WO2012145374A1

WO2012145374A1 - TARGETING miR-378 FAMILY MEMBERS FOR THE TREATMENT OF METABOLIC DISORDERS

Info

Publication number: WO2012145374A1
Application number: PCT/US2012/034039
Authority: WO
Inventors: Christine Esau
Original assignee: Regulus Therapeutics Inc.
Priority date: 2011-04-19
Filing date: 2012-04-18
Publication date: 2012-10-26

Abstract

Provided herein are methods and compositions for the treatment of metabolic disorders. Also provided herein are methods and compositions for improving insulin resistance, reducing blood glucose levels, and/or reducing blood insulin levels in a subject in need thereof. In certain embodiments, the methods comprise inhibiting the activity of one or more miR-378 family members. In certain embodiments, such methods comprise administering a compound comprising a modified oligonucleotide targeted to one or more miR-378 family members.

Description

TARGETING miR-378 FAMILY MEMBERS FOR THE TREATMENT OF METABOLIC

DISORDERS

This application claims the benefit of priority to U.S. Provisional Application Nos. 61/477,026, filed April 19, 201 1 , and 61 /549,141 , filed October 1 , 201 1 , which are incorporated herein by reference in their entireties for any purpose.

SEQUENCE LISTING

The Sequence Listing filed herewith as a text filed named "201 1 -04- 19 5766- 00005_REG0027USLSEQ" is incorporated herein by reference in its entirety.

FIELD OF INVENTION

Provided herein are methods and compositions for the treatment of metabolic disorders.

DESCRIPTION OF RELATED ART

MicroRNAs (miRNAs), also known as "mature miRNAs" are small (approximately 18-24 nucleotides in length), non-coding RNA molecules encoded in the genomes of plants and animals. In certain instances, highly conserved, endogenously expressed miRNAs regulate the expression of genes by binding to the 3'-untranslated regions (3'-UTR) of specific mRNAs. More than 1000 different miRNAs have been identified in plants and animals. Certain mature miRNAs appear to originate from long endogenous primary miRNA transcripts (also known as pri-miRNAs, pri-mirs, pri-miRs or pri-pre-miRNAs) that are often hundreds of nucleotides in length (Lee, et al., EMBO J., 2002, 21 (17), 4663-4670).

Functional analyses of miRNAs have revealed that these small non-coding RNAs contribute to different physiological processes in animals, including developmental timing, organogenesis, differentiation, patterning, embryogenesis, growth control and programmed cell death. Examples of particular processes in which miRNAs participate include stem cell differentiation, neurogenesis, angiogenesis, hematopoiesis, and exocytosis (reviewed by Alvarez-Garcia and Miska, Development, 2005, 132, 4653-4662).

Families of miRNAs can be characterized by nucleotide identity at positions 2-8 of the miR A, a region known as the seed sequence. Lewis et al. describe several miRNA families, as well as miRNA superfamilies, which are characterized by related seed sequences (Lewis et al. Cell, 2005, 120(1), 15-20).

SUMMARY OF INVENTION

Provided herein are methods for treating metabolic disorders, and conditions associated with metabolic disorders, comprising administering a compound comprising a modified oligonucleotide targeting miR-378 or a precursor or family member thereof.

Provided herein are methods for treating a metabolic disorder, comprising administering to a subject having a metabolic disorder a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and has a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member; thereby treating the metabolic disorder. Also provided herein are methods for preventing or delaying the onset of at least one metabolic disorder in a subject at risk for developing a metabolic disorder, comprising administering to the subject a compound comprising modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member; and thereby preventing or delaying the onset of a metabolic disorder in the subject. In certain embodiments, the metabolic disorder is selected from among pre-diabetes, diabetes, metabolic syndrome, obesity, diabetic dyslipidemia, hyperlipidemia, hypertension, hypertriglyceridemia,

hyperfattyacidemia, and hyperinsulinemia.

Provided herein are methods for improving insulin resistance in a subject comprising administering to the subject a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member; and thereby improving insulin resistance in the subject. In certain embodiments, the subject has impaired insulin resistance. In certain embodiments, the methods comprise selecting a subject having impaired insulin resistance.

Provided herein are methods for reducing a blood glucose level of a subject comprising

administering to the subject a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member; thereby reducing the blood glucose level of the subject.

Provided herein are methods for preventing or delaying the onset of an elevated blood glucose level in a subject at risk for developing an elevated glucose level comprising administering to the subject a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member; thereby preventing or delaying the onset of an elevated blood glucose level in the subject.

In certain embodiments, the methods provided herein comprise selecting a subject having an elevated blood glucose level. In certain embodiments, the subject has an elevated blood glucose level. In certain embodiments, the methods comprise measuring the blood glucose level of the subject. In certain embodiments, the blood glucose level is a fasted blood glucose level. In certain embodiments, the blood glucose level is a post-prandial blood glucose level. In certain embodiments, the blood glucose level is a whole blood glucose level. In certain embodiments, the blood glucose level is a plasma blood glucose level. In certain embodiments, the subject has a fasting blood glucose level between 100 and 125 mg/dL. In certain embodiments, the subject has a fasting blood glucose level at or above 125 mg dL. In certain embodiments, the subject has a two-hour post-prandial blood glucose level between 140 and 199 mg/dL. In certain embodiments, the subject has a two-hour post-prandial blood glucose level at or above 200 mg/dL.

In certain embodiments, the administering of the modified oligonucleotide reduces the blood glucose level to below 200 mg/dL. In certain embodiments, the administering of the modified

oligonucleotide reduces the blood glucose level to below 175 mg dL. In certain embodiments, the administering of the modified oligonucleotide reduces the blood glucose level to below 150 mg/dL. In certain embodiments, the administering of the modified oligonucleotide reduces the blood glucose level to below 125 mg dL. In certain embodiments, the administering of the modified oligonucleotide reduces the blood glucose level to below 120 mg/dL. In certain embodiments, the administering of the modified oligonucleotide reduces the blood glucose level to below 115 mg/dL. In certain embodiments, the administering of the modified oligonucleotide reduces the blood glucose level to below 1 10 mg/dL. In certain embodiments, the administering of the modified oligonucleotide reduces the blood glucose level to below 105 mg/dL. In certain embodiments, the administering of the modified oligonucleotide reduces the blood glucose level to below 100 mg/dL.

Provided herein are methods for improving glucose tolerance in a subject comprising administering to the subject a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member; and thereby improving glucose tolerance. In certain embodiments, the subject has impaired glucose tolerance.

In certain embodiments, the methods comprise selecting a subject having impaired glucose tolerance.

Provided herein are methods for improving insulin resistance in a cell or tissue comprising contacting the cell or tissue with a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to the nucleobase sequence of a miR- 378 family member. In certain embodiments, the cell or tissue is a liver, fat, or muscle cell or tissue. In certain embodiments, the cell or tissue is a fat cell or tissue.

Provided herein are methods for increasing insulin sensitivity in a cell comprising contacting a cell with a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member, wherein the cell has a decreased sensitivity to insulin. In certain embodiments, the cell is a liver, fat or muscle cell.

In certain embodiments, the modified oligonucleotide comprises a seed match sequence selected from SEQ ID NO: 18, 19, 20, 21 , 22, 23, 24, and 25.

In certain embodiments, the administration is parenteral administration. In certain embodiments, the administration is intravenous administration or subcutaneous administration. In certain embodiments, the administration is oral administration.

In certain embodiments, the methods provided herein may comprise administering at least one additional therapy. In certain embodiments, the at least one additional therapy is a glucose-lowering agent. In certain embodiments, the at least one additional therapy is a lipid-lowering agent. In certain embodiments, the glucose-lowering agent is selected from among a PPAR agonist (gamma, dual, or pan), a dipeptidyl peptidase (IV) inhibitor, a GLP-I analog, insulin or an insulin analog, an insulin secretagogue, a SGLT2 inhibitor, a human amylin analog, a biguanide, an alpha-glucosidase inhibitor, a meglitinide, a

thiazolidinedione, and sulfonylurea.

In certain embodiments, the methods provided herein may comprise administration of the compound in the form of a pharmaceutical composition.

In certain embodiments, the compound consists of the modified oligonucleotide. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 85% complementary to the nucleobase sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to the nucleobase sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 95% complementary to the nucleobase sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

In certain embodiments, the the nucleobase sequence of the modified oligonucleotide is 100%

complementary to the nucleobase sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

In certain embodiments, the modified oligonucleotide comprises at least one modified

internucleoside linkage. In certain embodiments, each internucleoside linkage of the modified

oligonucleotide is a modified internucleoside linkage. In certain embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

In certain embodiments, the modified oligonucleotide comprises at least one nucleoside comprising a modified sugar. In certain embodiments, each nucleoside of the modified oligonucleotide comprises a modified sugar. In certain embodiments, the modified sugar is independently selected from a 2'-0- methoxyethyl sugar, a 2'-fluoro sugar, 2'-0-methyl sugar, and a bicyclic sugar moiety.

In certain embodiments, the modified oligonucleotide consists of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or 22 linked nucleosides.

In certain embodiments, the methods provided herein comprise selecting a subject having elevated serum fatty acid levels relative to normal serum fatty acid levels.

Provided herein are compounds comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member, for use in therapy. In certain embodiments, the therapy is the treatment of a metabolic disorder. In certain embodiments, the therapy is the prevention or delay of onset of a metabolic disorder.

These and other embodiments of the present invention will become apparent in conjunction with the figures, description and claims that follow.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the arts to which the invention belongs. Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are the definitions which are well known and commonly used in the art. In the event that there is a plurality of definitions for terms herein, those in this section prevail. Standard techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of subjects. Certain such techniques and procedures may be found for example in "Carbohydrate

Modifications in Antisense Research" Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; and "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 18th edition, 1990. Both of these references are hereby incorporated by reference for any purpose. Where permitted, all patents, patent applications, published applications and publications, GENBANK sequences, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can change, but equivalent information can be found by searching archives of known past addresses or by performing new internet searches. Reference to a URL evidences the availability and public dissemination of such information.

Before the present compositions and methods are disclosed and described, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

Definitions

"Blood glucose level" means the concentration of glucose in the blood of a subject. In certain embodiments, blood glucose levels are expressed as milligrams of glucose per deciliter (dL) of blood. In certain embodiments, blood glucose levels are expressed as mmol of glucose per liter (L) of blood.

"Elevated blood glucose level" means a blood glucose level in a subject that is higher than normal according to standard medical criteria.

"Fasted blood glucose level" means a blood glucose level after a subject has fasted for a certain length of time. For example, a subject may fast for at least 8 hours prior to measurement of a fasted blood glucose level.

"Post-prandial blood glucose level" means a blood glucose level after a subject has eaten a meal. In certain embodiments, a post-prandial blood glucose level is measured two hours after a subject has eaten a meal.

"Whole blood glucose level" means the concentration of glucose in whole blood which has not been subjected to separation.

"Plasma blood glucose level" means the concentration of glucose in plasma following separation of whole blood into plasma and red blood cell fractions.

"Insulin sensitivity" means the ability of cells to take up glucose in response to insulin action.

"Insulin response index" (IRI) means a measure of insulin resistance calculated by multiplying a subject's amount of blood glucose (in mg/dl) and a subject's amount of blood insulin (in ng/ml). Insulin response index is analogous to the homeostatic model assessment (HOMA) method used to quantify insulin response and beta-cell function.

"Insulin resistance" means a condition in which normal amounts of insulin are inadequate to produce a normal insulin response from fat, muscle and liver cells. "Improving insulin resistance" means increasing the ability of cells to produce a normal insulin response. In certain embodiments, the cell is a fat cell. In certain embodiments, the cell is a liver cell. In certain embodiments, the cell is a muscle cell.

"Metabolic disorder" means a condition characterized by an alteration or disturbance in one or more metabolic processes in the body. Metabolic disorders include, but are not limited to, hyperglycemia, prediabetes, diabetes, type 1 diabetes, type 2 diabetes, obesity, diabetic dyslipidemia, metabolic syndrome, and hyperinsulinemia.

"Diabetes" or "diabetes mellitus" means a disease in which the body does not produce or properly use insulin, resulting in abnormally high blood glucose levels. In certain embodiments, diabetes is type 1 diabetes. In certain embodiments, diabetes is type 2 diabetes.

"Prediabetes" means a condition in which a subject's blood glucose levels are higher than in a subject with normal blood glucose levels but not high enough for a diagnosis of diabetes.

"Type 1 diabetes" means diabetes characterized by loss of the insulin-producing beta cells of the islets of Langerhans in the pancreas leading to a deficiency of insulin (also known as insulin-dependent diabetes mellitus or IDDM). Type I diabetes can affect children or adults, but typically appears between the ages of 10 and 16.

"Type 2 diabetes" means diabetes characterized by insulin resistance and relative insulin deficiency (also known as diabetes mellitus type 2, and formerly called diabetes mellitus type 2, non-insulin-dependent diabetes (NIDDM), obesity related diabetes, or adult-onset diabetes).

"Obesity" means an excessively high amount of body fat or adipose tissue in relation to lean body mass. The amount of body fat (or adiposity) includes both the distribution of fat throughout the body and the size of the adipose tissue deposits. Body fat distribution can be estimated by skin-fold measures, waist-to-hip circumference ratios, or techniques such as ultrasound, computed tomography, or magnetic resonance imaging. According to the Center for Disease Control and Prevention, individuals with a body mass index (BMI) of 30 or more are considered obese.

"Diabetic dyslipidemia" or "Type 2 diabetes with dyslipidemia" means a condition characterized by Type 2 diabetes, reduced HDL-C, elevated serum triglycerides, and elevated small, dense LDL particles.

"Metabolic syndrome" means a condition characterized by a clustering of lipid and nonlipid risk factors of metabolic origin. In certain embodiments, metabolic syndrome is identified by the presence of any 3 of the following factors: waist circumference of greater than 102 cm in men or greater than 88 cm in women; serum triglyceride levels of at least 150 mg/dL; HDL-C levels less than 40 mg/dL in men or less than 50 mg/dL in women; blood pressure of at least 130/85 mmHg; and fasting glucose of at least 1 10 mg/dL. These determinants can be readily measured in clinical practice (JAMA, 2001 , 285: 2486-2497).

"Steatosis" means a condition characterized by the excessive accumulation of triglycerides in hepatocytes.

"Steatohepatitis" means steatosis with inflammation. "Non-alcoholic fatty liver disease (NAFLD)" means a condition characterized by accumulation of fat in the liver in subjects who consume little to no alcohol. In certain embodiments, NAFLD is related to insulin resistance and the metabolic syndrome.

"Nonalcoholic steatohepatitis (NASH)" means a condition characterized by accumulation of fat in the liver, combined with inflammation and scarring in the liver. In certain embodiments NASH is a result of a worsening progression of NAFLD.

"Alcoholic steatohepatitis (ASH)" means an alcohol-induced condition characterized by accumulation of fat in the liver, combined with inflammation and scarring in the liver.

