Compositions including inhibitors of PTEN expression or activity to prevent or treat type 2 diabetes

Abstract

The disclosure provides methods of preventing or treating Type 2 diabetes in a mammalian subject, reducing risk factors associated with Type 2 diabetes, and / or reducing the likelihood or severity of Type 2 diabetes. The methods comprise administering to the subject an effective amount of an inhibitor of PTEN expression and / or activity.

Description

COMPOSITIONS INCLUDING INHIBITORS OF PTEN EXPRESSION OR ACTIVITY TO PREVENT OR TREAT TYPE 2 DIABETES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No.63 / 594,840, filed October 31, 2023, the contents of which are incorporated by reference in their entirety for any and all purposes. TECHNICAL FIELD

[0002] The present disclosure relates to compositions and methods for preventing, ameliorating or treating diabetes and / or reducing the severity of one or more risk factors, signs, or symptoms associated with Type 2 diabetes. Additionally, the present technology relates to administering an effective amount of an inhibitor of PTEN expression and / or activity to a subject suffering from or at risk for Type 2 diabetes. GOVERNMENT SUPPORT

[0003] This invention was made with government support under CA008748 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] Type 2 Diabetes (T2D) is a condition that is defined by insulin resistance, hyperglycemia, and hyperinsulinemia unless there has been pancreatic beta cell failure resulting in insulin deficiency1. One of the major causes of T2D are lifestyle and dietary changes of continuous, increased consumption of carbohydrates and fats resulting in obesity often accompanied by insulin resistance that later leads to T2D. According to the International Diabetes Federation the number people afflicted globally with Type 2 Diabetes is projected to be 592 million by 2035. Insulin resistance (IR) precedes and accompanies T2D and is characterized by the increased demand of insulin to activate the insulin receptor- PI3K-AKT signaling that is required to stimulate glucose uptake to maintain glucosehomeostasis2-5. Increased caloric intake leads to the initial departure from normal glucose and insulin levels resulting in hyperglycemia and hyperinsulinemia. This increase in insulin level is in a steady state with higher-than-normal glucose levels which do not return to normal until intervention. While the physiological state of pre-diabetes and insulin resistance is characterized by normal secretion of insulin from the islet cells, it is defined by the lack of sensitivity of the peripheral tissues like muscle, adipose and liver to insulin, resulting in insufficient absorption of glucose by these tissues from the blood. Beyond pre- diabetes, constant hyperglycemia and hyperinsulinemia results in exhausted pancreatic islet cells undergoing apoptosis and failing to expand under constant insulin demand. This results in full blown type-2 diabetes over time and may finally lead to insulin deficiency and an irreversibly altered glucose homeostasis which must be resolved by an external means of insulin administration5.

[0006] Accordingly, there is an urgent need for compositions that effectively prevent or reverse signs and symptoms of Type 2 Diabetes. SUMMARY OF THE PRESENT TECHNOLOGY

[0007] In one aspect, the present disclosure provides a method for treating or preventing the onset of Type 2 diabetes in a subject in need thereof comprising administering to the subject an effective amount of at least one agent that inhibits PTEN expression and / or activity. In some embodiments, the subject is diagnosed as having, suspected as having, or at risk of having Type 2 diabetes. Additionally or alternatively, in certain embodiments, Type 2 diabetes is characterized by one or more of insulin resistance, hyperglycemia, hyperinsulinemia, liver lipidosis, dyslipidemia, leptin resistance, and / or obesity. In some embodiments, the Type 2 diabetes is diet-induced. The agent may be administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, iontophoretically, transmucosally, or intramuscularly.

[0008] Additionally or alternatively, in some embodiments, administration of the agent prevents or reverses insulin resistance, hyperglycemia, and / or hyperinsulinemia in the subject. In certain embodiments, administration of the agent prevents or reverses liver lipidosis, dyslipidemia, and / or leptin resistance. Additionally or alternatively, in certain embodiments, administration of the agent prevents or reverses obesity in the subject. Insome embodiments, administration of the agent results in a decrease in epididymal white adipose fat, liver or muscle PTEN polypeptide levels in the subject compared to that observed prior to administration.

[0009] In any of the preceding embodiments of the methods disclosed herein, the agent is a small molecule PTEN inhibitor, or a PTEN-specific inhibitory nucleic acid. The PTEN- specific inhibitory nucleic acid may be a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA. Examples of small molecule PTEN inhibitors include, but are not limited to hydroxyl(oxo)vanadium 3-hydroxypiridine-2-carboxylic acid (VO-OHpic), bisperoxovanadium 1,10-phenantroline (bpV(phen)), bisperoxovanadium 5-hydroxipyridine (bpV(pic)), bisperoxovanadium 5-hydroxipyridine-2-carboxylic acid bpV(HOpic), bisperoxovanadium pyridin-2-squaramide (bpV(pis)), or N-(9,10-dioxo-9,10- dihydrophenanthren-2-yl) pivalamide (SF1670).

[0010] Additionally or alternatively, in some embodiments of the methods disclosed herein, the agent is a small molecule mTORC1 inhibitor, or a mTORC1-specific inhibitory nucleic acid. The mTORC1-specific inhibitory nucleic acid may be a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA. Examples of small molecule mTORC1 inhibitor include, but are not limited to rapamycin, everolimus, temsirolimus, INK128, OSI027, PP242, RapaLinks, Ku-0063794, PP30, XL 388, WYE-354, WAY-600, WYE-687, Torin-1, Torin-2, RMC-6272, or AZD8055.

[0011] In any of the above embodiments of the methods disclosed herein, the agent is administered for 4 weeks, 5 weeks, 6 weeks or more.

[0012] Additionally or alternatively, in certain embodiments, the methods of the present technology further comprise separately, sequentially or simultaneously administering one or more additional therapeutic agents to the subject. Examples of suitable additional therapeutic agents include, but are not limited to, insulin sensitizers (e.g., biguanides (e.g., metformin) and glitazones (e.g., rosiglitazone and pioglitazone)), DPP-4 inhibitors (e.g., sitagliptin, saxagliptin, and linagliptin), GLP-1 receptor agonists (e.g., exenatide, liraglutide and semaglutide), and SGLT2 inhibitors (e.g., canagliflozin, dapagliflozin and empagliflozin).

[0013] In another aspect, the present disclosure provides a method for selecting a patient suffering from or at risk for Type 2 diabetes for treatment comprising (a) detecting PTEN polypeptide expression and / or activity levels in epididymal white adipose fat, liver and / or muscle of the subject that are elevated compared to a control subject or a predetermined threshold; and (b) administering to the patient an effective amount of an agent that inhibits PTEN expression and / or activity. In any of the preceding embodiments of the methods disclosed herein, the agent is a small molecule PTEN inhibitor, or a PTEN-specific inhibitory nucleic acid. The PTEN-specific inhibitory nucleic acid may be a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA. Examples of small molecule PTEN inhibitors include, but are not limited to hydroxyl(oxo)vanadium 3-hydroxypiridine-2- carboxylic acid (VO-OHpic), bisperoxovanadium 1,10-phenantroline (bpV(phen)), bisperoxovanadium 5-hydroxipyridine (bpV(pic)), bisperoxovanadium 5-hydroxipyridine-2- carboxylic acid bpV(HOpic), bisperoxovanadium pyridin-2-squaramide (bpV(pis)), or N- (9,10-dioxo-9,10-dihydrophenanthren-2-yl) pivalamide (SF1670). Additionally or alternatively, in some embodiments of the methods disclosed herein, the agent is a small molecule mTORC1 inhibitor, or a mTORC1-specific inhibitory nucleic acid. The mTORC1-specific inhibitory nucleic acid may be a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA. Examples of small molecule mTORC1 inhibitor include, but are not limited to rapamycin, everolimus, temsirolimus, INK128, OSI027, PP242, RapaLinks, Ku-0063794, PP30, XL 388, WYE-354, WAY-600, WYE-687, Torin-1, Torin- 2, RMC-6272, or AZD8055. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGs.1A-1J Western Diet increases PTEN expression. FIGs.1A-1C: L C57BL / 6J mice were either fed with regular / control (CD) or western diet (WD) for 12 weeks and the weight, p>0.0001(n=16-10), insulin p>0.05 (n=3), glucose levels measured p>0.0055 (n=3). FIG.1D: After 2 weeks of control or western diet mice were fasted overnight and dosed with a bolus of glucose and their glucose levels measured for 2 hours at the indicated times for a glucose tolerance test (GTT) p>0.05 (n=5) Graphs were plots as SEM of n and p values were calculated by a two-way ANOVA and post Bonferroni tests. FIG.1E: Mice were either fed with control or western diet for 4 weeks and livers stained for H&E and Oil Red O (ORO) (n=3). Representative images shown. FIG.1F: Leptinlevels were measured in mice following 4 or 6 weeks of either control or western diet (n=3) g-i. FIG.1G:. Mice were either fed with control (CD) or western diet (WD) and proteins were extracted from eWAT, liver and muscle and analyzed by western blotting (n=3, protein from the eWAT from one control diet mouse could not be extracted) FIG.1H: PTEN from FIG.1G was quantified and normalized with Actin and the mean and S.E.M represented. p value was calculated using unpaired student’s t test i. eWAT and liver tissues from mice fed on CD or WD were immunostained for PTEN and representative images shown. FIG.1J: C57BL / 6J mice were either fed with control or western diet (high fat / high carb) for 6 weeks and liver sections stained with H&E. Representative images shown.

[0015] FIGs.2A-2D: PTEN increases while AKT activity declines over 12 weeks in western diet. FIGs.2A-2B: Mice were fed regular (CD) or western diet (WD) for 12 weeks and proteins were extracted from eWAT and muscle at the indicated times and analyzed by immunoblotting for PTEN expression and AKT activity (n=3 for every time point). FIGs.2C-2D: PTEN and pAKT from eWAT, muscle (FIGs.2A-2B) and liver (FIG.6B) were quantified and normalized to actin and fold change of animals on WD over CD represented. Note; Week 1 of eWAT and liver tissues and week 2 of muscle tissue are the same as used in FIG.1 since they were a part of the same long term kinetics study that was done in FIG.2.

[0016] FIGs.3A-3U: Inhibiting PTEN activity prevents and reverses insulin resistance. FIGs.3A-3C: Mice were fed on regular diet or western diet or western diet along with treatment with the PTEN inhibitor VO-OHpic (10mg / kg) once daily for 6 weeks and the weight (n=4-10), insulin, glucose (n=3) levels measured. FIG.3D glucose tolerance test (GTT) (n=5) after indicated diet and treatment. FIGs.3E-3G: Mice were fed on the indicated diets for 2 weeks and then treated with VO-OHpic (10mg / kg) once daily for 4 weeks and the weight (n=4-10), insulin (n=3), glucose levels measured (n=3). The CD and WD arms of FIGs.3A-3C and FIGs.3E-3G are the same datasets, done together for consistency. FIG.3H glucose tolerance test (GTT) (n=3) after indicated diet and treatment. FIGs.3I-3K. Mean and SEM of insulin (from FIGs.3B and 3F), glucose (from FIGs.3C and 3G) and leptin (n=3) of mice fed with the indicated diets and treatment at 4 or 6 weeks. FIG.3L: eWAT and livers from mice fed with the indicated diets and treatment stained for H&E and lipid OilRedO (n=3). Representative images are shown. FIG.3M: Violin plot ofmorphometric analysis of eWAT H&E from l (n=3). FIGs.3N-3Q: The mean and SEM of the liver parameters: liver weight, macrovesicular steatosis scores, MASH scores and cholesterol levels (n=3). p value was calculated using unpaired student’s t test in FIGs.3I- 3Q. FIGs.3R-3T: Proteins from eWAT, muscle and liver of mice fed and treated with indicated conditions analyzed by immunoblotting. CD, WD same as FIGs.2A-2D since the indicated experimental arms were run on same gels together. FIG.3U. Quantification of percent change in pAKT over control from FIGs.3R-3T.

[0017] FIGs.4A-4R: Inhibiting mTORC1 prevents and reverses obesity and insulin resistance. FIGs.4A-4D: Mice were fed on regular diet or western diet or western diet along with treatment with the mTORC1 inhibitor RMC-6272 (3mg / kg) once a week for 6 weeks and the weight (n=4-10), insulin (n=3), glucose levels(n=3) and GTT measured (n=5). FIGs.4E-4H: Mice were fed on the indicated diets for 2 weeks and then treated with RMC-6272 (3mg / kg) on western diet for 4 weeks, weight (n=4-10), insulin (n=3), glucose levels (n=3) and GTT measured. The CD and WD arms of FIGs.4A-4C and FIGs. 4F-4G are the same datasets as FIGs.3A-3C and FIGs.3F-3G. FIGs.4I-4K: Mean and SEM of insulin (from FIGs.4B and 4F) and glucose (from FIGs.4C and 4G) and leptin of mice fed with the indicated diet and treatment. FIG.4L: eWAT from mice treated with indicated conditions, stained for H&E, representative images shown (n=3). Violin plot (FIG.4M) and mean and SEM (FIG.4N) of morphometric analysis of FIG.4L (n=3). FIGs.4O-4P: The mean and SEM of the liver parameters weight, and MASH scores represented (n=3). p value was calculated using unpaired student’s t test in FIGs.4I-4O. FIG.4Q: Proteins from eWAT, liver and muscle of mice that were fed with indicated conditions were analyzed for PTEN and PI3K pathway activation by immunoblotting. CD, WD same as FIG.2 since the indicated experimental arms were run on the same gels together FIG.4R: Quantification of percent change in PTEN over control diet from FIG. 4Q.

