox / lox Mice were treated with tamoxifen to delete mir10b in ICCs. Both lines were injected with tamoxifen for 5 consecutive days, and mir-10b KO (Kit CreERT2 / + ;mir-10b - / -) Male mir-10b KO and male mir-10b WT mice 6 months after tamoxifen and oil injection are shown in Figure 2B. The mir-10b KO mice were heavier and contained more abdominal fat than the WT controls. mir-10b deletion in mir-10b KO mice was confirmed by qPCR (Figure 2C). miR-10b-5p expression was significantly decreased in smooth muscle of mir-10b KO jejunum and mir-10b KO colon. KIT protein expression was also decreased in smooth muscle of KO jejunum and KO colon (Figure 2D). miR-10b-5p is predicted by TargetScan to target KLF11. KLF11 was increased in KO tissues, suggesting that miR-10b-5p targets KLF11. In addition, the expression of HOXD3 and HOXD4 was unchanged in the KO tissues, suggesting that the deletion of Mir10b, which is encoded in the intronic region of these two overlapping genes, did not disrupt the expression of these two genes. + (KIT antibody) and CD45 - ICCs, as analyzed by hematopoietic cell marker selection, were significantly reduced in mir-10b KO small intestine and mir-10b KO colon (Figure 2E). Loss of ICCs in the jejunum and colon was confirmed by immunohistochemistry (Figure 2F). In the KO tissues, the ICC subtypes ICC DMP in the jejunum and ICC SMP in the colon appeared to be most reduced. The results are shown in Figure 2A-F.

[0207] Example 3 – Male mir-10b knockout mice develop diabetes Male mir-10b KO mice became moderately obese and developed type 2 diabetes, whereas female mice did not (Figure 3A-F). The body weight of mir-10b KO male mice increased progressively from 16 weeks (4 months) after tamoxifen injection, and at 30 weeks (6.5 months), they were significantly heavier than control male mir-10b WT mice (40.5 g vs. 32.7 g) (Figure 3A). Blood glucose levels of male mir-10b KO mice also increased progressively, and after 24 weeks (6 months), the mice became hyperglycemic (>200 mg / dL) (Figure 3B). Intraperitoneal glucose tolerance test (GTT) in mir-10b KO mice confirmed that while 1-7 month old WT control mice were able to clear the glucose load within 2 hours, mir-10b KO mice were progressively impaired in their response to glucose load from 1 to 7 months of age (Figure 3C). There is a significant increase in glucose intolerance in mir-10b KO mice at 6-7 months of age (Figure 3D). In addition, there is a progressive decrease in insulin sensitivity in mir-10b KO mice from 1 to 7 months of age (Figure 3E). There is a significant increase in insulin resistance in mir-10b KO mice after 4 months compared to mir-10b WT mice (Figure 3F). The results are shown in Figure 3A-F.

[0208] Example 4 – Male mir-10b knockout mice exhibit delayed gastrointestinal (GI) transit mir-10b KO mice also developed gastroparesis and constipation. KO mice showed a prolonged total GI transit beginning as early as 1 month of age, which progressively slowed until 3 months of age before stabilizing into constipation (Figure 4A). At 3 months of age, transit time was more than two-fold slower (125 min vs. 270 min). The delayed total GI transit was due to slower gastric emptying and slower colonic transit. IVIS in vivo imaging showed that gastric emptying in 7-month-old mir-10b KO mice was clearly delayed compared to WT controls (Figure 4B). Gastric emptying time in KO mice was almost two-fold slower at 3 months of age and even slower at 7 months of age (Figure 4C). Colonic transit in KO mice was also further delayed by more than three-fold (Figure 4D). Furthermore, the mean fecal pellet frequency and mean fecal pellet output in KO mice were significantly reduced (Figure 4E-F). All these data consistently suggest that mir-10b KO mice exhibited greatly reduced GI motility. More importantly, motility impairment preceded glucose and insulin intolerance in mir-10b KO mice. The results are shown in Figure 4A-F.