"Glucose tolerance test" or "GTT" means a test performed to determine how quickly glucose is cleared from the blood. Typically, the test involves administration of glucose, followed by measurement of glucose levels in blood at intervals over a period of time. "IPGTT" means a GTT performed following intraperitoneal injection of glucose. "OGTT" means a GTT performed following oral administration of glucose. In certain embodiments, a GTT is used to test for pre-diabetes. In certain embodiments, a GTT is used to identify a subject with diabetes. In certain embodiments, a GTT is used to identify a subject at risk for developing diabetes. In certain embodiments a GTT is used to identify a subject having insulin resistance.

"Insulin Tolerance Test (ITT)" means a test performed to measure insulin sensitivity through hormone response to the stress of a low blood sugar level. In certain embodiments, an ITT is used to test for pre-diabetes. In certain embodiments, an ITT is used to identify a subject with diabetes. In certain embodiments, an ITT is used to identify a subject at risk for developing diabetes. In certain embodiments an ITT is used to identify a subject having insulin resistance.

"Metabolic rate" means the rate of metabolism or the amount of energy expended in a given period. "Basal metabolic rate" means the amount of energy expended while at rest in a neutrally temperate environment, in the post-absorptive state (meaning that the digestive system is inactive, which requires about twelve hours of fasting in humans). The release of energy in this state is sufficient only for the functioning of the vital organs, such as the heart, lungs, brain and the rest of the nervous system, as well as the liver, kidneys, sex organs, muscles and skin.

"Anti-miR" means an oligonucleotide having a nucleobase sequence complementary to a microRNA. In certain embodiments, an anti-miR is a modified oligonucleotide.

"Subject" means a human or non-human animal selected for treatment or therapy.

"Subject in need thereof means a subject identified as in need of a therapy or treatment. In certain embodiments, a subject has a metabolic disorder. In embodiments, a subject has one or more clinical indications of a metabolic disorder or is at risk for developing a metabolic disorder.

"Administering" means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering.

"Parenteral administration," means administration through injection or infusion.

Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration. "Subcutaneous administration" means administration just below the skin.

"Intravenous administration" means administration into a vein.

"Administered concomitantly" refers to the administration of at least two agents to a subject in any manner in which the pharmacological effects of both are manifested in the subject at the same time.

Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The time during which the effects of the agents occur need not be identical. The effects need only be overlapping for a period of time and need not be coextensive.

"Duration" means the period of time during which an activity or event continues. In certain embodiments, the duration of treatment is the period of time during which doses of a pharmaceutical agent or pharmaceutical composition are administered.

"Therapy" means a disease treatment method. In certain embodiments, therapy includes, but is not limited to, chemotherapy, surgical resection, liver transplant, and/or chemoembolization.

"Treatment" means the application of one or more specific procedures used for the cure or amelioration of a disease. In certain embodiments, the specific procedure is the administration of one or more pharmaceutical agents.

"Amelioration" means a lessening of severity of at least one indicator of a condition or disease. In certain embodiments, amelioration includes a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art.

"At risk for developing" means a subject is predisposed to developing a condition or disease. In certain embodiments, a subject at risk for developing a condition or disease exhibits one or more symptoms of the condition or disease, but does not exhibit a sufficient number of symptoms to be diagnosed with the condition or disease. In certain embodiments, a subject at risk for developing a condition or disease exhibits one or more symptoms of the condition or disease, but to a lesser extent required to be diagnosed with the condition or disease.

"Prevent the onset of means to prevent the development a condition or disease in a subject who is at risk for developing the disease or condition. In certain embodiments, a subject at risk for developing the disease or condition receives treatment similar to the treatment received by a subject who already has the disease or condition.

"Delay the onset of means to delay the development of a condition or disease in a subject who is at risk for developing the disease or condition. In certain embodiments, a subject at risk for developing the disease or condition receives treatment similar to the treatment received by a subject who already has the disease or condition.

"Therapeutic agent" means a pharmaceutical agent used for the cure, amelioration or prevention of a disease.

"Dose" means a specified quantity of a pharmaceutical agent provided in a single administration. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual.

"Dosage unit" means a form in which a pharmaceutical agent is provided. In certain embodiments, a dosage unit is a vial containing lyophilized oligonucleotide. In certain embodiments, a dosage unit is a vial containing reconstituted oligonucleotide.

"Therapeutically effective amount" refers to an amount of a pharmaceutical agent that provides a therapeutic benefit to an animal.

"Pharmaceutical composition" means a mixture of substances suitable for administering to an individual that includes a pharmaceutical agent. For example, a pharmaceutical composition may comprise a sterile aqueous solution.

"Pharmaceutical agent" means a substance that provides a therapeutic effect when administered to a subject.

"Active pharmaceutical ingredient" means the substance in a pharmaceutical composition that provides a desired effect.

"Improved liver function" means the change in liver function toward normal limits. In certain embodiments, liver function is assessed by measuring molecules found in a subject's blood. For example, in certain embodiments, improved liver function is measured by a reduction in blood liver transaminase levels.

"Acceptable safety profile" means a pattern of side effects that is within clinically acceptable limits.

"Side effect" means a physiological response attributable to a treatment other than desired effects. In certain embodiments, side effects include, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies. Such side effects may be detected directly or indirectly. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.

"Injection site reaction" means inflammation or abnormal redness of skin at a site of injection in an individual.

"Subject compliance" means adherence to a recommended or prescribed therapy by a subject.

"Comply" means the adherence with a recommended therapy by a subject.

"Recommended therapy" means a treatment recommended by a medical professional for the treatment, amelioration, or prevention of a disease.

"Target nucleic acid" means a nucleic acid to which an oligomeric compound is designed to hybridize.

"Targeting" means the process of design and selection of nucleobase sequence that will hybridize to a target nucleic acid.

"Targeted to" means having a nucleobase sequence that will allow hybridization to a target nucleic acid. "Modulation" means to a perturbation of function or activity. In certain embodiments, modulation means an increase in gene expression. In certain embodiments, modulation means a decrease in gene expression.

"Expression" means any functions and steps by which coded information of a gene is converted into structures present and operating in a cell. Such structures, include, but are not limited to microRNAs, messenger R As, transfer RNAs, and proteins.

"5' target site" refers to the nucleobase of a target nucleic acid which is complementary to the 5'- most nucleobase of a particular oligonucleotide.

"3' target site" means the nucleobase of a target nucleic acid which is complementary to the 3'-most nucleobase of a particular oligonucleotide.

"Region" means a portion of linked nucleosides within a nucleic acid. In certain embodiments, an oligomeric compound has a nucleobase sequence that is complementary to a region of a target nucleic acid. For example, in certain embodiments an oligomeric compound is complementary to a region of a miRNA stem-loop sequence. In certain embodiments, an oligomeric compound is fully complementary to a region of a miRNA stem-loop sequence.

"Segment" means a smaller portion or sub-portion of a region.

"Nucleobase sequence" means the order of contiguous nucleobases, in a 5' to 3' orientation, independent of any sugar, linkage, and/or nucleobase modification.

"Contiguous nucleobases" means nucleobases immediately adjacent to each other in a nucleic acid.

"Nucleobase complementarity" means the ability of two nucleobases to pair non-covalently via hydrogen bonding.

"Complementary" means that an oligomeric compound is capable of hybridizing to a target nucleic acid under stringent hybridization conditions.

"Fully complementary" means each nucleobase of an oligomeric compound is capable of pairing with a nucleobase at each corresponding position in a target nucleic acid. For example, in certain embodiments, an oligomeric compound wherein each nucleobase has complementarity to a nucleobase within a region of a miRNA stem-loop sequence is fully complementary to the miRNA stem-loop sequence.

"Percent complementarity" means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound. In certain embodiments, percent complementarity of an oligomeric compound means the number of nucleobases that are complementary to the target nucleic acid, divided by the length of the modified oligonucleotide. For example, " 100% complementary" means that each nucleobase of an oligonucleotide is complementary to the nucleobase at the corresponding position in the target nucleic acid.

"Percent identity" means the number of nucleobases in a first nucleic acid that are identical to nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid. "Hybridize" means the annealing of complementary nucleic acids that occurs through nucleobase complementarity.

"Mismatch" in context of nucleobase sequences means a nucleobase of a first nucleic acid that is not capable of pairing with a nucleobase at a corresponding position of a second nucleic acid.

"Identical" in context of nucleobase sequences means having the same nucleobase sequence.

"MicroRNA" means a non-coding RNA between 18 and 25 nucleobases in length, which is the product of cleavage of a pre-miRNA by the enzyme Dicer. Examples of mature miRNAs are found in the miRNA database known as miRBase (http://microrna.sanger.ac.uk/). MicroRNA is abbreviated as "miRNA" or "miR."

"Pre-miRNA" or "pre-miR" means a non-coding RNA having a hairpin structure, which is the product of cleavage of a pri-miR by the double-stranded RNA-specific ribonuclease known as Drosha.

"Stem-loop sequence" means an RNA having a hairpin structure and containing a mature miRNA sequence. Pre-miRNA sequences and stem-loop sequences may overlap. Examples of stem-loop sequences are found in the miRNA database known as miRBase (http://microrna.sanger.ac.uk ).

"Pri-miRNA" or "pri-miR" means a non-coding RNA having a hairpin structure that is a substrate for the double-stranded RNA-specific ribonuclease Drosha.

"miRNA precursor" means a transcript that originates from a genomic DNA and that comprises a non-coding, structured RNA comprising one or more miRNA sequences. For example, in certain embodiments a miRNA precursor is a pre-miRNA. In certain embodiments, a miRNA precursor is a pri- miRNA.

"Monocistronic transcript" means a miRNA precursor containing a single miRNA sequence.

"Polycistronic transcript" means a miRNA precursor containing two or more miRNA sequences.

"Seed sequence" means a nucleobase sequence comprising from 6 to 8 contiguous nucleobases of nucleobases 1 to 9 of the 5'-end of a mature microRNA sequence.

"Seed match sequence" means a nucleobase sequence that is complementary to a seed sequence, and is the same length as the seed sequence.

"Compound comprising a modified oligonucleotide consisting of a number of linked nucleosides means a compound that includes a modified oligonucleotide having the specified number of linked nucleosides. Thus, the compound may include additional substituents or conjugates. Unless otherwise indicated, the compound does not include any additional nucleosides beyond those of the oligonucleotide.

"Oligomeric compound" means a compound comprising a polymer of linked monomeric subunits.

"Oligonucleotide" means a polymer of linked nucleosides, each of which can be modified or unmodified, independent from one another.

"Naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage between nucleosides.

"Natural sugar" means a sugar found in DNA (2'-H) or RNA (2'-OH).

"Natural nucleobase" means a nucleobase that is unmodified relative to its naturally occurring form. "Internucleoside linkage" means a covalent linkage between adjacent nucleosides. "Linked nucleosides" means nucleosides joined by a covalent linkage.

"Nucleobase" means a heterocyclic moiety capable of non-covalently pairing with another nucleobase.

"Nucleoside" means a nucleobase linked to a sugar.

"Nucleotide" means a nucleoside having a phosphate group covalently linked to the sugar portion of a nucleoside.

"Modified oligonucleotide" means an oligonucleotide having one or more modifications relative to a naturally occurring terminus, sugar, nucleobase, and/or intemucleoside linkage.

"Single-stranded modified oligonucleotide" means an oligonucleotide which is not hybridized to a complementary strand.

"Modified intemucleoside linkage" means any change from a naturally occurring intemucleoside linkage.

"Phosphorothioate intemucleoside linkage" means a linkage between nucleosides where one of the non-bridging atoms is a sulfur atom.

"Modified sugar" means substitution and/or any change from a natural sugar.

"Modified nucleobase" means any substitution and/or change from a natural nucleobase.

"5-methyIcytosine" means a cytosine modified with a methyl group attached to the 5' position.

"2'-0-methyl sugar" or "2'-OMe sugar" means a sugar having an O-methyl modification at the 2' position.

"2'-0-methoxyethyl sugar" or "2'-MOE sugar" means a sugar having an O-methoxyethyl modification at the 2' position.

"2'-0-fluoro" or "2'-F" means a sugar having a fluoro modification of the 2' position.

"Bicyclic sugar moiety" means a sugar modified by the bridging of two non-geminal ring atoms.

"2'-0-methoxyethyl nucleoside" means a 2'-modified nucleoside having a 2'-0-methoxyethyl sugar modification.

"2'-fluoro nucleoside" means a 2'-modified nucleoside having a 2'-fluoro sugar modification.

"2'-0-methyl" nucleoside means a 2'-modified nucleoside having a 2'-0-methyl sugar modification.

"Bicyclic nucleoside" means a 2'-modified nucleoside having a bicyclic sugar moiety.

"Motif means a pattern of modified and/or unmodified nucleobases, sugars, and/or intemucleoside linkages in an oligonucleotide.

A "fully-modified oligonucleotide" means each nucleobase, each sugar, and/or each intemucleoside linkage of an oligonucleotide is modified.

A "uniformly-modified oligonucleotide" means each nucleobase, each sugar, and/or each intemucleoside linkage of an oligonucleotide has the same modification throughout the modified oligonucleotide.

A "stabilizing modification" means a modification to a nucleoside that provides enhanced stability to a modified oligonucleotide, in the presence of nucleases, relative to that provided by 2'-deoxynucleosides linked by phosphodiester intemucleoside linkages. For example, in certain embodiments, a stabilizing modification is a stabilizing nucleoside modification. In certain embodiments, a stabilizing modification is an internucleoside linkage modification.

A "stabilizing nucleoside" means a nucleoside modified to provide enhanced nuclease stability to an oligonucleotide, relative to that provided by a 2'-deoxynucleoside. In one embodiment, a stabilizing nucleoside is a 2'-modified nucleoside.

A "stabilizing internucleoside linkage" means an internucleoside linkage that provides improved nuclease stability to an oligonucleotide relative to that provided by a phosphodiester internucleoside linkage. In one embodiment, a stabilizing internucleoside linkage is a phosphorothioate internucleoside linkage.

"miR-378" means the mature microRNA having the nucleobase sequence set forth in SEQ ID NO:

1.

"miR-378 stem-loop sequence" means the stem-loop sequence set forth in SEQ ID NO: 2.

"miR-422a" means the mature microRNA having the nucleobase sequence set forth in SEQ ID NO:

3.

"miR-422a stem-loop sequence" means the stem-loop sequence set forth in SEQ ID NO: 4 "miR-378 family" means a group of microRNAs including miR-378 and miR-422a. It will be understood by the skilled person that this definition is intended to include these yet-to-be-discovered family members as well.

"miR-378 seed sequence" means a seed sequence present in the nucleobase sequence of each member of the miR-378 family.

Overview

Metabolic disorders are characterized by one or more abnormalities in metabolic function in the body. Certain metabolic disorders are related to defects in how the body uses blood glucose, resulting in abnormally high levels of blood glucose. Metabolic disorders may also be characterized by a deficiency in insulin production, or a deficiency in sensitivity to insulin. Metabolic disorders affect millions of people worldwide, and can be life-threatening disorders. As such, there is a need for method and compositions to treat, prevent, or delay the onset of metabolic disorders.