[0018] FIGs.5A-5G Western Diet increases PTEN expression. FIG.5A: Fold increase in insulin of C57BL / 6J mice fed with western diet over mice fed with regular diet for 12 weeks (n=3). FIG.5B: GTT test was performed 2 days, 1, 2, 4 and 6 weeks after start of the diet (n=3) FIG.5C: Glucose levels at 30 min (peak glucose) of GTT from FIG. 5B was plotted over time for mice. on control or western diet. FIG: 5D: Glucose levels at 2hours (adapted glucose) of the GTT from FIG.5B was plotted over time for mice on control or western diet. FIG.5E: Liver and eWAT tissues from mice fed on CD or WD were immunostained for PTEN and representative images shown (images from replicate mice from FIG.1I) (n=3). FIGs.5F-5G: IR and IRS1 from FIG.1G was quantified and normalized with Actin and the mean and S.E.M represented (n=3). p value was calculated using unpaired student’s t test.

[0019] FIGs.6A-6B: PTEN increases while AKT activity declines over 12 weeks in western diet. FIG.6A: Mice were fed with regular (CD) or western diet (WD) for 2 days and protein from eWAT, muscle and liver were extracted and analyzed by western blotting for PTEN and PI3K pathway activity. FIG.6B: Mice were fed with regular (CD) or western diet (WD) for 10 weeks and protein from liver extracted and analyzed by western blotting for PTEN expression and AKT activation (n=3 for every time point). Note: Week 1 of liver tissue are the same as used in FIG.1 since they were part of the same long term kinetics study that was done in FIG.2.

[0020] FIGs.7A-7T: Inhibiting PTEN activity prevents and reverses insulin resistance FIGs.7A-7B: BT474 and MCF7 cells were treated with 1uM VO-OHpic for the indicated times, protein extracted and immunoblotted for PTEN and PI3K pathway activity. FIG.7C: 3T3L1 cells were treated with insulin alone or with insulin and VO-OHpic added at 20 hours of insulin treatment and immunoblotted for PTEN and PI3K pathway activity. FIG.7D: Weight of food eaten by mice for the indicated conditions was measured each day for 3 days, mean and SEM represented (n=3). FIGs.7E-7F: Mean and SEM of weight and glucose measured of mice for the indicated times and conditions (n=3). FIGs.7G-7H: GTT measured for the indicated times and treatment conditions (n=3). FIGs.7I-7J: Mice weight and leptin was measured for the indicated times and treatment conditions, mean and SEM represented (n=3). FIG.7K: Mean and SEM of area of eWAT adipocyte cells from FIGs.3L-3M was measured in the above dietary and treatment conditions (n=3) FIGs.7L- 7N: The mean and SEM represented of liver parameters microvesicular steatosis, hepatocellular hypertrophy, and lobular inflammation scores (n=3). p value was calculated using unpaired student’s t test in FIGs.7I-7N. FIGs.7O-7Q: Proteins from eWAT, muscle and liver of mice fed and treated with indicated conditions analyzed by immunoblotting. FIGs.7R-7T Quantification of pAKT normalized to actin from FIGs.3R-3T.

[0021] FIGs.8A-8N Inhibiting mTORC1 prevents and reverses obesity and insulin resistance. FIG.8A: MDA-MB-468 cell expressing an inducible PTEN with or without its 5’UTR was treated with 100ng / ml of doxycycline for the indicated times and analyzed for PTEN and 4E-BP1 expression. FIG.8B: 3T3L-1 cells were treated insulin alone or in combination with RMC-6272 (0.5nM) for the indicated times and immunoblotted for PTEN and PI3K pathway activity. FIG.8C: Mean and SEM of weight was measured in mice fed with control or western diet or treated with RMC-6272 (3mg / kg) at the start of the western diet or treated with the RMC-6272 after 2 weeks of western diet, for 4 weeks (n=3). FIG. 8D: Weight of food eaten by mice for the indicated time and conditions was measured each day for 3 days, mean and SEM represented (n=3). FIGs.8E-8F: Mean and SEM of weight and glucose measured of mice for the indicated times and conditions (n=3). FIG.8G: Mean and SEM of leptin was measured for the indicated times and treatments (n=3). FIG. 8H: Livers from mice with indicated diet and treatment stained for H&E and lipid OilRedO (n=3). Representative images are shown. FIG.8I: Mean and SEM of cholesterol was measured for the indicated times and treatments (n=3). FIGs.8J-8L: Weight, insulin and glucose was measured in mice fed with control diet, western diet or western diet with Rapamycin treatment (10mg / kg) for the indicated times (n=3). p value was calculated using unpaired student’s t test in FIGs.8C-8I and 8L. FIG.8M: Livers from mice fed with control diet or western diet with rapamycin treatment were stained for H&E. Representative images are displayed (n=3). FIG.8N: Proteins from eWAT, muscle and liver of mice fed and treated with indicated conditions analyzed by immunoblotting. DETAILED DESCRIPTION

[0022] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0023] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford UniversityPress); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0024] Very little is known about the molecular mechanisms underlying the events leading to the insulin resistance and type 2 diabetes state, especially in the changes that occur in key components of Insulin-PI3K-AKT signaling that has reduced functioning. Consideration of the Insulin-PI3K-AKT signaling maybe key to understanding and reversing the effects of insulin resistance in order to prevent the development of / treat full- blown Type 2 Diabetes.

[0025] The PI3K pathway is negatively regulated by PTEN, a phosphatase that dephosphorylates PIP3 on the 3-position and other phosphatases. PTEN is regulated by the Insulin-PI3K-mTOR signaling via mTORC1-4EBP1 dependent cap dependent translation6. This mechanism constitutes a negative feedback loop that serves to finely regulate the duration and amplitude of physiologic Insulin-PI3K-AKT signaling. As demonstrated herein, inhibition of PTEN was unexpectedly found to be sufficient in preventing or reversing obesity, hyperinsulinemia, hyperglycemia, liver lipidosis phenotypes and insulin resistance. Taken together, the therapeutic effects observed with direct PTEN inhibitors can be extrapolated to agents that target PI3K pathway components that modulate PTEN expression and / or activity, e.g., inhibition of mTORC1.Definitions

[0026] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0027] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0028] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, intrathecally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intraocularly, intradermally, transmucosally, iontophoretically, or topically. Administration includes self-administration and the administration by another.

[0029] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0030] “Diabetes” refers to high blood sugar or ketoacidosis, as well as chronic, general metabolic abnormalities arising from a prolonged high blood sugar status or a decrease in glucose tolerance. “Diabetes” encompasses both the type I and type II (Non InsulinDependent Diabetes Mellitus or NIDDM) forms of the disease. The risk factors for diabetes include the following factors: waistline of more than 40 inches for men or 35 inches for women, blood pressure of 130 / 85 mm Hg or higher, triglycerides above 150 mg / dl, fasting blood glucose greater than 100 mg / dl or high-density lipoprotein of less than 40 mg / dl in men or 50 mg / dl in women.

[0031] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0032] As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product, if required for proper expression and function.

[0033] The term “hyperinsulinemia” refers to a state in an individual in which the level of insulin in the blood is higher than a healthy control subject.

[0034] The term “insulin resistance” refers to a state in which a normal amount of insulin produces a subnormal biologic response relative to the biological response in a subject that does not have insulin resistance.

[0035] As used herein, “obese” individuals or individuals suffering from obesity are generally individuals having a body mass index (BMI) of at least 25 or greater. Obesity may or may not be associated with insulin resistance.

[0036] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art.

[0037] As used herein, “prevention” or “preventing” of a disorder or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0038] As used herein, a “sample” or “biological sample” refers to a body fluid or a tissue sample isolated from a subject. In some cases, a biological sample may consist of or comprise whole blood, platelets, red blood cells, white blood cells, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and / or chorionic villi, cultured cells, endothelial cells, synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid and the like. The term "sample" may also encompass the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, cerebrospinal fluid (CSF), saliva, mucus, sputum, semen, sweat, urine, or any other bodily fluids. Samples can be obtained from a subject by any means including, but not limited to, venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage, scraping, surgical incision, orintervention or other means known in the art. A blood sample can be whole blood or any fraction thereof, including blood cells (red blood cells, white blood cells or leukocytes, and platelets), serum and plasma.

[0039] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0040] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0041] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0042] As used herein, the terms “subject”, “patient”, or “individual” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the subject, patient or individual is a human.

[0043] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0044] It is also to be appreciated that the various modes of treatment of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.Therapeutic and Prophylactic Methods

[0045] The following discussion is presented by way of example only, and is not intended to be limiting.

[0046] One aspect of the present technology includes methods for treating or preventing the onset of Type 2 diabetes comprising administering to a subject a therapeutically effective amount of at least one agent that inhibits PTEN expression and / or activity. In some embodiments, the subject is diagnosed as having, suspected as having, or at risk of having Type 2 diabetes. Additionally or alternatively, in certain embodiments, Type 2 diabetes is characterized by one or more of insulin resistance, hyperglycemia, hyperinsulinemia, liver lipidosis, dyslipidemia, leptin resistance, and / or obesity. In some embodiments, the Type 2 diabetes is diet-induced. The agent may be administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, iontophoretically, transmucosally, or intramuscularly.

[0047] Additionally or alternatively, in some embodiments, administration of the agent prevents or reverses insulin resistance, hyperglycemia, and / or hyperinsulinemia in the subject. In certain embodiments, administration of the agent prevents or reverses liver lipidosis, dyslipidemia, and / or leptin resistance. Additionally or alternatively, in certain embodiments, administration of the agent prevents or reverses obesity in the subject. In some embodiments, administration of the agent results in a decrease in epididymal white adipose fat, liver or muscle PTEN polypeptide levels in the subject compared to that observed prior to administration.

[0048] In any of the preceding embodiments of the methods disclosed herein, the agent is a small molecule PTEN inhibitor, or a PTEN-specific inhibitory nucleic acid. The PTEN- specific inhibitory nucleic acid may be a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA.

[0049] Exemplary nucleic acid sequences of three human PTEN transcript variants, represented by SEQ ID NOs: 1-3.