[0209] Example 5 – Pathway analysis and target validation of miR-10b-5p in diabetes miR-10b-5p and its target genes related to diabetes were analyzed by Ingenuity Pathway Analysis (IPA) software. This analysis identified numerous miR-10b-5p targets associated with diabetes, including KLF4, KLF7, and KLF11 (Figure 5A). KLF11 is a transcriptional repressor and was selected for further study based on its interaction with INS. KLF11 binds to both GC and CACCC boxes in the insulin (INS) promoter (Niu 2007; Neve 2005) and represses the transcriptional activation of target genes (Niu 2007). The interacting genes (INSR, IRS1, IRS2, KIT, NEUROG3, SLC2A4, and GLP1R) contain GC-rich CpG islands, suggesting that these genes are potential KLF11 targets. Two mature miRNAs, miR-10b-5p and miR-10b-3p, are generated from the precursor (pre-miR-10b), which is highly conserved in both mice and humans, and the mature miRNAs are the same (Kozomara 2019). For miRNA target screening, six forms of chemically synthesized miR-10b-5p mimics (10b mimic 1, double-stranded RNA from Thermo Fisher Scientific; 2, double-stranded RNA from Dharmacon; 3, double-stranded RNA from QIAGEN; 4, single-stranded miR-10b-5p RNA from IDT; 5, annealed miR-10b-3p and miR-10b-5p RNA from IDT; 6, precursor miR-10b RNA from IDT) were tested in mouse NIT3T3 cells and human HEK293 along with antisense miR-10b-5p (single-stranded 10b inhibitor from Thermo Fisher Scientific, single-stranded miR-10b-3p from IDT), two mouse Klf11 siRNAs (siKlf11-1, siKlf11-2) and human KLF11 siRNAs (siKLF11-1 and siKLF11-2) (Thermo Fisher Scientific). Murine pre-miR-10b, 10b mimics and 10b inhibitors encoding miR-10b-5p and miR-10b-3p are shown in FIG. 13B.The effects on target genes in NIH3T3 and HEK293 cells transfected with 10b mimics and 10b inhibitors are shown in Figure 5C. Western blots show that miR-10b-5p efficiently targets Klf11 in transfected NIH3T3 and HEK293 cells (Figure 5C). Double-stranded RNA miR-10b mimic 1 most efficiently repressed KLF11 protein translation, followed by pre-miR-10b. The other two miR-10b mimics (2 and 3), single-stranded miR-10b-5p RNA, and annealed miR-10b-3p and miR-10b-5p RNAs induced much weaker protein repression than mimic 1 or no repression at all. In contrast, inhibitor miR-10b-3p RNA dramatically increased KLF11, suggesting that this inhibitor efficiently binds to and knocks down the levels of endogenous miR-10b-5p. In addition, KIT expression was negatively regulated by the amount of coexisting KLF11, suggesting that KLF11 suppresses KIT expression in NIH3T3 and HEK293 cells. miR-10b mimic 1 was selected for further targeting experiments. Diabetic genes suppressed by KLF11 were evaluated in four human cell lines (NIH3T3; HEK293; mouse β-cells NIT-1; human β-cells Panc10.05) using 10b mimics, 10b inhibitors, two mouse siKLF11s, and two human siKLF11s. KLF11 negatively regulated KIT, IRS1 and IRS2 in NIH3T3 and HEK293 (Figure 5D), and KIT, IRS1, IRS2, INS and ISNR in mouse and human β-cell lines (Figure 5E). The 10b mimic and the two siKLF11-1 and siKLF11-2 both reduced KLF11 expression. Introducing the 10b inhibitor increased KLF11 levels in all four cell lines, confirming the miR-10b-5p(mimic)-KLF11 pathway of diabetes gene regulation.For further target validation of the 10b mimic-KLF11, the miR-10b-5p target site in mouse Klf11 3'UTR (mKlf11 10b TS) and the miR-10b-5p target site in human KLF11 3'UTR (hKLF11 10b TS) were inserted at the end of the luciferase gene in a reporter gene plasmid (Figure 5F). Two reporter gene plasmids containing the mKlf11 10b TS mutant (mKlf11 10b TSM: four mutations in the seed sequence) and a scrambled sequence were also constructed and used as negative controls (Figure 5F). The mouse and human KLF11 10b TS sequences are highly conserved (Figure 5F). Four stable cell lines (NIH3T3, HEK293, mouse and human beta cell lines) transformed with each of these reporter gene plasmids were generated. NIH3T3-mKlf11 10b TS transfected with six serial amounts of 10b mimic and 10b inhibitor (1-200 pmol) showed dose-dependent reduction and induction of luciferase activity after 6, 12, 24 and 48 hours, with the greatest reduction and induction at the 12 hour time point (Figure 5G). NIH3T3-mKlf11 10b TSM transfected with 10b mimic / inhibitor showed no reduction or induction of luciferase activity (Figure 5G). Next, four cell lines harboring mKlf11 / hKLF11 10b TS, mKlf11 / hKLF11 10b TSM and / or scramble were transfected with 10b mimic and 10b inhibitor at a series of varying concentrations. All four cell lines harboring mKlf11 / hKLF11 10b TS showed dose-dependent inhibition of luciferase by the 10b mimic and induction of the enzyme by the 10b inhibitor (Figure 5H). However, cells harboring mKlf11 / hKLF11 10b TSM and / or scramble had no significant effect from the 10b mimic and 10b inhibitor. Taken together, these in vitro experiments demonstrated that miR-10b-5p negatively regulates both mouse KLF11 and human KLF11, which suppresses the expression of diabetes genes. The results are shown in Figure 5A-H.

[0210] Example 6 – Rescue of diabetes and gastroparesis in mir-10b knockout male mice by injection of miR-10b mimics To test the in vivo effect of the 10b mimetic, the 10b mimetic (263 ng / g) was delivered by intraperitoneal injection together with in vivo jetPEI into mir-10b KO diabetic mice. The body weights in the three groups (10b mimetic-injected and non-injected mir-10 KO mice and WT mice) were compared over a 10-week period after injection. The 10b mimetic-injected mice lost a small amount of weight over 4 weeks, whereas the non-injected KO mice gradually gained weight (Figure 6A). More importantly, the fasting glucose levels of the 10b mimetic-injected mice were immediately reduced to normal levels (~100 mg / dL) at 1 week and maintained until 10 weeks (Figure 6B). GTT and ITT showed that the glucose tolerance and insulin resistance of the mimetic-injected mice were improved to a similar or slightly better level than that of the WT mice (Figure 6C-D). Furthermore, GI function was gradually improved and restored in the mimic-injected mice. Fecal output gradually increased at 1 and 2 weeks and stabilized after 2 weeks (Figure 6E). Total GI transit time also decreased at 2 and 4 weeks and then became similar to that of WT mice (Figure 6F). Furthermore, gastric emptying time was significantly improved and completely restored to that of WT mice in 10b KO diabetic mice injected with 10b mimic (Figure 6G). ICCs in the jejunum and colon were lost in 10b KO mice, but were restored in mir-10 KO mice after miR-10b-5p mimic injection (Figure 6H). KIT protein expression was also restored in the pancreas and colon of 10b KO mice. Furthermore, an increase in miR-10b-5p after 10b mimic injection was confirmed in the blood, pancreas, jejunum, and colon (Figure 6J). The results are shown in Figure 6A-6J.

