Inhibitory nucleic acids and methods of use thereof

Abstract

The present disclosure provides inhibitory nucleic acids, compositions comprising the inhibitory nucleic acids, and methods of using the inhibitory nucleic acids to treat various disorders.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 427,254, filed November 22, 2022, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with government support under DK114277 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Submitted Material A Sequence Listing is provided herewith as Sequence Listing XML "BERK-478WO_SEQ_LIST" having a size of 11,512 bytes, created on November 10, 2023. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. [Background technology]

[0004] introduction Metabolic disorders, such as insulin resistance, hyperglycemia, type 2 diabetes, obesity, fatty liver disease, glucose intolerance, hyperinsulinemia, metabolic syndrome, and hypertension, are major health problems. Summary of the Invention

[0005] overview The present disclosure provides inhibitory nucleic acids, compositions comprising the inhibitory nucleic acids, and methods of using the inhibitory nucleic acids to treat various disorders. [Brief explanation of the drawings]

[0006] [Figure 1]Figures 1A-1B provide the nucleotide sequences of exemplary antisense oligonucleotides (ASOs) (Figure 1A; the nucleotide sequences of NRC0090, NRC0091, and NRC0119 are SEQ ID NOs: 1, 2, and 3, respectively), as well as the base-pairing positions of the ASOs within the target microRNA (miR-128-3p) (Figure 1B; SEQ ID NO: 7). [Figure 2] FIG. 2 shows a schematic representation of the effects of dystrophin loss in Duchenne muscular dystrophy (DMD). [Figure 3A] Figures 3A-3H show the in vivo effects of locked nucleic acid (LNA) antisense oligonucleotides (ASOs) targeting miR-128-3p. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 3F] See legend to Figure 3A. [Figure 3G] See legend to Figure 3A. [Figure 3H] See legend to Figure 3A. [Figure 4A] Figures 4A-4I show the effect of LNA ASOs targeting miR-128-3p on miR-128-3p levels in various tissues in vivo. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Figure 4F] See legend to Figure 4A. [Figure 4G] See legend to Figure 4A. [Figure 4H] See legend to Figure 4A. [Figure 4I] See legend to Figure 4A. [Figure 5]Figures 5A-5F show the effects of LNA ASO targeting miR-128-3p on body weight, body mass, and blood glucose levels. [Figure 6] 6A-6D show the genetic association of elevated circulating mature miR-128(3p) and miR-128-1 with reduced grip strength and lung function in mouse and canine models of DMD. [Figure 7] 7A-7E show the response of systemic or muscle-specific miR-128-3p knockout in mdx5cv mice. [Figure 8] Figures 8A-8C show the in vivo ASO dose response in mdx5cv mice. [Figure 9] Figures 9A-9N show the effect of miR-128-1 LNA ASO ("anti-miR-128-1 ASO") on pathological phenotypes in 6-week-old or 9-week-old mdx5cv DMD mouse models, demonstrating the effect of miR-128-1 LNA ASO on muscle fiber size, necrosis, and fibrosis. [Figure 10] 10A-10F show the effect of ASO administration on indicators of liver and kidney function. [Figure 11A] Figures 11A-11K show RNAseq analysis of WT mice and mdx5cv mice treated with control and anti-miR-128-1 ASOs, demonstrating the effect of miR-128-1 LNA ASO on dystrophic muscle fiber type switching in mdx5cv mice. [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 11D] See legend to Figure 11A. [Figure 11E] See legend to Figure 11A. [Figure 11F] See legend to Figure 11A. [Figure 11G] See legend to Figure 11A. [Figure 11H] See legend to Figure 11A. [Figure 11I] See legend to Figure 11A. [Figure 11J] See legend to Figure 11A. [Figure 11K] See legend to Figure 11A. [Figure 12A] 12A-12C show metabolic analysis of WT mice and mdx5cv mice treated with control ASO and anti-miR-128-1 ASO. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 13A] Figures 13A-13F show the effect of miR-128-1 LNA ASO on mitochondrial health in mdx5cv mice. [Figure 13B] See legend to Figure 13A. [Figure 13C] See legend to Figure 13A. [Figure 13D] See legend to Figure 13A. [Figure 13E] See legend to Figure 13A. [Figure 13F] See legend to Figure 13A. [Figure 14A] Figures 14A-14E show the effects of miR-128-1 LNA ASO on inflammation, ferroptosis, and oxidative stress. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 14D] See legend to Figure 14A. [Figure 14E] See legend to Figure 14A. [Figure 15A] 15A-15D show the histology of small intestinal sections from wild-type (WT) mice and mdx5cv mice treated with control ASO or anti-miR-128-1 ASO. [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 15D] See legend to Figure 15A. [Figure 16A]16A-16B show evaluation of miR-128 expression and its downstream targets in the myocardium of 6-week-old mdx5cv mice. [Figure 16B] See legend to Figure 16A. [Figure 17A] Figures 17A-17L show the effects of anti-miR-128-1 ASO in a myocardial infarction (MI) mouse model and a preclinical DMDY / - pig model. [Figure 17B] See legend to Figure 17A. [Figure 17C] See legend to Figure 17A. [Figure 17D] See legend to Figure 17A. [Figure 17E] See legend to Figure 17A. [Figure 17F] See legend to Figure 17A. [Figure 17G] See legend to Figure 17A. [Figure 17H] See legend to Figure 17A. [Figure 17I] See legend to Figure 17A. [Figure 17J] See legend to Figure 17A. [Figure 17K] See legend to Figure 17A. [Figure 17L] See legend to Figure 17A. [Figure 18A] Figures 18A-18C show the effect of anti-miR-128-1 ASO on fibrosis in an MI mouse model. [Figure 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. DETAILED DESCRIPTION OF THE INVENTION

[0007] definition As used herein, "antisense oligonucleotide" refers to a nucleic acid sequence that is complementary to a DNA or RNA sequence, for example, a microRNA sequence.

[0008] "RNA" refers to a molecule containing at least one or more ribonucleotide residues. A "ribonucleotide" is a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranose moiety. As used herein, the term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, essentially pure RNA, synthetic RNA, and recombinantly produced RNA, and also includes modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. The nucleotides of an RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides, or chemically synthesized nucleotides or deoxynucleotides.

[0009] "MicroRNAs" (miRNAs) are single-stranded RNA molecules approximately 21-23 nt in length. Generally, miRNAs regulate gene expression. They are encoded by genes and transcribed from the DNA of those genes, but miRNAs are not translated into proteins. Each primary miRNA transcript is processed into a short stem-loop structure, which is then further processed into a functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their primary function is to downregulate gene expression.

[0010] As used herein, "interfering RNA" refers to a double-stranded or single-stranded RNA sequence that can directly or indirectly (i.e., upon translation) inhibit or downregulate gene expression by mediating RNA interference. Interfering RNA includes, but is not limited to, toenail interfering RNA ("siRNA") and short hairpin RNA ("shRNA"). "RNA interference" refers to the selective degradation of sequence-compatible messenger RNA transcripts.

[0011] As used herein, "shRNA" (short hairpin RNA) refers to an RNA molecule comprising an antisense region, a loop portion, and a sense region, wherein the sense region has complementary nucleotides that base pair with the antisense region to form a double-stranded stem. After post-transcriptional processing, the short hairpin RNA is converted into a small interfering RNA by a cleavage event mediated by the enzyme Dicer, a member of the RNase III family.

[0012] "Small interfering RNA" or "siRNA," as used herein, refers to a small RNA molecule that can inhibit or downregulate gene expression by mediating RNA interference in a sequence-specific manner. Small RNAs can be, for example, about 18 to 21 nucleotides in length.

[0013] As used herein, "antagomir" refers to a small synthetic RNA that is complementary to a specific microRNA target, either with a mispairing at the cleavage site or with one or more base modifications to inhibit cleavage.

[0014] As used herein, the phrase "post-transcriptional processing" refers to mRNA processing that occurs after transcription, for example, mediated by the enzymes Dicer and / or Drosha.

[0015] "Effective amount" refers to the amount of an agent (e.g., an inhibitory nucleic acid of the present disclosure) or a composition comprising an agent (e.g., a composition comprising an inhibitory nucleic acid of the present disclosure) required to ameliorate the symptoms of a disease compared to an untreated patient. The effective amount of an agent or composition used to carry out therapeutic treatment of a disease or disorder will vary depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and administration regimen. Such an amount is referred to as an "effective amount."

[0016] As used herein, " cholesterol homeostasis " refers to the control of cholesterol intake, cholesterol biosynthesis, cholesterol conversion into bile acid and bile acid excretion, and these processes occur in the subject with healthy blood LDL, HDL and cholesterol levels (for example, this healthy level is also referred to herein as " reference standard ").Therefore, the subject who needs cholesterol homeostasis needs to improve the control of blood LDL, HDL and / or cholesterol, which will restore to healthy levels.

[0017] The terms "diabetes" and "diabetic" refer to a progressive disease of carbohydrate metabolism, often characterized by hyperglycemia and glycosuria, involving inadequate production or utilization of insulin. The terms "prediabetes" and "prediabetic" refer to a state in which a subject does not have the characteristics, symptoms, etc. typically observed in diabetes, but has characteristics, symptoms, etc. that may progress to diabetes if left untreated. The existence of these conditions can be determined, for example, using either fasting plasma glucose (FPG) test or oral glucose tolerance test (OGTT). Both generally require the subject to fast for at least 8 hours before the start of the test. In the FPG test, the subject's blood glucose is measured after the end of fasting; generally, the subject fasts overnight, and blood glucose is measured in the morning before the subject eats. Healthy subjects typically have FPG concentrations of about 90 to about 100 mg / dl, subjects with "prediabetes" typically have FPG concentrations of about 100 to about 125 mg / dl, and subjects with "diabetes" typically have FPG levels above about 126 mg / dl. In an OGTT, a subject's blood glucose is measured after fasting and again two hours after consuming a glucose-rich beverage. Two hours after consuming the glucose-rich beverage, healthy subjects typically have blood glucose concentrations below about 140 mg / dl, prediabetic subjects typically have blood glucose concentrations of about 140 to about 199 mg / dl, and diabetic subjects typically have blood glucose concentrations above about 200 mg / dl. The blood glucose levels listed above are for human subjects; the scales for normoglycemia, moderate hyperglycemia, and frank hyperglycemia in mouse subjects are different. After a 4-hour fast, healthy mouse subjects generally have FPG concentrations of about 100 to about 150 mg / dl, mouse subjects with "pre-diabetes" generally have FPG concentrations of about 175 to about 250 mg / dl, and mouse subjects with "diabetes" generally have FPG concentrations greater than about 250 mg / dl.

[0018] The term "insulin resistance," as used herein, refers to a condition in which normal amounts of insulin fail to produce a normal physiological or molecular response. In some cases, supraphysiological amounts of insulin, either endogenously produced or exogenously administered, can completely or partially overcome insulin resistance and produce a biological response.

[0019] The term "metabolic syndrome" refers to a cluster of related traits, including, but not limited to, hyperinsulinemia, impaired glucose tolerance, obesity, redistribution of fat to the abdomen or upper body, hypertension, impaired fibrinolysis, and dyslipidemia characterized by high triglycerides, low high-density lipoprotein (HDL) cholesterol, and high small dense low-density lipoprotein (LDL) particle levels. Subjects with metabolic syndrome are at risk for developing type 2 diabetes and / or other disorders (e.g., atherosclerosis).

[0020] The term "glucose metabolism disorder" encompasses disorders characterized by a clinical symptom or combination of clinical symptoms associated with elevated levels of glucose and / or elevated levels of insulin in a subject compared to a healthy individual. Elevated glucose and / or insulin levels can be manifested in, among other diseases, disorders, and conditions: hyperglycemia, type 2 diabetes, gestational diabetes, type 1 diabetes, insulin resistance, impaired glucose tolerance, hyperinsulinemia, impaired glucose metabolism, prediabetes, other metabolic disorders (e.g., metabolic syndrome, also known as syndrome X), and obesity. Polypeptides and compositions thereof of the present disclosure can be used, for example, to achieve and / or maintain glucose homeostasis, e.g., to reduce glucose levels in the bloodstream and / or reduce insulin levels to the range found in healthy subjects.

[0021] The term "hyperglycemia," as used herein, refers to a condition in which an elevated amount of glucose circulates in the plasma of a subject compared to a healthy individual. Hyperglycemia can be diagnosed using methods known in the art, such as measuring fasting blood glucose levels as described herein.

[0022] The term "hyperinsulinemia" as used herein refers to a state in which circulating insulin levels are elevated and blood glucose levels are elevated or normal.Hyperinsulinemia can be caused by dyslipidemia, such as insulin resistance associated with high triglycerides, high cholesterol, high low-density lipoprotein (LDL) levels, and low high-density lipoprotein (HDL) levels; high uric acid levels; polycystic ovary syndrome; type 2 diabetes, and obesity.Hyperinsulinemia can be diagnosed as having a plasma insulin level higher than about 2 μU / mL.

[0023] As used herein, the phrase "weight disorder" refers to a condition associated with excess weight and / or increased appetite. To determine whether a subject is overweight compared to a reference healthy individual, various parameters, such as the subject's age, height, sex, and health status, are used. For example, a subject can be considered overweight or obese by assessing the subject's body mass index (BMI), which is calculated by dividing the subject's weight (kilograms) by the square of the subject's height (meters). Approximately 18.5 kg / m 2 ~Approx. 24.9kg / m 2 Adults with a BMI in the range of about 25 kg / m are considered to have normal weight; 2 ~Approx. 29.9kg / m 2 An adult with a BMI of about 30 kg / m would be considered overweight (pre-obese). 2Adults with BMI above 100% can be considered obese.Increased appetite often contributes to overweight.There are several conditions associated with increased appetite, including night-time eating disorder syndrome, which is characterized by morning loss of appetite and nighttime overeating, and is often associated with insomnia, but may also be related to hypothalamus damage.