[0050] NM_000314.8 Homo sapiens phosphatase and tensin homolog (PTEN), transcript variant 1, mRNA (SEQ ID NO: 1) GTTCTCTCCTCTCGGAAGCTGCAGCCATGATGGAAGTTTGAGAGTTGAGCCGCTGTGAGGCGAGGCCGGGCTCAGGCGAGGGAGATGAGAGACGGCGGCGGCCGCGGCCCGGAGCCCCTCTCAGCGCCTGTGAGCAGCCG CGGGGGCAGCGCCCTCGGGGAGCCGGCCGGCCTGCGGCGGCGGCAGCGGCGGCGTTTCTCGCCTCCTCTT CGTCTTTTCTAACCGTGCAGCCTCTTCCTCGGCTTCTCCTGAAAGGGAAGGTGGAAGCCGTGGGCTCGGG CGGGAGCCGGCTGAGGCGCGGCGGCGGCGGCGGCACCTCCCGCTCCTGGAGCGGGGGGGAGAAGCGGCGG CGGCGGCGGCCGCGGCGGCTGCAGCTCCAGGGAGGGGGTCTGAGTCGCCTGTCACCATTTCCAGGGCTGG GAACGCCGGAGAGTTGGTCTCTCCCCTTCTACTGCCTCCAACACGGCGGCGGCGGCGGCTGGCACATCCA GGGACCCGGGCCGGTTTTAAACCTCCCGTGCGCCGCCGCCGCACCCCCCGTGGCCCGGGCTCCGGAGGCC GCCGGCGGAGGCAGCCGTTCGGAGGATTATTCGTCTTCTCCCCATTCCGCTGCCGCCGCTGCCAGGCCTC TGGCTGCTGAGGAGAAGCAGGCCCAGTCGCTGCAACCATCCAGCAGCCGCCGCAGCAGCCATTACCCGGC TGCGGTCCAGAGCCAAGCGGCGGCAGAGCGAGGGGCATCAGCTACCGCCAAGTCCAGAGCCATTTCCATC CTGCAGAAGAAGCCCCGCCACCAGCAGCTTCTGCCATCTCTCTCCTCCTTTTTCTTCAGCCACAGGCTCC CAGACATGACAGCCATCATCAAAGAGATCGTTAGCAGAAACAAAAGGAGATATCAAGAGGATGGATTCGA CTTAGACTTGACCTATATTTATCCAAACATTATTGCTATGGGATTTCCTGCAGAAAGACTTGAAGGCGTA TACAGGAACAATATTGATGATGTAGTAAGGTTTTTGGATTCAAAGCATAAAAACCATTACAAGATATACA ATCTTTGTGCTGAAAGACATTATGACACCGCCAAATTTAATTGCAGAGTTGCACAATATCCTTTTGAAGA CCATAACCCACCACAGCTAGAACTTATCAAACCCTTTTGTGAAGATCTTGACCAATGGCTAAGTGAAGAT GACAATCATGTTGCAGCAATTCACTGTAAAGCTGGAAAGGGACGAACTGGTGTAATGATATGTGCATATT TATTACATCGGGGCAAATTTTTAAAGGCACAAGAGGCCCTAGATTTCTATGGGGAAGTAAGGACCAGAGA CAAAAAGGGAGTAACTATTCCCAGTCAGAGGCGCTATGTGTATTATTATAGCTACCTGTTAAAGAATCAT CTGGATTATAGACCAGTGGCACTGTTGTTTCACAAGATGATGTTTGAAACTATTCCAATGTTCAGTGGCG GAACTTGCAATCCTCAGTTTGTGGTCTGCCAGCTAAAGGTGAAGATATATTCCTCCAATTCAGGACCCAC ACGACGGGAAGACAAGTTCATGTACTTTGAGTTCCCTCAGCCGTTACCTGTGTGTGGTGATATCAAAGTA GAGTTCTTCCACAAACAGAACAAGATGCTAAAAAAGGACAAAATGTTTCACTTTTGGGTAAATACATTCT TCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAG CATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATATCTAGTACTTACTTTAACAAAAAATGATCTT GACAAAGCAAATAAAGACAAAGCCAACCGATACTTTTCTCCAAATTTTAAGGTGAAGCTGTACTTCACAA AAACAGTAGAGGAGCCGTCAAATCCAGAGGCTAGCAGTTCAACTTCTGTAACACCAGATGTTAGTGACAA TGAACCTGATCATTATAGATATTCTGACACCACTGACTCTGATCCAGAGAATGAACCTTTTGATGAAGAT CAGCATACACAAATTACAAAAGTCTGAATTTTTTTTTATCAAGAGGGATAAAACACCATGAAAATAAACT TGAATAAACTGAAAATGGACCTTTTTTTTTTTAATGGCAATAGGACATTGTGTCAGATTACCAGTTATAG GAACAATTCTCTTTTCCTGACCAATCTTGTTTTACCCTATACATCCACAGGGTTTTGACACTTGTTGTCC AGTTGAAAAAAGGTTGTGTAGCTGTGTCATGTATATACCTTTTTGTGTCAAAAGGACATTTAAAATTCAA TTAGGATTAATAAAGATGGCACTTTCCCGTTTTATTCCAGTTTTATAAAAAGTGGAGACAGACTGATGTG TATACGTAGGAATTTTTTCCTTTTGTGTTCTGTCACCAACTGAAGTGGCTAAAGAGCTTTGTGATATACT GGTTCACATCCTACCCCTTTGCACTTGTGGCAACAGATAAGTTTGCAGTTGGCTAAGAGAGGTTTCCGAA GGGTTTTGCTACATTCTAATGCATGTATTCGGGTTAGGGGAATGGAGGGAATGCTCAGAAAGGAAATAAT TTTATGCTGGACTCTGGACCATATACCATCTCCAGCTATTTACACACACCTTTCTTTAGCATGCTACAGT TATTAATCTGGACATTCGAGGAATTGGCCGCTGTCACTGCTTGTTGTTTGCGCATTTTTTTTTAAAGCAT ATTGGTGCTAGAAAAGGCAGCTAAAGGAAGTGAATCTGTATTGGGGTACAGGAATGAACCTTCTGCAACA TCTTAAGATCCACAAATGAAGGGATATAAAAATAATGTCATAGGTAAGAAACACAGCAACAATGACTTAA CCATATAAATGTGGAGGCTATCAACAAAGAATGGGCTTGAAACATTATAAAAATTGACAATGATTTATTA AATATGTTTTCTCAATTGTAACGACTTCTCCATCTCCTGTGTAATCAAGGCCAGTGCTAAAATTCAGATG CTGTTAGTACCTACATCAGTCAACAACTTACACTTATTTTACTAGTTTTCAATCATAATACCTGCTGTGG ATGCTTCATGTGCTGCCTGCAAGCTTCTTTTTTCTCATTAAATATAAAATATTTTGTAATGCTGCACAGA AATTTTCAATTTGAGATTCTACAGTAAGCGTTTTTTTTCTTTGAAGATTTATGATGCACTTATTCAATAG CTGTCAGCCGTTCCACCCTTTTGACCTTACACATTCTATTACAATGAATTTTGCAGTTTTGCACATTTTT TAAATGTCATTAACTGTTAGGGAATTTTACTTGAATACTGAATACATATAATGTTTATATTAAAAAGGAC ATTTGTGTTAAAAAGGAAATTAGAGTTGCAGTAAACTTTCAATGCTGCACACAAAAAAAAGACATTTGAT TTTTCAGTAGAAATTGTCCTACATGTGCTTTATTGATTTGCTATTGAAAGAATAGGGTTTTTTTTTTTTT TTTTTTTTTTTTTTTTAAATGTGCAGTGTTGAATCATTTCTTCATAGTGCTCCCCCGAGTTGGGACTAGG GCTTCAATTTCACTTCTTAAAAAAAATCATCATATATTTGATATGCCCAGACTGCATACGATTTTAAGCG GAGTACAACTACTATTGTAAAGCTAATGTGAAGATATTATTAAAAAGGTTTTTTTTTCCAGAAATTTGGT GTCTTCAAATTATACCTTCACCTTGACATTTGAATATCCAGCCATTTTGTTTCTTAATGGTATAAAATTC CATTTTCAATAACTTATTGGTGCTGAAATTGTTCACTAGCTGTGGTCTGACCTAGTTAATTTACAAATAC AGATTGAATAGGACCTACTAGAGCAGCATTTATAGAGTTTGATGGCAAATAGATTAGGCAGAACTTCATC TAAAATATTCTTAGTAAATAATGTTGACACGTTTTCCATACCTTGTCAGTTTCATTCAACAATTTTTAAA TTTTTAACAAAGCTCTTAGGATTTACACATTTATATTTAAACATTGATATATAGAGTATTGATTGATTGCTCATAAGTTAAATTGGTAAAGTTAGAGACAACTATTCTAACACCTCACCATTGAAATTTATATGCCACCT TGTCTTTCATAAAAGCTGAAAATTGTTACCTAAAATGAAAATCAACTTCATGTTTTGAAGATAGTTATAA ATATTGTTCTTTGTTACAATTTCGGGCACCGCATATTAAAACGTAACTTTATTGTTCCAATATGTAACAT GGAGGGCCAGGTCATAAATAATGACATTATAATGGGCTTTTGCACTGTTATTATTTTTCCTTTGGAATGT GAAGGTCTGAATGAGGGTTTTGATTTTGAATGTTTCAATGTTTTTGAGAAGCCTTGCTTACATTTTATGG TGTAGTCATTGGAAATGGAAAAATGGCATTATATATATTATATATATAAATATATATTATACATACTCTC CTTACTTTATTTCAGTTACCATCCCCATAGAATTTGACAAGAATTGCTATGACTGAAAGGTTTTCGAGTC CTAATTAAAACTTTATTTATGGCAGTATTCATAATTAGCCTGAAATGCATTCTGTAGGTAATCTCTGAGT TTCTGGAATATTTTCTTAGACTTTTTGGATGTGCAGCAGCTTACATGTCTGAAGTTACTTGAAGGCATCA CTTTTAAGAAAGCTTACAGTTGGGCCCTGTACCATCCCAAGTCCTTTGTAGCTCCTCTTGAACATGTTTG CCATACTTTTAAAAGGGTAGTTGAATAAATAGCATCACCATTCTTTGCTGTGGCACAGGTTATAAACTTA AGTGGAGTTTACCGGCAGCATCAAATGTTTCAGCTTTAAAAAATAAAAGTAGGGTACAAGTTTAATGTTT AGTTCTAGAAATTTTGTGCAATATGTTCATAACGATGGCTGTGGTTGCCACAAAGTGCCTCGTTTACCTT TAAATACTGTTAATGTGTCATGCATGCAGATGGAAGGGGTGGAACTGTGCACTAAAGTGGGGGCTTTAAC TGTAGTATTTGGCAGAGTTGCCTTCTACCTGCCAGTTCAAAAGTTCAACCTGTTTTCATATAGAATATAT ATACTAAAAAATTTCAGTCTGTTAAACAGCCTTACTCTGATTCAGCCTCTTCAGATACTCTTGTGCTGTG CAGCAGTGGCTCTGTGTGTAAATGCTATGCACTGAGGATACACAAAAATACCAATATGATGTGTACAGGA TAATGCCTCATCCCAATCAGATGTCCATTTGTTATTGTGTTTGTTAACAACCCTTTATCTCTTAGTGTTA TAAACTCCACTTAAAACTGATTAAAGTCTCATTCTTGTCATTGTGTGGGTGTTTTATTAAATGAGAGTTT ATAATTCAAATTGCTTAAGTCCATTGAAGTTTTAATTAATGGGCAGCCAAATGTGAATACAAAGTTTTCA GTTTTTTTTTTTCCTGCTGTCCTTCAAAGCCTACTGTTTAAAAAAAAAAAAAAAAAAAAACATGGCCTGA GAGTAGAGTATCTGTCTACTCATGTTTAATTAAGGAAAAACACTTATTTTTAGGGCTTTAGTCATCACTT CATAAATTGTATAAGCACATTAAATAGCGTTCTAGTCCTGAAAAAGTCCAAGATTCTTAGAAAATTGTGC ATATTTTTATTATGACAGATGTTTGAAGATAATTCCCCAGAATGGATTTGATACTTTAGATTTCAATTTT GTGGCTTTTGTCTATTATTCTGTACTCTGCCATCAGCATATGGAAAGCTTCATTTACTCATCATGACTTG TGCCATATAAAAATTGATATTTCGGAATAGTCTAAAGGACTTTTTGTACTTGAATTTAATCATGTTGTTT CTAATATTCTTAAAAGCTTGAAGACTAAAGCATATCCTTTCAACAAAGCATAGTAAGGTAATAAGAAAGT GTAGTTTGTACAAGTGTTAAAAAAATAAAGTAGACAATGTTACAGTGGGACTTATTATTTCAAGTTTACA TTTTCTCCATGTAATTTTTTAAAAAGTAAATGAAAAAATGTGCAATAATGTAAAATATGAAGTGTATGTG TACACACATTTTATTTTTCGGTATCTTGGGTATACGTATGGTTGAAAACTATACTGGAGTCTAAAAGTAT TCTAATTTATAAGAAGACATTTTGGTGATGTTTGAAAAATAGAAATGTGCTAGTTTTGTTTTTATATCAT GTCCTTTGTACGTTGTAATATGAGCTGGCTTGGTTCAGTAAATGCCATCACCATTTCCATTGAGAATTTA AAACTCACCAGTGTTTAATATGCAGGCTTCCAAAGGCTTATGAAAAAAATCAAGACCCTTAAATCTAGTT AATTTGCTGCTAACATGAAACTCTTTGGTTCTTTTATTTTTGCCAGATAATTAGACACACATCTAAAGCT TAGTCTTAAATGGCTTAAGTGTAGCTATTGATTAGTGCTGTTGCTAGTTCAGAAAGAAATGTTTGTGAAT GGAAACAAGAATATTCAGTCCAAACTGTTGTAAGGACAGTACCTGAAAACCAGGAAACAGGATAATGGAA AAAGTCTTTTAAAGATGAAATGTTGGAGCCAACTTTCTTATAGAATTAATTGTATGTGGCTATAGAAAGC CTAATGATTGTTGCTTATTTTTGAGAGCATATTATTCTTTTATGACCATAATCTTGCTGTTTTTCCATCT TCCAAAAGATCTTCCTTCTAATATGTATATCAGAATGTGGGTAGCCAGTCAGACAAATTCATATTGGTTG GTAGCTTTAAAAAGTTTGTAATGTGAAGACAGGAAAGGACAAAATAGTTTGCTTTGGTGGTAGTACTCTG GTTGTTAAGCTAGGTATTTTGAGACTACTTCCCCATCACAACAACAATAAAATAATCACTCATAATCCTA TCACCTGGAGACATAGCCATCGTTAATATGTTAGTGACTATACAATCATGTTTTCTTCTGTATATCCATG TATATTCTTTAAAAATGAAATTTATACTGTACCTGATCTCAAAGCTTTTTAGCTTAGTATATCTGTCATG AATTTGTAGGATGTTCCATTGCATCAGAAAACGGACAGTGATTTGATTACTTTCTAATGCCACAGATGCA GATTACATGTAGTTATTGAGAATCCTTTCGAATTCAGTGGCTTAATCATGAATGTCTAAATATTGTTGAC ATTAGGATGATACATGTAAATTAAAGTTACATTTGTTTAGCATAGACAAGCTTAACATTGTAGATGTTTC TCTTCAAAAATCATCTTAAACATTTGCATTTGGAATTGTGTTAAATAGAATGTGTGAAACACTGTATTAG TAAACTTCATCACCTTTCTACTTCCTTATAGTTTGAACTTTTCAGTTTTTGTAGTTCCCAAACAGTTGCT CAATTTAGAGCAAATTAATTTAACACCTGCCAAAAAAAGGCTGCTGTTGGCTTATCAGTTGTCTTTAAAT TCAAATGCTCATGTGACTTTTATCACATCAAAAAATATTTCATTAATGATTCACCTTTAGCTCTGAAAAT TACCGCGTTTAGTAATTATAGTGGGCTTATAAAAACATGCAACTCTTTTTGATAGTTATTTGAGAATTTT GGTGAAAAATATTTAGCTGAGGGCAGTATAGAACTTATAAACCAATATATTGATATTTTTAAAACATTTT TACATATAAGTAAACTGCCATCTTTGAGCATAACTACATTTAAAAATAAAGCTGCATATTTTTAAATCAA GTGTTTAACAAGAATTTATATTTTTTATTTTTTAAAATTAAAAATAATTTATATTTCCTCTGTTGCATGA GGATTCTCATCTGTGCTTATAATGGTTAGAGATTTTATTTGTGTGGAATGAAGTGAGGCTTGTAGTCATG GTTCTAGTGTTTCAGTTTGCCAAGTCTGTTTACTGCAGTGAAATTCATCAAATGTTTCAGTGTGGTTTTC TGTAGCCTATCATTTACTGGCTATTTTTTTATGTACACCTTTAGGATTTTCTGCCTACTCTATCCAGTTGTCCAAATGATATCCTACATTTTACAAATGCCCTTTCAGTTTCTATTTTCTTTTTCCATTAAATTGCCCTC ATGTCCTAATGTGCAGTTTGTAAGTGTGTGTGTGTGTGTCTGTGTGTGTGTGAATTTGATTTTCAAGAGT GCTAGACTTCCAATTTGAGAGATTAAATAATTTAATTCAGGCAAACATTTTTCATTGGAATTTCACAGTT CATTGTAATGAAAATGTTAATCCTGGATGACCTTTGACATACAGTAATGAATCTTGGATATTAATGAATT TGTTAGTAGCATCTTGATGTGTGTTTTAATGAGTTATTTTCAAAGTTGTGCATTAAACCAAAGTTGGCAT ACTGGAAGTGTTTATATCAAGTTCCATTTGGCTACTGATGGACAAAAAATAGAAATGCCTTCCTATGGAG AGTATTTTTCCTTTAAAAAATTAAAAAGGTTAATTATTTTGACTA