[0211] Example 7 – miR-10b-5p mimics rescue diabetes and slow GI transit in high-fat, high-sucrose diet (HFHSD) mice Next, we designed an experiment to test whether the 10b mimic could rescue HFHSD-induced diabetic mice. copGFP / +Mice were fed either a HFHS diet or a normal diet for 4 months. Compared to normal diet mice (29 g and ∼107 mg / dL), HFHS diet mice (∼43 g) gained significantly more body weight and blood glucose (∼200 mg / dL). Both groups of mice were intraperitoneally injected twice with 10b mimic (500 ng / g) and negative control RNA (500 ng / g) together with in vivo jetPEI (first injection followed by second injection 5 weeks apart) or no injection. Body weight, blood glucose levels and GI function were compared over 15 weeks after the two injections in three groups of HFHSD and normal diet mice (10b mimic injection, negative control RNA injection and no injection). Among the HFHSD mice, those that received the first and second 10b mimetic injections did not gain weight over the course of 11 weeks, whereas the negative control-injected and uninjected HFHSD-fed mice gradually gained weight (Figure 7A). The three groups of normal-diet mice also gradually gained weight, but the increase was much slower than that of the HFHSD-fed mice. The HFHS-diet mice injected with the 10b mimetic showed an immediate and marked decline in fasting glucose levels to normal levels (90 mg / dL) at 1 week, as seen in the 10b mimetic-injected 10b KO mice (Figure 7B). However, glucose levels rebounded progressively, reaching prediabetic levels (150 mg / dL) at 5 weeks. After the second injection, glucose levels dropped to normal levels and remained at 100–140 mg / dL for 10 weeks. The three groups of normal-diet mice maintained glucose at normal levels. GTT and ITT showed that glucose tolerance and insulin resistance were improved in mimic-injected HFHSD mice to be comparable or superior to those in normal-diet mice, especially after the second injection (Figure 7C-7D). In addition, GI function in the mimic-injected HFHSD group improved and recovered progressively at 2 and 4 weeks in the first and second injection groups (Figure 7E). Four weeks after the second injection, GI transit time was almost comparable to that in normal-diet mice. In the first mimic-injected HFHSD mice, mean fecal pellet frequency and fecal output gradually increased from 1 to 4 weeks and decreased at 5 weeks (Figure 7F).However, after the second injection, the frequency and excretion rate at week 5 were greatly improved and were comparable to those of normal diet mice. The results are shown in Figures 7A to 7F.

[0212] Example 8 - Restoration of miR-10b, its regulatory proteins, ICC, and β-cells in diabetic mice treated with 10b mimetics The reduction of miR-10b-5p in the blood of HFHSD diabetic mice was confirmed by qPCR (Figure 8A). miRNAs were dramatically reduced in diabetic mice compared to chow-fed healthy controls. With 10b mimic injection, miRNAs were restored by around 63% at 1 week and gradually decreased at 2-4 weeks. Surprisingly, the miRNA levels were paralleled with the blood insulin levels in mice after 6 hours of fasting followed by glucose loading (Figure 8B). In addition, 10b mimic injected mice had higher insulin than healthy control mice at 1 week. The hemoglobin A1C percentage was the inverse of the insulin and C-peptide data (Figure 8C). 10b mimic injection reduced A1C%, which slowly rebounded at 1-4 weeks. KLF11 protein changes in blood, pancreas, stomach and colon of 10b mimic injected mice were examined by Western blotting (Figure 8D). KLF11 was significantly increased in uninjected HFHSD-fed mice, whereas it was expressed at much lower levels in normal diet-fed mice. 10b mimic injection into HFHSD mice efficiently depleted KLF11 in tissues at 1 and 3 weeks after injection (Figure 8D). In addition, KIT was decreased in HFHSD tissues and increased by 10b mimic at 1 week and further increased at 3 weeks (Figure 8D). Moreover, ICC was lost in HFHSD, but was readily detected in 10b mimic-injected stomach, jejunum, colon, and pancreas (Figure 8E). The results are shown in Figures 8A-G.

[0213] Example 9 –Preventive effects of miR-10b-5p mimics on diabetes and slow GI transit in HFHSD mice To determine whether miR-10b-5p can prevent the development of diabetes and gastroparesis in HFHSD-fed mice, miR-10b-5p mimics or miR-10b-5p inhibitors were injected monthly into HFHSD- or ND-fed healthy C57 mice. HFHSD-fed non-injected control mice gained weight rapidly, whereas ND-fed control mice gained weight at a slower rate. (Figure 39A and Figure 39B) HFHSD-fed mice injected with miR-10b-5p mimics gained weight slowly over 5 months, similar to ND-fed control mice, whereas mice injected with miR-10b-5p inhibitors gained weight rapidly in both HFHSD-fed and ND-fed groups. (Figure 39C-E) Blood glucose levels increased significantly in mice injected with miR-10b-5p inhibitors, reaching diabetic levels (>180 mg / dL) in HFHSD-fed mice and prediabetic levels (121-179 mg / dL) in ND-fed mice, whereas mice injected with miR-10b-5p mimics maintained healthy blood glucose levels over a 5-month period. (Figure 39G) Monthly injections of miR-10b-5p mimics restored and maintained blood miR-10b-5p levels, whereas levels rapidly declined after injection of miR-10b-5p inhibitors. (Figure 9H) Consistent with this, insulin and C-peptide levels also followed a similar pattern to those in mice injected with miR-10b-5p mimics or miR-10b-5p inhibitors. (Figure 39I and Figure 39J) In both diets, GI function was severely impaired in mice injected with miR-10b-5p inhibitors, whereas miR-10b-5p mimic injections protected HFHSD-fed mice from motor impairment. The antagonistic targeting effects of miR-10b-5p mimics and miR-10b-5p inhibitors in mice confirm that miR-10b-5p can regulate the development of diabetes and slow GI transit. Furthermore, restoration of miR-10b-5p by miR-10b-5p mimic injections prevents the development of these pathological conditions in HFHSD-fed mice.