[0024] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, referring to the complete or partial prevention of a disease or its symptoms, and / or therapeutic, referring to the partial or complete cure of a disease and / or adverse effects resulting from a disease. "Treatment," as used herein, includes the treatment of disease in a mammal, e.g., a human, and includes (a) preventing the onset of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) alleviating the disease, i.e., causing regression of the disease.

[0025] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to individual organisms, e.g., mammals, including, but not limited to, murines, simians, humans, non-human primates, ungulates, felines, canines, bovines, ovines, mammalian livestock, mammalian sport animals, and mammalian pets. In some cases, the "individual" is a human.

[0026] Before the present invention is further described, it is to be understood that this invention is not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and not for purposes of limitation, since the scope of the present invention will be limited only by the appended claims.

[0027] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.Although any method and material similar or equivalent to those described herein can also be used in carrying out or testing this invention, preferred methods and materials are described herein.All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials related to which the publication is cited.

[0029] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "an antisense oligonucleotide" includes a plurality of such oligonucleotides, and a reference to "the nucleic acid modification" includes a reference to one or more nucleic acid modifications and equivalents thereof known to those skilled in the art, and so forth. It should be further noted that the claims may be drafted to exclude optional elements. Accordingly, this statement shall serve as an antecedent basis for use of exclusive terminology, such as "solely," "only," and the like, or for use of a "negative" limitation in reciting claim elements.

[0030] The use of the terms "a," "an," and "the" and similar referents with respect to the description of this disclosure (particularly with respect to the claims that follow) should be construed to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended (i.e., meaning "including, but not limited to"), unless otherwise noted. The recitation of ranges of values ​​herein, unless otherwise indicated herein, merely serves as a shorthand method for referring individually to each separate value within the range, and each separate value is incorporated herein as if individually set forth herein. For example, if a range of 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to further clarify aspects of the disclosure and do not limit the scope of the disclosure unless otherwise recited in the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of aspects of the disclosure.

[0031] As used herein, the term "about" when used in reference to a quantity indicates that the quantity may vary by 10% of the stated amount. For example, "about 100" means an amount of 90 to 110. When "about" is used in reference to a range, "about" when used in reference to the lower limit of a range means that an amount 10% lower than the lower limit of the range is included in the lower limit, and "about" when used in reference to the upper limit of a range means that an amount 10% higher than the upper limit of the range is included in the upper limit. For example, about 100 to about 1000 means that the range spans 90 to 1100.

[0032] As used herein, the term "and / or," e.g., the phrase "A and / or B," is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or," e.g., the phrase "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0033] It is understood that the aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0034] It is understood that certain features of the present disclosure that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments of the present disclosure are specifically embraced by the present disclosure and are disclosed herein as if each and every combination were individually and expressly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0035] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0036] Detailed Description The present disclosure provides inhibitory nucleic acids that reduce intracellular levels of miR-128-1, and compositions comprising the inhibitory nucleic acids. The present disclosure provides methods for treating various disorders using the inhibitory nucleic acids of the present disclosure.

[0037] inhibitory nucleic acid The present disclosure provides an inhibitory nucleic acid that reduces the level of miR-128-1 in a cell (e.g., a cell in an individual).In some cases, the inhibitory nucleic acid is an antisense oligonucleotide (ASO).The present disclosure provides an ASO that reduces the level of miR-128-1 in a cell (e.g., a cell in an individual).

[0038] In some cases, miR-128 is represented by the nucleotide sequence In some cases, miR-128 is identified by the nucleotide sequence In some cases, miR-128 is identified by the nucleotide sequence In some cases, miR-128 is identified by the nucleotide sequence In some cases, miR-128 is represented by the nucleotide sequence In some cases, miR-128 is identified by the nucleotide sequence Includes TIFF2025538360000006.tif9151.

[0039] In some cases, an inhibitory nucleic acid (e.g., ASO) of the disclosure reduces the level of miR-128-1 in a cell more potently than an inhibitory nucleic acid (e.g., ASO) comprising the nucleotide sequence 5'-TTCACTGTG-3'. In some cases, an inhibitory nucleic acid (e.g., ASO) of the disclosure is at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 40-fold more potent than an inhibitory nucleic acid (e.g., ASO) comprising the nucleotide sequence 5'-TTCACTGTG-3'. For example, in some cases, an inhibitory nucleic acid (e.g., ASO) of the present disclosure reduces the level of miR-128-1 in a cell to a degree at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, or greater than 90% greater than the degree to which an inhibitory nucleic acid (e.g., ASO) comprising the nucleotide sequence 5'-TTCACTGTG-3' reduces the level of miR-128-1 in a cell when administered in the same amount as the inhibitory nucleic acid (e.g., ASO) of the present disclosure. In some cases, an inhibitory nucleic acid (e.g., ASO) of the disclosure achieves at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 50%, at least a 75%, or at least a 90% reduction in the level of miR-128-1 in cells within an individual when the inhibitory nucleic acid (e.g., ASO) of the disclosure is administered to the individual in an amount that is at least 10%, at least a 15%, at least a 20%, at least a 25%, at least a 50%, at least a 75%, or at least a 90% less amount than the amount of an inhibitory nucleic acid (e.g., ASO) comprising the nucleotide sequence 5'-TTCACTGTG-3' required to achieve the same reduction in the level of miR-128-1.

[0040] In some cases, an inhibitory nucleic acid (e.g., ASO) of the disclosure reduces the level of miR-128-1 in a cell more potently than an inhibitory nucleic acid (e.g., ASO) comprising the nucleotide sequence 5'-GGTTCACTGTG-3' (SEQ ID NO: 14). In some cases, an inhibitory nucleic acid (e.g., ASO) of the disclosure is at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 40-fold more potent than an inhibitory nucleic acid (e.g., ASO) comprising the nucleotide sequence 5'-GGTTCACTGTG-3' (SEQ ID NO: 14). For example, in some cases, an inhibitory nucleic acid (e.g., ASO) of the present disclosure reduces the level of miR-128-1 in a cell to a degree at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, or greater than 90% greater than the degree to which an inhibitory nucleic acid (e.g., ASO) comprising the nucleotide sequence 5'-GGTTCACTGTG-3' (SEQ ID NO:14) reduces the level of miR-128-1 in a cell when administered in the same amount as the inhibitory nucleic acid (e.g., ASO) of the present disclosure. In some cases, an inhibitory nucleic acid (e.g., ASO) of the disclosure achieves at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 50%, at least a 75%, or at least a 90% reduction in the level of miR-128-1 in cells within an individual when the inhibitory nucleic acid (e.g., ASO) of the disclosure is administered to the individual in an amount that is at least 10%, at least a 15%, at least a 20%, at least a 25%, at least a 50%, at least a 75%, or at least a 90% less amount than the amount of an inhibitory nucleic acid (e.g., ASO) comprising the nucleotide sequence 5'-GGTTCACTGTG-3' (SEQ ID NO:14) required to achieve the same reduction in the level of miR-128-1.

[0041] Inhibitory nucleic acids useful in the methods and compositions of the present invention include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single-stranded or double-stranded RNA interference (RNAi) compounds, such as siRNA compounds, modified bases / locked nucleic acids (LNA), antagomir, peptide nucleic acids (PNA), and other oligomeric compounds or oligonucleotide mimics, which hybridize to at least a portion of target nucleic acid (i.e., miR-128) and modulate its function.In some embodiments, inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides containing modified bonds, interfering RNA (RNAi), small interfering RNA (siRNA); microinterfering RNA (miRNA); small transient RNA (stRNA); or small hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNA (saRNA), or combinations thereof.See, for example, WO 2010040112. In some cases, the inhibitory nucleic acids of the present disclosure are ASOs.

[0042] In some cases, the inhibitory nucleic acid is 10-50, 13-50, or 13-30 nucleotides in length. Those skilled in the art will understand that this specifically refers to oligonucleotides having antisense portions that are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any range therein. In some cases, the inhibitory nucleic acid of the present disclosure is 15 nucleotides in length. In some cases, the inhibitory nucleic acid of the present disclosure is 12-30 or 13-30 nucleotides in length. Those skilled in the art will understand that this specifically refers to inhibitory nucleic acids having a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0043] In some cases, the inhibitory nucleic acid is a chimeric oligonucleotide containing two or more chemically distinct regions, each composed of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confer one or more beneficial properties (e.g., increased nuclease resistance, increased cellular uptake, increased binding affinity to the target), and a region that is a substrate for an enzyme that can cleave RNA:DNA hybrids or RNA:RNA hybrids. The chimeric inhibitory nucleic acid of the present disclosure may be formed as a composite structure of two or more oligonucleotides, modified oligonucleotides, oligonucleosides, and / or oligonucleotide mimetics, as described above. Such compounds are also referred to in the art as hybrids or gapmers.

[0044] As one non-limiting example, in some cases, a suitable inhibitory nucleic acid comprises the following nucleotide sequence: 5'-ACCGGTTCACTGTG-3' (SEQ ID NO:1) and has a length of 14 to 20 nucleotides. In some cases, the inhibitory nucleic acid comprises the following nucleotide sequence: 5'-ACCGGTTCACTGTG-3' (SEQ ID NO:1) and has a length of 14 nucleotides. In some cases, the inhibitory nucleic acid comprises one or more of: (i) locked nucleic acid (LNA); (ii) a modified backbone; and (iii) 5-methyldeoxycytosine. In some cases, a suitable inhibitory nucleic acid comprises all of the nucleotide sequences and modifications shown in Figure 1A and designated "NRC0090." The ASO designated NRC0090 in Figure 1A is +A * +C * / iMe-dC / * G * G * +T * +T * +C * A * C * +T * G * +T * +G, where "+" precedes LNA; * precedes a phosphorothioated base, and / iMe-dC / indicates an internal 5-methyldeoxycytosine.

[0045] As another non-limiting example, in some cases, a suitable inhibitory nucleic acid comprises the following nucleotide sequence: 5'-GACCGGTTCACTGT-3' (SEQ ID NO:2) and has a length of 14 to 20 nucleotides. In some cases, the inhibitory nucleic acid comprises the following nucleotide sequence: 5'-GACCGGTTCACTGT-3' (SEQ ID NO:2) and has a length of 14 nucleotides. In some cases, the inhibitory nucleic acid comprises one or more of: (i) locked nucleic acid (LNA); (ii) a modified backbone; and (iii) 5-methyldeoxycytosine. In some cases, a suitable inhibitory nucleic acid comprises all of the nucleotide sequences and modifications shown in Figure 1A and designated "NRC0091." The ASO designated NRC0091 in Figure 1A is a +G * +A * C * / iMe-dC / * +G * G * T * T * +C * +A * C * +T * +G * +T, where "+" precedes LNA; * precedes a phosphorothioated base, and / iMe-dC / indicates an internal 5-methyldeoxycytosine.

[0046] As another non-limiting example, in some cases, a suitable inhibitory nucleic acid comprises the following nucleotide sequence: 5'-AGACCGGTTCACTGTG-3' (SEQ ID NO:3) and has a length of 14 to 20 nucleotides. In some cases, the inhibitory nucleic acid comprises the following nucleotide sequence: AGACCGGTTCACTGTG-3' (SEQ ID NO:3) and has a length of 14 nucleotides. In some cases, the inhibitory nucleic acid comprises one or more of: (i) locked nucleic acid (LNA); (ii) a modified backbone; and (iii) 5-methyldeoxycytosine. In some cases, a suitable inhibitory nucleic acid comprises all of the nucleotide sequences and modifications shown in Figure 1A and designated "NRCO119." The ASO designated NRC0119 in Figure 1A is a +A * +G * A * +C * / iMe-dC / * +G * G * +T * T * +C * A * C * +T * G * +T * +G, where "+" precedes LNA; * precedes a phosphorothioated base, and / iMe-dC / indicates an internal 5-methyldeoxycytosine.

[0047] In some embodiments, the inhibitory nucleic acid contains at least one nucleotide modified at the 2' position of the sugar, such as a 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro modified nucleotide. In other cases, RNA modifications include 2'-fluoro, 2'-amino, and 2'-O-methyl modifications on the ribose of pyrimidines, abasic residues or inverted bases at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides, and these oligonucleotides have been shown to have higher Tm (i.e., higher target binding affinity) for a given target than 2'-deoxyoligonucleotides.

[0048] A number of nucleotide and nucleoside modifications have been shown to render the oligonucleotides into which they are incorporated more resistant to nuclease digestion than native oligodeoxynucleotides; these modified oligonucleotides remain intact for longer periods of time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those containing modified backbones, such as phosphorothioates, phosphotriesters, methylphosphonates, short alkyl or cycloalkyl intersugar linkages, or short heteroatom or heterocyclic intersugar linkages. In some cases, inhibitory nucleic acids include oligonucleotides having phosphorothioate backbones, as well as heteroatom backbones, specifically CH2--NH--O--CH3, CH3--N(CH3)--O--CH2 (known as methylene (methylimino) or MMI backbones), CH2--O--N(CH3)--CH2, CH2--N(CH3)--N(CH3)--CH2, and O--N(CH3)--CH2--CH2 backbones (the native phosphodiester backbone is represented as O--P--O--CH); amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbone structures (Summerton and Weller, U.S. Pat. No. 5,034,506); or oligonucleotides having a peptide nucleic acid (PNA) backbone (in which the phosphodiester backbone of an oligonucleotide is replaced with a polyamide backbone and the nucleotides are bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone; see Nielsen et al., Science 1991, 254, 1497).Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters; aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates, e.g., 3' alkylene phosphonates, and chiral phosphonates, phosphinates, phosphoramidates, e.g., 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with opposite polarity where pairs of adjacent nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0049] Modified oligonucleotide backbones that do not contain internal phosphorus atoms have backbones formed by short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and methylenethioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 moieties.