[0051] NM_001304717.5 Homo sapiens phosphatase and tensin homolog (PTEN), transcript variant 2, mRNA (SEQ ID NO: 2) GTTCTCTCCTCTCGGAAGCTGCAGCCATGATGGAAGTTTGAGAGTTGAGCCGCTGTGAGGCGAGGCCGGG CTCAGGCGAGGGAGATGAGAGACGGCGGCGGCCGCGGCCCGGAGCCCCTCTCAGCGCCTGTGAGCAGCCG CGGGGGCAGCGCCCTCGGGGAGCCGGCCGGCCTGCGGCGGCGGCAGCGGCGGCGTTTCTCGCCTCCTCTT CGTCTTTTCTAACCGTGCAGCCTCTTCCTCGGCTTCTCCTGAAAGGGAAGGTGGAAGCCGTGGGCTCGGG CGGGAGCCGGCTGAGGCGCGGCGGCGGCGGCGGCACCTCCCGCTCCTGGAGCGGGGGGGAGAAGCGGCGG CGGCGGCGGCCGCGGCGGCTGCAGCTCCAGGGAGGGGGTCTGAGTCGCCTGTCACCATTTCCAGGGCTGG GAACGCCGGAGAGTTGGTCTCTCCCCTTCTACTGCCTCCAACACGGCGGCGGCGGCGGCGGCACATCCAG GGACCCGGGCCGGTTTTAAACCTCCCGTCCGCCGCCGCCGCACCCCCCGTGGCCCGGGCTCCGGAGGCCG CCGGCGGAGGCAGCCGTTCGGAGGATTATTCGTCTTCTCCCCATTCCGCTGCCGCCGCTGCCAGGCCTCT GGCTGCTGAGGAGAAGCAGGCCCAGTCGCTGCAACCATCCAGCAGCCGCCGCAGCAGCCATTACCCGGCT GCGGTCCAGAGCCAAGCGGCGGCAGAGCGAGGGGCATCAGCTACCGCCAAGTCCAGAGCCATTTCCATCC TGCAGAAGAAGCCCCGCCACCAGCAGCTTCTGCCATCTCTCTCCTCCTTTTTCTTCAGCCACAGGCTCCC AGACATGACAGCCATCATCAAAGAGATCGTTAGCAGAAACAAAAGGAGATATCAAGAGGATGGATTCGAC TTAGACTTGACCTATATTTATCCAAACATTATTGCTATGGGATTTCCTGCAGAAAGACTTGAAGGCGTAT ACAGGAACAATATTGATGATGTAGTAAGGTTTTTGGATTCAAAGCATAAAAACCATTACAAGATATACAA TCTTTGTGCTGAAAGACATTATGACACCGCCAAATTTAATTGCAGAGTTGCACAATATCCTTTTGAAGAC CATAACCCACCACAGCTAGAACTTATCAAACCCTTTTGTGAAGATCTTGACCAATGGCTAAGTGAAGATG ACAATCATGTTGCAGCAATTCACTGTAAAGCTGGAAAGGGACGAACTGGTGTAATGATATGTGCATATTT ATTACATCGGGGCAAATTTTTAAAGGCACAAGAGGCCCTAGATTTCTATGGGGAAGTAAGGACCAGAGAC AAAAAGGGAGTAACTATTCCCAGTCAGAGGCGCTATGTGTATTATTATAGCTACCTGTTAAAGAATCATC TGGATTATAGACCAGTGGCACTGTTGTTTCACAAGATGATGTTTGAAACTATTCCAATGTTCAGTGGCGG AACTTGCAATCCTCAGTTTGTGGTCTGCCAGCTAAAGGTGAAGATATATTCCTCCAATTCAGGACCCACA CGACGGGAAGACAAGTTCATGTACTTTGAGTTCCCTCAGCCGTTACCTGTGTGTGGTGATATCAAAGTAG AGTTCTTCCACAAACAGAACAAGATGCTAAAAAAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTT CATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGC ATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATATCTAGTACTTACTTTAACAAAAAATGATCTTG ACAAAGCAAATAAAGACAAAGCCAACCGATACTTTTCTCCAAATTTTAAGGTGAAGCTGTACTTCACAAA AACAGTAGAGGAGCCGTCAAATCCAGAGGCTAGCAGTTCAACTTCTGTAACACCAGATGTTAGTGACAAT GAACCTGATCATTATAGATATTCTGACACCACTGACTCTGATCCAGAGAATGAACCTTTTGATGAAGATC AGCATACACAAATTACAAAAGTCTGAATTTTTTTTTATCAAGAGGGATAAAACACCATGAAAATAAACTT GAATAAACTGAAAATGGACCTTTTTTTTTTTAATGGCAATAGGACATTGTGTCAGATTACCAGTTATAGG AACAATTCTCTTTTCCTGACCAATCTTGTTTTACCCTATACATCCACAGGGTTTTGACACTTGTTGTCCA GTTGAAAAAAGGTTGTGTAGCTGTGTCATGTATATACCTTTTTGTGTCAAAAGGACATTTAAAATTCAAT TAGGATTAATAAAGATGGCACTTTCCCGTTTTATTCCAGTTTTATAAAAAGTGGAGACAGACTGATGTGT ATACGTAGGAATTTTTTCCTTTTGTGTTCTGTCACCAACTGAAGTGGCTAAAGAGCTTTGTGATATACTG GTTCACATCCTACCCCTTTGCACTTGTGGCAACAGATAAGTTTGCAGTTGGCTAAGAGAGGTTTCCGAAG GGTTTTGCTACATTCTAATGCATGTATTCGGGTTAGGGGAATGGAGGGAATGCTCAGAAAGGAAATAATT TTATGCTGGACTCTGGACCATATACCATCTCCAGCTATTTACACACACCTTTCTTTAGCATGCTACAGTT ATTAATCTGGACATTCGAGGAATTGGCCGCTGTCACTGCTTGTTGTTTGCGCATTTTTTTTTAAAGCATA TTGGTGCTAGAAAAGGCAGCTAAAGGAAGTGAATCTGTATTGGGGTACAGGAATGAACCTTCTGCAACAT CTTAAGATCCACAAATGAAGGGATATAAAAATAATGTCATAGGTAAGAAACACAGCAACAATGACTTAAC CATATAAATGTGGAGGCTATCAACAAAGAATGGGCTTGAAACATTATAAAAATTGACAATGATTTATTAA ATATGTTTTCTCAATTGTAACGACTTCTCCATCTCCTGTGTAATCAAGGCCAGTGCTAAAATTCAGATGC TGTTAGTACCTACATCAGTCAACAACTTACACTTATTTTACTAGTTTTCAATCATAATACCTGCTGTGGATGCTTCATGTGCTGCCTGCAAGCTTCTTTTTTCTCATTAAATATAAAATATTTTGTAATGCTGCACAGAA ATTTTCAATTTGAGATTCTACAGTAAGCGTTTTTTTTCTTTGAAGATTTATGATGCACTTATTCAATAGC TGTCAGCCGTTCCACCCTTTTGACCTTACACATTCTATTACAATGAATTTTGCAGTTTTGCACATTTTTT AAATGTCATTAACTGTTAGGGAATTTTACTTGAATACTGAATACATATAATGTTTATATTAAAAAGGACA TTTGTGTTAAAAAGGAAATTAGAGTTGCAGTAAACTTTCAATGCTGCACACAAAAAAAAGACATTTGATT TTTCAGTAGAAATTGTCCTACATGTGCTTTATTGATTTGCTATTGAAAGAATAGGGTTTTTTTTTTTTTT TTTTTTTTTTTTTTTAAATGTGCAGTGTTGAATCATTTCTTCATAGTGCTCCCCCGAGTTGGGACTAGGG CTTCAATTTCACTTCTTAAAAAAAATCATCATATATTTGATATGCCCAGACTGCATACGATTTTAAGCGG AGTACAACTACTATTGTAAAGCTAATGTGAAGATATTATTAAAAAGGTTTTTTTTTCCAGAAATTTGGTG TCTTCAAATTATACCTTCACCTTGACATTTGAATATCCAGCCATTTTGTTTCTTAATGGTATAAAATTCC ATTTTCAATAACTTATTGGTGCTGAAATTGTTCACTAGCTGTGGTCTGACCTAGTTAATTTACAAATACA GATTGAATAGGACCTACTAGAGCAGCATTTATAGAGTTTGATGGCAAATAGATTAGGCAGAACTTCATCT AAAATATTCTTAGTAAATAATGTTGACACGTTTTCCATACCTTGTCAGTTTCATTCAACAATTTTTAAAT TTTTAACAAAGCTCTTAGGATTTACACATTTATATTTAAACATTGATATATAGAGTATTGATTGATTGCT CATAAGTTAAATTGGTAAAGTTAGAGACAACTATTCTAACACCTCACCATTGAAATTTATATGCCACCTT GTCTTTCATAAAAGCTGAAAATTGTTACCTAAAATGAAAATCAACTTCATGTTTTGAAGATAGTTATAAA TATTGTTCTTTGTTACAATTTCGGGCACCGCATATTAAAACGTAACTTTATTGTTCCAATATGTAACATG GAGGGCCAGGTCATAAATAATGACATTATAATGGGCTTTTGCACTGTTATTATTTTTCCTTTGGAATGTG AAGGTCTGAATGAGGGTTTTGATTTTGAATGTTTCAATGTTTTTGAGAAGCCTTGCTTACATTTTATGGT GTAGTCATTGGAAATGGAAAAATGGCATTATATATATTATATATATAAATATATATTATACATACTCTCC TTACTTTATTTCAGTTACCATCCCCATAGAATTTGACAAGAATTGCTATGACTGAAAGGTTTTCGAGTCC TAATTAAAACTTTATTTATGGCAGTATTCATAATTAGCCTGAAATGCATTCTGTAGGTAATCTCTGAGTT TCTGGAATATTTTCTTAGACTTTTTGGATGTGCAGCAGCTTACATGTCTGAAGTTACTTGAAGGCATCAC TTTTAAGAAAGCTTACAGTTGGGCCCTGTACCATCCCAAGTCCTTTGTAGCTCCTCTTGAACATGTTTGC CATACTTTTAAAAGGGTAGTTGAATAAATAGCATCACCATTCTTTGCTGTGGCACAGGTTATAAACTTAA GTGGAGTTTACCGGCAGCATCAAATGTTTCAGCTTTAAAAAATAAAAGTAGGGTACAAGTTTAATGTTTA GTTCTAGAAATTTTGTGCAATATGTTCATAACGATGGCTGTGGTTGCCACAAAGTGCCTCGTTTACCTTT AAATACTGTTAATGTGTCATGCATGCAGATGGAAGGGGTGGAACTGTGCACTAAAGTGGGGGCTTTAACT GTAGTATTTGGCAGAGTTGCCTTCTACCTGCCAGTTCAAAAGTTCAACCTGTTTTCATATAGAATATATA TACTAAAAAATTTCAGTCTGTTAAACAGCCTTACTCTGATTCAGCCTCTTCAGATACTCTTGTGCTGTGC AGCAGTGGCTCTGTGTGTAAATGCTATGCACTGAGGATACACAAAAATACCAATATGATGTGTACAGGAT AATGCCTCATCCCAATCAGATGTCCATTTGTTATTGTGTTTGTTAACAACCCTTTATCTCTTAGTGTTAT AAACTCCACTTAAAACTGATTAAAGTCTCATTCTTGTCATTGTGTGGGTGTTTTATTAAATGAGAGTTTA TAATTCAAATTGCTTAAGTCCATTGAAGTTTTAATTAATGGGCAGCCAAATGTGAATACAAAGTTTTCAG TTTTTTTTTTTCCTGCTGTCCTTCAAAGCCTACTGTTTAAAAAAAAAAAAAAAAAAAAACATGGCCTGAG AGTAGAGTATCTGTCTACTCATGTTTAATTAAGGAAAAACACTTATTTTTAGGGCTTTAGTCATCACTTC ATAAATTGTATAAGCACATTAAATAGCGTTCTAGTCCTGAAAAAGTCCAAGATTCTTAGAAAATTGTGCA TATTTTTATTATGACAGATGTTTGAAGATAATTCCCCAGAATGGATTTGATACTTTAGATTTCAATTTTG TGGCTTTTGTCTATTATTCTGTACTCTGCCATCAGCATATGGAAAGCTTCATTTACTCATCATGACTTGT GCCATATAAAAATTGATATTTCGGAATAGTCTAAAGGACTTTTTGTACTTGAATTTAATCATGTTGTTTC TAATATTCTTAAAAGCTTGAAGACTAAAGCATATCCTTTCAACAAAGCATAGTAAGGTAATAAGAAAGTG TAGTTTGTACAAGTGTTAAAAAAATAAAGTAGACAATGTTACAGTGGGACTTATTATTTCAAGTTTACAT TTTCTCCATGTAATTTTTTAAAAAGTAAATGAAAAAATGTGCAATAATGTAAAATATGAAGTGTATGTGT ACACACATTTTATTTTTCGGTATCTTGGGTATACGTATGGTTGAAAACTATACTGGAGTCTAAAAGTATT CTAATTTATAAGAAGACATTTTGGTGATGTTTGAAAAATAGAAATGTGCTAGTTTTGTTTTTATATCATG TCCTTTGTACGTTGTAATATGAGCTGGCTTGGTTCAGTAAATGCCATCACCATTTCCATTGAGAATTTAA AACTCACCAGTGTTTAATATGCAGGCTTCCAAAGGCTTATGAAAAAAATCAAGACCCTTAAATCTAGTTA ATTTGCTGCTAACATGAAACTCTTTGGTTCTTTTATTTTTGCCAGATAATTAGACACACATCTAAAGCTT AGTCTTAAATGGCTTAAGTGTAGCTATTGATTAGTGCTGTTGCTAGTTCAGAAAGAAATGTTTGTGAATG GAAACAAGAATATTCAGTCCAAACTGTTGTAAGGACAGTACCTGAAAACCAGGAAACAGGATAATGGAAA AAGTCTTTTAAAGATGAAATGTTGGAGCCAACTTTCTTATAGAATTAATTGTATGTGGCTATAGAAAGCC TAATGATTGTTGCTTATTTTTGAGAGCATATTATTCTTTTATGACCATAATCTTGCTGTTTTTCCATCTT CCAAAAGATCTTCCTTCTAATATGTATATCAGAATGTGGGTAGCCAGTCAGACAAATTCATATTGGTTGG TAGCTTTAAAAAGTTTGTAATGTGAAGACAGGAAAGGACAAAATAGTTTGCTTTGGTGGTAGTACTCTGG TTGTTAAGCTAGGTATTTTGAGACTACTTCCCCATCACAACAACAATAAAATAATCACTCATAATCCTAT CACCTGGAGACATAGCCATCGTTAATATGTTAGTGACTATACAATCATGTTTTCTTCTGTATATCCATGT ATATTCTTTAAAAATGAAATTTATACTGTACCTGATCTCAAAGCTTTTTAGCTTAGTATATCTGTCATGAATTTGTAGGATGTTCCATTGCATCAGAAAACGGACAGTGATTTGATTACTTTCTAATGCCACAGATGCAG ATTACATGTAGTTATTGAGAATCCTTTCGAATTCAGTGGCTTAATCATGAATGTCTAAATATTGTTGACA TTAGGATGATACATGTAAATTAAAGTTACATTTGTTTAGCATAGACAAGCTTAACATTGTAGATGTTTCT CTTCAAAAATCATCTTAAACATTTGCATTTGGAATTGTGTTAAATAGAATGTGTGAAACACTGTATTAGT AAACTTCATCACCTTTCTACTTCCTTATAGTTTGAACTTTTCAGTTTTTGTAGTTCCCAAACAGTTGCTC AATTTAGAGCAAATTAATTTAACACCTGCCAAAAAAAGGCTGCTGTTGGCTTATCAGTTGTCTTTAAATT CAAATGCTCATGTGACTTTTATCACATCAAAAAATATTTCATTAATGATTCACCTTTAGCTCTGAAAATT ACCGCGTTTAGTAATTATAGTGGGCTTATAAAAACATGCAACTCTTTTTGATAGTTATTTGAGAATTTTG GTGAAAAATATTTAGCTGAGGGCAGTATAGAACTTATAAACCAATATATTGATATTTTTAAAACATTTTT ACATATAAGTAAACTGCCATCTTTGAGCATAACTACATTTAAAAATAAAGCTGCATATTTTTAAATCAAG TGTTTAACAAGAATTTATATTTTTTATTTTTTAAAATTAAAAATAATTTATATTTCCTCTGTTGCATGAG GATTCTCATCTGTGCTTATAATGGTTAGAGATTTTATTTGTGTGGAATGAAGTGAGGCTTGTAGTCATGG TTCTAGTGTTTCAGTTTGCCAAGTCTGTTTACTGCAGTGAAATTCATCAAATGTTTCAGTGTGGTTTTCT GTAGCCTATCATTTACTGGCTATTTTTTTATGTACACCTTTAGGATTTTCTGCCTACTCTATCCAGTTGT CCAAATGATATCCTACATTTTACAAATGCCCTTTCAGTTTCTATTTTCTTTTTCCATTAAATTGCCCTCA TGTCCTAATGTGCAGTTTGTAAGTGTGTGTGTGTGTGTCTGTGTGTGTGTGAATTTGATTTTCAAGAGTG CTAGACTTCCAATTTGAGAGATTAAATAATTTAATTCAGGCAAACATTTTTCATTGGAATTTCACAGTTC ATTGTAATGAAAATGTTAATCCTGGATGACCTTTGACATACAGTAATGAATCTTGGATATTAATGAATTT GTTAGTAGCATCTTGATGTGTGTTTTAATGAGTTATTTTCAAAGTTGTGCATTAAACCAAAGTTGGCATA CTGGAAGTGTTTATATCAAGTTCCATTTGGCTACTGATGGACAAAAAATAGAAATGCCTTCCTATGGAGA GTATTTTTCCTTTAAAAAATTAAAAAGGTTAATTATTTTGACTA