[0214] Example 10 - Kit Cre-ERT2 / + ;Rosa26 DTA / + KIT in male mice + Conditional removal of cells leads to diabetes and slow GI transit KIT in mice + To examine the loss of cell function, we injected tamoxifen into the Kit Cre-ERT2 / + ;Rosa26 DTA / + In (Kit-DTA), cells were conditionally removed using DTA, and in (Kit-DTA), Cre-ERT2 / + ;Rosa26 tdTom / + In (Kit-tdTom), cells were labeled with tdTomato. Kit-DTA mice gained weight faster than WT controls from 4 weeks onwards (Figure 10A). Blood glucose increased significantly from 6 weeks onwards after tamoxifen injection, resulting in hyperglycemic levels at 24 weeks (Figure 10B). GTT and ITT showed that glucose tolerance and insulin sensitivity in Kit-DTA mice were gradually impaired at 2 and 5 months (Figure 10C-D). From 7 days (7D) onwards after tamoxifen injection, the pancreas of Kit-tdTom mice contained numerous KIT + At 7 days, many KIT cells were identified (Fig. 10E). + The cells (tdTom) were transformed into beta cells (INS + ), but at 5 months, KIT + This is because β cells are highly sensitive to KIT + At 5 months, the cells were derived from KIT + This suggests that the majority of precursor-derived β cells are lost and replaced by newly derived β cells, and this is consistent with the KIT expression profile, which is preserved for up to 5 months. + These results also suggest that there are fewer progenitor-derived cells. +At 5 months, Kit-DTA mice had lost β cells. The number and size of islets in the pancreas of Kit-DTA mice were similar, but β cells were smaller in size and degenerated. miR-10b was significantly reduced in the blood of Kit-DTA mice at 5 days and 5 months compared to Kit-tdTom mice (Figure 10F). Consistent with the reduction in miR-10b, both insulin and C-peptide were slightly reduced at 5 days and significantly at 5 months (Figure 10G). Furthermore, KLF11 was increased in the blood, pancreas, and colon of Kit-DTA mice at 7 days and 5 months, while KIT was correspondingly reduced (Figure 10H). Furthermore, total GI transit time was significantly delayed in Kit-DTA mice at 2 and 5 months (Figure 10I). The results are shown in Figures 10A-I.

[0215] Example 11 – Expression of miR-10b-5p, KLF11, and KIT in human patients with T2D and gastroparesis We next examined samples from human patients with diabetes and idiopathic gastroparesis to see whether the abnormal expression patterns of miR-10b-5p, KLF11, and KIT seen in the diabetic mouse model were similar in human patient samples. Fifteen adult subjects (aged 18–65 years) diagnosed with gastroparesis based on established guidelines and abnormal gastric emptying scintigraphy and 15 control subjects were used for gene expression analysis. Based on miR-10b-5p expression levels, the samples could be divided into four distinct groups: high expression group (HC), corresponding to healthy control samples; intermediate expression group (IG-H and IG-L), corresponding to idiopathic gastroparesis (IG) samples and divided into high and low expression groups; and low expression group (DG), corresponding to diabetic gastroparesis samples (Figure 11A). Consistent with the data from mice, insulin and C-peptide levels showed a pattern correlating with miR-10b-5p expression in human samples (Figure 11B and Figure 11C). The levels of miR-10b-5p, insulin and C-peptide were significantly lower in the IG-L group compared to the HC group, suggesting that the IG-L group was in the pre-diabetic stage. The A1C levels of the IG-H and IG-H groups were similar to the HC group in the normal range (>5.7) (Figure 11D), suggesting that A1C cannot distinguish the pre-diabetic state from the HC group. KLF11 protein levels in serum and antrum were also higher in the DG group than in the HC group, while KIT levels were lower in the DG group (Figure 11E). Consistent with the results seen in mice, samples from human patients with T2D and / or gastroparesis showed decreased miR-10b-5p, increased KLF11 and decreased KIT. Deep sequencing of miRNAs from two individual blood samples of HC, IG-H, IG-L and DG identified differentially expressed miRNAs. The expression patterns of the top 70 most dynamically regulated miRNAs show clustering between HC and IG-H and between IG-L and DG (Figures S11F and S11G). miR-10a-5 and miR-10b-5p levels were highest in HC, followed by IG-H, IG-L, and DG, respectively (Figure S11G).Taken together, the expression levels of miR-10b-5p, KLF11 and KIT in HC and DG human patient samples are highly similar to the mouse data obtained in this study. In addition, the expression levels of miR-10b-5p, insulin and C-peptide in IG-H and IG-L suggest that these groups are in a prediabetic state progressing to diabetes.

[0216] Example 12 – Diabetes model of miR-10b regulation Without wishing to be bound by theory, data from this transgenic animal study and human studies suggest that miR-10b appears to regulate diabetes, GI motility, and obesity (Figure 12). miR-10b-5p targets and represses two metabolic transcription factors, KLF7 and KLF11. These transcription factors negatively regulate the expression of metabolic genes (LEP, ADIPOQ, GLP1R, KIT, INS, INSR, SLC2A2, SLC2A4, IRS1, and IRS2) that direct cell proliferation, differentiation, insulin production, and insulin sensitivity in adipocytes, ICCs, pancreatic progenitor cells, β-cells, and skeletal muscle cells. miR-10b is highly expressed under normal glucose level conditions, which represses KLF7 and KLF11, thereby enabling high expression of metabolic genes. However, under high glucose conditions, miR-10b is silenced, thereby allowing the two transcription factors to escape and become overexpressed, which downregulates metabolic gene expression and leads to the development of diabetes, GI dysmotility and obesity.

[0217] Example 13: miR-10a-5p mimics rescue obesity and diabetes phenotypes Figure 13A shows the sequences and structures of the mouse miR-10a precursor encoding miR-10a-5p and miR-10a-3p, the synthetic miR-10a-5p molecule (miR-10a-5p mimic) and the synthetic miR-10a-5p antisense molecule (miR-10a-5p inhibitor). Figure 13B shows the sequences and structures of the mouse miR-10b precursor encoding miR-10b-5p and miR-10b-3p, the synthetic miR-10b-5p molecule (miR-10b-5p mimic) and the synthetic miR-10b-5p antisense molecule (miR-10b-5p inhibitor). Note that single nucleotide base differences between miR-10a-5p mimic (U) and miR-10b-5p mimic (A) and between miR-10a-5p inhibitor (A) and miR-10b-5p inhibitor (U) are shown in bold. These constructs were then used in subsequent studies. miR-10a-5p mimic rescues obesity and diabetes phenotypes in mice fed a high-fat high-sucrose diet (HFHSD) or normal diet (ND). (Figure 14A and Figure 14B) Comparison of body weight and fasting blood glucose. Male C57 mice were fed a HFHSD throughout the experiment (4-43 weeks) and injected twice with miR-10a-5p mimic or negative control (scrambled RNA) (first injection at 22 weeks and second injection at 31 weeks), or no injection at 22 weeks, and then monitored for 21 weeks post-injection (PI). n = 3–6 in each group. *p<0.05 and **p<0.01 (miR-10a-5p mimic vs. no injection in HFHSD).