[0050] One or more substituted sugar moieties may be included at the 2' position, for example, one of the following: OH, SH, SCH, F, OCN, OCHOCH, OCH, O(CH)CH, O(CH)NH, or O(CH)CH, where n is 1 to about 10; C-C lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl, or aralkyl; Cl; Br; CN; CF; OCF; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH; SOCH; ONO; NO; N; NH; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleaving group; reporter group; intercalator; group for improving the pharmacokinetic properties of oligonucleotides; or group for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Suitable modifications include 2'-methoxyethoxy [2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl)] (Martin et al., Helv. Chim. Acta, 1995, 78, 486). Other modifications include 2'-methoxy (2'-O-CH3), 2'-propoxy (2'-OCH2CH2CH3), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the oligonucleotide, specifically the 3' position of the sugar on the 3'-terminal nucleotide and the 5' position of the 5'-terminal nucleotide. Oligonucleotides can also have sugar mimetics, such as cyclobutyls, in place of the pentofuranosyl group.

[0051] Inhibitory nucleic acids may additionally or alternatively contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases that are found only rarely or transiently in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidines, specifically 5-methylcytosine (also known as 5-methyl-2'deoxycytosine, often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentobiosyl HMC, as well as synthetic nucleobases such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine, or other hetero-substituted alkyl adenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, and 2,6-diaminopurine. "Universal" bases known in the art, such as inosine, may also be included. 5-Me-C substitutions increase nucleic acid duplex stability by 0.6-12. <0> It has been shown that 5-Me-C increases the C. In some cases, the inhibitory nucleic acids of the disclosure include one or more 5-Me-C.

[0052] It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the above modifications may be incorporated into a single oligonucleotide, or even at a single nucleoside within an oligonucleotide.

[0053] In some embodiments, both the sugar and internucleoside linkage, i.e., the backbone, of a nucleotide unit are replaced with novel groups. The base unit is maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an oligonucleotide is replaced with an amide-containing backbone, such as an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference. Further teachings on PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0054] Inhibitory nucleic acids may also contain one or more nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6-azocytosine, and 6- These include azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted forms of adenine and guanine, 5-halo, specifically 5-bromo, 5-trifluoromethyl, and other 5-substituted forms of uracil and cytosine, 7-methylquanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine and adenine.

[0055] Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in 'The Concise Encyclopedia of Polymer Science and Engineering', pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, those disclosed in English et al., Angewandle Chemie, International Edition, 1991, 30, page 613, and those disclosed in Sanghvi, YS, Chapter 15, 'Antisense Research and Applications', pages 289-302, Crooke, ST and Lebleu, B. ea., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of inhibitory nucleic acids. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2-, N-6-, and O-6-substituted purines, such as 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine, with 5-methylcytosine substitutions increasing nucleic acid duplex stability by 0.6 to 1.2. <0> It has been shown to increase C by 2'-O-methoxyethyl sugar modifications (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., 'Antisense Research and Applications', CRC Press, Boca Raton, 1993, pp. 276-278), making it suitable for inclusion in inhibitory nucleic acids, for example, alone or in combination with 2'-O-methoxyethyl sugar modifications.

[0056] In some cases, inhibitory nucleic acid is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of oligonucleotide.Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties; cholic acid; thioethers, such as hexyl-S-tritylthiol; thiocholesterol; aliphatic chains, such as dodecanediol or undecyl residues; phospholipids; for example, di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate; polyamines or polyethylene glycol chains; adamantane acetic acid; palmityl moieties; octadecylamine moieties; or hexylamino-carbonyl-t-oxycholesterol moieties.

[0057] These moieties or conjugates can include functional groups, such as conjugate groups covalently bonded to primary or secondary hydroxyl groups. Suitable conjugate groups for use include intercalators, importer molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterol, lipids, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. Groups that enhance pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and / or strengthen sequence-specific hybridization with target nucleic acids. Groups that enhance pharmacokinetic properties include groups that improve uptake, distribution, metabolism, or excretion of inhibitory nucleic acids. Representative conjugate groups are disclosed in International Patent Application No. PCT / US92 / 09196, filed October 23, 1992, and U.S. Patent No. 6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acid, thioethers such as hexyl-5-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0058] The inhibitory nucleic acid useful in the method of the present invention is sufficiently complementary to all or a portion of miR-128, i.e., hybridizes sufficiently well and with sufficient specificity, to produce the desired effect. "Complementary" refers to the ability of pairing through hydrogen bonds between two sequences containing naturally occurring or non-naturally occurring bases or their analogs. For example, if a base at a certain position in the inhibitory nucleic acid can hydrogen bond with a base at a corresponding position in the miR-128 sequence, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.

[0059] In the present disclosure, hybridization refers to hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleic acid bases that pair through the formation of hydrogen bonds. "Complementary" as used herein refers to the ability of precise pairing between two nucleotides. An inhibitory nucleic acid and miR-128 are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other. Therefore, "specifically hybridizable" and "complementary" are terms used to indicate a sufficient degree of complementarity or precise pairing for stable and specific binding between an inhibitory nucleic acid and a miR-128 target sequence. For example, if a base at a certain position in an inhibitory nucleic acid can hydrogen bond with a base at a corresponding position in a miR-128 molecule, then the bases at that position are considered to be complementary to each other.

[0060] Although 100% complementarity is desirable in some embodiments, it is understood in the art that a complementary nucleic acid sequence need not be 100% complementary to the sequence of its target nucleic acid to be specifically hybridizable. Complementary nucleic acid sequences for purposes of the methods of the present invention are specifically hybridizable if binding of the sequence to the target miR-128 molecule interferes with the normal function of the target miR-128, causing loss of activity, and are sufficiently complementary to avoid non-specific binding of these sequences to non-target miR-128 sequences under conditions where specific binding is desired, e.g., physiological conditions in the case of in vivo assays or therapeutic treatments, and under stringency conditions under which the assay is performed in the case of in vitro assays. For example, stringent salt concentrations are usually less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide or at least about 50% formamide. Stringent temperature conditions usually include a temperature of at least 30°C, at least about 37°C, or at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergents, such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. As a non-limiting example, in some cases hybridization is performed in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30° C. As another non-limiting example, in some cases hybridization is performed in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C.As another non-limiting example, in some cases, hybridization is carried out in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations of these conditions will be readily apparent to one of skill in the art.

[0061] Generally, inhibitory nucleic acids useful in the methods described herein have at least 80% sequence complementarity to a target region within a target nucleic acid, e.g., 90%, 95%, or 100% sequence complementarity to a target region within miR-128 (e.g., a target region containing a seed sequence). For example, an antisense compound in which 18 of the 20 nucleobases of an antisense oligonucleotide are complementary to the target region and therefore specifically hybridizes exhibits 90% complementarity. The percent complementarity between an inhibitory nucleic acid and a region of a target nucleic acid can be routinely determined using the Basic Local Alignment Search Tool (BLAST program) (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). Inhibitory nucleic acids (e.g., ASOs) that hybridize to miR-128 target sequences can be identified through routine experimentation. Generally, inhibitory nucleic acids must retain specificity for their targets, i.e., they must not directly bind to or directly significantly affect the expression levels of transcripts other than the intended target.

[0062] antisense As mentioned above, in some cases, the inhibitory nucleic acid of the present disclosure is ASO.ASO is typically designed to block the expression of DNA or RNA target by binding to target and stopping expression at the level of transcription, translation or splicing.The ASO of the present disclosure is a complementary nucleic acid sequence designed to hybridize with miR-128 target sequence under stringent conditions.Therefore, to produce the desired effect, the oligonucleotide is selected to be sufficiently complementary to target, i.e., hybridize sufficiently well and with sufficient specificity.

[0063] Modified bases / locked nucleic acids (LNA) In some cases, the inhibitory nucleic acid of the present disclosure contains one or more modified linkages or bases. Modified bases include phosphorothioates, methylphosphonates, peptide nucleic acids, or locked nucleic acid (LNA) molecules. For example, in some cases, the modified nucleotide is a locked nucleic acid molecule, such as [α]-L-LNA. LNA is a ribonucleic acid analog in which the ribose ring is "locked" by a methylene bridge between the 2'-oxygen and the 4'-carbon, i.e., an oligonucleotide containing at least one LNA monomer, i.e., one 2'-O,4'-C-methylene-β-D-ribofuranosyl nucleotide. LNA bases form standard Watson-Crick base pairs, but the locked configuration increases the rate and stability of the base pairing reaction (Jepsen et al., Oligonucleotides, 14, 130-146 (2004)). LNA also has an increased affinity for base pairing with RNA compared to DNA. These properties make LNAs particularly useful as probes for fluorescence in situ hybridization (FISH) and comparative genomic hybridization, as miRNA knockdown tools, and as antisense oligonucleotides for targeting mRNA or other RNAs.

[0064] LNA molecules can include molecules in which one strand contains 10 to 30, e.g., 12 to 24, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, to a miR-128 target sequence, with, e.g., 3, 2, 1, or 0 mismatched nucleotides. LNA molecules can be chemically synthesized using methods known in the art.

[0065] Antagomir In some cases, the inhibitory nucleic acid is an antagomir. An antagomir is a chemically modified antisense oligonucleotide that targets a miR-128 target sequence. For example, an antagomir for use in the methods described herein may contain a nucleotide sequence sufficiently complementary to hybridize to a miR-128 target sequence of about 12 to 25 nucleotides or about 15 to 23 nucleotides.

[0066] Generally, antagomir contains a cholesterol moiety, for example, at the 3' end. In some embodiments, antagomir has various modifications for protection from RNase and pharmacological properties, such as enhanced uptake into tissues and cells. For example, in addition to the modifications described above for antisense oligos, antagomir can have one or more of full or partial 2'-O-methylation of the sugar and / or a phosphorothioate backbone. The phosphorothioate modification provides protection against RNase activity, and its lipophilicity contributes to enhanced uptake into tissues. In some embodiments, antagomir can contain six phosphorothioate backbone modifications (two phosphorothioates at the 5' end and four at the 3' end). An antagomir useful in the methods of the present invention may be modified in terms of its length or the number of nucleotides comprising the antagomir. An antagomir must retain specificity for its target, i.e., it must not directly bind to or directly significantly affect the expression level of transcripts other than the intended target. In some embodiments, the inhibitory nucleic acid is locked and includes a cholesterol moiety (eg, locked antagomir).

[0067] siRNA / shRNA In some cases, the inhibitory nucleic acid of the present disclosure is an interfering RNA, for example, but not limited to, a small interfering RNA (" siRNA ") or a short hairpin RNA (" shRNA "). Methods for constructing an interfering RNA are well known in the art. For example, an interfering RNA can be assembled from two separate oligonucleotides, where one strand is a sense strand and the other is an antisense strand, and the antisense strand and the sense strand are self-complementary (i.e., each strand comprises a nucleotide sequence that is complementary to the nucleotide sequence of the other strand; for example, the antisense strand and the sense strand form a duplex or double-stranded structure); the antisense strand comprises a nucleotide sequence that is complementary to the nucleotide sequence of a target nucleic acid molecule or a part thereof (i.e., an undesired gene), and the sense strand comprises a nucleotide sequence that corresponds to the target nucleic acid sequence or a part thereof. Alternatively, an interfering RNA can be assembled from a single oligonucleotide, where the self-complementary sense region and the antisense region are connected by a nucleic acid-based or non-nucleic acid-based linker. Interfering RNA can be the polynucleotide of double-stranded, asymmetric double-stranded, hairpin or asymmetric hairpin secondary structure, with self-complementary sense region and antisense region, wherein antisense region comprises the nucleotide sequence that is complementary to the nucleotide sequence of another target nucleic acid molecule or a part thereof, and sense region has the nucleotide sequence that corresponds to target nucleic acid sequence or a part thereof.Interference can also be the circular single-stranded polynucleotide of the stem, which comprises two or more loop structures and comprises self-complementary sense region and antisense region, wherein antisense region comprises the nucleotide sequence that is complementary to the nucleotide sequence of target nucleic acid molecule or a part thereof, and sense region has the nucleotide sequence that corresponds to target nucleic acid sequence or a part thereof, and circular polynucleotide can be processed in vivo or in vitro to produce the active siRNA molecule that can mediate RNA interference.

[0068] In some cases, the interfering RNA coding region encodes a self-complementary RNA molecule having a sense region, an antisense region, and a loop region. Such RNA molecules, when expressed, desirably form a "hairpin" structure and are referred to herein as "shRNAs." The loop region is generally about 2 to about 10 nucleotides in length. In some embodiments, the loop region is about 6 to about 9 nucleotides in length. In some embodiments, the sense and antisense regions are about 15 to about 20 nucleotides in length. After post-transcriptional processing, the short hairpin RNA is converted into siRNA by a cleavage event mediated by the enzyme Dicer, a member of the RNase III family. The siRNA can then inhibit expression of genes with which it shares homology.

[0069] The target RNA cleavage reaction induced by siRNA is highly sequence-specific.Generally, siRNA containing the same nucleotide sequence as a part of target nucleic acid is used for inhibition.However, 100% sequence identity between siRNA and target gene is not required.Therefore, the present disclosure has the advantage that it can tolerate sequence variations that can be expected due to genetic mutation, lineage polymorphism, or evolutionary divergence.For example, siRNA sequences with insertions, deletions, and single point mutations compared to target sequences have also been found to be effective for inhibition.Alternatively, siRNA sequences containing substitutions or insertions by nucleotide analogs can be effective for inhibition.Generally, siRNA must maintain its specificity for its target, i.e., it must not directly bind to or directly significantly affect the expression level of transcripts other than the intended target.

[0070] Preparation of inhibitory nucleic acids Inhibitory nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, for example, as described in Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.

[0071] Pharmaceutical Compositions The present disclosure provides compositions, e.g., pharmaceutical compositions, comprising the inhibitory nucleic acids of the present disclosure. The inhibitory nucleic acids of the present disclosure are also referred to hereinafter as "drugs" or "active agents."

[0072] In some cases, the composition is formulated with a pharmaceutically acceptable carrier. Pharmaceutical compositions and formulations can be administered parenterally, topically, orally, or by local administration, for example, by aerosol, or transdermally. Pharmaceutical compositions can be formulated in any manner and can be administered in a variety of unit dosage forms depending on the condition or disease and the extent of the disease, the overall medical condition of each patient, the resulting preferred method of administration, etc. Details regarding techniques for pharmaceutical formulation and administration are fully described in the scientific and patent literature; see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

[0073] The inhibitory nucleic acid can be administered alone or as a component of a pharmaceutical preparation (composition).The inhibitory nucleic acid can be formulated for administration in any convenient way for use in human medicine or veterinary medicine.Wetting agents, emulsifying agents, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants can also be present in the composition.