[0052] NM_001304718.2 Homo sapiens phosphatase and tensin homolog (PTEN), transcript variant 3, mRNA (SEQ ID NO: 3) GTTCTCTCCTCTCGGAAGCTGCAGCCATGATGGAAGTTTGAGAGTTGAGCCGCTGTGAGGCGAGGCCGGG CTCAGGCGAGGGAGATGAGAGACGGCGGCGGCCGCGGCCCGGAGCCCCTCTCAGCGCCTGTGAGCAGCCG CGGGGGCAGCGCCCTCGGGGAGCCGGCCGGCCTGCGGCGGCGGCAGCGGCGGCGTTTCTCGCCTCCTCTT CGTCTTTTCTAACCGTGCAGCCTCTTCCTCGGCTTCTCCTGAAAGGGAAGGTGGAAGCCGTGGGCTCGGG CGGGAGCCGGCTGAGGCGCGGCGGCGGCGGCGGCACCTCCCGCTCCTGGAGCGGGGGGGAGAAGCGGCGG CGGCGGCGGCCGCGGCGGCTGCAGCTCCAGGGAGGGGGTCTGAGTCGCCTGTCACCATTTCCAGGGCTGG GAACGCCGGAGAGTTGGTCTCTCCCCTTCTACTGCCTCCAACACGGCGGCGGCGGCGGCGGCACATCCAG GGACCCGGGCCGGTTTTAAACCTCCCGTCCGCCGCCGCCGCACCCCCCGTGGCCCGGGCTCCGGAGGCCG CCGGCGGAGGCAGCCGTTCGGAGGATTATTCGTCTTCTCCCCATTCCGCTGCCGCCGCTGCCAGGCCTCT GGCTGCTGAGGAGAAGCAGGCCCAGTCGCTGCAACCATCCAGCAGCCGCCGCAGCAGCCATTACCCGGCT GCGGTCCAGAGCCAAGCGGCGGCAGAGCGAGGGGCATCAGCTACCGCCAAGTCCAGAGCCATTTCCATCC TGCAGAAGAAGCCCCGCCACCAGCAGCTTCTGCCATCTCTCTCCTCCTTTTTCTTCAGCCACAGGCTCCC AGACATGACAGCCATCATCAAAGAGATCGTTAGCAGAAACAAAAGGAGATATCAAGAGGATGGATTCGAC TTAGACTTGACCTATATTTATCCAAACATTATTGCTATGGGATTTCCTGCAGAAAGACTTGAAGGCGTAT ACAGGAACAATATTGATGATGTAGTAAGTTGTGCTGAAAGACATTATGACACCGCCAAATTTAATTGCAG AGTTGCACAATATCCTTTTGAAGACCATAACCCACCACAGCTAGAACTTATCAAACCCTTTTGTGAAGAT CTTGACCAATGGCTAAGTGAAGATGACAATCATGTTGCAGCAATTCACTGTAAAGCTGGAAAGGGACGAA CTGGTGTAATGATATGTGCATATTTATTACATCGGGGCAAATTTTTAAAGGCACAAGAGGCCCTAGATTT CTATGGGGAAGTAAGGACCAGAGACAAAAAGGCAGATCCTACAGGAGGTATTCCAGATAAAGGCATTATT GTCATAGGAGATGGCAGCTCCATGGATGTTATTGCCCCTTAAGACCTTCCAGTGGGACAAGATGTATAGG TGGAAGACAGTGATATTGATGATCCTGACCTTGTAGAGGCCAAGGCTAAAGGAGTAACTATTCCCAGTCA GAGGCGCTATGTGTATTATTATAGCTACCTGTTAAAGAATCATCTGGATTATAGACCAGTGGCACTGTTG TTTCACAAGATGATGTTTGAAACTATTCCAATGTTCAGTGGCGGAACTTGCAATCCTCAGTTTGTGGTCT GCCAGCTAAAGGTGAAGATATATTCCTCCAATTCAGGACCCACACGACGGGAAGACAAGTTCATGTACTT TGAGTTCCCTCAGCCGTTACCTGTGTGTGGTGATATCAAAGTAGAGTTCTTCCACAAACAGAACAAGATG CTAAAAAAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAG AAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAA TGACAAGGAATATCTAGTACTTACTTTAACAAAAAATGATCTTGACAAAGCAAATAAAGACAAAGCCAAC CGATACTTTTCTCCAAATTTTAAGGTGAAGCTGTACTTCACAAAAACAGTAGAGGAGCCGTCAAATCCAG AGGCTAGCAGTTCAACTTCTGTAACACCAGATGTTAGTGACAATGAACCTGATCATTATAGATATTCTGACACCACTGACTCTGATCCAGAGAATGAACCTTTTGATGAAGATCAGCATACACAAATTACAAAAGTCTGA ATTTTTTTTTATCAAGAGGGATAAAACACCATGAAAATAAACTTGAATAAACTGAAAATGGACCTTTTTT TTTTTAATGGCAATAGGACATTGTGTCAGATTACCAGTTATAGGAACAATTCTCTTTTCCTGACCAATCT TGTTTTACCCTATACATCCACAGGGTTTTGACACTTGTTGTCCAGTTGAAAAAAGGTTGTGTAGCTGTGT CATGTATATACCTTTTTGTGTCAAAAGGACATTTAAAATTCAATTAGGATTAATAAAGATGGCACTTTCC CGTTTTATTCCAGTTTTATAAAAAGTGGAGACAGACTGATGTGTATACGTAGGAATTTTTTCCTTTTGTG TTCTGTCACCAACTGAAGTGGCTAAAGAGCTTTGTGATATACTGGTTCACATCCTACCCCTTTGCACTTG TGGCAACAGATAAGTTTGCAGTTGGCTAAGAGAGGTTTCCGAAGGGTTTTGCTACATTCTAATGCATGTA TTCGGGTTAGGGGAATGGAGGGAATGCTCAGAAAGGAAATAATTTTATGCTGGACTCTGGACCATATACC ATCTCCAGCTATTTACACACACCTTTCTTTAGCATGCTACAGTTATTAATCTGGACATTCGAGGAATTGG CCGCTGTCACTGCTTGTTGTTTGCGCATTTTTTTTTAAAGCATATTGGTGCTAGAAAAGGCAGCTAAAGG AAGTGAATCTGTATTGGGGTACAGGAATGAACCTTCTGCAACATCTTAAGATCCACAAATGAAGGGATAT AAAAATAATGTCATAGGTAAGAAACACAGCAACAATGACTTAACCATATAAATGTGGAGGCTATCAACAA AGAATGGGCTTGAAACATTATAAAAATTGACAATGATTTATTAAATATGTTTTCTCAATTGTAACGACTT CTCCATCTCCTGTGTAATCAAGGCCAGTGCTAAAATTCAGATGCTGTTAGTACCTACATCAGTCAACAAC TTACACTTATTTTACTAGTTTTCAATCATAATACCTGCTGTGGATGCTTCATGTGCTGCCTGCAAGCTTC TTTTTTCTCATTAAATATAAAATATTTTGTAATGCTGCACAGAAATTTTCAATTTGAGATTCTACAGTAA GCGTTTTTTTTCTTTGAAGATTTATGATGCACTTATTCAATAGCTGTCAGCCGTTCCACCCTTTTGACCT TACACATTCTATTACAATGAATTTTGCAGTTTTGCACATTTTTTAAATGTCATTAACTGTTAGGGAATTT TACTTGAATACTGAATACATATAATGTTTATATTAAAAAGGACATTTGTGTTAAAAAGGAAATTAGAGTT GCAGTAAACTTTCAATGCTGCACACAAAAAAAAGACATTTGATTTTTCAGTAGAAATTGTCCTACATGTG CTTTATTGATTTGCTATTGAAAGAATAGGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAAATGTGCAGT GTTGAATCATTTCTTCATAGTGCTCCCCCGAGTTGGGACTAGGGCTTCAATTTCACTTCTTAAAAAAAAT CATCATATATTTGATATGCCCAGACTGCATACGATTTTAAGCGGAGTACAACTACTATTGTAAAGCTAAT GTGAAGATATTATTAAAAAGGTTTTTTTTTCCAGAAATTTGGTGTCTTCAAATTATACCTTCACCTTGAC ATTTGAATATCCAGCCATTTTGTTTCTTAATGGTATAAAATTCCATTTTCAATAACTTATTGGTGCTGAA ATTGTTCACTAGCTGTGGTCTGACCTAGTTAATTTACAAATACAGATTGAATAGGACCTACTAGAGCAGC ATTTATAGAGTTTGATGGCAAATAGATTAGGCAGAACTTCATCTAAAATATTCTTAGTAAATAATGTTGA CACGTTTTCCATACCTTGTCAGTTTCATTCAACAATTTTTAAATTTTTAACAAAGCTCTTAGGATTTACA CATTTATATTTAAACATTGATATATAGAGTATTGATTGATTGCTCATAAGTTAAATTGGTAAAGTTAGAG ACAACTATTCTAACACCTCACCATTGAAATTTATATGCCACCTTGTCTTTCATAAAAGCTGAAAATTGTT ACCTAAAATGAAAATCAACTTCATGTTTTGAAGATAGTTATAAATATTGTTCTTTGTTACAATTTCGGGC ACCGCATATTAAAACGTAACTTTATTGTTCCAATATGTAACATGGAGGGCCAGGTCATAAATAATGACAT TATAATGGGCTTTTGCACTGTTATTATTTTTCCTTTGGAATGTGAAGGTCTGAATGAGGGTTTTGATTTT GAATGTTTCAATGTTTTTGAGAAGCCTTGCTTACATTTTATGGTGTAGTCATTGGAAATGGAAAAATGGC ATTATATATATTATATATATAAATATATATTATACATACTCTCCTTACTTTATTTCAGTTACCATCCCCA TAGAATTTGACAAGAATTGCTATGACTGAAAGGTTTTCGAGTCCTAATTAAAACTTTATTTATGGCAGTA TTCATAATTAGCCTGAAATGCATTCTGTAGGTAATCTCTGAGTTTCTGGAATATTTTCTTAGACTTTTTG GATGTGCAGCAGCTTACATGTCTGAAGTTACTTGAAGGCATCACTTTTAAGAAAGCTTACAGTTGGGCCC TGTACCATCCCAAGTCCTTTGTAGCTCCTCTTGAACATGTTTGCCATACTTTTAAAAGGGTAGTTGAATA AATAGCATCACCATTCTTTGCTGTGGCACAGGTTATAAACTTAAGTGGAGTTTACCGGCAGCATCAAATG TTTCAGCTTTAAAAAATAAAAGTAGGGTACAAGTTTAATGTTTAGTTCTAGAAATTTTGTGCAATATGTT CATAACGATGGCTGTGGTTGCCACAAAGTGCCTCGTTTACCTTTAAATACTGTTAATGTGTCATGCATGC AGATGGAAGGGGTGGAACTGTGCACTAAAGTGGGGGCTTTAACTGTAGTATTTGGCAGAGTTGCCTTCTA CCTGCCAGTTCAAAAGTTCAACCTGTTTTCATATAGAATATATATACTAAAAAATTTCAGTCTGTTAAAC AGCCTTACTCTGATTCAGCCTCTTCAGATACTCTTGTGCTGTGCAGCAGTGGCTCTGTGTGTAAATGCTA TGCACTGAGGATACACAAAAATACCAATATGATGTGTACAGGATAATGCCTCATCCCAATCAGATGTCCA TTTGTTATTGTGTTTGTTAACAACCCTTTATCTCTTAGTGTTATAAACTCCACTTAAAACTGATTAAAGT CTCATTCTTGTCATTGTGTGGGTGTTTTATTAAATGAGAGTTTATAATTCAAATTGCTTAAGTCCATTGA AGTTTTAATTAATGGGCAGCCAAATGTGAATACAAAGTTTTCAGTTTTTTTTTTTCCTGCTGTCCTTCAA AGCCTACTGTTTAAAAAAAAAAAAAAAAAAAAACATGGCCTGAGAGTAGAGTATCTGTCTACTCATGTTT AATTAAGGAAAAACACTTATTTTTAGGGCTTTAGTCATCACTTCATAAATTGTATAAGCACATTAAATAG CGTTCTAGTCCTGAAAAAGTCCAAGATTCTTAGAAAATTGTGCATATTTTTATTATGACAGATGTTTGAA GATAATTCCCCAGAATGGATTTGATACTTTAGATTTCAATTTTGTGGCTTTTGTCTATTATTCTGTACTC TGCCATCAGCATATGGAAAGCTTCATTTACTCATCATGACTTGTGCCATATAAAAATTGATATTTCGGAA TAGTCTAAAGGACTTTTTGTACTTGAATTTAATCATGTTGTTTCTAATATTCTTAAAAGCTTGAAGACTA AAGCATATCCTTTCAACAAAGCATAGTAAGGTAATAAGAAAGTGTAGTTTGTACAAGTGTTAAAAAAATAAAGTAGACAATGTTACAGTGGGACTTATTATTTCAAGTTTACATTTTCTCCATGTAATTTTTTAAAAAGT AAATGAAAAAATGTGCAATAATGTAAAATATGAAGTGTATGTGTACACACATTTTATTTTTCGGTATCTT GGGTATACGTATGGTTGAAAACTATACTGGAGTCTAAAAGTATTCTAATTTATAAGAAGACATTTTGGTG ATGTTTGAAAAATAGAAATGTGCTAGTTTTGTTTTTATATCATGTCCTTTGTACGTTGTAATATGAGCTG GCTTGGTTCAGTAAATGCCATCACCATTTCCATTGAGAATTTAAAACTCACCAGTGTTTAATATGCAGGC TTCCAAAGGCTTATGAAAAAAATCAAGACCCTTAAATCTAGTTAATTTGCTGCTAACATGAAACTCTTTG GTTCTTTTATTTTTGCCAGATAATTAGACACACATCTAAAGCTTAGTCTTAAATGGCTTAAGTGTAGCTA TTGATTAGTGCTGTTGCTAGTTCAGAAAGAAATGTTTGTGAATGGAAACAAGAATATTCAGTCCAAACTG TTGTAAGGACAGTACCTGAAAACCAGGAAACAGGATAATGGAAAAAGTCTTTTAAAGATGAAATGTTGGA GCCAACTTTCTTATAGAATTAATTGTATGTGGCTATAGAAAGCCTAATGATTGTTGCTTATTTTTGAGAG CATATTATTCTTTTATGACCATAATCTTGCTGTTTTTCCATCTTCCAAAAGATCTTCCTTCTAATATGTA TATCAGAATGTGGGTAGCCAGTCAGACAAATTCATATTGGTTGGTAGCTTTAAAAAGTTTGTAATGTGAA GACAGGAAAGGACAAAATAGTTTGCTTTGGTGGTAGTACTCTGGTTGTTAAGCTAGGTATTTTGAGACTA CTTCCCCATCACAACAACAATAAAATAATCACTCATAATCCTATCACCTGGAGACATAGCCATCGTTAAT ATGTTAGTGACTATACAATCATGTTTTCTTCTGTATATCCATGTATATTCTTTAAAAATGAAATTTATAC TGTACCTGATCTCAAAGCTTTTTAGCTTAGTATATCTGTCATGAATTTGTAGGATGTTCCATTGCATCAG AAAACGGACAGTGATTTGATTACTTTCTAATGCCACAGATGCAGATTACATGTAGTTATTGAGAATCCTT TCGAATTCAGTGGCTTAATCATGAATGTCTAAATATTGTTGACATTAGGATGATACATGTAAATTAAAGT TACATTTGTTTAGCATAGACAAGCTTAACATTGTAGATGTTTCTCTTCAAAAATCATCTTAAACATTTGC ATTTGGAATTGTGTTAAATAGAATGTGTGAAACACTGTATTAGTAAACTTCATCACCTTTCTACTTCCTT ATAGTTTGAACTTTTCAGTTTTTGTAGTTCCCAAACAGTTGCTCAATTTAGAGCAAATTAATTTAACACC TGCCAAAAAAAGGCTGCTGTTGGCTTATCAGTTGTCTTTAAATTCAAATGCTCATGTGACTTTTATCACA TCAAAAAATATTTCATTAATGATTCACCTTTAGCTCTGAAAATTACCGCGTTTAGTAATTATAGTGGGCT TATAAAAACATGCAACTCTTTTTGATAGTTATTTGAGAATTTTGGTGAAAAATATTTAGCTGAGGGCAGT ATAGAACTTATAAACCAATATATTGATATTTTTAAAACATTTTTACATATAAGTAAACTGCCATCTTTGA GCATAACTACATTTAAAAATAAAGCTGCATATTTTTAAATCAAGTGTTTAACAAGAATTTATATTTTTTA TTTTTTAAAATTAAAAATAATTTATATTTCCTCTGTTGCATGAGGATTCTCATCTGTGCTTATAATGGTT AGAGATTTTATTTGTGTGGAATGAAGTGAGGCTTGTAGTCATGGTTCTAGTGTTTCAGTTTGCCAAGTCT GTTTACTGCAGTGAAATTCATCAAATGTTTCAGTGTGGTTTTCTGTAGCCTATCATTTACTGGCTATTTT TTTATGTACACCTTTAGGATTTTCTGCCTACTCTATCCAGTTGTCCAAATGATATCCTACATTTTACAAA TGCCCTTTCAGTTTCTATTTTCTTTTTCCATTAAATTGCCCTCATGTCCTAATGTGCAGTTTGTAAGTGT GTGTGTGTGTGTCTGTGTGTGTGTGAATTTGATTTTCAAGAGTGCTAGACTTCCAATTTGAGAGATTAAA TAATTTAATTCAGGCAAACATTTTTCATTGGAATTTCACAGTTCATTGTAATGAAAATGTTAATCCTGGA TGACCTTTGACATACAGTAATGAATCTTGGATATTAATGAATTTGTTAGTAGCATCTTGATGTGTGTTTT AATGAGTTATTTTCAAAGTTGTGCATTAAACCAAAGTTGGCATACTGGAAGTGTTTATATCAAGTTCCAT TTGGCTACTGATGGACAAAAAATAGAAATGCCTTCCTATGGAGAGTATTTTTCCTTTAAAAAATTAAAAA GGTTAATTATTTTGACTA