[0218] Example 14: miR-10a-5p mimic injections result in significant weight loss and reduce BMI and waist circumference Male C57 mice were fed a HFHSD throughout the study and treated with two injections of miR-10a-5p mimic or negative control or no injection as in previous studies. Figure 15A shows the gross anatomical changes of HFHSD-induced obese diabetic mice injected with miR-10a-5p mimic or negative control (scrambled RNA) or no injection as shown in Figure 14 (mice 12W PI after first injection at 43W). (Figure 15B) Gross anatomical changes of the same HFHSD-induced obese diabetic mice injected with miR-10a-5p mimic at 22 weeks, pre-injection and 4 weeks PI. Changes in mean BMI and mean waist circumference (Figure 15A and Figure 15B) were also evaluated. n=3 per group. *p<0.05 and **p<0.01. In Figure 14, mean BMI and mean waist circumference changes in HFHSD-induced obese diabetic mice injected with miR-10a-5p mimic or negative control (scrambled RNA) or without injection compared to healthy mice fed ND and not injected (Figure 16A and Figure 16B) (measured after the first injection at 23 weeks, 8 weeks PI, and after the second injection at 31 weeks, 1 week PI). n=6-12 per group. *p<0.05 and **p<0.01. Overall, this study demonstrated a 40% weight loss and improved glucose and insulin tolerance in diabetic mice administered the 10a mimic.

[0219] Example 15: miR-10a-5p mimic injection restores insulin levels in HFHSD-induced diabetic mice Since miR-10b-5p mimic injection increases insulin levels, we investigated the effect of miR-10a-5p on insulin levels. Changes in insulin levels were assessed after 6 hours of fasting (measured 2 weeks PI after the second injection at 31 weeks) in male mice fed HFHSD or normal diet (ND) and injected with either miR-10a-5p mimic, miR-10b-5p mimic or negative control (scrambled RNA) or no injection (Figure 17). n=6-12 per condition per experiment. Follow-up studies then assessed changes in insulin over 4 weeks after injection of either miR-10b-5p or miR-10a-5p. Inverse regulation of insulin was observed between miR-10a-5p mimic and miR-10b-5p mimic (Figure 17). miR-10a-5p mimic injection slightly reduces insulin levels in HFHSD-induced diabetic mice, followed by a slow recovery, whereas miR-10b-5p mimic injection transiently increases insulin levels, followed by a gradual decrease. Changes in insulin levels after 6 hours of fasting over 4 weeks in male mice fed HFHSD or ND and injected with either miR-10a-5p mimic or miR-10b-5p mimic or no injection. n=6-12 per condition per experiment. **p<0.01 (miR-10a-5p mimic vs. no injection in HFHSD);##p<0.01 (miR-10b-5p mimic vs. no injection in HFHSD). We next assessed the ability of miR-10a-5p mimic to improve glucose and insulin tolerance in HFHSD-induced diabetic mice. (Figure 18A) Intraperitoneal (IP) glucose tolerance test (GTT) 4 weeks after or without miR-10a-5b mimic injection. (Figure 18B) Area under the curve (AUC) plot of GTT. n=6-12 per group. **p<0.01. Insulin resistance was assessed in a similar study. (Figure 19A) IP insulin tolerance test (ITT) 4 weeks after the first injection of miR-10a-5p mimic. (Figure 19B) Area under the curve (AUC) plot of ITT. n=6-12 per group. **p<0.01. Effect of miR-10a-5p treatment on insulin-related metabolic regulatory hormones GLP-1 and leptin.miR-10a-5p mimic injection was found to increase (Figure 20A) GLP-1 and (Figure 20B) leptin in HFHSD-induced diabetic mice. miR-10a-5p mimic injection increased and restored (Figure 20A) GLP-1 and (Figure 20B) leptin in HFHSD-induced diabetic mice to hormone levels close to those of ND-fed healthy mice over 4 weeks. Increased signaling of both of these hormones is known to reduce body weight. n=4 per group per experiment. **p<0.01 (miR-10a-5p mimic vs. no injection on HFHSD). Taken together, these results demonstrate that insulin levels in miR-10a-5p mimic-treated mice were significantly restored to levels similar to nondiabetic mice. Also, without wishing to be bound by theory, these results suggest that treatment of diabetic obesity with miR-10a-5p mimics may reverse multiple symptoms of this condition.

[0220] Example 16: miR-10a-5p mimic injection improves GI function HFHSD-fed male C57BL / 6 mice were evaluated for GI function after treatment with miR-10a-5p mimic or scrambled negative control, or without injection. (Figures 21A-C) Total GI transit time, fecal pellet output, and colonic transit time were evaluated (Figures 21A and 21B, measured 8 weeks PI after the first injection at 23W; Figure 21C, measured 2 weeks PI after the second injection at 31W). A follow-up study evaluated total GI transit time (Figure 21A), fecal pellet output (Figure 21B), and colonic transit time (Figure 21C) in HFHSD-fed male diabetic mice or ND-fed healthy mice, injected with or without miR-10a-5p mimic, at 4 weeks PI. (Figures 21D-F) Changes in total GI transit time, fecal pellet output, and colonic transit time in individual mice in Figures 21A-C. n=6-18 per group. **p<0.01. As an additional measure of gastric function, gastric emptying rates were measured in HFHSD-fed male diabetic mice or ND-fed male healthy mice with or without injection of miR-10a-5p mimics. Food mixed with fluorescent GastroSense 750 (GS) was applied to the stomach by fluorescent gavage and then imaged with an IVIS Lumina III system at 0 and 60 min post-gavage (Figure 22). These data demonstrated that treatment with 10a mimics can reduce GI transit time, increase fecal pellet emptying, increase gastric emptying, and significantly reduce colonic transit time. Overall, these observations suggest that treatment with miR-10a-5p mimics not only results in amelioration of diabetic obesity, but also improves GI function.