As illustrated herein, the inhibition of miR-378 in an experimental model of impaired glucose tolerance and type 2 diabetes influences the metabolism of mice with diet-induced obesity. In this model, inhibition of miR-378 reduced blood glucose levels, reduced blood insulin levels, reduced insulin excursion and improved insulin response index during oral glucose tolerance tests. Decreased levels of fatty acids and increased levels of beta-hydroxybutyrate, a metabolic oxidation product of fatty acids were also observed. Also observed was an improvement in the liver health of the animals in which miR-378 activity was inhibited.

Accordingly, provided herein are methods and compositions to inhibit miR-378. Such methods result in the improvement of insulin resistance, the reduction of blood glucose levels, the reduction of blood insulin levels, improvements in insulin sensitivity, and improvements in liver health. Also provided herein are methods to treat, prevent, or delay the onset of metabolic disorders that are related to elevated blood glucose levels, elevated insulin levels, and/or impaired insulin sensitivity, through the use of a composition that inhibits miR-378. In certain embodiments, metabolic disorders include, but are not limited to, prediabetes, diabetes, including Type 1 or Type 2 diabetes, metabolic syndrome, obesity, diabetic dyslipidemia, hyperglycemia, hypoglycemia, and hyperinsulinemia.

Also provided herein are methods and compositions for inhibiting two or more miR-378 family members. Such methods may comprise a modified oligonucleotide having 100% overall complementarity to two or more miR-378 family members. Alternatively, such methods may comprise the administration of one or more modified oligonucleotides, each having 100% overall complementarity to a single member of the miR-378 family. For example, the methods provided herein comprise administration of two compounds, one of which has a nucleobase sequence complementary to miR-378 and the other having a nucleobase sequence complementary to miR-422.

Certain Conditions and Treatments

As described herein, inhibition of miR-378 results in desirable outcomes, such as improved insulin resistance and reduced blood glucose levels. Accordingly, provided herein are compositions and methods for the inhibition of a member of the miR-378 family in a subject having a metabolic disorder or symptoms of a metabolic disorder. Such methods comprise administering a compound comprising a modified

oligonucleotide having a nucleobase sequence complementary to a member of the miR-378 family, or a precursor thereof. In any of the embodiments described herein, a member of the miR-378 family is miR-378. In any of the embodiments described herein, a member of the miR-378 family is miR-422a.

In certain embodiments, provided herein are methods for treating a metabolic disorder in a subject comprising administering to the subject a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to a member of the miR-378 family. In certain embodiments, the subject has a metabolic disorder. In certain embodiments, the subject is identified as having a metabolic disorder. In certain embodiments, a metabolic disorder includes, without limitation, prediabetes, diabetes (including Type 1 or Type 2 diabetes), metabolic syndrome, obesity, or diabetic dyslipidemia, hyperglycemia, hypoglycemia, hyperfattyacidemia and hyperinsulinemia. In certain embodiments, the subject is diagnosed with one or more metabolic disorders. A subject may be diagnosed with a metabolic disorder following the administration of medical tests well-known to those in the medical profession.

In certain embodiments, provided herein are methods for preventing the onset of a metabolic disorder in a subject comprising administering to the subject a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to a member of the miR-378 family, or a precursor thereof. In certain embodiments, the subject is at risk for developing a metabolic disorder. In certain embodiments, the subject is identified being at risk for developing a metabolic disorder. In certain embodiments, a metabolic disorder is prediabetes, diabetes (including Type 1 or Type 2 diabetes), metabolic syndrome, obesity, or diabetic dyslipidemia,

hyperglycemia, hypoglycemia, hyperinsulinemia, hyperfattyacidemia, ketoacidosis and celiac disease. In certain embodiments, provided herein are methods for delaying the onset of a metabolic disorder in a subject comprising administering to the subject a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to a member of the miR-378 family, or a precursor thereof. In certain embodiments, the subject is at risk for developing a metabolic disorder. In certain embodiments, the subject is identified being at risk for developing a metabolic disorder. In certain embodiments, a metabolic disorder includes, without limitation, prediabetes, diabetes (including Type 1 or Type 2 diabetes), metabolic syndrome, obesity, or diabetic dyslipidemia, hyperglycemia, hypoglycemia, hyperfattyacidemia and hyperinsulinemia.

In certain embodiments, a subject has one or more metabolic disorders. In certain embodiments, a subject has been diagnosed with one or more metabolic disorders. A subject may be diagnosed with a metabolic disorder following the administration of medical tests well-known to those in the medical profession.

A subject's response to treatment may be evaluated by tests similar to those used to diagnosis the metabolic disorder, including blood glucose level tests, glucose tolerance tests, and HbA l c tests. Response to treatment may also be assessed by comparing post-treatment test results to pre-treatment test results.

In certain embodiments, a subject having elevated blood glucose levels is insulin resistant.

One of the main functions of insulin is to lower blood glucose levels. A subject whose cells are sensitive to the effects of insulin needs only a relatively small amount of insulin to keep blood glucose levels in the normal range. A subject who is insulin resistant requires more insulin to get the same blood glucose- lowering effects. Insulin resistance in muscle and fat cells results in reduced glucose uptake, while insulin resistance in liver reduces glucose storage and a failure to suppress glucose production. Other insulin- dependent functions can be affected in insulin resistant state, for example insulin resistance in fat cells results in increased hydrolysis of stored triglycerides, which elevates free fatty acids in the blood.

Insulin resistance may cause hyperinsulinemia. Hyperinsulinemia may be associated with high blood pressure, heart disease and heart failure, obesity (particularly abdominal obesity), osteoporosis, and certain types of cancer, such as colon, breast, and prostate cancer.

Provided herein are methods and compositions for treating subjects having insulin resistance. In certain embodiments, a subject having elevated blood glucose levels has insulin resistance. In certain embodiments, a subject having diabetes has insulin resistance. In certain embodiments, a subject having type 2 diabetes has insulin resistance. In certain embodiments, a subject having type 1 diabetes has insulin resistance.

In certain embodiments, provided herein are methods for improving insulin resistance in a subject comprising administering a subject to the subject a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence complementary to a member of the miR-378 family, or precursor thereof. In certain embodiments, the subject has insulin resistance. In certain embodiments, the methods comprise selecting a subject having insulin resistance. In certain embodiments, insulin resistance is improved in muscle cells, leading to an increased uptake of glucose in muscle cells. In certain embodiments, insulin resistance is improved in liver cells, leading to increased glucose storage in liver cells. In certain embodiments, insulin resistance is improved in fat cells, leading to reduced hydrolysis of triglycerides, and consequently reduced free fatty acid in the blood.

Insulin resistance may be detected using a procedure known as the hyperinsulinemic euglycemic clamp, which measures the amount of glucose necessary to compensate for an increased insulin level without causing hypoglycemia. During the procedure, insulin is infused at 10- 120 mU per m² per minute. In order to compensate for the insulin infusion, a 20% solution of glucose is infused to maintain blood sugar levels between 5 and 5.5 mmol/L. The rate of glucose infusion is determined by checking the blood sugar levels every 5 to 10 minutes. Low-dose insulin infusions are more useful for assessing the response of the liver, whereas high-dose insulin infusions are useful for assessing peripheral (i.e., muscle and fat) insulin action. The rate of glucose infusion during the last 30 minutes of the test determines insulin sensitivity. If high levels (7.5 mg/min or higher) are required, the subject is insulin-sensitive. Very low levels (4.0 mg/min or lower) indicate that the subject is resistant to insulin action. Levels between 4.0 and 7.5 mg/min are not definitive and suggest impaired glucose tolerance. Impaired glucose tolerance may be an early sign of insulin resistance. Glucose tracers, such as 3-³H glucose, 6,6-²H-glucose, or 1 -^I3C glucose, may be used in the procedure. Other radioactive forms of glucose may be employed in a research setting. Prior to beginning the hyperinsulinemic period, a 3 hour tracer infusion enables the determination of the basal rate of glucose production. During the clamp procedure, the plasma tracer concentrations enable the calculation of whole- body insulin-stimulated glucose metabolism, as well as the production of glucose by the body (i.e., endogenous glucose production).

In certain embodiments, provided herein are methods for reducing blood glucose levels in a subject comprising administering to the subject a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to a member of the miR-378 family.

In certain embodiments, the methods provided herein comprise measuring blood glucose levels. Blood glucose levels may be measured before and/or after administration of a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to a member of the miR-378 family. Blood glucose levels may be measured in whole blood, or may be measured in plasma. Blood glucose levels may be measured in a clinical laboratory, or may be measured using a blood glucose meter.

In certain embodiments, blood glucose levels are measured in a subject when the subject has fasted for at least 8 hours. In certain embodiments, blood glucose levels are measured at random times, and the measurement is not timed according to the intake of food or drink. In certain embodiments, blood glucose levels are measured in the post-prandial state, i.e. after the subject has eaten a meal. In certain embodiments, blood glucose levels are measured in a subject two hours after the subject has eaten a meal. In certain embodiments, blood glucose levels are measured at timed intervals following administration of glucose to the subject, in order to determine how quickly the subject's body clears glucose from the blood. Any measurements of blood glucose levels may be made in whole blood or in plasma. In certain embodiments, the subject has elevated blood glucose levels. In certain embodiments, a subject is identified as having elevated blood glucose levels. Such identification is typically made by a medical professional. In certain embodiments, an elevated blood glucose levels is a fasting blood glucose level between 100 and 125 mg/dL. In certain embodiments, an elevated blood glucose level is a fasting blood glucose level above 126 mg dL. In certain embodiments, an elevated blood glucose level is a two-hour post-prandial glucose level between 140 and 199 mg/dL. In certain embodiments, an elevated blood glucose level is a two-hour post-prandial glucose level at 200 mg/dL or higher.

In certain embodiments, a subject having elevated blood glucose levels has pre-diabetes. In certain embodiments, a subject is identified as having pre-diabetes. In certain embodiments, the subject has a fasting blood glucose level between 100 and 125 mg/dL. In certain embodiments, the subject has a two-hour postprandial blood glucose level between 140 and 199 mg/dL. A diagnosis of pre-diabetes is typically made by a medical professional, who may consider factors in addition to blood glucose levels when determining whether a subject has pre-diabetes.

In certain embodiments, a subject having elevated blood glucose levels has diabetes. In certain embodiments, a subject is identified as having diabetes according to the subject's blood glucose levels. In certain embodiments, the subject has a fasting blood glucose level above 126 mg/dL. In certain

embodiments, the subject has a two-hour post-prandial blood glucose level at or above 200 mg/dL. A diagnosis of diabetes is typically made by a medical professional, who may consider factors in addition to blood glucose levels when determining whether a subject has diabetes.

In certain embodiments, the method provided herein comprise monitoring blood glucose levels before administration of a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to a member of the miR-378 family. In certain embodiments, the methods provided herein comprise measuring blood glucose levels after administration of a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to a member of the miR-378 family. In certain embodiments, a subject measures blood glucose levels one or more times daily.

In certain embodiments, methods for reducing blood glucose levels comprise reducing a subject's blood glucose levels to blood glucose levels determined as desirable by medical organizations, such as the American Diabetes Association or the World Health Organization. In certain embodiments, blood glucose levels are reduced below 130 mg/dL when measured before a subject has had a meal. In certain

embodiments, blood glucose levels are reduced to below 180 mg/dL when measured after a subject has had a meal.

In certain embodiments, the administration occurs at least once per week. In certain embodiments, the administration occurs once every two weeks. In certain embodiments, the administration occurs once every three weeks. In certain embodiments, the administration occurs once every four weeks. The frequency of administration may be set by a medical professional to achieve a desirable blood glucose level in a subject. The frequency of administration may be dependent upon a subject's blood glucose levels. For example, in certain embodiments, administration may be more frequent when a subject has elevated blood glucose levels.

Measurements of HbAlc levels may be used to determine how well a subject's blood glucose levels are controlled over time. HbAlc levels are an indication of the amount of glycated hemoglobin in the blood, and can provide an estimate of how well a subject's blood glucose levels have been managed over 2-3 month period prior to the measurement of HbA l c levels. High HbA l c levels may put a subject at risk for developing complications related to diabetes, such as eye disease, heart disease, kidney disease, nerve damage, or stroke. As such, in certain embodiments it is desirable that a subject's HbAl c levels be within ranges that are considered normal by a medical professional. In certain embodiments, an HbAlc level of 6% or less is normal. In certain embodiments, a medical professional may recommend that a subject's HbAl c level be 7% or less. In certain embodiments, the administering results in reduced HbAlc levels.

In certain embodiments, the activity of a member of the miR-378 family is inhibited by use of a microRNA sponge, which comprises one or more sequences having nucleobase complementarity to a member of the miR-378 family. "MicroRNA sponge" means a competitive inhibitor of a microRNA in the form of a transcript expressed from a strong promoter, containing multiple, tandem binding sites to a microRNA of interest. When vectors encoding these sponges are introduced into cells, sponges de-repress microRNA targets at least as strongly as chemically modified antisense oligonucleotides. They specifically inhibit microRNAs with a complementary heptameric seed, such that a single sponge can be used to block an entire microRNA seed family. In certain embodiments, the microRNA family comprises miR-378 and miR-422a.