[0053] In one aspect, the present disclosure provides PTEN-specific inhibitory nucleic acids comprising a nucleic acid molecule which is complementary to a portion of a PTEN nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3.

[0054] The present disclosure also provides an antisense nucleic acid comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 1-3 (PTEN mRNA), thereby reducing or inhibiting PTEN expression. The antisense nucleic acid may be antisense RNA, or antisense DNA. Antisense nucleic acids based on the known PTEN gene sequence can be readily designed and engineered using methods known in the art.

[0055] Antisense nucleic acids are molecules which are complementary to a sense nucleic acid strand, e.g., complementary to the coding strand of a double-stranded DNA molecule (or cDNA) or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can form hydrogen bonds with a sense nucleic acid. The antisense nucleic acid can be complementary to an entire PTEN coding strand, or to a portion thereof, e.g., all or part of the protein coding region (or open reading frame). In some embodiments, the antisense nucleic acid is an oligonucleotide which is complementary to only a portion of the coding region of PTEN mRNA. In certain embodiments, an antisense nucleic acid molecule can be complementary to a noncoding region of the PTEN coding strand. In some embodiments, the noncoding region refers to the 5′ and 3′ untranslated regions that flank the coding region and are not translated into amino acids. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of PTEN. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.

[0056] An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- hodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5- carboxymethylaminomethyl-2-thouridine, 5-carboxymethylaminometh-yluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-metnylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopenten-yladenine, uracil-5-oxyacetic acid (v), wybutosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2- thiouracil, 2-thlouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester,uracil-5-cxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).

[0057] The antisense nucleic acid molecules may be administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and / or genomic DNA encoding the protein of interest to thereby inhibit expression of the protein, e.g., by inhibiting transcription and / or translation. The hybridization can occur via Watson-Crick base pairing to form a stable duplex, or in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.

[0058] In some embodiments, the antisense nucleic acid molecules are modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. In some embodiments, the antisense nucleic acid molecule is an alpha-anomeric nucleic acid molecule. An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al., Nucleic Acids. Res.15:6625- 6641(1987)). The antisense nucleic acid molecule can also comprise a 2′-O - methylribonucleotide (Inoue et al., Nucleic Acids Res.15:6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

[0059] The present disclosure also provides a short hairpin RNA (shRNA) or small interfering RNA (siRNA) comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 1-3 (PTEN mRNA), thereby reducing or inhibiting PTEN expression. In some embodiments, the shRNA or siRNA is about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 base pairs in length. Double- stranded RNA (dsRNA) can induce sequence-specific post-transcriptional gene silencing (e.g., RNA interference (RNAi)) in many organisms such as C. elegans, Drosophila, plants, mammals, oocytes and early embryos. RNAi is a process that interferes with or significantly reduces the number of protein copies made by an mRNA. For example, adouble-stranded siRNA or shRNA molecule is engineered to complement and hydridize to a mRNA of a target gene. Following intracellular delivery, the siRNA or shRNA molecule associates with an RNA-induced silencing complex (RISC), which then binds and degrades a complementary target mRNA (such as PTEN mRNA).

[0060] The present disclosure also provides a ribozyme comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 1-3 (PTEN mRNA), thereby reducing or inhibiting PTEN expression. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a complementary single-stranded nucleic acid, such as an mRNA. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach, Nature 334:585-591 (1988))) can be used to catalytically cleave PTEN transcripts, thereby inhibiting translation of PTEN.

[0061] A ribozyme having specificity for a PTEN-encoding nucleic acid can be designed based upon a PTEN nucleic acid sequence disclosed herein. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a PTEN-encoding mRNA. See, e.g., U.S. Pat. No.4,987,071 and U.S. Pat. No.5,116,742. Alternatively, PTEN mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261:1411-1418, incorporated herein by reference.

[0062] The present disclosure also provides a synthetic guide RNA (sgRNA) comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 1-3 (PTEN mRNA). Guide RNAs for use in CRISPR-Cas systems are typically generated as a single guide RNA comprising a crRNA segment and a tracrRNA segment. The crRNA segment and a tracrRNA segment can also be generated as separate RNA molecules. The crRNA segment comprises the targeting sequence that binds to a portion of any one of SEQ ID NOs: 1-3, and a stem portion that hybridizes to a tracrRNA. The tracrRNA segment comprises a nucleotide sequence that is partially or completely complementary to the stem sequence of the crRNA and a nucleotide sequence that binds to the CRISPR enzyme. In some embodiments, the crRNA segment and the tracrRNA segment are provided as a single guide RNA. In some embodiments, thecrRNA segment and the tracrRNA segment are provided as separate RNAs. The combination of the CRISPR enzyme with the crRNA and tracrRNA make up a functional CRISPR-Cas system. Exemplary CRISPR-Cas systems for targeting nucleic acids, are described, for example, in WO2015 / 089465.