[0221] Example 17: Both miR-10a-5p and miR-10b-5p mimics confer protection against obesity and diabetes phenotypes To see whether treatment with miR-10a-5p and miR-10b-5p mimics could also prevent the development of obesity and diabetes in addition to treating existing conditions, male C57BL / 6 mice were fed a HFHSD diet and then injected twice (first and second injections) with miR-10a-5p mimics, miR-10b-5p mimics, or negative control (scrambled RNA), or no injection, and fed a HFHSD or ND diet. (Figures 23A and 23B) Body weight and fasting blood glucose in ND-fed mice. (Figures 23C and 23D) Body weight and fasting blood glucose in HFHSD-fed mice. n=3 per group. *p<0.05 and **p<0.01 (miR-10a-5p mimic vs. no injection); #p<0.05 and ##p<0.01 (miR-10b-5p mimic vs. no injection). These data showed that pretreatment with both mimics significantly reduced the onset of obesity and maintained low blood glucose levels compared to controls. In normal diet-fed mice, only miR-10b-5p treatment resulted in a significant delay in weight gain. However, this was not accompanied by differences in blood glucose levels. Further studies of ND-fed mice revealed that injection of miR-10a-5p mimic and / or miR-10b-5p mimic into ND-fed mice did not alter fasting glucose levels at 2W PI after the second injection at 9W. n=3 per group (Figure 23B).

[0222] Example 18: Treatment with miR-10a-5p and miR-10b-5p inhibitors triggers and exacerbates obesity and diabetes phenotypes in mice Having observed that miR-10a-5p and miR-10b-5p mimic treatment could reduce and prevent obesity and diabetes phenotypes in mice, we next conducted a study to see whether treatment with inhibitors based on the miR-10a-5p and miR-10b-5p sequences could have the opposite effect (shown in Figure 23). Male C57BL / 6 mice were fed a ND or HSHSD and then injected twice (1st and 2nd injections) at 4W and 9W with either a miR-10a-5p inhibitor, a miR-10b-5p inhibitor, both miR-10a-5p and miR-10b-5p inhibitors (miR-10a / b-5p inhibitors), or a negative control (scrambled RNA), or no injection, and then fed a HFHSD or normal diet (ND). (Figure 24A and Figure 24B) Body weight and fasting blood glucose in mice fed ND. (Figure 24C and Figure 24D) Body weight and fasting blood glucose in mice fed HFHSD. n=3 for each group. *p<0.05 and **p<0.01 (miR-10a-5p inhibitor vs. no injection), #p<0.05 and ##p<0.01 (miR-10b-5p inhibitor vs. no injection), ^p<0.05 and ^^p<0.01 (miR-10a / b-5p inhibitor vs. no injection). Next, the effect of the inhibitor on fasting insulin levels was evaluated. In ND-fed male healthy mice (Figure 25A) and HFHSD-fed male diabetic mice (Figure 25B), miR-10a-5p inhibitors increase fasting insulin levels, while miR-10b-5p inhibitors decrease fasting insulin levels at 2 weeks PI after the second injection at 9 weeks. **p<0.01. In addition, gastric emptying was observed in mice treated with the inhibitors. (Figure 26) Images in ND-fed mice at 4 weeks PI or without injection at 10 weeks after the second injection of either miR-10a-5p inhibitor, miR-10b-5p inhibitor or negative control (scrambled RNA). GS stands for GastroSense 750. Overall, these data demonstrated accelerated diabetes onset and weight gain in mice fed either type of diet.Treatment with miR-10a-5p and miR-10b-5p inhibitors triggers and exacerbates the obese and diabetic phenotype in mice.

[0223] Example 19: Gene targets of miR-10a-5p and miR-10b-5p Next, a series of studies were undertaken to investigate the effects of miR-10a-5p and miR-10b-5p mimics on target gene expression. (Figure 27) Observations showed that miR-10a-5p and miR-10b-5p differentially targeted key proteins (LGR5, REG4, PDX1, NEROG3 and PDGFRA) in regulating stem or progenitor cells in colonic mucosa, pancreas and white adipocytes of ND-fed mice at 4 weeks PI after the first injection. GAPDH was used as an endogenous control in this Western blot. Subsequent studies demonstrated that miR-10a-5p and miR-10b-5p differentially targeted the same obesity- and diabetes-related proteins. Comparison of the targeting effect of miR-10a-5p mimic, miR-10b-5p mimic, miR-10a-5p inhibitor and miR-10b-5p inhibitor on obesity and diabetes related proteins. Mouse pancreatic β cells (NIT-1 cells) or mouse adipocytes (LM cells) were transfected with miR-10a-5p mimic, miR-10b-5p mimic, miR-10a-5p inhibitor, miR-10b-5p inhibitor or negative control (scrambled RNA) or no transfection. (Figure 28A) Western blot of KLF11, KIT, LEP, GLU4, ADIPOQ, IRS-1, IRS-2 and GAPDH control in NIT-1 cells. Protein markers (M) are shown with the corresponding molecular weight (kDa). (Figure 28B) Quantification of proteins in A, normalized with GAPDH. (FIG. 28C) Western blot of LEP, LEPR and GAPDH controls in LM cells. n=3 for each group. *p<0.05 and **p<0.01.