According to a publically available human gene target location tool (http://www.targetscan.org), the family currently defined by miR-378 and miR-422a has 106 conserved targets: transducer of ERBB2, 2, IAA1522, SDA1 domain containing 1 , methyltransferase like 4, cell division cycle 40 homolog (S.

cerevisiae), polyhomeotic homolog 3 (Drosop ila), sulfatase 1 , RNA binding motif single stranded interacting protein, ribosomal RNA processing 1 homolog B (S. cerevisiae), bone morphogenetic protein 2, neurogenic differentiation 1 , vang-like 1 (van gogh, Drosophila), AP2 associated kinase 1 , karyopherin alpha 6 (importin alpha 7), PAP associated domain containing 5, mediator complex subunit 12-like, mitogen-activated protein kinase 1 , pleckstrin homology domain containing, family G (with RhoGef domain) member 2, Cas-Br-M (murine) ecotropic retroviral transforming sequence, G-rich RNA sequence binding factor 1 , purine-rich element binding protein B, solute carrier family 38, member 1 , regulating synaptic membrane exocytosis 4, golgi transport 1 homolog A (5. cerevisiae), pleomorphic adenoma genelike 2, insulin-like growth factor 1 receptor, SRY (sex determining region Y)-box 7, forkhead box G l , doublecortex; lissencephaly, X-linked (doublecortin), dapper, antagonist of beta-catenin, homolog 1 (Xenopus laevis), solute carrier family 39 (zinc transporter), member 9, mitogen-activated protein kinase 1 interacting protein 1 -like, mediator complex subunit 19, IQ motif and Sec7 domain 2, KIAA1219, transducin (beta)-like 1 X-linked receptor I , speckle-type POZ protein-like, dual-specificity tyrosine-(Y)- phosphorylation regulated kinase 1 A, potassium voltage-gated channel, Shal-related subfamily, member 1 , calcium channel, voltage-dependent, alpha 2/delta subunit 4, importin 9, IAA1576 protein, FUS interacting protein (serine/arginine-rich) 1 , glutamine and serine rich 1 , NIMA (never in mitosis gene a)-related kinase 4, tousled-like kinase 2, melanoma-derived leucine zipper, extra-nuclear factor, arrestin domain containing 2, paired box 8, , poly(A) polymerase alpha, PDLIM 1 interacting kinase 1 like, ladybird homeobox 2, M L/myocardin-like 2, v-maf musculoaponeurotic fibrosarcoma oncogene homolog G (avian), dihydrolipoamide S-acetyltransferase, chromosome 1 open reading frame 21 , FERM and PDZ domain containing 4, pleckstrin and Sec7 domain containing 3, leukocyte receptor cluster (LRC) member 9, splicing factor, arginine/serine-rich 3, histone deacetylase 4, FK506 binding protein 5, SARI gene homolog A (S. cerevisiae), calneuron 1 , chromobox homolog 1 (HP1 beta homolog Drosophila), H3 histone, family 3B (H3.3B), Ras-like without CAAX 1 , zinc finger, DHHC-type containing 9, exportin 5, enabled homolog (Drosophila), growth factor receptor-bound protein 2, glutaminase, ankyrin repeat domain 52, clock homolog (mouse), WD repeat domain 37, WD repeat domain 40A, DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked, IAA0644 gene product, ubiquitin-conjugating enzyme E2W (putative), transmembrane protein 129, casein kinase 1 , gamma 2, frizzled homolog 5 (Drosophila), coenzyme Q 10 homolog B (S. cerevisiae), sterile alpha and TIR motif containing 1 , MAX binding protein, solute carrier family 30 (zinc transporter), member 8, kinase suppressor of ras 1 , BCL2-like 2, NSFL1 (p97) cofactor (p47), spindlin family, member 3, nuclear transcription factor, X-box binding 1, chromosome 17 open reading frame 74, bone morphogenetic protein 8b, regulator of chromosome condensation 2,

dihydrolipoamide branched chain transacylase E2, nuclear fragile X mental retardation protein interacting protein 2, chromosome 6 open reading frame 89, myocyte enhancer factor 2D, RNA binding motif protein 14, ring finger 144B, M-phase phosphoprotein 8, wingless-type MMTV integration site family, member 10A, metal-regulatory transcription factor 1, tetraspanin 17, extracellular leucine-rich repeat and fibronectin type 111 domain containing 2, and ribosomal protein L23a. It is expected that inhibition of protein expression of one or more of these targets by administration of a modified oligonucleotide targeting a miR-378 family member will be useful for treatment of metabolic disorders.

Certain Nucleobase Sequences

In certain embodiments, an oligonucleotide has a sequence that is complementary to a miRNA or a precursor thereof. Nucleobase sequences of mature miRNAs and their corresponding stem-loop sequences described herein are the sequences found in miRBase, an online searchable database of miRNA sequences and annotation, found at http://microrna.sanger.ac.uk/. Entries in the miRBase Sequence database represent a predicted hairpin portion of a miRNA transcript (the stem-loop), with information on the location and sequence of the mature miRNA sequence. The miRNA stem-loop sequences in the database are not strictly precursor miRNAs (pre-miRNAs), and may in some instances include the pre-miRNA and some flanking sequence from the presumed primary transcript. The miRNA nucleobase sequences described herein encompass any version of the miRNA, including the sequences described in Release 10.0 of the miRBase sequence database and sequences described in any earlier release of the miRBase sequence database. A sequence database release may result in the re-naming of certain miRNAs. The compositions of the present invention encompass modified oligonucleotides that are complementary to any nucleobase sequence version of the miR As described herein.

In certain embodiments, a modified oligonucleotide has a nucleobase sequence that is complementary to a miRNA or a precursor thereof. Accordingly, in certain embodiments, the nucleobase sequence of a modified oligonucleotide may have one or more mismatched base pairs with respect to its target miRNA or precursor sequence, and remains capable of hybridizing to its target sequence. In certain embodiments, a modified oligonucleotide has a nucleobase sequence that is fully complementary to a miRNA or precursor thereof.

In certain embodiments, a modified oligonucleotide has a nucleobase sequence that is

complementary to a region of the miR-378 stem-loop sequence (SEQ ID NO: 2). In certain embodiments, a modified oligonucleotide has a nucleobase sequence that is complementary to the region of nucleobases 43- 63 of SEQ ID NO: 2. In certain embodiments, a modified oligonucleotide has a nucleobase sequence that is complementary to the nucleobase sequence of miR-378 (ACUGGACUUGGAGUCAGAAGG; SEQ ID NO: 1). In certain embodiments, a modified oligonucleotide has a nucleobase sequence comprising the nucleobase sequence CCTTCTGACTCCAAGTCCAGT (SEQ ID NO: 5). In certain embodiments, a modified oligonucleotide has a nucleobase sequence consisting of the nucleobase sequence

CCTTCTGACTCCAAGTCCAGT (SEQ ID NO: 5).

complementary to a region of the miR-422a stem-loop sequence (SEQ ID NO: 4). In certain embodiments, a modified oligonucleotide has a nucleobase sequence that is complementary to the region of nucleobases 10- 31 of SEQ ID NO: 4. In certain embodiments, a modified oligonucleotide has a nucleobase sequence that is complementary to the nucleobase sequence of miR-422a (ACUGGACUUAGGGUCAGAAGGC; SEQ ID NO: 3). In certain embodiments, a modified oligonucleotide has a nucleobase sequence comprising the nucleobase sequence GCCTTCTGACCCTAAGTCCAGT (SEQ ID NO: 6). In certain embodiments, a modified oligonucleotide has a nucleobase sequence consisting of the nucleobase sequence

GCCTTCTGACCCTAAGTCCAGT (SEQ ID NO: 6)

As noted herein, miR-378 and miR-422a are members of a microRNA family and thus share an identical seed sequence. For example, nucleobases 2 through 9 of miR-378 are identical to nucleobases 2 through 9 of miR-422a. In certain embodiments, a modified oligonucleotide comprises a nucleobase sequence that is complementary to a miR-378 seed sequence selected from Table 1. Thus, the modified oligonucleotide is complementary to more than one miR-378 family member. For example, a modified oligonucleotide having a nucleobase sequence comprising SEQ ID NO: 20 is complementary to the nucleobase sequence of both miR-378 andmiR-422a. As such, the modified oligonucleotide may hybridize to and inhibit the activity of both miR-378 and miR-422a. In certain embodiments, a miR-378 family member comprises a seed sequence selected from Table 1. In certain embodiments, the modified oligonucleotide comprises a miR-378 seed match sequence selected from Table I .

Table 1: Seed and seed match sequences for the miR-378 family Seed Sequence Seed Sequence SEQ Seed Match Sequence SEQ Type (5' to 3') ID NO: (5' to 3') ID NO:

Nonamer 1-9 acuggacuu 8 aaguccagu 18

Octamer 1-8 acuggacu 9 aaguccag 19

Octamer 2-9 cuggacuu 10 aguccagu 20

Heptamer 1 -7 acuggac 1 1 aagucca 21

Heptamer 2-8 cuggacu 12 aguccag 22

Heptamer 3-9 uggacuu 13 guccagu 23

Hexamer 1-6 acugga 14 aagucc 24

Hexamer 2-7 cuggac 15 agucca 25

Hexamer 3-8 uggacu 16 guccag 26

Hexamer 4-9 ggacuu 17 uccagu 27

Oligonucleotides having any length described herein may comprise a seed-match sequence. In certain embodiments, the modified oligonucleotide consists of 7 linked nucleosides and comprises a seed- match sequence selected from SEQ ID NOs: 21 , 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 8 linked nucleosides and comprises a seed-match sequence selected from SEQ ID NOs: 19, 20, 21 , 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified

oligonucleotide consists of 9 linked nucleosides and comprises a seed-match sequence selected from SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide . consists of 10 linked nucleosides selected from SEQ ID NOs: 18, 19, 20, 21 , 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 1 1 linked nucleosides and comprises a seed- match sequence selected from SEQ ID NOs: 18, 19, 20, 21 , 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 12 linked nucleosides and comprises a seed-match sequence selected from SEQ ID NOs: 18, 19, 20, 21 , 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 13 linked nucleosides and comprises a seed-match sequence selected from SEQ ID NOs: 18, 19, 20, 21 , 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 14 linked nucleosides and comprises a seed-match sequence selected from SEQ ID NOs: 18, 19, 20, 21 , 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 15 linked nucleosides and comprises a seed-match sequence selected from SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides and comprises a seed-match sequence selected from SEQ ID NOs: 18, 19, 20, 21 , 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides and comprises a seed-match sequence selected from SEQ ID NOs: 1 8, 19, 20, 21 , 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 18 linked nucleosides and comprises a seed- match sequence selected from SEQ ID NOs: 18, 19, 20, 21 , 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 19 linked nucleosides and comprises a seed-match sequence selected from SEQ ID NOs: 18, 19, 20, 21 , 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides and comprises a seed-match sequence selected from SEQ ID NOs: 18, 19, 20, 21 , 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 21 linked nucleosides and comprises a seed-match sequence selected from SEQ ID NOs: 18, 19, 20, 21 , 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 22 linked nucleosides and comprises a seed-match sequence selected from SEQ ID NOs: 18, 19, 20, 21 , 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 23 linked nucleosides and comprises a seed-match sequence selected from SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 24 linked nucleosides and comprises a seed-match sequence selected from SEQ ID NOs: 18, 19, 20, 21 , 22, 23, 24, 25, 26 and 27. In certain embodiments, the modified oligonucleotide consists of 25 linked nucleosides and comprises a seed- match sequence selected from SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27.

Modified oligonucleotides consisting of 7, 8, 9, 10, or 1 1 linked nucleosides and complementary to nucleotides 2 through 9, 2 through 8 or 2 through 7 of a miRNA have been shown to inhibit activity of the miRNA. Such inhibitory activity is described in PCT Publication No. WO/2009/043353, which is herein incorporated by reference in its entirety for its description of modified oligonucleotides targeting miRNA seed sequences.

complementary to a nucleobase sequence having at least 80% identity to a nucleobase sequence of a miR stem-loop sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In certain embodiments, a modified

oligonucleotide has a nucleobase sequence that is complementary to a nucleobase sequence having at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% identity, or 100% identity to a nucleobase sequence of a miR stem-loop sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

complementary to a nucleobase sequence having at least 80% identity to a nucleobase sequence of a miRNA having a nucleobase sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In certain embodiments, a modified oligonucleotide has a nucleobase sequence that is complementary to a nucleobase sequence having at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% identity, or 100% identity to a nucleobase sequence of a miRNA nucleobase sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

In certain embodiments, a nucleobase sequence of a modified oligonucleotide is fully

complementary to a miRNA nucleobase sequence listed herein, or a precursor thereof. In certain embodiments, a modified oligonucleotide has a nucleobase sequence having one mismatch with respect to the nucleobase sequence of the mature miRNA, or a precursor thereof. In certain embodiments, a modified oligonucleotide has a nucleobase sequence having two mismatches with respect to the nucleobase sequence of the miRNA, or a precursor thereof. In certain embodiments, a modified oligonucleotide has a nucleobase sequence having no more than two mismatches with respect to the nucleobase sequence of the mature miRNA, or a precursor thereof. In certain embodiments, the mismatched nucleobases are contiguous. In certain embodiments, the mismatched nucleobases are not contiguous.

In certain embodiments, a modified oligonucleotide consists of a number of linked nucleosides that is equal to the length of the mature miR to which it is complementary.

In certain embodiments, the number of linked nucleosides of a modified oligonucleotide is less than the length of the mature miRNA to which it is complementary. In certain embodiments, the number of linked nucleosides of a modified oligonucleotide is one less than the length of the mature miR to which it is complementary. In certain embodiments, a modified oligonucleotide has one less nucleoside at the 5' terminus. In certain embodiments, a modified oligonucleotide has one less nucleoside at the 3' terminus. In certain embodiments, a modified oligonucleotide has two fewer nucleosides at the 5' terminus. In certain embodiments, a modified oligonucleotide has two fewer nucleosides at the 3' terminus. A modified oligonucleotide having a number of linked nucleosides that is less than the length of the miRNA, wherein each nucleobase of a modified oligonucleotide is complementary to each nucleobase at a corresponding position in a miRNA, is considered to be a modified oligonucleotide having a nucleobase sequence that is fully complementary to a portion of a miRNA sequence.

In certain embodiments, the number of linked nucleosides of a modified oligonucleotide is greater than the length of the miRNA to which it is complementary. In certain embodiments, the nucleobase of an additional nucleoside is complementary to a nucleobase of a miRNA stem-loop sequence. In certain embodiments, the number of linked nucleosides of a modified oligonucleotide is one greater than the length of the miRNA to which it is complementary. In certain embodiments, the additional nucleoside is at the 5' terminus of a modified oligonucleotide. In certain embodiments, the additional nucleoside is at the 3' terminus of a modified oligonucleotide. In certain embodiments, the number of linked nucleosides of a modified oligonucleotide is two greater than the length of the miRNA to which it is complementary. In certain embodiments, the two additional nucleosides are at the 5' terminus of a modified oligonucleotide. In certain embodiments, the two additional nucleosides are at the 3' terminus of a modified oligonucleotide. In certain embodiments, one additional nucleoside is located at the 5' terminus and one additional nucleoside is located at the 3' terminus of a modified oligonucleotide.

In certain embodiments, a portion of the nucleobase sequence of a modified oligonucleotide is fully complementary to the nucleobase sequence of the miRNA, but the entire modified oligonucleotide is not fully complementary to the miRNA. In certain embodiments, the number of nucleosides of a modified oligonucleotide having a fully complementary portion is greater than the length of the miRNA. For example, a modified oligonucleotide consisting of 24 linked nucleosides, where the nucleobases of nucleosides 1 through 23 are each complementary to a corresponding position of a miRNA that is 23 nucleobases in length, has a 23 nucleoside portion that is fully complementary to the nucleobase sequence of the miRNA and approximately 96% overall complementarity to the nucleobase sequence of the miRNA.

In certain embodiments, the nucleobase sequence of a modified oligonucleotide is fully complementary to a portion of the nucleobase sequence of a miRNA. By way of non-limiting example, a modified oligonucleotide consisting of 22 linked nucleosides, where the nucleobases of nucleosides 1 through 22 are each complementary to a corresponding position of a miRNA that is 23 nucleobases in length, is fully complementary to a 22 nucleobase portion of the nucleobase sequence of a miRNA. Such a modified oligonucleotide has approximately 96% overall complementarity to the nucleobase sequence of the entire miRNA, and has 100% complementarity to a 22 nucleobase portion of the miRNA.