[0063] In some embodiments, a synthetic guide RNA is a single RNA represented as comprising the following elements: 5ʹ-X1-X2-Y-Z-3ʹ where X1 and X2 represent the crRNA segment, where X1 is the targeting sequence that binds to a portion of any one of SEQ ID NOs: 1-3, X2 is a stem sequence the hybridizes to a tracrRNA, Z represents a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to X2, and Y represents a linker sequence. In some embodiments, the linker sequence comprises two or more nucleotides and links the crRNA and tracrRNA segments. In some embodiments, the linker sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. In some embodiments, the linker is the loop of the hairpin structure formed when the stem sequence hybridized with the tracrRNA.

[0064] In some embodiments, a synthetic guide RNA is provided as two separate RNAs where one RNA represents a crRNA segment: 5ʹ-X1-X2-3ʹ where X1 is the targeting sequence that binds to a portion of any one of SEQ ID NOs: 1-3, X2 is a stem sequence the hybridizes to a tracrRNA, and one RNA represents a tracrRNA segment, Z, that is a separate RNA from the crRNA segment and comprises a nucleotide sequence that is partially or completely complementary to X2 of the crRNA.

[0065] Exemplary crRNA stem sequences and tracrRNA sequences are provided, for example, in WO / 2015 / 089465, which is incorporated by reference herein. In general, a stem sequence includes any sequence that has sufficient complementarity with a complementary sequence in the tracrRNA to promote formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the stem sequence hybridized to the tracrRNA. In general, degree of complementarity is with reference to the optimal alignment of the stem and complementary sequence in the tracrRNA, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self- complementarity within either the stem sequence or the complementary sequence in thetracrRNA. In some embodiments, the degree of complementarity between the stem sequence and the complementary sequence in the tracrRNA along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the stem sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the stem sequence and complementary sequence in the tracrRNA are contained within a single RNA, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. In some embodiments, the tracrRNA has additional complementary sequences that form hairpins. In some embodiments, the tracrRNA has at least two or more hairpins. In some embodiments, the tracrRNA has two, three, four or five hairpins. In some embodiments, the tracrRNA has at most five hairpins.

[0066] In a hairpin structure, the portion of the sequence 5ʹ of the final “N” and upstream of the loop corresponds to the crRNA stem sequence, and the portion of the sequence 3ʹ of the loop corresponds to the tracrRNA sequence. Further non-limiting examples of single polynucleotides comprising a guide sequence, a stem sequence, and a tracr sequence are as follows (listed 5ʹ to 3ʹ), where “N” represents a base of a guide sequence (e.g. a modified oligonucleotide provided herein), the first block of lower case letters represent stem sequence, and the second block of lower case letters represent the tracrRNA sequence, and the final poly-T sequence represents the transcription terminator: (a) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataa ggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 4); (b) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccg aaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 5); (c) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccg aaatcaacaccctgtcattttatggcagggtgtTTTTTT (SEQ ID NO: 6); (d) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaactt gaaaaagtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 7); (e) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaac ttgaaaaagtgTTTTTTT (SEQ ID NO: 8); and (f)NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTT TTTTTT (SEQ ID NO: 9).

[0067] Selection of suitable oligonucleotides for use in as a targeting sequence in a CRISPR Cas system depends on several factors including the particular CRISPR enzyme to be used and the presence of corresponding proto-spacer adjacent motifs (PAMs) downstream of the target sequence in the target nucleic acid. The PAM sequences direct the cleavage of the target nucleic acid by the CRISPR enzyme. In some embodiments, a suitable PAM is 5'- NRG or 5'-NNGRR (where N is any Nucleotide) for SpCas9 or SaCas9 enzymes (or derived enzymes), respectively. Generally the PAM sequences should be present between about 1 to about 10 nucleotides of the target sequence to generate efficient cleavage of the target nucleic acid. Thus, when the guide RNA forms a complex with the CRISPR enzyme, the complex locates the target and PAM sequence, unwinds the DNA duplex, and the guide RNA anneals to the complementary sequence on the opposite strand. This enables the Cas9 nuclease to create a double-strand break.

[0068] A variety of CRISPR enzymes are available for use in conjunction with the disclosed guide RNAs of the present disclosure. In some embodiments, the CRISPR enzyme is a Type II CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzes DNA cleavage. In some embodiments, the CRISPR enzyme catalyzes RNA cleavage. In some embodiments, the CRISPR enzyme is any Cas9 protein, for instance any naturally-occurring bacterial Cas9 as well as any chimeras, mutants, homologs or orthologs. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified variants thereof. In some embodiments, the CRISPR enzyme cleaves both strands of the target nucleic acid at the Protospacer Adjacent Motif (PAM) site. In some embodiments, the CRISPR enzyme is a nickase, which cleaves only one strand of the target nucleic acid.

[0069]

[0070] Examples of small molecule PTEN inhibitors include, but are not limited to hydroxyl(oxo)vanadium 3-hydroxypiridine-2-carboxylic acid (VO-OHpic), bisperoxovanadium 1,10-phenantroline (bpV(phen)), bisperoxovanadium 5-hydroxipyridine (bpV(pic)), bisperoxovanadium 5-hydroxipyridine-2-carboxylic acid bpV(HOpic), bisperoxovanadium pyridin-2-squaramide (bpV(pis)), or N-(9,10-dioxo-9,10- dihydrophenanthren-2-yl) pivalamide (SF1670).

[0071] Additionally or alternatively, in some embodiments of the methods disclosed herein, the agent is a small molecule mTORC1 inhibitor, or a mTORC1-specific inhibitory nucleic acid. The mTORC1-specific inhibitory nucleic acid may be a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA. Examples of small molecule mTORC1 inhibitor include, but are not limited to rapamycin, everolimus, temsirolimus, INK128, OSI027, PP242, RapaLinks, Ku-0063794, PP30, XL 388, WYE-354, WAY-600, WYE-687, Torin-1, Torin-2, RMC-6272, or AZD8055.

[0072] In therapeutic applications, compositions or medicaments comprising an agent that inhibits PTEN expression and / or activity as disclosed herein are administered to a subject suspected of, or already suffering from Type 2 diabetes, in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease, including its complications and intermediate pathological phenotypes in development of the disease.

[0073] Subjects diagnosed with Type 2 diabetes can be identified by any or a combination of diagnostic or prognostic assays known in the art. For example, typical symptoms of Type 2 diabetes include, but are not limited to, frequent urination, excess thirst, fatigue, unexplained weight loss, itchiness around the genital area, regular bouts of thrush (a yeast infection), cuts or wounds that heal slowly and blurred vision. Oral glucose tolerance tests (OGTT) are used to measure how well the body can process a larger amount of sugar and serve to determine risk of diabetes in a subject.

[0074] In some embodiments, subjects suffering from Type 2 diabetes that are treated with an agent that inhibits PTEN expression and / or activity will show amelioration or elimination of one or more of the following symptoms: frequent urination, excess thirst, fatigue, unexplained weight loss, itchiness around the genital area, regular bouts of thrush (a yeast infection), cuts or wounds that heal slowly and blurred vision.

[0075] Subjects at risk or susceptible to Type 2 diabetes include those who are obese, have a family history of diabetes, a sedentary lifestyle, or are of African American, American Indian, Asian American, Hispanic / Latino, or Pacific Islander. Such subjects can be identified by, e.g., any or a combination of diagnostic or prognostic assays known in the art.

[0076] In prophylactic applications, pharmaceutical compositions or medicaments comprising an agent that inhibits PTEN expression and / or activity as disclosed herein are administered to a subject susceptible to, or otherwise at risk of Type 2 diabetes, in an amount sufficient to eliminate or reduce the risk, or delay the onset of the disease, including biochemical, histologic and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.

[0077] Prophylactic administration of an agent that inhibits PTEN expression and / or activity can occur prior to the manifestation of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression.

[0078] In some embodiments, treatment with the agent that inhibits PTEN expression and / or activity will prevent or delay the onset of one or more of the following symptoms: frequent urination, excess thirst, fatigue, unexplained weight loss, itchiness around the genital area, regular bouts of thrush (a yeast infection), cuts or wounds that heal slowly and blurred vision.

[0079] In one aspect, the present disclosure provides a method for selecting a patient suffering from or at risk for Type 2 diabetes for treatment comprising (a) detecting PTEN polypeptide expression and / or activity levels in epididymal white adipose fat, liver and / or muscle of the subject that are elevated compared to a control subject or a predetermined threshold; and (b) administering to the patient an effective amount of an agent that inhibits PTEN expression and / or activity. In any of the preceding embodiments of the methods disclosed herein, the agent is a small molecule PTEN inhibitor, or a PTEN-specific inhibitory nucleic acid. The PTEN-specific inhibitory nucleic acid may be a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA. Examples of small molecule PTEN inhibitors include, but are not limited to hydroxyl(oxo)vanadium 3-hydroxypiridine-2-carboxylic acid (VO-OHpic), bisperoxovanadium 1,10-phenantroline (bpV(phen)), bisperoxovanadium 5-hydroxipyridine (bpV(pic)), bisperoxovanadium 5-hydroxipyridine-2- carboxylic acid bpV(HOpic), bisperoxovanadium pyridin-2-squaramide (bpV(pis)), or N- (9,10-dioxo-9,10-dihydrophenanthren-2-yl) pivalamide (SF1670). Additionally or alternatively, in some embodiments of the methods disclosed herein, the agent is a small molecule mTORC1 inhibitor, or a mTORC1-specific inhibitory nucleic acid. The mTORC1-specific inhibitory nucleic acid may be a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA. Examples of small molecule mTORC1 inhibitor include, but are not limited to rapamycin, everolimus, temsirolimus, INK128, OSI027, PP242, RapaLinks, Ku-0063794, PP30, XL 388, WYE-354, WAY-600, WYE-687, Torin-1, Torin- 2, RMC-6272, or AZD8055.

[0080] For therapeutic and / or prophylactic applications, a composition comprising an agent that inhibits PTEN expression and / or activity as disclosed herein, is administered to the subject. In some embodiments, the agent that inhibits PTEN expression and / or activity is administered one, two, three, four, or five times per day. In some embodiments, the agent that inhibits PTEN expression and / or activity is administered more than five times per day. Additionally or alternatively, in some embodiments, the agent that inhibits PTEN expression and / or activity is administered every day, every other day, every third day, every fourth day, every fifth day, or every sixth day. In some embodiments, the agent that inhibits PTEN expression and / or activity is administered weekly, bi-weekly, tri-weekly, or monthly. In some embodiments, the agent that inhibits PTEN expression and / or activity is administered for a period of one, two, three, four, or five weeks. In some embodiments, the agent that inhibits PTEN expression and / or activity is administered for six weeks or more. In some embodiments, the agent that inhibits PTEN expression and / or activity is administered for twelve weeks or more. In some embodiments, the agent that inhibits PTEN expression and / or activity is administered for a period of less than one year. In some embodiments, the agent that inhibits PTEN expression and / or activity is administered for a period of more than one year. In some embodiments, the agent that inhibits PTEN expression and / or activity is administered throughout the subject’s life.

[0081] In some embodiments of the methods of the present technology, the agent that inhibits PTEN expression and / or activity is administered daily for 1 week or more. In someembodiments of the methods of the present technology, the agent that inhibits PTEN expression and / or activity is administered daily for 2 weeks or more. In some embodiments of the methods of the present technology, the agent that inhibits PTEN expression and / or activity is administered daily for 3 weeks or more. In some embodiments of the methods of the present technology, the agent that inhibits PTEN expression and / or activity is administered daily for 4 weeks or more. In some embodiments of the methods of the present technology, the agent that inhibits PTEN expression and / or activity is administered daily for 6 weeks or more. In some embodiments of the methods of the present technology, the agent that inhibits PTEN expression and / or activity is administered daily for 12 weeks or more. In some embodiments, the agent that inhibits PTEN expression and / or activity is administered daily throughout the subject’s life.Determination of the Biological Effect of Agents that Inhibit PTEN Expression and / or Activity

[0082] In various embodiments, suitable in vitro or in vivo assays are performed to determine the effect of a specific agent that inhibits PTEN expression and / or activity and whether its administration is indicated for treatment. In various embodiments, in vitro assays can be performed with representative animal models, to determine if a given agent that inhibits PTEN expression and / or activity exerts the desired effect on reducing or eliminating signs and / or symptoms of Type 2 diabetes. Compounds for use in therapy can be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model system known in the art can be used prior to administration to human subjects. In some embodiments, in vitro or in vivo testing is directed to the biological function of one or more agents that inhibit PTEN expression and / or activity.

[0083] Animal models of Type 2 diabetes may be generated using techniques known in the art. Such models may be used to demonstrate the biological effect of agents that inhibit PTEN expression and / or activity in the prevention and treatment of Type 2 diabetes, and for determining what comprises a therapeutically effective amount of the one or more agents that inhibit PTEN expression and / or activity as disclosed herein in a given context.Modes of Administration and Effective Dosages

[0084] Any method known to those in the art for contacting a cell, organ or tissue with one or more agents that inhibit PTEN expression and / or activity as disclosed herein may be employed. Suitable methods include in vitro, ex vivo, or in vivo methods. In vivo methods typically include the administration of one or more agents that inhibit PTEN expression and / or activity to a mammal, suitably a human. When used in vivo for therapy, the one or more agents that inhibit PTEN expression and / or activity as described herein are administered to the subject in effective amounts (i.e., amounts that have desired therapeutic effect). The dose and dosage regimen will depend upon the degree of the disease state of the subject, the characteristics of the particular agent used, e.g., its therapeutic index, and the subject’s history.

[0085] The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. An effective amount of one or more agents that inhibit PTEN expression and / or activity useful in the methods may be administered to a mammal in need thereof by any of a number of well-known methods for administering pharmaceutical compounds. The agents that inhibit PTEN expression and / or activity may be administered systemically or locally.

[0086] The one or more agents that inhibit PTEN expression and / or activity as described herein can be incorporated into pharmaceutical compositions for administration, singly or in combination, to a subject for the treatment or prevention of Type 2 diabetes. Such compositions typically include the active agent and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0087] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. For convenience of the patient or treating physician, the dosing formulation can be provided in a kit containing all necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g., 7 days of treatment).