[0224] Example 20: miR-10a-5p rescues diabetes-associated non-alcoholic fatty liver disease Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in type 2 diabetes mellitus patients and correlates with concomitant obesity and insulin resistance. Using the HFHS mouse model from the above example, a series of studies were undertaken to determine whether treatment with miR-10a-5p mimics could prevent or reverse the fatty, inflammatory and fibrotic liver phenotype in diabetic mice fed a HFHSD. (Figure 29) Male diabetic C57BL / 6 mice fed a HFHSD for 18 weeks (4-22 weeks) or male healthy mice fed a ND diet were injected once at 22 weeks with miR-10a-5p mimics or miR-10a-5p inhibitors or continued to be fed the same diet without injection. Macroscopic and stained images of the livers of these mice are shown. Liver tissues were dissected from HFHSD and / or ND mice at 2 weeks PI and stained with H&E, Oil Red O (lipids), Picrosirius Red (fibrosis) and CD64 (macrophages). Similar studies were performed to evaluate the levels of liver injury-related markers in treated and untreated mice. (Figure 30A and Figure 30B) Blood tests for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in HFHSD-fed male diabetic or ND-fed male healthy mice for 18 weeks, injected with miR-10a-5p mimic once at 22 weeks or not and continued to be fed the same diet. AST and ALT were measured in HFHSD and / or ND mice at 2 weeks PI. n=3 per group. *p<0.05 and **p<0.01.

[0225] Example 21: miR-10a-5p treatment rescues high cholesterol and reduces inflammation in diabetic mice In addition to insulin and glucose dysregulation, abnormal GI function and development of fatty liver disease, diabetes is known to be associated with high levels of "bad" LDL cholesterol. Treatment with miR-10a-5p mimics was found to rescue high cholesterol in HFHSD-fed diabetic mice. (Figure 31A and Figure 31B) Blood tests for high-density lipoprotein (HDL) and low-density lipoprotein (LDL) / very low-density lipoprotein (VLDL) cholesterol in HFHSD-fed male diabetic mice or ND-fed male healthy mice for 18 weeks, injected with miR-10a-5p mimics once at 22 weeks or continued to feed the same diet without injection. HDL and LDL / VLDL were measured in HFHSD and / or ND mice at 2 weeks PI. n=6 per group. **p<0.01. Studies have also found that the levels of inflammation-related cytokines are often high in patients with type 2 diabetes. In particular, these patients tend to have higher levels of the proinflammatory cytokine IL-6 and lower levels of the anti-inflammatory cytokine IL-10. Treatment of HFHSD mice with miR-10a-5p mimics was found to reduce inflammation in HFHSD-fed diabetic mice. (Figure 32A and Figure 32B) Blood tests for the proinflammatory cytokine IL-6 and the anti-inflammatory cytokine IL-10 in HFHSD-fed male diabetic mice or ND-fed male healthy mice for 18 weeks, injected with miR-10a-5p mimics once at 22 weeks or not, and continued to feed the same diet. IL-6 and IL-10 were measured in HFHSD and / or ND mice at 2 weeks PI. n=6-8 per group. **p<0.01.

[0226] Example 22: Improved cardiac function in diabetic mice treated with miR-10b-5p Abnormal cardiac function and diabetes are closely related. Patients with diabetes are at increased risk of developing heart disease and heart failure. Similarly, patients with heart failure often develop diabetes as a complication. To determine whether treatment with miR-10b-5p mimics improves cardiac function, a series of studies were performed in HFHSD-induced diabetic mice. Cardiac function in HFHSD-fed male diabetic mice and ND-fed male healthy mice was assessed using echocardiography with or without injection of miR-10b-5p mimics or miR-10b-5p inhibitors. (Figure 33A) Ejection fraction (EF). (Figure 33B) Fractional shortening (FS). (Figure 33C) Stroke volume (SV). (Figure 33D) Heart rate (HR). HFHS-fed diabetic mice (no injection) showed reduced EF, FS and SV compared to ND-fed mice (no injection). Injection of miR-10b-5p mimic in ND-fed mice did not change EF and FS but improved SV. Injection of miR-10b-5p inhibitor in ND-fed mice reduced EF, FS and SV. Injection of miR-10b-5p mimic or miR-10b-5p inhibitor in HFHSD-fed mice did not significantly change EF, FS and SV. Heart rates of 400-500 beats / min were maintained in all mice during echocardiography. n=3 per group. *p<0.05 and **p<0.01.

[0227] Example 23: Comparison of miR-10a-5p and miR-10b-5p treatments with established diabetes drugs Because diabetes and diabetes-related diseases are relatively common, several drugs designed to combat aspects of the disease have been developed. The relative potency of miR-10a-5p and miR-10b-5p mimics compared to established chemotherapeutic drugs may be an indication of clinical usefulness. Therefore, we undertook a series of studies to compare the drug effects of miR-10a-5p, miR-10b-5p, and an investigational miR-103 / 107 inhibitor (RG-125) on body weight and blood glucose. miR-10a-5p mimics rescue obese and diabetic phenotypes in HFHSD-fed mice. (Figure 34A and Figure 34B) Comparison of body weight and fasting blood glucose. Male C57BL / 6 mice were fed HFHSD or ND throughout the experiment and were injected at 22 weeks with either a miR-10a-5p mimic, a miR-10b-5p mimic, or a miR-103 / 107 inhibitor or no injection and then monitored for 9 weeks PI. AstraZeneca, in collaboration with Regulus Therapeutics, is completing phase 1 and 2 clinical trials with a miR-103 / 107 inhibitor (RG-125) in patients with type 2 diabetes and non-alcoholic fatty liver disease. miR-10a-5p and miR-10b-5p have a significantly improved and durable effect on lowering blood glucose and body weight compared to miR-103 / 107 inhibitors. n=3–6 per group. *p<0.05 and **p<0.01 (miR-10a-5p mimic vs. miR-103 / 7 inhibitor), #p<0.05 and ##p<0.01 (miR-10b-5p mimic vs. miR-103 / 7 inhibitor).