In certain embodiments, a portion of the nucleobase sequence of a modified oligonucleotide is fully complementary to a portion of the nucleobase sequence of a miRNA, or a precursor thereof. In certain embodiments, 15 contiguous nucleobases of a modified oligonucleotide are each complementary to 15 contiguous nucleobases of a miRNA, or a precursor thereof. In certain embodiments, 16 contiguous nucleobases of a modified oligonucleotide are each complementary to 16 contiguous nucleobases of a miRNA, or a precursor thereof. In certain embodiments, 17 contiguous nucleobases of a modified oligonucleotide are each complementary to 17 contiguous nucleobases of a miRNA, or a precursor thereof. In certain embodiments, 18 contiguous nucleobases of a modified oligonucleotide are each complementary to 18 contiguous nucleobases of a miRNA, or a precursor thereof. In certain embodiments, 19 contiguous nucleobases of a modified oligonucleotide are each complementary to 19 contiguous nucleobases of a miRNA, or a precursor thereof. In certain embodiments, 20 contiguous nucleobases of a modified oligonucleotide are each complementary to 20 contiguous nucleobases of a miRNA, or a precursor thereof. In certain embodiments, 22 contiguous nucleobases of a modified oligonucleotide are each complementary to 22 contiguous nucleobases of a miRNA, or a precursor thereof. In certain embodiments, 23 contiguous nucleobases of a modified oligonucleotide are each complementary to 23 contiguous nucleobases of a miRNA, or a precursor thereof. In certain embodiments, 24 contiguous nucleobases of a modified oligonucleotide are each complementary to 24 contiguous nucleobases of a miRNA, or a precursor thereof.

The nucleobase sequences set forth herein, including but not limited to those found in the Examples and in the sequence listing, are independent of any modification to the nucleic acid. As such, nucleic acids defined by a SEQ ID NO may comprise, independently, one or more modifications to one or more sugar moieties, to one or more internucleoside linkages, and/or to one or more nucleobases.

Although the sequence listing accompanying this filing identifies each nucleobase sequence as either "RNA" or "DNA" as required, in practice, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as "RNA" or "DNA" to describe modified oligonucleotides is somewhat arbitrary. For example, a modified

oligonucleotide comprising a nucleoside comprising a 2'-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2'-OH for the natural 2'-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligomeric compound having the nucleobase sequence

"ATCGATCG" encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence "AUCGAUCG" and those having some DNA bases and some RNA bases such as "AUCGATCG" and oligomeric compounds having other modified bases, such as "AT^CGAUCG," wherein ^meC indicates a cytosine base comprising a methyl group at the 5-position.

Certain Modified Oligonucleotides

In certain embodiments, an oligonucleotide consists of 8 to 25 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 7 to 1 1 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 12 to 30 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 12 to 25 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 15 to 30 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 15 to 25 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 19 to 24 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 21 to 24 linked nucleosides.

In certain embodiments, a modified oligonucleotide consists of 7 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 8 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 9 linked nucleosides. In certain embodiments, a modified

oligonucleotide consists of 10 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 1 1 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 12 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 13 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 14 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 15 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 16 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 17 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 18 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 19 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 20 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 21 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 22 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 23 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 24 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 25 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 26 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 27 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 28 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 29 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 30 linked nucleosides.

Certain Compounds

The compounds provided herein are useful for the treatment of metabolic disorders. In certain embodiments, the compound comprises a modified oligonucleotide. In certain embodiments, the compound consists of a modified oligonucleotide. In certain embodiments, the oligonucleotide is a modified oligonucleotide.

In certain embodiments, the compound comprises an oligonucleotide hybridized to a complementary strand, i.e. the compound comprises a double-stranded oligomeric compound. In certain embodiments, the hybridization of an oligonucleotide to a complementary strand forms at least one blunt end. In certain embodiments, the hybridization of an oligonucleotide to a complementary strand forms a blunt end at each terminus of the double-stranded oligomeric compound. In certain embodiments, a terminus of an oligonucleotide comprises one or more additional linked nucleosides relative to the number of linked nucleosides of the complementary strand. In certain embodiments, the one or more additional nucleosides are at the 5' terminus of an oligonucleotide. In certain embodiments, the one or more additional nucleosides are at the 3' terminus of an oligonucleotide. In certain embodiments, at least one nucleobase of a nucleoside of the one or more additional nucleosides is complementary to the target RNA. In certain embodiments, each nucleobase of each one or more additional nucleosides is complementary to the target RNA. In certain embodiments, a terminus of the complementary strand comprises one or more additional linked nucleosides relative to the number of linked nucleosides of an oligonucleotide. In certain embodiments, the one or more additional linked nucleosides are at the 3' terminus of the complementary strand. In certain embodiments, the one or more additional linked nucleosides are at the 5' terminus of the complementary strand. In certain embodiments, two additional linked nucleosides are linked to a terminus. In certain embodiments, one additional nucleoside is linked to a terminus.

In certain embodiments, the compound comprises an oligonucleotide conjugated to one or more moieties which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. In certain embodiments, the moiety is a cholesterol moiety or a lipid moiety. Additional moieties for conjugation include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. In certain embodiments, a conjugate group is attached directly to an oligonucleotide. In certain embodiments, a conjugate group is attached to an oligonucleotide by a linking moiety selected from amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N- maleimidomethyl) cyclohexane-l-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C 10 alkynyl. In certain embodiments, a substituent group is selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, the compound comprises an oligonucleotide having one or more stabilizing groups that are attached to one or both termini of an oligonucleotide to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect an oligonucleotide from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap), or at the 3'-terminus (3'-cap), or can be present on both termini. Cap structures include, for example, inverted deoxy abasic caps.

Suitable cap structures include a 4',5'-methylene nucleotide, a l-(beta-D-erythrofuranosyl) nucleotide, a 4'-thio nucleotide, a carbocyclic nucleotide, a 1 ,5-anhydrohexitol nucleotide, an L-nucleotide, an alpha-nucleotide, a modified base nucleotide, a phosphorodithioate linkage, a threo-pentofuranosyl nucleotide, an acyclic 3',4'-seco nucleotide, an acyclic 3,4-dihydroxybutyl nucleotide, an acyclic 3,5- dihydroxypentyl nucleotide, a 3'-3'-inverted nucleotide moiety, a 3'-3'-inverted abasic moiety, a 3'-2'- inverted nucleotide moiety, a 3'-2'-inverted abasic moiety, a 1 ,4-butanediol phosphate, a 3'-phosphoramidate, a hexylphosphate, an aminohexyl phosphate, a 3'-phosphate, a 3'-phosphorothioate, a phosphorodithioate, a bridging methylphosphonate moiety, a non-bridging methyl phosphonate moiety, 5'-amino-alkyl phosphate, a l ,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1 ,2- aminododecyl phosphate, a hydroxypropyl phosphate, a 5'-5'-inverted nucleotide moiety, a 5'-5'-inverted abasic moiety, a 5'-phosphoramidate, a 5'-phosphorothioate, a 5'-amino, a bridging and or non-bridging 5'- phosphoramidate, a phosphorothioate, and a 5'-mercapto moiety.

Certain Modifications

In certain embodiments, oligonucleotides provided herein may comprise one or more modifications to a nucleobase, sugar, and/or internucleoside linkage, and as such is a modified oligonucleotide. A modified nucleobase, sugar, and/or internucleoside linkage may be selected over an unmodified form because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets and increased stability in the presence of nucleases.

In certain embodiments, a modified oligonucleotide comprises one or more modified nucleosides. In certain embodiments, a modified nucleoside is a stabilizing nucleoside. An example of a stabilizing nucleoside is a sugar-modified nucleoside.

In certain embodiments, a modified nucleoside is a sugar-modified nucleoside. In certain embodiments, the sugar-modified nucleosides can further comprise a natural or modified heterocyclic base moiety and/or a natural or modified internucleoside linkage and may include further modifications independent from the sugar modification. In certain embodiments, a sugar modified nucleoside is a 2'- modified nucleoside, wherein the sugar ring is modified at the 2' carbon from natural ribose or 2'-deoxy- ribose.

In certain embodiments, a 2'-modified nucleoside has a bicyclic sugar moiety. In certain embodiments, the bicyclic sugar moiety is a D sugar in the alpha configuration. In certain embodiments, the bicyclic sugar moiety is a D sugar in the beta configuration. In certain embodiments, the bicyclic sugar moiety is an L sugar in the alpha configuration. In certain embodiments, the bicyclic sugar moiety is an L sugar in the beta configuration.

In certain embodiments, the bicyclic sugar moiety comprises a bridge group between the 2' and the 4'-carbon atoms. In certain embodiments, the bridge group comprises from 1 to 8 linked biradical groups. In certain embodiments, the bicyclic sugar moiety comprises from 1 to 4 linked biradical groups. In certain embodiments, the bicyclic sugar moiety comprises 2 or 3 linked biradical groups. In certain embodiments, the bicyclic sugar moiety comprises 2 linked biradical groups. In certain embodiments, a linked biradical group is selected from -0-, -S-, -N(R,)-, -C(R,)(R₂)-, -C(R,)=C(R,)-, -C(R,)=N-, -C(=NR,)-, -Si(R,)(R₂)-, - S(=0)₂-, -S(=0)-, -C(=0)- and -C(=S)-; where each R_} and R₂ is, independently, H, hydroxyl, C_rCi₂ alkyl, substituted CrC_l2 alkyl, C₂-C_l2 alkenyl, substituted C₂-C₁₂ alkenyl, C₂-Ci₂ alkynyl, substituted C₂-C,₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, a heterocycle radical, a substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, substituted oxy (-0-), amino, substituted amino, azido, carboxyl, substituted carboxyl, acyl, substituted acyl, CN, thiol, substituted thiol, sulfonyl (S(=0)₂-H), substituted sulfonyl, sulfoxyl (S(=0)-H) or substituted sulfoxyl; and each substituent group is, independently, halogen, Ci-C|₂ alkyl, substituted Ci-C₎₂ alkyl, C₂- ]₂ alkenyl, substituted C₂-C_]2 alkenyl, C₂-C|₂ alkynyl, substituted C₂-C₎₂ alkynyl, amino, substituted amino, acyl, substituted acyl, Ci-C_]2 aminoalkyl, C_rCi₂ aminoalkoxy, substituted C1-C12 aminoalkyl, substituted C Ci2 aminoalkoxy or a protecting group.

In some embodiments, the bicyclic sugar moiety is bridged between the 2' and 4' carbon atoms with a biradical group selected from -0-(CH₂)_p-, -0-CH₂-,-0-CH₂CH₂-, -O-CH(alkyl)-, -NH-(CH₂)_P-, -N(alkyl)- (CH₂)--, -O-CH(alkyl)-, -(CH(alkyl))-(CH₂)_p-, -NH-0-(CH₂)_p-, -N(alkyl)-0-(CH₂)_p-, or -0-N(alkyl)-(CH₂)_p-, wherein p is 1 , 2, 3, 4 or 5 and each alkyl group can be further substituted. In certain embodiments, p is 1 , 2 or 3.

In certain embodiments, the bicyclic sugar moiety has a CH(CH₃)-0 bridge between the 4' and the 2' furanose ring atoms. In certain embodiments, the CH(CH₃)-0 bridge is constrained in the S orientation. In certain embodiments, the (CH₂)₂-0 is constrained in the R orientation.

In certain embodiments, a 2'-modifled nucleoside comprises a 2'-substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF₃, OCF₃, 0-, S-, or N(R_n,)-alkyl; 0-, S-, or N(R_m)-alkenyl; 0-, S- or N(R_m)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, 0(CH₂)₂SCH_3> O- (CH^-O-N R- iR,,) or 0-CH₂-C(=0)-N(R_m)(R_n), where each R,„ and R„ is, independently, H, an amino protecting group or substituted or unsubstituted Ci-Cio alkyl. These 2'-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (N0₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, a 2'-modified nucleoside comprises a 2'-substituent group selected from F, NH₂, N₃, OCF₃, 0-CH₃, 0(CH₂)₃NH₂, CH₂-CH=CH₂, 0-CH₂-CH=CH₂, OCH₂CH₂OCH₃, 0(CH₂)₂SCH₃, O- (CH₂)₂-0-N(R_m)(R_n), -0(CH₂)₂0(CH₂)₂N(CH₃)_2> and N-substituted acetamide (0-CH₂-C(=0)-N(R_m)(R_n) where each R™ and R_n is, independently, H, an amino protecting group or substituted or unsubstituted C|-C|₀ alkyl.

In certain embodiments, a 2'-modified nucleoside comprises a 2'-substituent group selected from F, OCF_3, 0-CH₃₎ OCH₂CH₂OCH₃, 2·-Ο(0Η₂)₂5αΗ₃, 0-(CH₂)₂-0-N(CH₃)₂, -0(CH₂)₂0(CH₂)₂N(CH₃)₂₎ and O- CH₂-C(=0)-N(H)CH₃.

In certain embodiments, a 2'-modified nucleoside comprises a 2'-substituent group selected from F, 0-CH₃, and OCH₂CH₂OCH₃.

In certain embodiments, a sugar-modified nucleoside is a 4'-thio modified nucleoside. In certain embodiments, a sugar-modified nucleoside is a 4'-thio-2'-modified nucleoside. A 4'-thio modified nucleoside has a β-D-ribonucleoside where the 4'-0 replaced with 4'-S. A 4'-thio-2'-modified nucleoside is a 4'-thio modified nucleoside having the 2'-OH replaced with a 2'-substituent group. Suitable 2'-substituent groups include 2'-OCH₃, 2'-0-(CH₂)₂-OCH₃, and 2'-F.

In certain embodiments, a modified oligonucleotide comprises one or more internucleoside modifications. In certain embodiments, each internucleoside linkage of an oligonucleotide is a modified internucleoside linkage. In certain embodiments, a modified internucleoside linkage comprises a phosphorus atom. In certain embodiments, a modified oligonucleotide comprises at least one phosphorothioate intemucleoside linkage. In certain embodiments, each intemucleoside linkage of a modified oligonucleotide is a phosphorothioate intemucleoside linkage.

In certain embodiments, a modified intemucleoside linkage does not comprise a phosphorus atom. In certain embodiments, an intemucleoside linkage is formed by a short chain alkyl intemucleoside linkage. In certain embodiments, an intemucleoside linkage is formed by a cycloalkyi intemucleoside linkages. In certain embodiments, an intemucleoside linkage is formed by a mixed heteroatom and alkyl intemucleoside linkage. In certain embodiments, an intemucleoside linkage is formed by a mixed heteroatom and cycloalkyi intemucleoside linkages. In certain embodiments, an intemucleoside linkage is formed by one or more short chain heteroatomic intemucleoside linkages. In certain embodiments, an intemucleoside linkage is formed by one or more heterocyclic intemucleoside linkages. In certain embodiments, an intemucleoside linkage has an amide backbone. In certain embodiments, an intemucleoside linkage has mixed N, O, S and CH₂ component parts.