[0074] Formulations of the inhibitory nucleic acids of the present disclosure include those suitable for intradermal, inhalation, oral / nasal, topical, parenteral, rectal, and / or vaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient (e.g., inhibitory nucleic acid of the present disclosure) that can be combined with a carrier material to produce a single dosage form will vary depending on the host treated and the particular mode of administration, e.g., intradermal or inhalation. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of compound that produces a therapeutic effect, e.g., an antigen-specific T cell response or humoral response.

[0075] Pharmaceutical preparations can be prepared according to any method known to those skilled in the art for the manufacture of pharmaceuticals. Such drugs can contain sweeteners, flavorings, colorings, and preservatives. Preparations can be mixed with non-toxic pharmaceutically acceptable excipients suitable for manufacturing. Preparations can contain one or more diluents, emulsifiers, preservatives, buffers, excipients, etc., and can be provided in the form of liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled-release preparations, tablets, pills, gels, patches, implants, etc.

[0076] Pharmaceutical preparations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate dosage amounts. Such carriers allow the formulation of medicines into unit dosage forms suitable for patient ingestion, such as tablets, pills, powders, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc. Pharmaceutical preparations for oral use can be formulated as solid excipients, optionally by grinding the resulting mixture and processing the granular mixture to obtain tablets or dragee cores, optionally after adding suitable additional compounds. Suitable solid excipients include carbohydrate or protein fillers, such as sugars, e.g., lactose, sucrose, mannitol, or sorbitol; starches derived from corn, wheat, rice, potato, or other plants; celluloses, e.g., methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; and gums, e.g., gum arabic and tragacanth; and proteins, e.g., gelatin and collagen. Disintegrants or solubilizers, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or its salts, such as sodium alginate, may be added. Push-fit capsules may contain the active agent mixed with a filler or binder, such as lactose or starch, a lubricant, such as talc or magnesium stearate, and optionally, a stabilizer. In soft capsules, the active agent may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol, with or without a stabilizer.

[0077] Aqueous suspensions may contain the active agent (e.g., an inhibitory nucleic acid of the present disclosure) mixed with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injection. Such excipients include suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia, as well as dispersing or wetting agents, such as naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, aspartame, or saccharin. The preparations may be adjusted for osmotic pressure.

[0078] In some cases, oily pharmaceuticals are used for the administration of inhibitory nucleic acids.Oily suspensions can be prepared by suspending active agents in vegetable oils, such as peanut oil, olive oil, sesame oil, or coconut oil, or mineral oils, such as liquid paraffin; or mixtures thereof.See, for example, U.S. Patent No. 5,716,928, which describes the use of essential oils or essential oil components to increase the bioavailability of orally administered hydrophobic pharmaceutical compounds and reduce inter- and intra-individual variability (see also U.S. Patent No. 5,858,401).Oily suspensions can contain thickeners, such as beeswax, hard paraffin, or cetyl alcohol.Sweeteners, such as glycerol, sorbitol, or sucrose, can be added to provide a palatable oral preparation.These preparations can be preserved by adding antioxidants, such as ascorbic acid. For examples of injectable oil vehicles, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.

[0079] Pharmaceutical preparations may be in the form of an oil-in-water emulsion. The oil phase may be the aforementioned vegetable oil or mineral oil, or a mixture thereof. Suitable emulsifiers include naturally occurring gums, such as gum arabic and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Emulsions may also contain sweeteners and flavoring agents, for example, in the case of syrup and elixir formulations. Such formulations may also contain demulcents, preservatives, or coloring agents. In an alternative embodiment, the injectable oil-in-water emulsion contains paraffin oil, sorbitan monooleate, ethoxylated sorbitan monooleate, and / or ethoxylated esbitan trioleate.

[0080] Pharmaceutical compositions can be administered via intranasal, intraocular, and intravaginal routes, such as suppositories, insufflation, powders, and aerosol formulations (for examples of steroid inhalants, see, e.g., Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75:107-111). Suppository formulations can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at body temperature, thus melting and releasing the drug in the body. Such materials include cocoa butter and polyethylene glycol.

[0081] In some cases, pharmaceutical compositions formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols may be delivered by topical routes, transdermally.

[0082] In some cases, pharmaceutical compositions can be delivered as microspheres for sustained release in the body.For example, microspheres can be administered by subcutaneously slowly releasing drug through intradermal injection (see Rao (1995) J.Biomater Sci.Polym.Ed.7:623-645); as biodegradable and injectable gel formulation (see, for example, Gao (1995) Pharm.Res.12:857-863 (1995)); or as oral administration microspheres (see, for example, Eyles (1997) J.Pharm.Pharmacol.49:669-674).

[0083] In some cases, pharmaceutical compositions may be administered parenterally, for example, by intravenous (IV) administration or administration into a body cavity or lumen of an organ. These formulations may contain a solution of an active agent (e.g., an inhibitory nucleic acid of the present disclosure) in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that may be used include water and Ringer's solution, isotonic sodium chloride. Additionally, sterile, fixed oils may be used as a solvent or suspending medium. For this purpose, any bland, fixed oil, such as synthetic monoglycerides or diglycerides, may be used. Additionally, fatty acids, such as oleic acid, may also be used in the preparation of injectable solutions. These solutions are sterile and generally free of undesirable substances. These formulations may be sterilized by conventional, well-known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the active agent in these formulations can vary widely and is selected primarily based on the volume, viscosity, body weight, etc. of the liquid, depending on the particular mode of administration selected and the patient's needs. Formulations for intravenous administration can be sterile injectable preparations, such as sterile injectable aqueous or oily suspensions. The suspensions can be formulated using appropriate dispersing or wetting agents and suspending agents. The sterile injectable preparations can also be suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions of 1,3-butanediol. Administration can be by bolus injection or continuous infusion (e.g., substantially uninterrupted introduction into the blood vessels for a specified period of time).

[0084] In some cases, the pharmaceutical composition can be a lyophilized product. A stable lyophilized composition containing an inhibitory nucleic acid can be produced by lyophilizing a solution containing the pharmaceutical composition of the present disclosure and a bulking agent, such as mannitol, trehalose, raffinose, and sucrose, or a mixture thereof. The process for preparing a stable lyophilized formulation can include lyophilizing a solution containing about 2.5 mg / mL of nucleic acid, about 15 mg / mL of sucrose, about 19 mg / mL of NaCl, and a sodium citrate buffer solution having a pH greater than 5.5 and less than 6.5. See, for example, US20040028670.

[0085] Compositions and formulations can be delivered by using liposomes.By using liposomes, the delivery of active agents (such as the inhibitory nucleic acids of the present disclosure) can be focused to target cells in vivo, especially when the liposome surface carries a ligand specific to target cells or is otherwise preferentially directed to a specific organ.See, for example, U.S. Patent No. 6,063,400; 6,007,839; Al-Muhammed (1996) J.Microencapsul.13:293-306; Chonn (1995) Curr.Opin.Biotechnol.6:698-708; Ostro (1989) Am.J.Hosp.Pharm.46:1576-1587.As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged as a bilayer. Liposomes are unilamellar or multilamellar vesicles with a membrane made of lipophilic material and an inner aqueous phase containing the composition to be delivered.Cationic liposomes are positively charged liposomes that are thought to interact with negatively charged DNA molecules to form stable complexes.pH-sensitive liposomes or negatively charged liposomes are thought to capture DNA rather than form complexes with DNA.Both cationic and non-cationic liposomes have been used to deliver DNA to cells.

[0086] Liposomes may also include "sterically stabilized" liposomes, i.e., liposomes containing one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation life compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the lipid moiety forming the liposome vesicle contains one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Liposomes and their uses are further described in U.S. Patent No. 6,287,860.

[0087] The preparation of the present disclosure can be administered for preventive treatment and / or therapeutic treatment.In some cases, the composition for therapeutic application is administered to the subject in need thereof (for example, the individual who has the risk of the disorder described herein (for example, the risk higher than the general population) or has the disorder described herein) in an amount sufficient to cure, alleviate or partially stop the clinical manifestation of disorder or its complications; this can be called therapeutically effective amount.

[0088] The amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose. Effective administration schedules and amounts, i.e., dosing regimens, for this use depend on a variety of factors, such as the stage of the disease or condition, the severity of the disease or condition, the patient's general health, the patient's physical condition, age, etc. In calculating the dosing regimen for a patient, the mode of administration is also taken into consideration.

[0089] The dosing regimen also takes into account pharmacokinetic parameters well known in the art, i.e., the rate of absorption, bioavailability, metabolism, clearance, etc., of the active agent (see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005). The state of the art allows physicians to determine the dosing regimen for each individual patient, active agent (e.g., inhibitory nucleic acid), and disease or condition being treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance for determining the dosing regimen, i.e., the dosing schedule and dosage level.

[0090] For example, single or multiple administrations of the formulation may be given, depending on the dosage and frequency required and tolerated by the patient, the extent and amount of therapeutic effect (e.g., effect on blood glucose levels) occurring after each administration, etc. The formulation should provide a sufficient amount of the active agent (e.g., inhibitory nucleic acid) to effectively treat, prevent, or ameliorate a condition, disease, or symptom.

[0091] In an alternative embodiment, the daily dosage of a pharmaceutical formulation for oral administration is about 1 μg to about 100 mg of nucleic acid per kilogram of body weight per day. In contrast to oral administration, lower dosages may be used for the bloodstream, body cavities, or organ lumens. Substantially higher dosages may be used for topical or oral administration, or for administration by powder, spray, or inhalation. Actual methods for preparing formulations suitable for parenteral or oral administration are known or apparent to those skilled in the art and are described in more detail in publications such as Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

[0092] In some embodiments, the methods described herein can include co-administration with other drugs or medications, such as compositions for lowering blood glucose levels. For example, the inhibitory nucleic acids can be co-administered with drugs for treating or reducing the risk of a disorder described herein.

[0093] Treatment methods - metabolic disorders The present disclosure provides methods of using the inhibitory nucleic acids of the present disclosure in subjects suffering from, for example, metabolic syndrome (e.g., high LDL, low HDL, high triglycerides, obesity, non-alcoholic fatty liver disease, insulin resistance, and / or hypertension), type 2 diabetes, and / or cardiovascular disease (CVD). The treatment methods of the present disclosure provide one or more of the following: (a) lowering circulating LDL; (b) increasing HDL; (c) lowering triglycerides; (d) reducing obesity; (e) reducing insulin resistance and type 2 diabetes; (f) ameliorating non-alcoholic fatty liver disease; and (g) reducing atherosclerosis / CVD. The methods include administering an effective amount of the inhibitory nucleic acid of the present disclosure or a composition (e.g., a pharmaceutical composition) comprising the same to an individual in need thereof. In some cases, the inhibitory nucleic acid of the present disclosure is administered as a lipid nanoparticle. In some cases, the inhibitory nucleic acid of the present disclosure is administered as a liposome.

[0094] A "therapeutically effective amount" refers to the administration of an agent (e.g., an inhibitory nucleic acid) to a subject, either as a single dose or as part of a series of doses, alone or as part of a pharmaceutical composition, in an amount that, when administered to a patient, is capable of having a detectable positive effect on any symptom, aspect, or characteristic of a disease, disorder, or condition. A therapeutically effective amount can be ascertained by measuring the relevant physiological effect. In some cases, for example, in the case of a hyperglycemic condition, a drop or decrease in blood glucose or an improvement in a glucose tolerance test can be used to determine whether the amount of agent is effective to treat the hyperglycemic condition. A therapeutically effective amount can be adjusted in relation to the dosing regimen, diagnostic analysis of the subject's condition, etc.

[0095] In some cases, a therapeutically effective amount of an inhibitory nucleic acid of the disclosure is an amount that, when administered in one or more doses, is sufficient to lower or decrease any level (e.g., baseline level) of fasting plasma glucose (FPG), e.g., the amount is sufficient to reduce an FPG level greater than 200 mg / dl to less than 200 mg / dl, the amount is sufficient to reduce an FPG level between 175 mg / dl and 200 mg / dl to below the starting level, the amount is sufficient to reduce an FPG level between 150 mg / dl and 175 mg / dl to below the starting level, the amount is sufficient to reduce an FPG level between 125 mg / dl and 150 mg / dl to below the starting level, etc. (e.g., reducing FPG levels to less than 125 mg / dl, less than 120 mg / dl, less than 115 mg / dl, less than 110 mg / dl, etc.).

[0096] In some cases, a therapeutically effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered in one or more doses, is sufficient to lower or reduce hemoglobin A1c (HbA1c) levels by about 10% to 9% or more, about 9% to 8% or more, about 8% to 7% or more, about 7% to 6% or more, about 6% to 5% or more, etc. In some cases, a therapeutically effective amount of an inhibitory nucleic acid of the present disclosure is an amount that is sufficient to lower or reduce HbA1c levels by about 0.1%, 0.25%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 33%, 35%, 40%, 45%, 50% or more.

[0097] In some cases, a therapeutically effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered in one or more doses, is sufficient to result in insulin levels within the normal range.

[0098] In some cases, a therapeutically effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered in one or more doses, is sufficient to bring serum alanine transaminase (ALT) levels within the normal range. In some cases, a therapeutically effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered in one or more doses, is sufficient to reduce serum ALT levels by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or more than 50% compared to serum ALT levels before treatment. In some cases, a therapeutically effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered in one or more doses, is sufficient to bring serum aspartate transaminase (AST) levels within the normal range. In some cases, a therapeutically effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered in one or more doses, is sufficient to reduce serum AST levels by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or more than 50% compared to serum AST levels before treatment.

[0099] In some cases, an effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered to a subject in one or more doses, produces a desired result compared to a healthy subject. For example, an effective dose can be one that, when administered to a subject with elevated plasma glucose and / or plasma insulin, achieves a desired reduction by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more than 80% compared to a healthy subject.