[0228] Figure 35 illustrates the study design to compare the drug effects of miR-10a-5p mimics and miR-10b-5p mimics with popular antidiabetic drug treatments and FDA-approved GI motility enhancers on blood glucose, body weight and GI function in diabetic mice fed HFHSD for 8 weeks after treatment or healthy mice fed ND without injection as a control. miR-10a-5p (500ng / g body weight), miR-10b-5p (500ng / g body weight) or scrambled RNA (500ng / g body weight) were injected twice, once at week 1 and once at week 2, by IP injection. Metformin (250mg / kg body weight) or sitagliptin (DPP4 inhibitor) (10mg / kg body weight) were administered orally (PO) daily for 4 weeks. Liraglutide (GLP-1 receptor agonist) (0.2 mg / kg body weight) was injected twice daily by subcutaneous (SC) injection for 2 weeks. Insulin (0.75 U / kg body weight) was injected once daily by IP injection for 4 weeks. Prucalopride (5-HT4 receptor agonist) (2 mg / kg body weight) was administered PO daily for 4 weeks. Drug effects are shown in Figures 36-38. Treatment with miR-10a-5p mimics and miR-10b-5p mimics had better and longer effects on lowering (Figure 36A) blood glucose and (Figure 36B) body weight than the antidiabetic medications and gastrointestinal motility enhancers listed in Figure 35. n=5 for each group. Similarly, miR-10a-5p mimics and miR-10b-5p mimics have better and longer effects on improving GI motility than antidiabetic medications and prucalopride, which is used to treat chronic constipation (Figures 37-38). After treatment, total GI transit time was measured for each group at 2, 4, 6 and 8 weeks (Figure 37). n=5 per group. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. The frequency of drug treatments is shown in Figure 35. miR-10a-5p mimics, miR-10b-5p mimics and prucalopride improved total GI transit, whereas the four antidiabetic medications did not (Figure 37).However, prucalopride had a transient effect on GI transit during treatment for 4 weeks compared to miR-10a-5p and miR-10b-5p mimics, which had a sustained effect for 8 weeks. Finally, delayed gastric emptying in control scrambled RNA HFHSD-fed diabetic mice was improved by miR-10a-5p mimics, miR-10b-5p mimics and prucalopride at 4 weeks, whereas liraglutide did not improve delayed gastric emptying (Figure 38A-B). Normal gastric emptying rates were maintained at 8 weeks in mice injected with miR-10a-5p and miR-10b-5p mimics, but were delayed in prucalopride-treated mice, suggesting that both miRNAs had a much more sustained effect than prucalopride in improving gastric emptying. The frequency of drug treatment is shown in Figure 35. Overall, these data demonstrate that treatment of diabetes with miR-10a-5p and / or miR-10b-5p mimics can provide equivalent or better clinical outcomes than established pharmacological treatments and may represent promising investigational agents.

[0229] Other aspects: The recitation of a listing of elements in any definition of a variable herein includes definition of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0230] The disclosures of all patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that others skilled in the art may devise other embodiments and modifications of the present invention without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent modifications.

Claims

1. A pharmaceutical composition comprising a miR-10a-5p mimetic or a miR-10b-5p mimetic for treating diabetes in a subject in need thereof, wherein the miR-10a-5p mimetic comprises the nucleic acid sequence set forth in SEQ ID NO:1 and the nucleic acid sequence set forth in SEQ ID NO:2, and the miR-10b-5p mimetic comprises the nucleic acid sequence set forth in SEQ ID NO:6 and the nucleic acid sequence set forth in SEQ ID NO:

7.

2. A pharmaceutical composition comprising the miR-10a-5p mimic of claim 1, wherein the diabetes is type 2 diabetes, or a pharmaceutical composition comprising the miR-10b-5p mimic of claim 1, wherein the diabetes is type 1 or type 2 diabetes.

3. A pharmaceutical composition comprising a miR-10a-5p mimetic or a miR-10b-5p mimetic for reducing weight, lowering blood glucose, increasing insulin sensitivity, treating diabetes-related fatty liver disease, or reducing diabetes-related inflammation in a subject, wherein the miR-10a-5p mimetic comprises the nucleic acid sequence shown in SEQ ID NO:1 and the nucleic acid sequence shown in SEQ ID NO:2, and the miR-10b-5p mimetic comprises the nucleic acid sequence shown in SEQ ID NO:6 and the nucleic acid sequence shown in SEQ ID NO:

7.

4. A pharmaceutical composition comprising a miR-10b-5p mimic for increasing interstitial cells of Cajal (ICC) proliferation in a subject, wherein the miR-10b-5p mimic comprises a nucleic acid sequence shown in SEQ ID NO:6 and a nucleic acid sequence shown in SEQ ID NO:

7.

5. The pharmaceutical composition of claim 4, wherein increasing ICC proliferation in the subject restores function of ICC in the subject, preferably wherein the ICC is located within the smooth muscle of the gastrointestinal tract of the subject, more preferably wherein the smooth muscle is located within the stomach, small intestine or colon of the subject, and even more preferably wherein the ICC comprises an ICC precursor, ICC-MY, ICC-IM, ICC-DMP, ICC-SM or ICC-SMP.

6. A pharmaceutical composition comprising a miR-10b-5p mimic for increasing KIT expression in ICCs in a subject, preferably wherein increasing KIT expression in the subject restores function of ICCs in the subject, wherein the miR-10b-5p mimic comprises a nucleic acid sequence as shown in SEQ ID NO:6 and a nucleic acid sequence as shown in SEQ ID NO:

7.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein said miR-10a-5p mimic and said miR-10b-5p mimic are mammalian, preferably human.

8. A pharmaceutical composition comprising a miR-10a-5p mimic or a miR-10b-5p mimic and a pharma- ceutically acceptable carrier or adjuvant, wherein the miR-10a-5p mimic comprises the nucleic acid sequence shown in SEQ ID NO:1 and the nucleic acid sequence shown in SEQ ID NO:2, and the miR-10b-5p mimic comprises the nucleic acid sequence shown in SEQ ID NO:6 and the nucleic acid sequence shown in SEQ ID NO:

7.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, wherein the miR-10a-5p mimic is a miR-10a-5p duplex, a chemically-modified double-stranded miR-10a-5p, an unmodified double-stranded miR-10a-5p, a single-stranded chemically-modified miR-10a-5p, or a single-stranded unmodified miR-10a-5p; and the miR-10b-5p mimic is a miR-10b-5p duplex, a chemically-modified double-stranded miR-10b-5p, an unmodified double-stranded miR-10b-5p, a single-stranded chemically-modified miR-10b-5p, or a single-stranded unmodified miR-10b-5p.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein said miR-10a-5p mimic and said miR-10b-5p mimic are engineered.

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