In certain embodiments, a modified oligonucleotide comprises one or more modified nucleobases. In certain embodiments, a modified oligonucleotide comprises one or more 5-meth lcytosines. In certain embodiments, each cytosine of a modified oligonucleotide comprises a 5-methylcytosine.

In certain embodiments, a modified nucleobase is selected from 5-hydroxymethyl cytosine, 7- deazaguanine and 7-deazaadenine. In certain embodiments, a modified nucleobase is selected from 7-deaza- adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. In certain embodiments, a modified nucleobase is selected from 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, a modified nucleobase comprises a polycyclic heterocycle. In certain embodiments, a modified nucleobase comprises a tricyclic heterocycle. In certain embodiments, a modified nucleobase comprises a phenoxazine derivative. In certain embodiments, the phenoxazine can be further modified to form a nucleobase known in the art as a G-cIamp.

Certain Oligonucleotide Motifs

Suitable motifs for modified oligonucleotides of the present invention include, but are not limited to, fully modified, uniformly modified, and positionally modified. Modified oligonucleotides having a fully modified motif, including a uniformly modified motif, may be designed to target mature miRNAs.

Alternatively, modified oligonucleotides having a fully modified motif, including a uniformly modified motif, may be designed to target certain sites of pri-miRNAs or pre-miR As, to block the processing of miRNA precursors into mature miRNAs. Modified oligonucleotides having a fully modified motif or uniformly modified motif are effective inhibitors of miRNA activity.

In certain embodiments, a fully modified oligonucleotide comprises a sugar modification at each nucleoside. In certain embodiments, pluralities of nucleosides are 2'-0-methoxy ethyl nucleosides and the remaining nucleosides are 2'-fluoro nucleosides. In certain embodiments, each of a plurality of nucleosides is a 2'-0-methoxyethyl nucleoside and each of a plurality of nucleosides is a bicyclic nucleoside. In certain embodiments, a fully modified oligonucleotide further comprises at least one modified internucleoside linkage. In certain embodiments, each internucleoside linkage of a fully sugar-modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, a fully sugar-modified oligonucleotide further comprises at least one phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage of a fully sugar-modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a fully modified oligonucleotide is modified at each internucleoside linkage. In certain embodiments, each internucleoside linkage of a fully modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a uniformly modified oligonucleotide comprises the same sugar modification at each nucleoside. In certain embodiments, each nucleoside of a modified oligonucleotide comprises a 2'-0-methoxyethyl sugar modification. In certain embodiments, each nucleoside of a modified oligonucleotide comprises a 2'-0-methyl sugar modification. In certain embodiments, each nucleoside of a modified oligonucleotide comprises a 2'-fluoro sugar modification. In certain embodiments, a uniformly modified oligonucleotide further comprises at least one modified internucleoside linkage. In certain embodiments, each internucleoside linkage of a uniformly sugar-modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, a uniformly sugar-modified oligonucleotide further comprises at least one phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage of a uniformly sugar-modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a uniformly modified oligonucleoside comprises the same internucleoside linkage modifications throughout. In certain embodiments, each internucleoside linkage of a uniformly modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a positionally modified oligonucleotide comprises regions of linked nucleosides, where each nucleoside of each region comprises the same sugar moiety, and where each nucleoside of each region comprises a sugar moiety different from that of an adjacent region.

In certain embodiments, a positionally modified oligonucleotide comprises at least one modified internucleoside linkage. In certain embodiments, each internucleoside linkage of a positionally modified oligonucleoside is a modified internucleoside linkage. In certain embodiments, at least one internucleoside linkage of a positionally modified oligonucleotide is a phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage of a positionally modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a modified oligonucleotide consisting of linked nucleosides is represented by Formula I:

T_l-(Nu₁)„₁-( u₂)_n2-(Nu₃)_n3-( u₄)_n4-(Nu₅)_n5-T₂, wherein:

Nui and Nu₅ are, independently, 2' stabilizing nucleosides;

u₂ and Nu₄ are 2'-fluoro nucleosides;

Nu₃ is a 2'-modified nucleoside; each of ni and ns is, independently, from 0 to 3;

the sum of n₂ plus r is between 10 and 25;

n₃ is from 0 and 5; and

each T| and T₂ is, independently, H, a hydroxy! protecting group, an optionally linked conjugate group or a capping group.

In certain embodiments, Nui and Nu₅ are, independently, 2'-modified nucleosides.

In certain embodiments, Nu, is 0-(CH₂)₂-OCH₃, Nu₃ is 0-(CH₂)₂-OCH₃, and Nu₅ 0-(CH₂)₂-OCH₃.

In certain embodiments, each intemucleoside linkage is a modified internucleoside linkage. In certain embodiments, each internucleoside is a phosphorothioate linkage.

In certain embodiments, a nucleoside comprises a modified nucleobase. In certain embodiments, where a 2'-0-methoxyethyl nucleoside comprises cytosine, the cytosine is a 5-methylcytosine.

In certain embodiments, Nui is 0-(CH₂)₂-OCH₃, Nu₃ is 0-(CH₂)₂-OCH₃, Nu_s 0-(CH₂)₂-OCH₃, T, is H and T₂ is H.

In certain embodiments, T| and T₂ are each, independently, H or a hydroxyl protecting group. In certain embodiments, at least one of T| and T₂ is 4,4'-dimethoxytrityl. In certain embodiments, at least one of T| and T₂ is an optionally linked conjugate group. In certain embodiments, at least one of Ti and T₂ is a capping group. In certain embodiments, the capping group is an inverted deoxy abasic group.

In certain embodiments, the sum of n₂ and a» is 13. In certain embodiments, the sum of n₂ and n₄ is 14. In certain embodiments, the sum of n₂ and n₄ is 15. In certain embodiments, the sum of n₂ and , is 16. In certain embodiments, the sum of n₂ and a» is 17. In certain embodiments, the sum of n₂ and at is 18.

In certain embodiments, ni , n₂, and n₃ are each, independently, from 1 to 3. In certain embodiments, ni, n₂, and n₃ are each, independently, from 2 to 3. In certain embodiments, nj is 1 or 2; n₂ is 2 or 3; and n₃ is 1 or 2. In certain embodiments, ni is 2; n₃ is 2 or 3; and n₅ is 2. In certain embodiments, ni is 2; n₃ is 3; and n₅ is 2. In certain embodiments, Π| is 2; n₃ is 2; and n₅ is 2.

In certain embodiments, a modified oligonucleotide consists of 20 linked nucleosides. In certain embodiments, the sum of n₂ and n₄ is 13; ni is 2; n₃ is 3; and n₅ is 2. In certain embodiments, the sum of n₂ and Oi is 14; ni is 2; n₃ is 2; and n₅ is 2.

In certain embodiments, a modified oligonucleotide consists of 21 linked nucleosides. In certain embodiments, the sum of n₂ and n₄ is 14; ni is 2; n₃ is 3; and n₅ is 2. In certain embodiments, the sum of n₂ and Oi is 15; ni is 2; n₃ is 2; and n₅ is 2.

In certain embodiments, a modified oligonucleotide consists of 22 linked nucleosides. In certain embodiments, the sum of n₂ and n is 15; Π| is 2; n₃ is 3; and n₅ is 2. In certain embodiments, the sum of n₂ and ¾ is 16; n, is 2; n₃ is 2; and ns is 2.

In certain embodiments, a modified oligonucleotide consists of 23 linked nucleosides. In certain embodiments, the sum of n₂ and n₄ is 16; ni is 2; n₃ is 3; and n₅ is 2. In certain embodiments, the sum of n₂ and at is 17; n, is 2; n₃ is 2; and n₅ is 2. In certain embodiments, a modified oligonucleotide consists of 24 linked nucleosides. In certain embodiments, the sum of n₂ and n₄ is 17; ni is 2; n₃ is 3; and n₅ is 2. In certain embodiments, the sum of n₂ and ni is 18; n, is 2; n₃ is 2; and n₅ is 2.

In certain embodiments, a modified oligonucleotide consists of 22 linked nucleosides; n, is 2; n₂ is 9; n₃ is 3; n₄ is 6; n₅ is 2; Nu, is 0-(CH₂)₂-OCH₃; Nu₃ is 0-(CH₂)₂-OCH₃; and Nu₅ 0-(CH₂)₂-OCH₃.

In certain embodiments, a modified oligonucleotide consists of 22 linked nucleosides; n_t is 2; n₂ is 9; n₃ is 3; n₄ is 6; n_s is 2; Nu, is 0-(CH₂)₂-OCH₃; Nu₃ is 0-(CH₂)₂-OCH₃; Nu₅ 0-(CH₂)₂-OCH₃; and each internucleoside linkage is a phosphorothioate linkage.

In certain embodiments, a modified oligonucleotide consists of 21 linked nucleosides; has the nucleobase sequence selected from a nucleobase sequence as shown in SEQ ID NO: 5; n, is 2; n₂ is 8; n₃ is 3; Oi is 6; n₅ is 2; Nu, is 0-(CH₂)₂-OCH₃; Nu₃ is 0-(CH₂)₂-OCH₃; Nu₅ 0-(CH₂); each internucleoside linkage is a phosphorothioate linkage; the cytosines at nucleobases 1 , 2, 1 1 and 12 are 5-methylcytosines.

In certain embodiments, a compound is represented by the following formula II:

In certain embodiments, Q is a 2'-0-methyl modified nucleoside. In certain embodiments, x is phosphorothioate. In certain embodiments, y is phosphodiester. In certain embodiments, each of zl , z2, z3, and z4 is, independently phosphorothioate or phosphodiester. In certain embodiments, n is 6 to 17. In certain embodiments, L is cholesterol. In certain embodiments, n is 12 to 17.

In certain embodiments, x is

One of A and B is S while the other is O;

y is

Each of zl , z2, z3, and z4 is independently x or y;

n = 6-17

L is

Wherein:

X is N(CO)R⁷, or NR⁷;

Each of R¹, R³ and R⁹, is independently, H, OH, or -CH₂OR^b provided that at least one of R¹, R³ and R⁹ is OH and at least one of R¹, R³ and R⁹ is -CH₂OR^b;

R⁷ is R^d or C,-C₂₀ alkyl substituted with NR^cR^d or NHC(0)R^d;

R^c is H or Ci-Q alkyl;

R^d is a carbohydrate radical; or a steroid radical, which is optionally tethered to at least one carbohydrate radical; and

R^b is

with one of A and B is S while the other is O.

In certain embodiments, R^d is cholesterol. In certain embodiments each of z¹, z², z³, and z⁴ is

one of A and B is S while the other is O.

In certain embodiments, R¹ is -CH₂OR^b. In certain embodiments, R⁹ is OH. In certain embodiments, R¹ and R⁹ are trans. In certain embodiments, R⁹ is OH. In certain embodiments, R¹ and R³ are trans. In certain embodiments, R3 is -CH₂OR^b. In certain embodiments, R¹ is OH. In certain embodiments, R¹ and R³ are trans. In certain embodiments, R⁹ is OH. In certain embodiments, R³ and R⁹ are trans. In certain embodiments, R⁹ is CH₂OR^b. In certain embodiments, R¹ is OH. In certain embodiments, R¹ and R⁹ are trans. In certain embodiments, X is NC(0)R⁷. In certain embodiments, R⁷ is -CH2(CH₂)₃CH₂NHC(0)R^d.

In certain embodiments, a modified oligonucleotide having a positionally modified motif comprises LNA. In certain embodiments, a modified oligonucleotide has a motif selected from among one of the motifs listed below, wherein L = an LNA nucleoside, d= a DNA nucleoside, M = a 2'-MOE nucleoside, and F = a 2'-Fluoro nucleoside. In certain embodiments, nucleosides in parentheses are optionally included in the modified oligonucleotide, in other words, the motif encompasses modified oligonucleotides of varying lengths depending upon how many nucleosides in parentheses are included.

LdLddLLddLdLdLL Ld Ld LLLd d LLLd LL

LMLMM LLMMLMLMLL

LMLMLLLMMLLLMLL

LFLFFLLFFLFLFLL

LFLFLLLFFLLLFLL

LddLddLddL(d)(d)(L)(d)(d)(L)(d)

dLddLddLdd(L)(d)(d)(L)(d)(d)(L)

ddLddLddLd(d)(L)(d)(d)(L)(d)(d)

LMMLMMLMML(M)(M)(L)(M)(M)(L)(M)

MLMMLMMLMM(L)(M)(M)(L)(M)(M)(L)

MMLMMLMMLM( )(L)(M)(M)(L)(M)(M)

LFFLFFLFFL(F)(F)(L)(F)(F)(L)(F)

FLFFLFFLFF(L)(F)(F)(L)(F)(F)(L)

FFLFFLFFLF(F)(L)(F)(F)(L)(F)(F)

dLdLdLdLdL(d)(L)(d)(L)(d)(L)(d)

LdLdLdLdL(d)(L)(d)(L)(d)(L)(d)(L)

MLMLMLMLML(M)(L)(M)(L)(M)(L)(M)

LMLMLMLML(M)(L)(M)(L)(M)(L)(M)(L)

FLFLFLFLFL(F)(L)(F)(L)(F)(L)(F)

LFLFLFLFL(F)(L)(F)(L)(F)(L)(F)(L)

Additional motifs are disclosed in PCT Publication No. WO/2007/1 12754, which is herein incorporated by reference in its entirety for the description of oligonucleotide modifications and patterns of oligonucleotide modifications.

Certain Additional Therapies

Treatments for metabolic disorders may comprise more than one therapy. As such, in certain embodiments the present invention provides methods for treating metabolic disorders comprising administering to a subject in need thereof a compound comprising a modified oligonucleotide complementary to a member of the miR-378 family, or a precursor thereof, and further comprising administering at least one additional pharmaceutical agent.

In certain embodiments, the additional pharmaceutical agent is a glucose-lowering agent.

In certain embodiments, the glucose-lowering agent is a PPAR agonist (gamma, dual, or pan), a dipeptidyl peptidase (IV) inhibitor, a GLP-I analog, insulin or an insulin analog, an insulin secretagogue, a SGLT2 inhibitor, a human amylin analog, a biguanide, an alpha-glucosidase inhibitor, a meglitinide, a thiazolidinedione, or a sulfonylurea.

In certain embodiments, the glucose-lowering agent is a GLP-I analog. In certain embodiments, the GLP-I analog is exendin-4 or liraglutide. In certain embodiments, the glucose-lowering agent is a sulfonylurea. In certain embodiments, the sulfonylurea is acetohexamide, chlorpropamide, tolbutamide, tolazamide, glimepiride, a glipizide, a glyburide, or a gliclazide.

In certain embodiments, the glucose-lowering agent is a biguanide. In certain embodiments, the biguanide is metformin. In certain embodiments, blood glucose levels are decreased without increased lactic acidosis as compared to the lactic acidosis observed after treatment with metformin alone.