[0100] In some cases, an effective amount of an inhibitory nucleic acid of the disclosure, when administered to a subject in one or more doses, will increase a BMI within the normal range, e.g., 18.5 kg / m 2 ~Approx. 24.9kg / m 2In some cases, an effective amount of an inhibitory nucleic acid of the present disclosure is an amount that reduces a starting BMI within 30 kg / m 2 If higher, it is an amount that, when administered to a subject in one or more doses, reduces BMI by at least 5%, at least 10%, or at least 15%.

[0101] Route of administration Suitable routes of administration include oral, rectal, nasal, pulmonary, topical, subcutaneous, intramuscular, intraperitoneal, intravenous, intradermal, intrathecal, and epidural. In some cases, the route of administration is intramuscular. In some cases, the route of administration is intravenous.

[0102] Combination therapy The present disclosure contemplates the use of the inhibitory nucleic acid of the present disclosure in combination with one or more additional drugs (for example, one or more additional active therapeutic agents) or other preventive or therapeutic modalities.In such combination therapy, various active drugs often have different mechanisms of action.Such combination therapy can be particularly advantageous by allowing for the reduction of the dose of one or more drugs, thereby reducing or eliminating the adverse effects associated with one or more drugs; Moreover, such combination therapy can exert synergistic therapeutic or preventive effects on the underlying disease, disorder, or condition.

[0103] As used herein, "combination" is intended to include therapies that may be administered separately, e.g., formulated separately for separate administration (e.g., as may be provided in a kit), and therapies that may be administered together in a single formulation (i.e., a "combination").

[0104] In certain cases, the inhibitory nucleic acid of the present disclosure and at least one additional agent are administered or applied sequentially, for example, one agent is administered before one or more other agents. In other cases, the inhibitory nucleic acid of the present disclosure and at least one additional agent are administered simultaneously, for example, two or more agents are administered simultaneously or nearly simultaneously; the two or more agents may be present in two or more separate formulations or may be combined into a single formulation (i.e., a combination formulation). For the purposes of this disclosure, two or more agents are considered to be administered in combination, regardless of whether they are administered sequentially or simultaneously.

[0105] The inhibitory nucleic acids of the present disclosure can be used in combination with other agents useful in the treatment of the disorders or conditions described herein, such as those normally administered to subjects suffering from obesity, eating disorders, hyperglycemia, hyperinsulinemia, glucose intolerance, and other glucose metabolism disorders.

[0106] The present disclosure contemplates combination therapy with numerous agents (and classes thereof), including: (1) insulin, insulin mimetics, and agents involving stimulation of insulin secretion, such as sulfonylureas (e.g., chlorpropamide, tolazamide, acetohexamide, tolbutamide, glyburide, glimepiride, glipizide) and meglitinides (e.g., mitiglinide, repaglinide, and nateglinide); (2) biguanides (e.g., metformin and pharmaceutically acceptable salts thereof, specifically metformin hydrochloride and its extended-release formulations, e.g., Glumetza™, Fortamet™, and GlucophageXR™), and other agents that act by promoting glucose utilization, reducing hepatic glucose production, and / or decreasing intestinal glucose release; (3) α-glucosidase inhibitors (e.g., acarbose, voglibose, and miglitol), etc. and other drugs that delay carbohydrate digestion, thereby delaying absorption from the intestinal tract and reducing postprandial hyperglycemia; (4) thiazolidinediones (e.g., rosiglitazone, troglitazone, pioglitazone, glipizide, balaglitazone, rivoglitazone, netoglitazone, AMG131, MBX2044, mitoglitazone, lobeglitazone, IDR-105, troglitazone, englitazone, ciglitazone, adaglitazone, darglitazone) that enhance (e.g., by insulin sensitization) the action of insulin, including insulin and insulin mimetics (e.g., insulin degludec, insulin glargine, insulin lispro, insulin detemir, insulin glulisine, and inhalable formulations of each), thereby promoting glucose utilization in peripheral tissues;(5) glucagon-like peptides, such as DPP-IV inhibitors (e.g., alogliptin, omarigliptin, linagliptin, vildagliptin, and sitagliptin), and glucagon-like peptide-1 (GLP-1) and GLP-1 agonists and analogs (e.g., exenatide (BYETTA and ITCA 650 (a subcutaneously inserted osmotic pump that delivers an exenatide analog over 12 months; Intarcia, Boston, Mass.)), and GLP-1 receptor agonists (e.g., dulaglutide, semaglutide, albiglutide, exenatide, liraglutide, lixisenatide, taspoglutide, CJC-1131, and BIM-51077, e.g., intranasal, transdermal, and once-weekly formulations thereof);and (6) DPP-IV resistance analogs (incretin mimetics), PPARγ agonists, PPARα agonists, such as fenofibric acid derivatives (e.g., gemfibrozil, clofibrate, ciprofibrate, fenofibrate, bezafibrate), dual-acting PPAR agonists (e.g., ZYH2, ZYH1, GFT505, chiglitazar, muraglitazar, aleglitazar, sodelglitazar, and naveglitazar), pan-acting PPAR agonists, PTP1B inhibitors (e.g., ISIS-113715 and TTP814), SGLT inhibitors (e.g., ASP1941, SGLT-3, empagliflozin, dapagliflozin, canagliflozin, B I-10773, PF-04971729, remogloflozin, TS-071, tofogliflozin, ipragliflozin, and LX-4211), insulin secretagogues, angiotensin-converting enzyme inhibitors (e.g., alacepril, benazepril, captopril, ceronapril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, imidapril, lisinopril, moveltipril, perindopril, quinapril, ramipril, spirapril, temocapril, or trandolapril), angiotensin II receptor antagonists (e.g., losartan, valsartan, candesartan, olmesartan, telmesartan), etc.;

[0107] subject Subjects suitable for treatment with the methods of the present disclosure include individuals with metabolic disorders. Subjects suitable for treatment with the methods of the present disclosure include obese individuals. Subjects suitable for treatment with the methods of the present disclosure include individuals with type 2 diabetes. Subjects suitable for treatment with the methods of the present disclosure include individuals with diabetic retinopathy. Subjects suitable for treatment with the methods of the present disclosure include individuals with non-alcoholic fatty liver disease (NAFLD). Subjects suitable for treatment with the methods of the present disclosure include individuals with non-alcoholic steatohepatitis (NASH).

[0108] Methods for treating muscular dystrophy The present disclosure provides a method for treating muscular dystrophy disorders.The method comprises administering an effective amount of the inhibitory nucleic acid of the present disclosure or a composition (e.g., a pharmaceutical composition) comprising the same to an individual in need thereof.In some cases, the inhibitory nucleic acid of the present disclosure is administered as a lipid nanoparticle.In some cases, the inhibitory nucleic acid of the present disclosure is administered as a liposome.

[0109] Muscular dystrophic disorders that can be treated by the methods of the present disclosure include, for example, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, myotonic muscular dystrophy, facioscapulohumeral muscular dystrophy, and limb-girdle muscular dystrophy.

[0110] In some cases, an effective amount of an inhibitory nucleic acid of the present disclosure, when administered to a subject in one or more doses, results in one or both of: (i) an improvement in the overall metabolic profile, including mitochondrial homeostasis; and (ii) a reduction in oxidative stress.

[0111] In some cases, an effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered to a subject in one or more doses, provides an increase in the subject's muscle strength. For example, in some cases, an effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered to a subject in one or more doses, provides an increase in the subject's muscle strength of at least 10%, at least 25%, at least 50%, at least 2-fold, or more than 2-fold compared to the subject's muscle strength level before treatment with the inhibitory nucleic acid.

[0112] In some cases, an effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered to a subject in one or more doses, provides a reduction in skeletal muscle fibrosis and necrosis. For example, in some cases, an effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered to a subject in one or more doses, provides a reduction in skeletal muscle fibrosis and necrosis of at least 10%, at least 25%, at least 50%, at least 60%, at least 70%, or more than 70% compared to the subject's level of skeletal muscle fibrosis and necrosis before treatment with the inhibitory nucleic acid.

[0113] Route of administration Suitable routes of administration include oral, rectal, nasal, pulmonary, topical, subcutaneous, intramuscular, intraperitoneal, intravenous, intradermal, intrathecal, and epidural. In some cases, the route of administration is intramuscular. In some cases, the route of administration is intravenous.

[0114] Suitable subjects for treatment Suitable subjects for treatment using the methods of the present disclosure include individuals with DMD, Becker muscular dystrophy, myotonic muscular dystrophy, facioscapulohumeral muscular dystrophy, or limb-girdle muscular dystrophy.

[0115] Examples of Non-Limiting Aspects of the Disclosure The above-mentioned aspects (e.g., embodiments) of the subject matter of the present invention can be useful alone or in combination with one or more other aspects or embodiments.Without limiting the above description, some non-limiting aspects of the present disclosure are provided below.As will be clear to those skilled in the art by referring to this disclosure, each individually numbered aspect can be used with or combined with any of the individually numbered aspects before and after it.This is to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below.

[0116] Embodiment 1. Nucleotide Sequence: (a) 5'-ACCGGTTCACTGTG-3' (SEQ ID NO: 1); or (b) 5'-GACCGGTTCACTGT-3' (SEQ ID NO:2); or (c)5'-AGACCGGTTCACTGTG-3'(SEQ ID NO:3) wherein the inhibitory nucleic acid comprises one or more locked nucleic acids (LNAs). Aspect 2. (i) a modified backbone; and / or (ii) one or more 5-methyldeoxycytosine residues 2. The inhibitory nucleic acid of embodiment 1, comprising: Aspect 3. The inhibitory nucleic acid of embodiment 1 or embodiment 2, having a length of 14 to 20 nucleotides. Aspect 4. The nucleotide sequence (including modifications) shown in FIG. 1A and designated "NRC0090" 4. The inhibitory nucleic acid of any one of embodiments 1 to 3, comprising: Aspect 5. The nucleotide sequence (including modifications) shown in FIG. 1A and designated "NRC-0091" 4. The inhibitory nucleic acid of any one of embodiments 1 to 3, comprising: Aspect 6. The nucleotide sequence (including modifications) shown in FIG. 1A and designated "NRC-0119" 4. The inhibitory nucleic acid of any one of embodiments 1 to 3, comprising: Aspect 7. (a) an inhibitory nucleic acid according to any one of embodiments 1 to 6; (b) a pharmaceutically acceptable excipient; 10. A pharmaceutical composition comprising: Aspect 8. The pharmaceutical composition of embodiment 7, wherein the pharmaceutically acceptable excipient comprises one or more lipids. Aspect 9. The pharmaceutical composition of embodiment 7, wherein the pharmaceutically acceptable excipient comprises a poly(amidoamine), a poly(propyleneimine), or a poly(L-lysine). Aspect 10. (a) an inhibitory nucleic acid according to any one of embodiments 1 to 6; (b) a pharmaceutically acceptable excipient; A lipid nanoparticle comprising: Aspect 11. A method of treatment comprising administering to an individual in need thereof an effective amount of an inhibitory nucleic acid according to any one of embodiments 1 to 6, a pharmaceutical composition according to any one of embodiments 7 to 9, or a lipid nanoparticle according to embodiment 10. Aspect 12. A method for treating a metabolic disorder in an individual, comprising administering to the individual an effective amount of an inhibitory nucleic acid according to any one of embodiments 1 to 6, a pharmaceutical composition according to any one of embodiments 7 to 9, or a lipid nanoparticle according to embodiment 10. Aspect 13. 13. The method of embodiment 12, wherein the metabolic disorder is insulin resistance, hyperglycemia, type 2 diabetes, obesity, fatty liver disease, glucose intolerance, hyperinsulinemia, metabolic syndrome, or hypertension. Aspect 14. The method of embodiment 12, wherein the metabolic disorder comprises insulin resistance. Aspect 15. The method of embodiment 12, wherein the metabolic disorder comprises metabolic syndrome. Aspect 16. The method of embodiment 12, wherein the metabolic disorder comprises type 2 diabetes. Aspect 17. 17. The method of any one of embodiments 12-16, wherein the individual has a body mass index greater than 30.0. Aspect 18. 18. The method of any one of aspects 12 to 17, wherein said administering step results in serum insulin levels within the normal range. Aspect 19. 18. The method of any one of aspects 12-17, wherein said administering step results in blood glucose levels within the normal range. Aspect 20. The method of any one of embodiments 12-19, further comprising administering at least one additional therapeutic agent. Aspect 21. The method of embodiment 20, wherein the at least one additional therapeutic agent is insulin, an insulin analog, a biguanidine, or a thiazolidinedione. Aspect 22. 21. The method of any one of aspects 11 to 20, wherein said administering step is via oral administration. Aspect 23. A method for treating muscular dystrophy in an individual, comprising administering to the individual an effective amount of an inhibitory nucleic acid according to any one of embodiments 1 to 6, a pharmaceutical composition according to any one of embodiments 7 to 9, or a lipid nanoparticle according to embodiment 10. Aspect 24. The method of embodiment 23, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. [Example]

[0117] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, such as bp, base pairs; kb, kilobases; pl, picoliters; s or sec, seconds; min, minutes; h or hr, hours (hours); aa, amino acids; kb, kilobases; bp, base pairs; nt, nucleotides; im, intramuscular (intramuscular); ip, intraperitoneal (intraperitoneal); sc, subcutaneous (subcutaneous); etc.

[0118] Example 1 Figure 1A shows the sequence information of the three ASOs. ID indicates the drug name, Oligo indicates the nucleotide sequence (uppercase letters indicate locked nucleic acids (LNAs) and lowercase letters indicate standard bases), and STRING indicates the modification (a + sign precedes LNAs and * (The symbol / iMe-dC / indicates an internal 5-methyldeoxycytosine; the ...

[0119] In this disclosure, we demonstrate that miR-128-1 miRNA is an interesting therapeutic target for DMD because it controls skeletal muscle mitochondrial metabolism and directly targets several key downstream regulators of dystrophin loss (Figure 2). To investigate the therapeutic potential and mechanism of action of miR-128-1 in DMD, we developed a locked nucleic acid antisense oligonucleotide (LNA ASO) targeting miR-128-1. Highly efficient depletion of miR-128-1 in skeletal muscle was achieved via weekly subcutaneous injection. Furthermore, miR-128-1 was identified to be involved in the regulation of numerous genes that have been shown to act as modifiers of the DMD phenotype, such as PGC-1α, AMPKα2, SIRT1, JAG1, and Wnt7A, as well as other metabolic regulators, such as PPARα, PPARγ, ULK1, CPT1β, and others.

[0120] Figure 2. Dystrophin loss in DMD myocytes results in abnormal mitochondrial function, and multiple regulators of mitochondrial biology and autophagy, as well as several developmental regulators, such as Notch and Wnt signaling components, can at least partially rescue DMD pathology. Interestingly, many of these regulators are predicted or validated targets of the miR-128-1 miRNA.

[0121] We developed a new generation of anti-miR-128-3p ASOs with significantly increased potency. An ASO design algorithm engineered the sequence, length, and LNA position to maximize binding potency and accessibility to the miR-128-3p seed region. Through this process, melting temperature and binding energy were also optimized, while self-complementarity and off-target binding were minimized. NRC0090, NRC0091, and NRC0119 performed best in the stringent screening process, and in vitro testing revealed that these ASOs were 20-50 times more potent than previous anti-miR-128-3p ASO prototypes. 1,2Importantly, the new ASO appears to be well tolerated and non-toxic when administered to mice at high doses (Figure 3), and short-term treatment of mice successfully results in potent inhibition of miR-128-3p levels in liver, muscle, eWAT, and BAT tissues (Figure 2).

[0122] Figure 3. New LNA ASOs targeting miR-128-3p are up to 50-fold more potent than the initial LNA ASO prototype, demonstrating on-target in vivo efficacy without toxicity. (A-C) Dose-response curves for candidate and initially reported ASOs. Huh-7 hepatocarcinoma cells were transfected with anti-miR-128-3p LNA ASOs or scrambled controls and assayed for miR-128-3p levels via RT-qPCR after (A) 72 h or (B-C) 24 h. miR-128-3p levels in treatment groups were normalized to U6 reference RNA and then to the scrambled control. (D-G) In vivo tolerability of LNA ASOs. Nine-week-old male C57BL / 6 mice (n = 5) were repeatedly injected sc with a 20 mg / kg dose of NRC0090, scrambled control ASO, or saline on days 0, 2, 4, and 6. Weight gain was monitored, and a terminal blood draw was performed on day 8 for measurement of liver toxicity markers (AST / ALT) and nephrotoxicity markers (BUN). (H) In vivo anti-miR-128-3p LNA ASO targeting study. Seven-week-old male C57BL / 6 mice (n = 5) were fed a high-fat diet and injected sc with 10 mg / kg of anti-miR-128-3p LNA ASO or scrambled control. Treatments were administered on days 0 and 7, and the indicated tissues were harvested on day 10 for miR-128-3p quantification by RT-qPCR. miR-128-3p in the treatment groups was normalized to U6 reference RNA and then to the scrambled control. Data are presented as mean and SEM.

[0123] LNA chemistry confers potent increases in affinity and specificity for the target and, when combined with the phosphorothioate backbone of ASOs, increases in vivo stability and pharmacokinetics. Multiple studies in mice and non-human primates have demonstrated the efficacy of LNA ASOs targeting miR-33. 18 Based on previous studies, the terminal half-life of LNA ASOs in the circulation is typically 2-3 weeks in mammals, and LNA ASOs can be detected in tissues up to 7 weeks after a single injection. Dosing studies in mice indicate that target inhibition is maintained 28 days after a single injection, thus allowing for weekly and even monthly dosing (Figure 4). Furthermore, dose-response studies demonstrate strong target engagement in all major metabolic tissues, including skeletal muscle, liver, subcutaneous and visceral WAT, and BAT (Figure 4).

[0124] Figure 4. Dose optimization study in mice. (A-D) Time course study. Eight-week-old C57BL / 6 mice (n = 5) fed a standard diet were injected sc with 10 mg / kg NRC0090 anti-miR-128-3p or scrambled control ASO on day 0. Tissues were harvested from cohorts on days 3, 7, 14, and 28 for miR-128-3p quantification by RT-qPCR. miR-128-3p in the treatment groups was normalized to U6 reference RNA and then normalized to the scrambled control. Data are presented as mean and SEM. (E-I) Dose-response study in mice. Seven-week-old C57BL / 6 mice (n = 5) were fed a high-fat diet and injected sc with the indicated doses of NRC0090 anti-miR-128-3p or scrambled control ASO on day 0. On day 28, tissues were harvested for miR-128-3p quantification by RT-qPCR. miR-128-3p in the treatment groups was normalized to U6 reference RNA and then normalized to the scrambled control. Data are presented as mean and SEM.

[0125] Example 2: Metabolic Syndrome NRC0090, NRC0091, and NRC0119 were tested in diet-induced obese mice and shown to have protective effects against conditions associated with MetS, including weight gain, fat mass accumulation, and glucose intolerance (Figure 5).

[0126] Figure 5. (A-B) Seven-week-old male C57BL / 6 mice (n = 3) were fed a 60% high-fat diet and scrambled control with 10 mg / kg / week of the indicated anti-miR-128-3p LNA ASO or scrambled control, and body weight was monitored over time. (C-D) Three-week-old male C57BL / 6 mice (n = 5) were fed a 60% high-fat diet and scrambled control with 10 mg / kg / week of NRC0090 anti-miR-128-3p LNA ASO or scrambled control. Body weight was monitored weekly (C), and fat mass and lean mass were measured via EchoMRI at the end of the study (D). (E-F) Six-week-old male C57BL / 6 mice (n = 10) were fed a 60% high-fat diet and scrambled control with 5 mg / kg / month of NRC0090 anti-miR-128-3p LNA ASO or scrambled control. After 15 weeks of treatment, mice were fasted for 6 hours and then injected i.p. with 1 g / kg D-glucose, and blood glucose levels were monitored over time (E). Area under the curve was calculated using GraphPad using a Y=0 baseline, and data were analyzed via one-way ANOVA (F).

[0127] Example 3: Duchenne muscular dystrophy Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, leading to progressive muscle degeneration and weakness, ultimately resulting in premature death from respiratory and cardiac failure. To date, no cure exists for DMD. Steroids are only intended to control symptoms and are associated with numerous serious adverse effects. Newly developed approaches focused on restoring partially functional dystrophin (e.g., exon-skipping antisense oligonucleotides (ASOs) and AAV-micro-dystrophin) have shown limited efficacy in a relatively small percentage of patients, with uncertain duration of efficacy. Therefore, additional efforts are being made to alleviate the disease by ameliorating the downstream deleterious effects of dystrophin loss, including metabolic abnormalities associated with mitochondrial dysfunction, elevated reactive oxygen species (ROS), fat infiltration, inflammation, and fibrosis.

[0128] miRNAs are short (18–24 nucleotide) regulatory non-coding RNAs that function in development, metabolism, and disease by epigenetically regulating specific target genes through their 3′ untranslated regions (3′UTRs). Numerous miRNAs have been shown to be upregulated in DMD muscle and are therefore termed "dystro-miRs," but confirmatory data demonstrating that therapeutic targeting of dystro-miRs ameliorates the DMD phenotype in animal models are lacking.

[0129] The mature miR-128-3p miRNA (derived from the miR-128-1 locus on human chromosome 2 and miR-128-2 on chromosome 3, also known as miR-128a and miR-128b, respectively) has been identified as a master regulator of metabolic homeostasis. Specifically, miR-128-3p expression negatively regulates mitochondrial respiration and biogenesis and represses the expression of key genes involved in mitochondrial function and homeostasis.

[0130] To block the action of miR-128-3p, we created a novel locked nucleic acid antisense oligonucleotide (LNA ASO). The miR-128-3p LNA ASO was modified to enable subcutaneous delivery and robustly targets miR-128-3p in skeletal and cardiac muscle. In this study, we investigated the effects of mdx 5cv Mouse models and DMD Y / - The efficacy of anti-miR-128-3p LNA ASO as a treatment was investigated in pig models. Near-complete rescue of skeletal muscle and cardiac dysfunction was demonstrated in these models. Furthermore, treatment significantly improved well-established metabolic disturbances associated with DMD, including glucose intolerance, mitochondrial dysfunction, and oxidative stress, to near-wild-type levels.

[0131] A mouse model of Duchenne muscular dystrophy (mdx 5cv ) found that anti-miR-128-1 treatment dramatically improved muscle strength and endurance, while also reducing skeletal muscle fibrosis and necrosis, with an excellent overall safety profile. This therapeutic effect was mediated through improvements in the overall metabolic profile, including mitochondrial homeostasis, reduced oxidative stress, and lipid oxidation. Interestingly, mdx 5cv The effect of ASO on the small intestine of mice is also demonstrated.

[0132] Elevated miR-128-3p expression is associated with muscle weakness in human DMD patients and animal models Using human data from the UK Biobank (n>300,000), we found a genetic association between the miR-128-1 and miR-128-2 loci encoding miR-128-3p and grip strength and respiratory function in humans (Figure 6A). Expression of miR-128-3p across DMD animal models and this data supports the mdx 5cvWe demonstrate that miR-128-3p is elevated in both the mouse model (Figure 6B, C) and the DE50-MD canine model (Figure 6D), consistent with literature from DMD patients showing that miR-128-3p is elevated in patient plasma and skeletal muscle tissue.

[0133] Figure 6. miR-128-3p expression is associated with muscle weakness in humans and DMD animal models. (A) The miR-128-3p locus is genetically linked to reduced grip strength and lung function (UK Biobank; n>300,000), and miR-128-3p is elevated in mouse and canine models of DMD. The miR-128-3p genomic locus on human chromosome 2 is associated with bilateral reduced grip strength and impaired pulmonary muscle function (forced expiratory volume and forced vital capacity). (B, C) MiR-128-3p levels in skeletal muscle and serum were measured in WT and mdx mice of various ages. 5cv (D) The levels of miR-128-3p were measured in DE50-MD1 mice, a 3- to 18-month-old DMD canine model.

[0134] mdx 5cv LNA ASO treatment targeting miR-128-3p ameliorates all pathological phenotypes in a DMD mouse model To assess whether miR-128-3p is a pathological modifier in DMD, 5cv Mice were crossed with whole-body miR-128-3p knockout mice or muscle-specific miR-128-3p knockout mice. Knockdown of miR-128-3p expression was observed in mdx mice. 5cv We hypothesized that miR-128-3p expression would improve skeletal muscle function in mice. These results show that miR-128-3p expression was downregulated by 36.6% and 22.9% in whole-body miR-128-3p knockout mice and muscle-specific miR-128-3p knockout mice, respectively, compared to wild-type mice (Figure 7A). Grip strength was significantly improved in both mdx mouse models. 5cvWhen exercise endurance was tested by treadmill running to exhaustion, the data show that knockout of miR-128-3p in skeletal muscle significantly improved endurance (Fig. 7C, D) and further reduced circulating creatine kinase levels (a muscle damage marker; Fig. 7E). Taken together, this suggests that miR-128-3p expression significantly improved endurance in mdx mice compared with mdx mice. 5cv These results suggest that IL-16 contributes to DMD pathology in mice.

[0135] Figure 7. Systemic or muscle-specific miR-128-3p knockout results in mdx 5cv Improves muscle function in mice. (A) miR-128-3p levels were measured in GA muscle. (B) Passive wire hanging was measured for grip strength. (C, D) Treadmill running to fatigue was used to measure exercise endurance. Time and distance were plotted, respectively. (E) Serum creatine kinase was measured to assess muscle damage.

[0136] To determine whether the use of LNA ASOs to suppress miR-128-3p could be applied as a treatment for DMD, we generated a state-of-the-art LNA ASO that could target miR-128-3p and robustly downregulate its expression. 5cv To evaluate the knockdown efficiency of this LNA ASO in mouse tissues, a dose-response study was performed. Seven days after a single subcutaneous injection at a range of doses, miR-128-3p expression levels were measured in skeletal muscle, diaphragm, and cardiac muscle. The data demonstrated dramatic inhibition of miR-128-3p in all of these tissues (Figure 8). Based on this data, a dose of 10 mg / kg delivered subcutaneously once weekly was selected for subsequent mouse studies.

[0137] Figure 8. mdx 5cvIn vivo ASO dose response in mice. Mice were injected with 10 mg / kg of control ASO 406 or anti-miR-128-3p ASO 90 at doses ranging from 1.25 mg / kg to 10 mg / kg. 7 days after the single injection, the TA, diaphragm, and heart were harvested. (A-C) miR-128-3p expression levels in the TA (A), diaphragm (B), and heart (C). Data are normalized to the control ASO 406 group.

[0138] Considering the early onset of DMD, mdx 5cv In mice, anti-miR-128-3p LNA ASO treatment was initiated at 3 weeks of age and continued until 6 or 9 weeks of age (Figure 9A). Analysis of miR-128-3p expression levels demonstrated robust (>95%) knockdown that persisted throughout the treatment period (Figure 9B). miR-128-3p knockdown significantly ameliorated many of the pathological findings of dystrophin loss. First, the data demonstrated a dramatic reduction in circulating creatine kinase (Figure 9C), a significant rescue of grip strength (Figure 9D), and a marked improvement in endurance exercise capacity (Figure 9E, F). Muscle histology revealed increased myofiber diameter, reduced myofiber necrosis, and reduced myofiber fibrosis (Figure 9G–L). Furthermore, the anti-miR-128-3p LNA ASO was well tolerated in these mice, showing no apparent hepatotoxicity or nephrotoxicity (Figures 10A–10F).