In certain embodiments, the glucose-lowering agent is a meglitinide. In certain embodiments, the meglitinide is nateglinide or repaglinide.

In certain embodiments, the glucose-lowering agent is a thiazolidinedione. In certain embodiments, the thiazolidinedione is pioglitazone, rosiglitazone, or troglitazone. In certain embodiments, blood glucose levels are decreased without greater weight gain than observed with rosiglitazone treatment alone.

In certain embodiments, the glucose-lowering agent is an alpha-glucosidase inhibitor. In certain embodiments, the alpha-glucosidase inhibitor is acarbose or miglitol.

In certain embodiments, the glucose-lowering agent is an antisense oligonucleotide targeted to

PTP1 B.

In certain embodiments, an additional therapy is an anti-obesity agent. In certain embodiments, an anti-obesity agent is Orlistat, Sibutramine, or Rimonabant.

In a certain embodiment, the additional therapy is therapeutic lifestyle change. In certain embodiments, the therapeutic lifestyle change includes an exercise regimen and/or diet.

In certain embodiments the dose of an additional pharmaceutical agent is the same as the dose that would be administered if the additional pharmaceutical agent was administered alone.

In certain embodiments the dose of an additional pharmaceutical agent is lower than the dose that would be administered if the additional pharmaceutical agent was administered alone. In certain embodiments the dose of an additional pharmaceutical agent is greater than the dose that would be administered if the additional pharmaceutical agent was administered alone.

Further examples of additional pharmaceutical agents include, but are not limited to, corticosteroids, including but not limited to prednisone; immunoglobulins, including, but not limited to intravenous immunoglobulin (IVIg); analgesics (e.g., acetaminophen); anti-inflammatory agents, including, but not limited to non-steroidal anti-inflammatory drugs (e.g., ibuprofen, COX-I inhibitors, and COX-2, inhibitors); salicylates; antibiotics; antivirals; antifungal agents; antidiabetic agents (e.g., biguanides, glucosidase inhibitors, insulins, sulfonylureas, and thiazolidenediones); adrenergic modifiers; diuretics; hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin, progestin, somatostan, and thyroid hormones);

immunomodulators; muscle relaxants; antihistamines; osteoporosis agents (e.g., biphosphonates, calcitonin, and estrogens); prostaglandins, antineoplastic agents; psychotherapeutic agents; sedatives; poison oak or poison sumac products; antibodies; and vaccines.

In certain embodiments, an additional therapy is a lipid-Iowering therapy. In certain embodiments, a lipid-lowering therapy is therapeutic lifestyle change. In certain embodiments, a lipid-lowering therapy is LDL apheresis. Certain Pharmaceutical Compositions

Provided herein are pharmaceutical compositions comprising oligonucleotides. In certain embodiments, such pharmaceutical compositions are used for the treatment of metabolic disorders, and associated conditions. In certain embodiments, a pharmaceutical composition provided herein comprises a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence complementary to one or more miR-378 family members, or a precursor thereof. In certain embodiments, a pharmaceutical composition provided herein comprises a compound consisting of a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence complementary to one or more miR-378 family members, or a precursor thereof.

Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intraventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intramedullary, and

subcutaneous). In certain embodiments, pharmaceutical intrathecals are administered to achieve local rather than systemic exposures. For example, pharmaceutical compositions may be injected directly in the area of desired effect (e.g., into the liver).

In certain embodiments, a pharmaceutical composition is administered in the form of a dosage unit (e.g., tablet, capsule, bolus, etc.). In certain embodiments, such pharmaceutical compositions comprise a modified oligonucleotide in a dose selected from 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 1 10 mg, 1 15 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 270 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg, 305 mg, 310 mg, 315 mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg, 345 mg, 350 mg, 355 mg, 360 mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg, 395 mg, 400 mg, 405 mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg, 440 mg, 445 mg, 450 mg, 455 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg, 485 mg, 490 mg, 495 mg, 500 mg, 505 mg, 510 mg, 515 mg, 520 mg, 525 mg, 530 mg, 535 mg, 540 mg, 545 mg, 550 mg, 555 mg, 560 mg, 565 mg, 570 mg, 575 mg, 580 mg, 585 mg, 590 mg, 595 mg, 600 mg, 605 mg, 610 mg, 615 mg, 620 mg, 625 mg, 630 mg, 635 mg, 640 mg, 645 mg, 650 mg, 655 mg, 660 mg, 665 mg, 670 mg, 675 mg, 680 mg, 685 mg, 690 mg, 695 mg, 700 mg, 705 mg, 710 mg, 715 mg, 720 mg, 725 mg, 730 mg, 735 mg, 740 mg, 745 mg, 750 mg, 755 mg, 760 mg, 765 mg, 770 mg, 775 mg, 780 mg, 785 mg, 790 mg, 795 mg, and 800 mg. In certain embodiments, a pharmaceutical composition of the comprises a dose of modified oligonucleotide selected from 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, and 800mg.

In certain embodiments, a pharmaceutical agent is a sterile lyophilized modified oligonucleotide that is reconstituted with a suitable diluent, e.g., sterile water for injection or sterile saline for injection. The reconstituted product is administered as a subcutaneous injection or as an intravenous infusion after dilution into saline. The lyophilized drug product consists of an oligonucleotide which has been prepared in water for injection, or in saline for injection, adjusted to pH 7.0-9.0 with acid or base during preparation, and then lyophilized. The lyophilized modified oligonucleotide may be 25-800 mg of an oligonucleotide. It is understood that this encompasses 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mg of modified lyophilized oligonucleotide. The lyophilized drug product may be packaged in a 2 mL Type I, clear glass vial (ammonium sulfate-treated), stoppered with a bromobuty! rubber closure and sealed with an aluminum FLIP-OFF® overseal.

In certain embodiments, the pharmaceutical compositions provided herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically- active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the oligonucleotide(s) of the formulation.

Lipid moieties have been used in nucleic acid therapies in a variety of methods. In one method, the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In another method, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.

In certain embodiments, INTRALIPID is used to prepare a pharmaceutical composition comprising an oligonucleotide. Intralipid is fat emulsion prepared for intravenous administration. It is made up of 10% soybean oil, 1.2% egg yolk phospholipids, 2.25% glycerin, and water for injection. In addition, sodium hydroxide has been added to adjust the pH so that the final product pH range is 6 to 8.9.

In certain embodiments, a pharmaceutical composition provided herein comprise a polyamine compound or a lipid moiety complexed with a nucleic acid. In certain embodiments, such preparations comprise one or more compounds each individually having a structure defined by formula (III) or a pharmaceutically acceptable salt thereof, r ^~~i

wherein each X^a and X^b, for each occurrence, is independently alkylene; n is 0, 1, 2, 3, 4, or 5; each R is independently H, wherein at least n + 2 of the R moieties in at least about 80% of the molecules of the compound of formula (I) in the preparation are not H; m is 1 , 2, 3 or 4; Y is O, NR², or S; R¹ is alkyl, alkenyl, or alkynyl; each of which is optionally substituted with one or more substituents; and R² is H, alkyl, alkenyl, or alkynyl; each of which is optionally substituted each of which is optionally substituted with one or more substituents; provided that, if n = 0, then at least n + 3 of the R moieties are not H. Such preparations are described in PCT publication WO/2008/042973, which is herein incorporated by reference in its entirety for the disclosure of lipid preparations. Certain additional preparations are described in Akinc et al., Nature Biotechnology 26, 561 - 569 (01 May 2008), which is herein incorporated by reference in its entirety for the disclosure of lipid preparations.

In certain embodiments, pharmaceutical compositions provided herein comprise one or more modified oligonucleotides and one or more excipients. In certain embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided herein is prepared using known techniques, including, but not limited to mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes.

In certain embodiments, a pharmaceutical composition provided herein is a liquid (e.g., a suspension, elixir and/or solution). In certain of embodiments, a liquid pharmaceutical composition is prepared using ingredients known in the art, including, but not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents.

In certain embodiments, a pharmaceutical composition provided herein is a solid (e.g., a powder, tablet, and/or capsule). In certain of embodiments, a solid pharmaceutical composition comprising one or more oligonucleotides is prepared using ingredients known in the art, including, but not limited to, starches, sugars, diluents, granulating agents, lubricants, binders, and disintegrating agents.

In certain embodiments, a pharmaceutical composition provided herein is formulated as a depot preparation. Certain such depot preparations are typically longer acting than non-depot preparations. In certain embodiments, such preparations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. In certain embodiments, depot preparations are prepared using suitable polymeric or hydrophobic materials (for example an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

In certain embodiments, a pharmaceutical composition provided herein comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided herein comprises a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co- solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co- solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied:for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

In certain embodiments, a pharmaceutical composition provided herein comprises a sustained- release system. A non-limiting example of such a sustained-release system is a semi-permeable matrix of solid hydrophobic polymers. In certain embodiments, sustained-release systems may, depending on their chemical nature, release pharmaceutical agents over a period of hours, days, weeks or months.

In certain embodiments, a pharmaceutical composition provided herein is prepared for oral administration. In certain of embodiments, a pharmaceutical composition is formulated by combining one or more compounds comprising an oligonucleotide with one or more pharmaceutically acceptable carriers. Certain of such carriers enable pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject. In certain embodiments, pharmaceutical compositions for oral use are obtained by mixing oligonucleotide and one or more solid excipient. Suitable excipients include, but are not limited to, fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). In certain embodiments, such a mixture is optionally ground and auxiliaries are optionally added. In certain embodiments, pharmaceutical compositions are formed to obtain tablets or dragee cores. In certain embodiments, disintegrating agents (e.g., cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate) are added.

In certain embodiments, dragee cores are provided with coatings. In certain embodiments, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to tablets or dragee coatings.

In certain embodiments, pharmaceutical compositions for oral administration are push-fit capsules made of gelatin. Certain of such push-fit capsules comprise one or more pharmaceutical agents of the present invention in admixture with one or more filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In certain embodiments, pharmaceutical compositions for oral administration are soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. In certain soft capsules, one or more pharmaceutical agents of the present invention are be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

In certain embodiments, pharmaceutical compositions are prepared for buccal administration. Certain of such pharmaceutical compositions are tablets or lozenges formulated in conventional manner.

In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, and the like). In certain of embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as

preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared for transmucosal administration. In certain of embodiments penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition is prepared for administration by inhalation. Certain of such pharmaceutical compositions for inhalation are prepared in the form of an aerosol spray in a pressurized pack or a nebulizer. Certain of such pharmaceutical compositions comprise a propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In certain embodiments using a pressurized aerosol, the dosage unit may be determined with a valve that delivers a metered amount. In certain embodiments, capsules and cartridges for use in an inhaler or insufflator may be formulated. Certain of such formulations comprise a powder mixture of a pharmaceutical agent of the invention and a suitable powder base such as lactose or starch.

In certain embodiments, a pharmaceutical composition is prepared for rectal administration, such as a suppositories or retention enema. Certain of such pharmaceutical compositions comprise known ingredients, such as cocoa butter and/or other glycerides. In certain embodiments, a pharmaceutical composition is prepared for topical administration. Certain of such pharmaceutical compositions comprise bland moisturizing bases, such as ointments or creams. Exemplary suitable ointment bases include, but are not limited to, petrolatum, petrolatum plus volatile silicones, and lanolin and water in oil emulsions. Exemplary suitable cream bases include, but are not limited to, cold cream and hydrophilic ointment. '

In certain embodiments, a pharmaceutical composition provided herein comprises an

oligonucleotide in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

In certain embodiments, one or more modified oligonucleotides provided herein is formulated as a prodrug. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form of an oligonucleotide. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in certain instances, a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form. In certain instances, a prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, a prodrug is an ester. In certain embodiments, the ester is metabolically hydrolyzed to carboxylic acid upon administration. In certain instances the carboxylic acid containing compound is the corresponding active form. In certain embodiments, a prodrug comprises a short peptide (polyaminoacid) bound to an acid group. In certain of embodiments, the peptide is cleaved upon administration to form the corresponding active form.

In certain embodiments, a prodrug is produced by modifying a pharmaceutically active compound such that the active compound will be regenerated upon in vivo administration. The prodrug can be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a

pharmaceutically active compound is known, can design prodrugs of the compound (see, e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388- 392).

Certain Kits

The present invention also provides kits. In some embodiments, the kits comprise one or more compounds of the invention comprising a modified oligonucleotide, wherein the nucleobase sequence of the modified oligonucleotide is complementary to one or more miR-378 family members. The compounds complementary to one or more miR-378 family members can be any of the compounds described herein, and can have any of the modifications described herein. In some embodiments, the compounds complementary to one or more miR-378 family members can be present within a vial. A plurality of vials, such as 10, can be present in, for example, dispensing packs. In some embodiments, the vial is manufactured so as to be accessible with a syringe. The kit can also contain instructions for using the compounds complementary to one or more miR-378 family members.

In some embodiments, the kits may be used for administration of the compound complementary to one or more miR-378 family members to a subject. In such instances, in addition to compounds complementary to one or more miR-378 family members, the kit can further comprise one or more of the following: syringe, alcohol swab, cotton ball, and/or gauze pad. In some embodiments, the compounds complementary to one or more members of the miR-378 family can be present in a pre- filled syringe (such as a single-dose syringes with, for example, a 27 gauge, ½ inch needle with a needle guard), rather than in a vial. A plurality of pre-filled syringes, such as 10, can be present in, for example, dispensing packs. The kit can also contain instructions for administering the compounds complementary to one or more miR-378 family members.

Certain Experimental Models

In certain embodiments, the present invention provides methods of using and/or testing modified oligonucleotides of the present invention in an experimental model. Those having skill in the art are able to select and modify the protocols for such experimental models to evaluate a pharmaceutical agent of the invention.

Generally, modified oligonucleotides are first tested in cultured cells. Suitable cell types include those that are related to the cell type to which delivery of an oligonucleotide is desired in vivo. For example, suitable cell types for the study of the methods described herein include primary hepatocytes, primary adipocytes, preadipocytes, differentiated adipocytes, HepG2 cells, Huh7 cells, 3T3L1 cells, and C2C 12 cells (murine myoblasts).

In certain embodiments, the extent to which an oligonucleotide interferes with the activity of a miRNA is assessed in cultured cells. In certain embodiments, inhibition of miRNA activity may be assessed by measuring the levels of the miRNA. Alternatively, the level of a predicted or validated miRNA target may be measured. An inhibition of miRNA activity may result in the increase in the mRNA and/or protein of a miRNA target. Further, in certain embodiments, certain phenotypic outcomes may be measured. For example, suitable phenotypic outcomes include insulin signaling.

Suitable experimental animal models for the testing of the methods described herein include: ob/ob mice (a model for diabetes, obesity and insulin resistance), db/db mice (a model for diabetes, obesity and insulin resistance), high-fat fed C57B16/J mice, agouti mice, Zucker diabetic rats, and aP2-SREBP transgenic mice.