[0139] Figure 9. Anti-miR-128-3p LNA ASO inhibits mdx 5cv The pathological phenotype of the DMD mouse model was ameliorated. (A) Overview of the experimental design. (B) WT mice and mdx mice treated with control and treatment ASOs. 5cvmiR-128-3p expression levels in mouse GA muscles. (C) Circulating creatine kinase activity, a marker of muscle damage, was measured. (D) Two-limb wire hanging was measured for muscle strength. (E, F) Treadmill running to fatigue was used to measure exercise endurance. Time and distance were plotted, respectively. (G) H&E images of TA muscle histology at 40x and 100x magnification. (H) Quantification of TA muscle fiber size in all three experimental groups. (I) Necrosis was detected by staining with an IgG antibody, and the fluorescence intensity was quantified as shown in (J). (K) TA muscle cryosections were stained with picrosirius red for fibrosis. (L) Quantification of fibrosis area in the three experimental groups. n = 3–5 per group. (M) Two-limb cage-top wire hanging was measured in all three groups. (N) H&E images of TA muscle histology at 100x magnification.

[0140] Figure 10. ASO injection is safe and non-toxic in the liver and kidney. (A, B) WT mice and mdx mice treated with control ASO and anti-miR-128-3p ASO. 5cv H&E images of liver and kidney from mice. (C, D) WT mice and mdx mice treated with control ASO and anti-miR-128-3p ASO. 5cv Serum enzyme levels of hepatic ALT and AST in mice. (E, F) Serum urea nitrogen (BUN) and creatinine were measured as indicators of renal function.

[0141] Anti-miR-128-3p LNA ASO treatment ameliorates metabolic dysfunction in DMD DMD patients and animal models exhibit significant metabolic dysfunction, including glucose intolerance, reduced expression of glycolytic and oxidative enzymes, impaired mitochondrial homeostasis and function, increased oxidative stress, and abnormal intrafibral and plasma lipid levels. To elucidate the mechanistic role of miR-128-3 in DMD pathology, we performed a multicenter study of mdx mice treated with anti-miR-128-3p LNA ASO or scrambled control LNA ASO compared to wild-type mice. 5cvRNA sequencing (RNA-seq) was performed on gastrocnemius muscles from mdx mice. 5cv We identified 3,142 genes that were differentially expressed in the disease model compared with wild-type controls, and the expression of 473 of these genes was altered in response to anti-miR-128-3p treatment (Figure 11A, B). The most significantly upregulated pathways included oxidative phosphorylation, the TCA cycle, pyruvate metabolism, fatty acid metabolism, adipogenesis, and myogenesis (Figure 11C). Significantly downregulated pathways included interferon response, IL6-JAK-STAT3 signaling, and inflammatory response (Figure 11D). RNA-seq data highlighted numerous alterations in metabolic pathways. To examine the effects of anti-miR-128-3p LNA ASO treatment on metabolites in dystrophic muscle tissue, we performed metabolomic analysis. LNA ASO treatment altered several classes of metabolites, with lipid species being the most prominent category (Figure 12A). Clustering of these metabolites demonstrates that toxic lipid species are elevated in the dystrophic state and are ameliorated by LNA ASO treatment (Fig. 11E, F). These species included ceramide, which, when elevated, is known to cause insulin resistance in skeletal muscle. To explore this further, we investigated the effects of anti-miR-128-3p LNA ASO-treated mdx mice. 5cv Intraperitoneal glucose tolerance tests were performed in mice, and the results revealed a significant improvement in glucose clearance with LNA ASO treatment (Figure 12B, C).

[0142] Mitochondrial dysfunction is also a well-established metabolic abnormality in DMD pathology. This RNA-seq analysis revealed that LNA ASO treatment increased the expression of several mitochondria-related pathways. To further investigate mitochondrial homeostasis, we examined the expression of key regulators of mitochondrial function, biogenesis, and dynamics. Several of these regulators, including AMPKα2, PGC-1α, and PPARα, were found to be significantly derepressed by anti-miR-128-3p LNA ASO treatment (Figure ​(Figure11G,13D,E). Anti-miR-128-3p LNA ASO treatment significantly increased mitochondrial DNA copy number, thereby suggesting increased mitochondrial biogenesis (Figure 11H). Electron microscopy analysis of mitochondrial ultrastructure revealed that anti-miR-128-3p LNA ASO-treated mdx mice significantly increased the expression of several mitochondria-related pathways. To further investigate mitochondrial homeostasis, we investigated the expression of several key regulators of mitochondrial function, biogenesis, and dynamics. Several of these regulators, including AMPKα2, PGC-1α, and PPARα, were found to be significantly derepressed by anti-miR-128-3p LNA ASO treatment (Figure ​(Figure11G,13D,E). 13D,E). Anti-miR-128-3p LNA ASO treatment significantly increased mitochondrial DNA copy number, thereby suggesting increased mitochondrial biogenesis (Figure 11H). Electron microscopy analysis of mitochondrial ultrastructure revealed that anti-miR-128-3p LNA ASO-treated mdx mice significantly increased the expression of several mitochondria-related pathways. 5cv We demonstrated improvement of abnormal mitochondrial integrity in mdx mice (Figure 11J). 5cv Soleus muscle showed perinuclear mitochondrial localization, which was resolved by anti-miR-128-3p LNA ASO treatment (Figure 13A). 5cv In soleus muscle, mitochondria were longitudinally misaligned, but treatment with anti-miR-128-3p LNA ASO restored correct mitochondrial alignment. Consistent with the findings of improved mitochondrial morphology, we also observed a reduction in hydrogen peroxide levels and lipid peroxidation with treatment, thereby improving the mitochondrial morphology in mdx muscles treated with anti-miR-128-3p LNA ASO. 5cv We demonstrated a reduction in oxidative stress in mice (Fig. 11I, Fig. 13B, C). Furthermore, muscle fiber composition shifted in favor of oxidative fiber types, with an increase in type IIa fibers and a decrease in type IIb fibers (Fig. 11K, Fig. 13F). This fiber type switching is consistent with the observed derepression of PGC-1α expression. Together, these data indicate an overall improvement in mitochondrial health.

[0143] Figure 11. Anti-miR-128-3p LNA ASO treatment ameliorates metabolic dysfunction in DMD. (A-D) WT mice and mdx mice treated with control and anti-miR-128-3p ASOs. 5cv RNA-seq analysis of mice. (A, B) Venn diagram and volcano plot representation of differentially expressed genes. (C, D) Pathway enrichment analysis shows upregulated (C) and downregulated (D) signaling cascades. (E, F) Wild-type mice (E) and mdx mice treated with control ASO or anti-miR-128-3p LNA ASO. 5cv mdx compared with mouse (F) 5cv Analysis of altered metabolites in the gastrocnemius muscle of a mouse model. (G) Derepression of genes controlling mitochondrial homeostasis was measured by RT-qPCR. (H) Mitochondrial DNA copy number was measured in 6-week-old soleus muscles. (I) Oxidative stress was quantified. (J) Longitudinal sections of soleus muscles showed mitochondrial rearrangement and loss of order at 9 weeks of age by electron microscopy. (K) Three muscle fiber types were stained by immunofluorescence.

[0144] Figure 12. Metabolomic analysis shows mdx improves glucose tolerance 5cv (A) mdx mice treated with miR-128-3p LNA ASO show reduction of lipotoxic species. 5cv Heatmap of fold changes in metabolite species upon miR-128-3p LNA ASO treatment in mice. (B, C) Intraperitoneal glucose tolerance tests were performed at 6 weeks of age. Area under the curve was quantified in (C).

[0145] Figure 13. Anti-miR-128-3p LNA ASO treatment improves mitochondrial health in DMD. (A) Electron microscopy of soleus muscle was imaged from transverse (top) and longitudinal (bottom) sections. (B, C) Lipid peroxidation, as indicated by MDA production, was measured in skeletal muscle and serum. (D) Western blot was performed to measure mitochondrial protein levels in TA muscle. (E) Quantification of mitochondrial protein levels. (F) Myofiber types (shown in Figure 3K) were quantified.

[0146] Figure 14. The effects of anti-miR-128-1 ASO on inflammation, ferroptosis, and oxidative stress were examined. (A) Expression levels of TNFα, IL-1b, and IL6 were measured by RT-qPCR. (B) Genes involved in ferroptosis were examined. (C and D) The lipid oxidation by-product MDA was measured in TA skeletal muscle and serum of all three experimental groups. (E) H2O2 levels were measured in TA muscle.

[0147] Figure 15. WT mice and mdx mice treated with control ASO or anti-miR-128-3p ASO 5cv Histology of mouse small intestine sections. (A-C) WT group, mdx 5cv Representative H&E images of the control and ASO-treated groups. The red line indicates the thickness of the intestinal wall, the black arrow indicates the height of the small intestinal villi, and the blue arrow indicates the depth of the small intestinal crypts. (D) Quantification of the thickness of the smooth muscle layer is plotted.

[0148] Figure 16. 6-week-old mdx 5cv Evaluation of miR-128-3p expression and its downstream targets in mouse myocardium. (A) WT group, mdx 5cv (B) Gene expression profiles of miR-128-3p downstream targets.

[0149] Example 4: Myocardial Infarction (MI) and Heart Failure miR-128-3p is a key regulator of intrinsic cardiac fibrosis and regeneration after myocardial infarction (MI). We hypothesized that miR-128-3p could be a therapeutic target for combating myocardial fibrosis and promoting cardiac regeneration. We used a state-of-the-art LNA ASO, which showed significantly improved stability and safety and demonstrated high efficacy in several tissues, including skeletal muscle and heart, to inhibit miR-128-3p.

[0150] Anti-miR-128-3p LNA ASO treatment rescues mouse and pig models of cardiac dysfunction Death in DMD patients ultimately results from cardiac and respiratory failure. Due to the robust improvement in skeletal muscle function with anti-miR-128-3p LNA ASO treatment, we sought to further explore whether the treatment could also rescue cardiac failure. 5cv The model does not reproduce the cardiac dysfunction observed in human patients. Therefore, other models of heart failure were investigated. Cardiac-selective knockout and AAV-decoy-mediated targeting of miR-128-3p have been shown to improve cardiac function after myocardial infarction (MI) in mice. First, we investigated whether anti-miR-128-3p LNA ASO could improve cardiac function in a model of heart failure induced by permanent ligation. Permanent ligation of the anterior descending coronary artery was performed, and then mice were immediately injected subcutaneously with 10 mg / kg of LNA ASO. Injections were repeated weekly, and tissues were harvested 28 days after MI. Anti-miR-128-3p LNA ASO treatment resulted in complete knockdown of miR-128-3p expression in the heart 28 days after MI (Figure 17A). Weekly subcutaneous injections significantly knocked down miR-128-3p levels, demonstrating high efficacy in targeting cardiac tissue. Furthermore, there was reduced fibrosis in the scar area in the treated group (Figure 18).

[0151] Echocardiography was used to measure cardiac function before and 28 days after MI. These results revealed a significant improvement in ejection fraction and a significant decrease in end-systolic volume after anti-miR-128-3p LNA ASO treatment, thereby indicating a marked improvement in cardiac function (Figures 17B, C). Furthermore, mice treated with anti-miR-128-3p LNA ASO showed an increased probability of survival compared with mice injected with a scrambled control LNA ASO (Figure 17D).

[0152] Due to the significant improvement in cardiac function in the mouse MI model, we further investigated the therapeutic potential of anti-miR-128-3p LNA ASO in a severely affected pig model of DMD. The cardiac physiology of pigs is relatively similar to that of humans, and we believe that this may contribute to the development of DMD. Y / - The pig model shows significant cardiac dysfunction. To investigate the effects of anti-miR-128-3p LNA ASO treatment, 6-week-old pigs were subcutaneously treated with 5 mg / kg body weight LNA ASO once a week for 2 months (Figure 17E). After LNA ASO treatment, these DMD Y / - Cardiac dysfunction in pigs was significantly rescued, as measured by LV ejection fraction and LV fractional shortening (Figure 17F, G). Serum troponin I levels, a biomarker of myocardial infarction and cardiac cell death, were also rescued by LNA ASO treatment (Figure 17J). To further elucidate the mechanism of cardiac dysfunction rescue, mass spectrometry-based quantitative proteomics analysis of myocardium was performed. Principal component analysis revealed that DMD Y / - When pigs were treated with anti-miR-128-3p LNA ASO, the proteomic profile was found to shift toward wild-type (Figure 17I). Compared to wild-type pigs, 17 differentially regulated proteins were observed in the DMD disease state (Figures 17H, K). However, DMD treated with anti-miR-128-3p LNA ASO showed a significant shift in the proteomic profile toward wild-type (Figure 17I). Y / - Pigs do not show altered expression patterns for 12 of these 17 proteins (Fig. 4h, k). Interestingly, troponin I was significantly higher in DMD compared to wild-type. Y / -DMD elevated in pigs but treated with anti-miR-128-3p LNA ASO Y / - DMD was not elevated in pigs (Fig. 17K) compared to wild-type samples. Y / - Proteins with decreased abundance in the samples belong to the functional clusters of cell adhesion, wound response regulation, protein maturation, protein activation, and extracellular structural order. Collectively, these results indicate that anti-miR-128-3p LNA ASO is an effective therapeutic approach for mouse and pig models of cardiac dysfunction.

[0153] Figure 17. Anti-miR128-3p LNA ASO inhibits the efficacy of anti-miR128-3p LNA ASO in mouse MI models and preclinical DMD Y / - Rescue of cardiac dysfunction in a porcine model. (A) MiR-128-3p levels were measured in a mouse MI model treated with LNA ASO. (B, C) Ejection fraction and end-systolic volume were measured in the MI model. (D) Kaplan-Meier estimates of survival. (E) DMD patients treated with anti-miR-128-3p ASO or control ASO. Y / - End-point miR-128-3p expression was measured in the myocardium of pigs. (F, G) Left ventricular fractional shortening and ejection fraction were measured by echocardiography. (H) Volcano plot visualization of proteome alterations: Proteins with significantly altered abundance in the DMD group (FDR<0.05) are colored blue and red for down- and up-regulation, respectively. (I) WT pig group, DMD Y / - Principal component analysis of the pig placebo group and the anti-miR-128-3p LNA ASO experimental group. (J) Serum levels of troponin I were measured. (h) For WT, n=4; DMD Y / - (K) Pathway analysis of differentially abundant proteins (FDR<0.05) in at least one condition is shown. (L) Echocardiographic assessment of end-diastolic volume in an MI mouse model. For control ASO, n=9; for anti-miR-128-1 ASO, n=11.

[0154] Figure 18. Assessment of fibrosis in an MI mouse model. (A) Picrosirius red staining is used to quantify fibrotic tissue in the heart. Image J is used to quantify the area of ​​fibrosis. (B) Fibrosis in the scar zone and interstitial fibrosis in the infarct border zone were measured. (C) Quantification of infarct size, fibrosis in the scar zone, and interstitial fibrosis in the border zone were measured and quantified. For control ASO, n=9; for anti-miR-128-1 ASO, n=11.

[0155] method Mouse breeding and experiments. (Herein, mdx 5cv (called) Dmd mdx-5Cv C57BL / 6J mice were purchased from the Jackson laboratory (strain number 002379) and crossed with C57BL / 6J mice for at least six generations. Wild-type C57BL / 6J mice were purchased from the Jackson laboratory as controls. Muscle-specific miR-128 knockout mice (MCK cre / miR-128 f / f / mdx 5cv ) is miR-128-1 f / f They were crossed with mice and 5cv (MCK cre / miR-128) was generated from MCK-Cre mice (Jackson Labs line number 006405) crossed with f / f / mdx 5cv ) mdx with whole-body miR-128 knockout 5cv Mouse (CMV cre / miR-128 f / f / mdx 5cv ) were generated using CMV-Cre mice (Jackson Labs line number 006054). Wild-type and mdx mice 5cvMice were injected with scrambled control or anti-miR-128-3p ASO once a week starting at 3 weeks of age. Behavioral testing was performed at weeks 6 and 9. Tissues and blood were collected at the end of weeks 6 and 9. Collected blood samples were centrifuged at 2,000 rpm for 10 minutes to obtain serum. Skeletal muscle tissue was used for morphological and molecular analysis. All experimental procedures were performed in accordance with IACUC regulations and approved by the University of California, Berkeley IACUC (AUP#2018-10-11513-1). All protocols complied with federal regulations, the National Research Council Guide for the Care and Use of Laboratory Animals, and the Public Health Service Policy on Humane Care and Use of Laboratory Animals.

[0156] Pig husbandry and analysis. All pig experiments were carried out in accordance with the German Animal Welfare Act and Directive 2010 / 63 / EU on the protection of animals used scientifically and were approved by the responsible animal welfare authority (Government of Upper Bavaria; permission 55.2-1-54-2532-163-2014). All pigs were maintained in a specific pathogen-free environment (Center for Innovative Medical Models) at the Ludwig-Maximilian University Munich. Food and water were provided ad libitum. DMD and WT pigs were littermates. To standardize the experimental groups, all piglets in the placebo and treatment groups were sired by the same male pig to a heterozygous carrier sow (DMD + / - ) was produced by crossing with

[0157] The generation of a pig model carrying a DMD exon 52 deletion has been described in detail elsewhere (Stirm, Fonteyne et al. 2021).

[0158] The first cardiac ultrasound examination and blood sampling were performed at 5 weeks of age (1 week after weaning). The first injection was performed 1 week later. Three DMD pigs each were randomly assigned to treatment and placebo groups and received 5 mg of LNA ASO against miR-128-3p or random LNA sequences per kg of body weight weekly for 2 months. Subcutaneous injections were performed in the subcutaneous fat just behind the ear. Furthermore, blood samples were collected 2 weeks and 2 months after the first injection, and final cardiac ultrasound examinations, necropsies, and tissue sampling were performed at the same time.

[0159] Locked nucleic acid antisense oligonucleotide (LNA ASO). This ASO targeting the microRNA miR-128 was designed and modified for stability and efficacy. The sequence is 14 residues long, ACcggTTCacTgTG (SEQ ID NO:1). The LNA ASO is 100% chemically synthesized by IDT DNA and is harmless, non-toxic, and non-infectious. It is also pharmaceutical grade and endotoxin-free.

[0160] Muscle Function Behavioral Testing. All mice were exercised twice a week for 3 weeks using a 30-minute treadmill run at 9 m / min (Kaczor et al., 2007; Hudecki et al., 1993). Behavioral tests of muscle function, including passive wire hanging, limb wire hanging, and treadmill fatigue tests, were performed according to the standard operating procedures of the Treat NMD Neuromuscular Network. For the treadmill fatigue test, mice were placed on the belt of a 6-lane motorized treadmill (Exer 3 / 6 Treadmill; Columbus Instruments, Columbus, OH) equipped with a shocker plate. The treadmill was operated at 5 m / min at a 0° incline for 5 minutes, after which the speed was increased by 1 m / min per minute. The test was stopped when the mouse remained on the shocker plate for 20 seconds without attempting to re-enter the treadmill, and the time to fatigue was determined. Distance was then calculated based on speed and running time.

[0161] Measurement of creatine kinase, alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine. Serum levels of creatine kinase, ALT, AST, BUN, and creatinine were measured using commercially available kits.

[0162] RT-qPCR. GA skeletal muscle was used for total RNA extraction. miR-128 levels were measured using a probe from Thermo Scientific. U6 was used as a housekeeping control.

[0163] cDNA was generated using a BioRad reverse transcriptase kit, and gene expression was then measured by qPCR.

[0164] RNAseq analysis. Total RNA was extracted using a Qiagen kit (catalog). A cDNA library was constructed from 1 μg of total RNA derived from mouse GA muscle tissue using the Stranded mRNA-seq kit (KAPA) according to the manufacturer's protocol. The library was sequenced on a NovaSeq 6000 (Novogene) targeting 40 million read pairs with paired-end reads for 150 cycles. STAR aligner was used to map sequencing reads to transcripts in the mouse mm10 reference genome. Read counts for individual transcripts were generated by HTSeq-count, followed by EdgeR to estimate expression values ​​and detect differentially expressed transcripts. Differentially expressed genes were defined by at least a 2-fold change with an FDR of less than 0.01.

[0165] Immunohistochemistry and histology. TA muscle tissue was embedded in OCT and snap-frozen in liquid nitrogen. Muscles were sectioned into 10 μM sections. Hematoxylin and eosin staining was performed. Myofiber size was measured from H&E sections using Image J. Picosirius red staining was performed to stain fibrotic tissue. IgG, IgM, and IgA were used to stain necrotic cells. For quantification, three images were analyzed for each sample to generate a combined average.

[0166] Blood collection and clinical chemistry. Blood was collected from the right jugular vein using Serum Monovettes® (Sarstedt, Numbrecht, Germany). To allow clotting, the Serum Monovettes® was kept at room temperature for 30 minutes, followed by centrifugation at 1800 g for 20 minutes at 4°C. Serum was aliquoted and stored at -80°C until further processing (storage period did not exceed 6 months). Creatine kinase levels were determined using the CKL ACN 057 kit on a Cobas 311 analyzer system (Sridhar, Roche et al.). Troponin I levels were measured by CMIA (Alinity, Abbott, Illinois, USA) in the laboratory of SYNLAB.vet GmbH Augsburg.

[0167] Echocardiography. For echocardiography, pigs were sedated with 20 mg / kg ketamine (Ursotamin®, Serumwerke Bernburg, Bernburg, Germany) and 2 mg / kg azaperone (Azaporc®, Serumwerke Bernburg) and subsequently anesthetized with 4 mg / kg / h propofol (Propofol 2%, Fresenius Kabi, Bad Homburg, Germany). After achieving a sufficient depth of anesthesia, the pigs were placed in right lateral recumbency, and cardiac function was measured by standard 2D transthoracic echocardiography (Esaote MyLab X8). Left ventricular ejection fraction and fractional shortening were determined by M-mode imaging. All measurements were performed by the same investigator. Genotype could not be blinded due to significant differences, but measurements of the placebo and treatment groups were blinded to the investigator.

[0168] Sample preparation for proteome analysis. Heart and skeletal muscle tissue samples were snap-frozen in liquid nitrogen and freeze-ground using a CP02 automatic dry grinder (Covaris, Woburn, MA, USA) according to the manufacturer's instructions. The tissue powder was dissolved in 8 M urea / 0.5 M NH4HCO3 by sonication (18 cycles of 10 seconds) using a Sonopuls HD3200 (Bandelin, Berlin, Germany). Protein was quantified using a Pierce 660 nm protein assay (Thermo Fisher Scientific, Rockford, IL, USA). 20 micrograms of protein were digested with Lys-C (FUJIFILM Wako Chemicals Europe GmbH, Neuss, Germany) for 4 hours at 37°C, followed by digestion with modified porcine trypsin (Promega, Madison, WI, USA) for 16 hours.

[0169] Nano-liquid chromatography (LC)-tandem mass spectrometry (MS) and bioinformatics. One microgram of digest was injected into an UltiMate 3000 nano-LC system connected online to a Q-Exactive HF-X instrument (Thermo Fisher Scientific). The sample was transferred to a PepMap 100 C18 trap column (100 μm x 2 cm, 5 μM particles, Thermo Fisher Scientific) and separated on an analytical column (PepMap RSLC C18, 75 μm x 50 cm, 2 μm particles, Thermo Fisher Scientific) at 250 nL / min using an 80-minute gradient of 5–20% solvent B, followed by a 9-minute ramp to 40% solvent B. Solvent A consisted of 0.1% formic acid in water, and solvent B consisted of 0.1% formic acid in acetonitrile. MS spectra were acquired on the Q Exactive HF-X mass spectrometer using the Top15 data-dependent acquisition method. Raw files were processed by Maxquant (v.1.6.7.0) [1] using the built-in search engine Andromeda [2] and the NCBI RefSeq Sus scrofa database (v.7-5-2020). Protein intensities were normalized using the MaxLFQ approach [3]. Statistical analysis and visualization were performed using Perseus [4] and the R framework [5]. Proteins detected in all replicates of at least one condition were retained for quantitative analysis. Missing values ​​were imputed using Perseus default parameters. To further account for fold changes, Volcano plots were generated using a two-tailed Student's t-test and a permutation-based FDR cutoff of 0.05 with an s0 parameter of 0.1 [6].

[0170] reference TIFF2025538360000007.tif198151TIFF2025538360000008.tif73151

[0171] While the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step(s), to the objective, spirit, and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

1. Nucleotide sequence: (a) 5'-ACCGGTTCACTGTG-3' (SEQ ID NO:1); or (b) 5'-GACCGGTTCACTGT-3' (SEQ ID NO:2); or (c) 5'-AGACCGGTTCACTGTG-3' (SEQ ID NO:3) wherein the inhibitory nucleic acid comprises one or more locked nucleic acids (LNAs).

2. (i) a modified backbone; and / or (ii) one or more 5-methyldeoxycytosine residues 2. The inhibitory nucleic acid of claim 1, comprising:

3. 3. The inhibitory nucleic acid of claim 1 or claim 2, having a length of 14 to 20 nucleotides.

4. The nucleotide sequence shown in Figure 1A and designated "NRC0090" The inhibitory nucleic acid of any one of claims 1 to 3, comprising:

5. The nucleotide sequence shown in FIG. 1A and designated "NRC-0091" The inhibitory nucleic acid of any one of claims 1 to 3, comprising:

6. The nucleotide sequence shown in FIG. 1A and designated "NRC-0119" The inhibitory nucleic acid of any one of claims 1 to 3, comprising:

7. (a) an inhibitory nucleic acid according to any one of claims 1 to 6; (b) a pharmaceutically acceptable excipient; 10. A pharmaceutical composition comprising:

8. 8. The pharmaceutical composition of claim 7, wherein the pharmaceutically acceptable excipient comprises one or more lipids.

9. 8. The pharmaceutical composition of claim 7, wherein the pharmaceutically acceptable excipient comprises poly(amidoamine), poly(propyleneimine), or poly(L-lysine).

10. (a) an inhibitory nucleic acid according to any one of claims 1 to 6; (b) a pharmaceutically acceptable excipient; A lipid nanoparticle comprising:

11. A method of treatment comprising administering an effective amount of an inhibitory nucleic acid according to any one of claims 1 to 6, a pharmaceutical composition according to any one of claims 7 to 9, or a lipid nanoparticle according to claim 10 to an individual in need thereof.

12. A method for treating a metabolic disorder in an individual, comprising administering to the individual an effective amount of an inhibitory nucleic acid described in any one of claims 1 to 6, a pharmaceutical composition described in any one of claims 7 to 9, or a lipid nanoparticle described in claim 10.

13. 13. The method of claim 12, wherein the metabolic disorder is insulin resistance, hyperglycemia, type 2 diabetes, obesity, fatty liver disease, glucose intolerance, hyperinsulinemia, metabolic syndrome, or hypertension.

14. 13. The method of claim 12, wherein the metabolic disorder comprises insulin resistance.

15. 13. The method of claim 12, wherein the metabolic disorder comprises metabolic syndrome.

16. 13. The method of claim 12, wherein the metabolic disorder comprises type 2 diabetes.

17. 17. The method of any one of claims 12 to 16, wherein the individual has a body mass index greater than 30.

0.

18. 18. The method of any one of claims 12 to 17, wherein said administering step results in serum insulin levels within the normal range.

19. 18. The method of any one of claims 12 to 17, wherein said administering step results in blood glucose levels within the normal range.

20. 20. The method of any one of claims 12 to 19, further comprising administering at least one additional therapeutic agent.

21. 21. The method of claim 20, wherein the at least one additional therapeutic agent is insulin, an insulin analog, a biguanidine, or a thiazolidinedione.

22. 21. The method of any one of claims 11 to 20, wherein said administering step is via oral administration.

23. A method for treating muscular dystrophy in an individual, comprising administering to the individual an effective amount of an inhibitory nucleic acid described in any one of claims 1 to 6, a pharmaceutical composition described in any one of claims 7 to 9, or a lipid nanoparticle described in claim 10.

24. 24. The method of claim 23, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.

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