Certain Quantitation Assays

The effects of antisense inhibition of a miRNA following the administration of modified oligonucleotides may be assessed by a variety of methods known in the art. In certain embodiments, these methods are be used to quantitate miRNA levels in cells or tissues in vitro or in vivo. In certain embodiments, changes in miRNA levels are measured by microarray analysis. In certain embodiments, changes in miRNA levels are measured by one of several commercially available PCR assays, such as the TaqMan® MicroRNA Assay (Applied Biosystems). In certain embodiments, antisense inhibition of a miRNA is assessed by measuring the mRNA and/or protein level of a target of a miRNA. Antisense inhibition of a miRNA generally results in the increase in the level of mRNA and/or protein of a target of the miRNA.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES

Example 1: Inhibition of miR-378 lowers insulin levels, glucose levels and improves the insulin response index in a mouse model of diabetes and obesity

High-fat fed obese mice (also called diet-induced obese mice or DIO mice), were used as a model of impaired glucose tolerance and type 2 diabetes according to methods known in the art. A modified oligonucleotide targeted to anti-miR-378 was tested in this model. The modified oligonucleotide has a nucleobase sequence of SEQ ID NO: 5 comprising phosphorothioate internucleoside linkages at each internucleoside linkage, 2'-0-methoxyethyl nucleosides at positions 1 , 2, 1 1 - 13, 20 and 21 ; 2'-fluoro nucleosides at positions 3- 10 and 14-19 (corresponding to Formula I, Table 2, row 20); 5-methylcytosine nucleobases at positions 1 , 2, 1 1 , and 12; and uracil at positions 3, 4, 6, 10 and 16. This oligonucleotide is hereinafter referred to as "anti-miR-378 oligonucleotide."

C57B1/6 male mice were maintained on a high fat diet for more than 16 weeks. The mice were then treated with an intraperitoneal (i.p.) injection with the anti-miR-378 oligonucleotide (n = 8) or a saline control (n = 6) at a dose of 5 mg/kg twice weekly for six weeks.

After five weeks of treatment, an oral glucose tolerance test (OGTT) was performed. The OGTT, also referred to as the glucose tolerance test, measures the body's ability to metabolize glucose, i.e. to clear it out of the bloodstream. The mice were subject to a four hour fast, after which glucose was administered and plasma insulin levels were measured 0, 30 and 90 min after administration of the glucose. The results are shown in Table 2.

Table 2

Effect of miR-378 inhibition by the anti-miR-378 oligonucleotide on plasma insulin levels during

OGTT

The 30 min and 90 min data demonstrate significantly lower insulin levels for the mice treated with the anti-miR-378 oligonucleotide relative to the saline-treated control. The differences are statistically significant (p < 0.05, Student's test).

Glucose concentrations were also measured in the same mice 0, 30 and 90 minutes after administration of glucose. The results are shown in Table 3.

Table 3

Effect of miR-378 inhibition by an anti-miR-378 oligonucleotide on glucose levels during OGTT

The data presented in Tables 2 and 3 were used to calculate insulin response indices (IRI, an estimate of insulin resistance by multiplying the insulin levels by the glucose levels. The results are shown in Table 4.

Table 4

Effect of miR-378 inhibition by an anti-miR-378 oligonucleotide on the insulin response index (IRI)

The 30 min and 90 min data demonstrate significantly lower insulin response indices (IRJs) for the mice treated with the anti-miR-378 oligonucleotide relative to the saline-treated control. The differences are statistically significant (p < 0.05, Student's test). The insulin response index was lowered by 72% and 73% at the time points 30 and 60 min after the glucose injection, respectively.

These data indicate that administration of the anti-miR-378 oligonucleotide has a significant effect on the insulin response to the glucose injection. The insulin levels which are boosted by the glucose injection do not attain the high levels reached in the control -treated mice. These data provide an indication that an oligomeric compound targeted to miR-378 may be useful for treating, preventing or delaying the onset of metabolic disorders; improving insulin resistance or increasing insulin sensitivity; delaying the onset of elevated glucose levels; and improving glucose tolerance. Example 2: Inhibition of miR-378 lowers free fatty acids and increases ketone bodies in the blood serum of mouse model of diabetes and obesity

The same groups of mice described in Example 1 treated with the same oligonucleotide and saline control were analyzed by known methods to determine levels of free fatty acids in the blood serum. The mean fatty acid levels of the mice treated with the anti-miR-378 oligonucleotide were 1.76 mM, and mean fatty acid levels of the mice treated with the saline control were0.94 mM. The 47% reduction in free fatty acid levels was found to be statistically significant (p < 0.05, Student's t-test).

Levels of beta-hydroxybutyrate (ketone bodies) in the blood serum were measured by known methods. This compound is a metabolic product of the oxidation of fatty acids. The mean level of beta- hydroxybutyrate in the mice treated with the anti-miR-378 oligonucleotide was 36 μΜ. Beta- hydroxybutyrate was not detected in the blood serum of the mice treated with the saline control.

In a similar experiment using an additional model of diabetes, obesity and insulin resistance, ob/ob mice were treated with the anti-miR-378 oligonucleotide ( 15 mg/kg) or saline control. Serum free fatty acids were reduced in a statistically significant manner in the ob/ob mice.

The results of these measurements provide evidence that the anti-miR-378 oligonucleotide is influencing the metabolism of free fatty acids. The reduction in free fatty acids suggests improved insulin resistance in adipose tissue, which in turn indicates an improvement in diabetes. The increased beta- hydroxybutyrate indicates an increase in fatty acid oxidation in the liver, which suggests and improvement in insulin sensitivity of the liver and an improvement in fatty liver. Therefore, an oligomeric compound targeted to miR-378 may be appropriate for treating metabolic disorders characterized by

hyperfattyacidemia, such as diabetes.

Example 3: Effects of Inhibition of miR-378 on liver health in a mouse model of diabetes and obesity

The presence of elevated serum transaminases such as alanine transaminase (ALT) and aspartate transaminase (AST) are commonly used as indicators of liver damage or dysfunction. After six weeks of treatment of the mice with the anti-miR-378 oligonucleotide and the saline control as described in Examples 1 and 2, ALT levels were measured by known methods. The mean ALT levels of the mice treated with the anti-miR-378 oligonucleotide were 70 U/L and the mean ALT levels of the mice treated with saline control werel 26 U/L. The oligonucleotide-treated mice had a mean ALT level 44% lower than that of the mice treated with the saline control. This difference was found to be statistically significant (p < 0.05, Student's t- test).

As elevated ALT is an indicator of liver damage or dysfunction, a reduction in ALT following administration of the anti-miR-378 oligonucleotide suggests improved liver function relative to the mice treated with the saline control. Example 4: Effects of inhibition of miR-378 on whole body weight and organ weights in a mouse model of diabetes and obesity

After the 6 week treatment period, the mice described in Examples 1 to 3 were sacrificed by established methods. Whole body weights were measured. Spleen, liver and kidney were resected and their weights were measured to determine the effects of the anti-miR-378 oligonucleotide on the weight gain of the mice and the selected organs during the study period. Based on the measurements of body and organ weights, no obvious toxicities resulted from treatment with the anti-miR-378 oligonucleotide.

The most pronounced weight gain (saline group vs. the oligonucleotide-treated group) was observed in the liver (12%), and in the spleen (27%). Although not statistically significant, these results indicate a trend toward having less weight gain the spleen, liver and kidney when the anti-miR-378 oligonucleotide is administered to the mouse model of diabetes and obesity.

Example 5: Effects of inhibition of miR-378 on gene regulation

At the end of the 6 week treatment period, RNA was isolated from adipose and liver tissues from the DIO mice described in Examples 1 to 4. Microarray analysis was performed to evaluate changes in the expression of mRNA transcripts that contain miR-378 target sites (i.e., transcripts that contain a miR-378 seed match sequence). Changes in transcript expression were evaluated among the following groups of RNA transcripts: (1) transcripts containing a 6-base match to the miR-378 seed sequence ('6-mers' in Figure 1 ); (2) transcripts containing a 7-base mismatch to the miR-378 seed sequence ('7-mers' in Figure 1); (3) transcripts containing an 8-base mismatch to the miR-378 seed sequence ('8-mers' in Figure 1); and (4) transcripts without a miR-378 seed match sequence ('Non-seed' in Figure 1 ). The data are analyzed to determine whether the fold-change distributions of the miR-378 seed-matched transcripts are significantly different from the fold-change distribution of the non-seed match transcripts; a significant fold change difference indicates an effect specific to miR-378. Cumulative density function (CDF) plots in Figure I show the distribution of fold-changes (log2) of differential gene expression after anti-miR-378 treatment, compared to control samples. A two-sample Kolmogorov-Smirnov (KS) test is used to determine whether there is a difference, or relative shift between two distributions. A significant p-value confirms the presence of such a shift. The data shown in Figure 1 indicate that, based on this particular analysis, there are no discernible "global" effects on transcript expression in liver, while effects on transcript expression are clearly detected in adipose. This does not, however, necessarily mean a lack of effects in liver; there may still be effects on a small set of targets that are not captured by the CDF. The extent of gene regulation was greater in adipose tissue than in liver tissue, suggesting that the phenotypes observed following inhibition of miR-378 are achieved by targeting tissues other than liver tissue, for example adipose tissue.

The microarray data was additionally analyzed to identify cellular pathways that are regulated following inhibition of miR-378. This analysis included all genes, and was not restricted to transcripts containing a miR-378 seed match sequence. Accordingly, this analysis revealed changes in the level of both direct and indirect mRNA targets of miR-378. Shown in Table 5 are fold changes in mRNA transcripts involved in oxidation, lipogenesis, and insulin sensitivity. These data demonstrate that inhibition of miR- 378 enhances the oxidative gene expression, but suppresses the lipogenic and gene expression in the adipose tissue of DIO mice, suggesting an improved insulin sensitivity in adipose tissue of these mice. The expression of two genes involved in insulin sensitivity was also decreased.

Table 5

Inhibition of miR-378 increases oxidative genes and decreases lipogenic genes in adipose tissue

CONCLUDING STATEMENTS

The foregoing description of the specific embodiments so fully reveals the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

What is claimed is:

1. A method of treating a metabolic disorder, comprising administering to a subject having a metabolic disorder a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and has a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member; thereby treating the metabolic disorder.

2. A method of preventing or delaying the onset of at least one metabolic disorder in a subject at risk for developing a metabolic disorder, comprising administering to the subject a compound comprising modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member; and thereby preventing or delaying the onset of a metabolic disorder in the subject.

3. The method of claims 1 or 2, wherein at least one metabolic disorder is selected from among prediabetes, diabetes, metabolic syndrome, obesity, diabetic dyslipidemia, hyperlipidemia, hypertension, hypertriglyceridemia, hyperfattyacidemia, and hyperinsulinemia.

4. A method for improving insulin resistance in a subject comprising administering to the subject a

compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member; and thereby improving insulin resistance in the subject.

5. The method of claim 4, wherein the subject has impaired insulin resistance.

6. The method of claim 4, comprising selecting a subject having impaired insulin resistance.

7. A method for reducing a blood glucose level of a subject comprising administering to the subject a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member; thereby reducing the blood glucose level of the subject.

8. A method for preventing or delaying the onset of an elevated blood glucose level in a subject at risk for developing an elevated glucose level comprising administering to the subject a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member; thereby preventing or delaying the onset of an elevated blood glucose level in the subject.

9. The method of any of the above claims, comprising selecting a subject having an elevated blood glucose level.

10. The method of any of the above claims, wherein the subject has an elevated blood glucose level.

11. The method of any of the above claims, comprising measuring the blood glucose level of the subject.

12. The method of any of claims 7-11, wherein the blood glucose level is a fasted blood glucose level, a post-prandial blood glucose level, a whole blood glucose level, or a plasma blood glucose level.

13. The method of any of the above claims, wherein the subject has a fasting blood glucose level between 100 and 125 mg/dL or a fasting blood glucose level at or above 125 mg/dL.

14. The method of any of claims 1-14, wherein the subject has a two-hour post-prandial blood glucose level between 140 and 199 mg/dL or a two-hour post-prandial blood glucose level at or above 200 mg/dL.

15. The method of any of the above claims, wherein the modified oligonucleotide reduces the blood glucose level to below 200 mg/dL, to below 175 mg/dL, to below 150 mg/dL, to below 125 mg/dL, to below 120 mg/dL, to below 115 mg/dL, to below 110 mg/dL, to below 105 mg/dL, or to below 100 mg/dL.

16. A method for improving glucose tolerance in a subject comprising administering to the subject a

compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member; and thereby improving glucose tolerance.

17. The method of claim 29, wherein the subject has impaired glucose tolerance.

18. The method of claim 29, comprising selecting a subject having impaired glucose tolerance.

19. A method of improving insulin resistance in a cell or tissue comprising contacting the cell or tissue with a compound comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member.

20. The method of claim 19 wherein the cell or tissue is a liver, fat, or muscle cell or tissue.

21. A method of increasing insulin sensitivity in a cell comprising contacting a cell with a compound

comprising a modified oligonucleotide consisting of 8 to 25 linked nucleosides and having a nucleobase sequence complementary to the nucleobase sequence of a miR-378 family member, wherein the cell has a decreased sensitivity to insulin.

22. The method of claim 21 wherein the cell is a liver, fat or muscle cell.

23. The method of any of the above claims, wherein the administering comprises parenteral administration, intravenous administration, subcutaneous administration, or oral administration.

24. The method of any of the above claims, further comprising administering at least one additional therapy.

25. The method of claim 25, wherein the at least one additional therapy is a glucose-lowering agent or a lipid-lowering agent.

26. The method of any of the above claims, wherein the compound is administered in the form of a pharmaceutical composition.

27. The method of any of the above claims, wherein the compound consists of the modified oligonucleotide.

28. The method of any of the above claims, wherein the nucleobase sequence of the modified

oligonucleotide is at least 85% complementary to the nucleobase sequence of SEQ ID NO: 2 or SEQ ID NO: 4, is at least 90% complementary to the nucleobase sequence of SEQ ID NO: 2 or SEQ ID NO: 4, is at least 95% complementary to the nucleobase sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or is 100% complementary to the nucleobase sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

29. The method of any of the above claims, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage or wherein each internucleoside linkage of the modified

oligonucleotide is a modified internucleoside linkage.

30. The method of claim 29, wherein the modified internucleoside linkage is a phosphorothioate

internucleoside linkage.

31. The method of any of the above claims, wherein the modified oligonucleotide comprises at least one nucleoside comprising a modified sugar, or wherein each nucleoside of the modified oligonucleotide comprises a modified sugar.

32. The method of claim 31, wherein the modified sugar is independently selected from a 2'-0- methoxyethyl sugar, a 2'-fluoro sugar, 2'-0-methyl sugar, and a bicyclic sugar moiety.

33. The method of any the above claims wherein the modified oligonucleotide consists of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 linked nucleosides.

34. The method of any of the above claims comprising selecting a subject having elevated serum fatty acid levels relative to normal serum fatty acid levels.