[0088] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0089] The pharmaceutical compositions having one or more agents that inhibit PTEN expression and / or activity as disclosed herein can include a carrier, which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like. Glutathione and other antioxidants can be included to prevent oxidation. In many cases, it will be advantageous to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0090] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0091] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0092] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.

[0093] Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In one embodiment, transdermal administration may be performed by iontophoresis.

[0094] A therapeutic agent can be formulated in a carrier system. The carrier can be a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle. In one embodiment, the therapeutic agent is encapsulated in a liposome while maintaining the agent’s structural integrity. One skilled in the art would appreciate that there are a variety of methods to prepare liposomes. (See Lichtenberg, et al., Methods Biochem. Anal., 33:337-462 (1988); Anselem, et al., Liposome Technology, CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)). An active agent can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems.

[0095] The carrier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix. In one embodiment, the therapeutic agent can be embedded in the polymer matrix, while maintaining the agent’s structural integrity. The polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly α-hydroxy acids. Examples include carriers made of, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is poly-lactic acid (PLA) or copoly lactic / glycolic acid (PGLA). The polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)).A polymer formulation for human growth hormone (hGH) has been used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).

[0096] Examples of polymer microsphere sustained release formulations are described in PCT publication WO 99 / 15154 (Tracy, et al.), U.S. Pat. Nos.5,674,534 and 5,716,644 (both to Zale, et al.), PCT publication WO 96 / 40073 (Zale, et al.), and PCT publication WO 00 / 38651 (Shah, et al.). U.S. Pat. Nos.5,674,534 and 5,716,644 and PCT publication WO 96 / 40073 describe a polymeric matrix containing particles of erythropoietin that are stabilized against aggregation with a salt.

[0097] In some embodiments, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using known techniques. The materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0098] The therapeutic compounds can also be formulated to enhance intracellular delivery. For example, liposomal delivery systems are known in the art, see, e.g., Chonn and Cullis, “Recent Advances in Liposome Drug Delivery Systems,” Current Opinion in Biotechnology 6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: Selecting Manufacture and Development Processes,” Immunomethods, 4(3):201-9 (1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery: Progress and Problems,” Trends Biotechnol., 13(12):527-37 (1995). Mizguchi, et al., Cancer Lett., 100:63-69 (1996), describes the use of fusogenic liposomes to deliver a protein to cells both in vivo and in vitro.

[0099] Dosage, toxicity and therapeutic efficacy of any therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are advantageous. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0100] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to determine useful doses in humans accurately. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0101] Typically, an effective amount of the one or more agents that inhibit PTEN expression and / or activity as disclosed herein sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Suitably, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example dosages can be 1 mg / kg body weight or 10 mg / kg body weight every day, every two days or every three days or within the range of 1-10 mg / kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of the therapeutic compound ranges from 0.001-10,000 micrograms per kg body weight. In one embodiment, one or more agent concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced orterminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0102] In some embodiments, a therapeutically effective amount of one or more agents that inhibit PTEN expression and / or activity may be defined as a concentration of inhibitor at the target tissue of 10-32to 10-6molar, e.g., approximately 10-7molar. This concentration may be delivered by systemic doses of 0.001 to 100 mg / kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, such as by single daily or weekly administration, but also including continuous administration (e.g., parenteral infusion or transdermal application).

[0103] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.

[0104] The mammal treated in accordance with the present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits. In some embodiments, the mammal is a human. Combination Therapy

[0105] In some embodiments, the agents that inhibit PTEN expression and / or activity may be combined with one or more additional therapies for the prevention or treatment of Type 2 diabetes. Additional therapeutic agents include, but are not limited to, insulin sensitizers (e.g., biguanides (e.g., metformin) and glitazones (e.g., rosiglitazone and pioglitazone)), DPP-4 inhibitors (e.g., sitagliptin, saxagliptin, and linagliptin), GLP-1 receptor agonists (e.g., exenatide, liraglutide and semaglutide), and SGLT2 inhibitors (e.g., canagliflozin, dapagliflozin and empagliflozin).

[0106] In some embodiments, the one or more agents that inhibit PTEN expression and / or activity disclosed herein may be separately, sequentially or simultaneously administered with at least one additional therapeutic agent selected from the groupconsisting of insulin sensitizers (e.g., biguanides (e.g., metformin) and glitazones (e.g., rosiglitazone and pioglitazone)), DPP-4 inhibitors (e.g., sitagliptin, saxagliptin, and linagliptin), GLP-1 receptor agonists (e.g., exenatide, liraglutide and semaglutide), and SGLT2 inhibitors (e.g., canagliflozin, dapagliflozin and empagliflozin).

[0107] In certain embodiments, an additional therapeutic agent is administered to a subject in combination with the one or more agents that inhibit PTEN expression and / or activity such that a synergistic therapeutic effect is produced. For example, administration of one or more agents that inhibit PTEN expression and / or activity with one or more additional therapeutic agents for the prevention or treatment of Type 2 diabetes will have greater than additive effects in the prevention or treatment of the disease. For example, lower doses of one or more of the therapeutic agents may be used in treating or preventing Type 2 diabetes resulting in increased therapeutic efficacy and decreased side-effects. In some embodiments, the one or more agents that inhibit PTEN expression and / or activity are administered in combination with any of the at least one additional therapeutic agents described above, such that a synergistic effect in the prevention or treatment of Type 2 diabetes results.

[0108] In any case, the multiple therapeutic agents may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing between the multiple doses may vary from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents. Kits

[0109] The present disclosure also provides kits for the prevention and / or treatment of Type 2 diabetes or symptoms thereof (e.g., insulin resistance, hyperglycemia, and / or hyperinsulinemia) comprising one or more agents that inhibit PTEN expression and / or activity (e.g., PTEN-specific inhibitory nucleic acids or small molecule PTEN inhibitors) disclosed herein. Optionally, the above described components of the kits of the present technology are packed in suitable containers and labeled for the prevention and / or treatmentof Type 2 diabetes or symptoms thereof (e.g., insulin resistance, hyperglycemia, and / or hyperinsulinemia). In some embodiments, the agents that inhibit PTEN expression and / or activity are direct or indirect inhibitors of PTEN expression and / or activity

[0110] The above-mentioned components may be stored in unit or multi-dose containers, for example, sealed ampoules, vials, bottles, syringes, and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution. The kit may further comprise a second container which holds a diluent suitable for diluting the pharmaceutical composition towards a higher volume. Suitable diluents include, but are not limited to, the pharmaceutically acceptable excipient of the pharmaceutical composition and a saline solution. Furthermore, the kit may comprise instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition, whether diluted or not. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper which may be pierced by a hypodermic injection needle). The kit may further comprise more containers comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable hosts. The kits may optionally include instructions customarily included in commercial packages of therapeutic or diagnostic products, that contain information about, for example, the indications, usage, dosage, manufacture, administration, contraindications and / or warnings concerning the use of such therapeutic or diagnostic products.

[0111] The kit can also comprise, e.g., a buffering agent, a preservative or a stabilizing agent. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. The kits of the present technology may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit. Incertain embodiments, the use of the reagents can be according to the methods of the present technology. EXAMPLES

[0112] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way. Example 1: Materials and Methods

[0113] Animal studies-weight measurements and blood parameters: For the western diet induced obesity and insulin resistance phenotypes, C57BL / 6J mice at 8 weeks of age were placed on either a standard laboratory rodent chow or western diet (D12079B, Research Diets) and allowed to eat ad libitum and their weights measured as indicated. Mice were allowed to eat ad libitum and food withdrawn for 3 hours and blood serum was collected and measured for glucose, cholesterol and liver function tests using chemical analyzers and insulin and leptin using ELISA at the indicated times.

[0114] Glucose tolerance test: Mice were fasted for 6 hours, and glucose (2g / kg) was intraperitoneally injected into the mice and blood collected from the tail vein and glucose concentrations were determined at the 30 minutes, 1 hr and 2 hrs.

[0115] Tissue collection and western blotting: Mice were allowed to eat ad libitum and food withdrawn for 3 hours and epididymal white adipose tissue, liver, leg and arm muscles were collected from mice at the end of each time point (as indicated) and flash frozen. They were homogenized in SDS lysis buffer (50mM Tris-HCL pH 7.4, 10% Glycerol, 2% SDS) and boiled at 95 C for five minutes. Lysates were then briefly sonicated, boiled again for 5 minutes, before clearing by centrifugation at 14,000rpm for 10 minutes at room temperature. The supernatant was collected, and protein concentration was determined using the BCA kit (Pierce) per manufacturer’s instructions. Protein samples were diluted in SDS sample buffer (final concentration: 62.5mM TrisHCL pH 6.8, 2% SDS, 10% Glycerol, 15.5mg / mL DTT, 0.02mg / mL Bromophenol blue). 25-50 mg of protein was loaded onto each lane of a 4%–12% BisTris mini gel or midi gel (Invitrogen) for immunoblotting. Transfer was onto nitrocellulose membranes (0.2 mm, GE Health Care) before blocking for 1h at room temperature and incubating with primary antibodies of the indicated protein targets overnight at 4 C. Membranes were incubated with secondaryrabbit antibody (Sigma) or secondary mouse antibody (GE Health Care) for 1h at room temperature. Blots were developed in Perkin-Elmer’s Western Lightning ECL or Millipore’s Immobilon HRP reagents per manufacturer’s instructions.

[0116] Cell lines and Antibodies and drugs: BT474, MCF7, MDA-MB-468 cells were acquired from ATCC and cultured in DMEM-F12 and 3T3L-1 was cultured in DMEM. All cell lines were supplemented with 10% Fetal Bovine serum (FBS) and 1% penicillin and streptomycin and 4mM Glutamine. Antibodies used were PTEN (CST #9559), pAKT T308 (CST #2965), pAKT S473 (CST #4060), pPRAS40 (CST #2997), p4E-BP1 T37 / 46 (CST#2855), p4E-BPs S65 (CST #9451), IRS1 (CST# 2382), IR-ß (CST #3025), ß-Actin (CST #4970).

[0117] Histological analysis: Representative sections of the liver, pancreas, brain, epididymal adipose tissue, retroperitoneal white adipose tissue, skeletal muscle from forelimbs, and skeletal muscle from the hindlimbs were fixed in 10% neutral-buffered formalin, processed in alcohol and xylene, embedded in paraffin, sectioned (5-μm-thick) and stained with hematoxylin and eosin. Oil red O staining was performed on formalin fixed, OCT-embedded frozen sections (5-μm-thick) of liver. For histopathological analysis, hematoxylin–eosin-stained or ORO-stained tissue specimens were evaluated by a board- certified veterinary pathologist (S.E.C.). Liver sections were evaluated and scored, using a semiquantitative histopathology scoring system, with slight modifications, for mouse model of metabolic dysfunction associated fatty liver disease56. Briefly, macrovesicular steatosis, microvesicular steatosis and hepatocellular hypertrophy were separately scored, and the extent and severity of the lesions were graded, into the following categories: 0 (<5%), 1 (5- 10%), 2 (10–25%), 3 (25-75%) and 4 (>75%). Inflammation was evaluated by counting the number of inflammatory foci per five 100x fields using the following categories: normal (<0.5 foci), minimal (0.5-1.0 foci), mild (1.0-2.0 foci), moderate (2.0-5.0 foci), severe (>5.0 foci). An Olympus BX45 light microscope was used to capture images with a DP26 camera using cellSens. Dimension software (v1.16).

[0118] Immunohistochemistry: Immunolabeling of PTEN in liver and epididymal white adipose sections was performed at the MSK Biobank and Pathology Core facility using standard protocols. Formalin-fixed, paraffin-embedded sections were stained using an automated staining platform. Briefly, following deparaffinization and heat- inducedepitope retrieval, the primary antibody against PTEN (1:200, Cat. No 9559, clone 138G6, Cell Signaling Technologies) 57.

[0119] Morphometric analysis of eWAT: Cell size distribution in hematoxylin-eosin (H&E)-stained sections of epididymal white adipose tissue was analyzed from triplicates of 40X images per group and cell size was quantified using Adiposoft Software (Image J)58

[0120] Treatment with PTEN inhibitor VO-OHpic: VO-OHpic was suspended in 2% DMSO, 40% PEG 300, 5% Tween-80, ddH2O and administered intraperitonially at a dose of 10mg / kg, every day, once a day. This was done either at the same time as the start of the western diet in mice for 6 weeks or after 2 weeks of western diet feeding for 4 weeks

[0121] Treatment with mTORC1 inhibitor RMC-6272 or Rapamycin: RMC-6272 was suspended in 1:1 (v / w) Transcutol / Solutol HS 15 and administered intraperitonially at a dose of 3mg / kg, once a week. This was done either at the same time as the start of the western diet in mice for 6 weeks or after 2 weeks of western diet feeding for 4 weeks. Rapamycin was dissolved in 100% DMSO and administered intraperitonially at a dose of 10mg / kg, three times a week along with western diet. Example 2: Western Diet increases PTEN expression

[0122] It was hypothesized that a high fat high carbohydrate diet that causes hyperinsulinemia will also increase PTEN expression in insulin sensitive tissues. A mouse model of diet induced insulin resistance and obesity25-27was used in which C57BL / 6J 6 week old male mice were fed ad libitum with a “western diet” in which 41% of the calories are from fat and 43% from carbohydrates or regular chow (control diet). The former simulates the modern dietary pattern in western countries characterized by high intake of processed foods rich in refined sugars, oils, and saturated fats. This diet when given to rodents mimics a variety of human metabolic syndromes including insulin resistance and obesity28-30.

[0123] Mice on the western diet (WD) and control diet (CD) started gaining weight within a week and, the former, gained significantly more weight than mice on CD (65% increase in WD and 31% increase in CD after 12 weeks) (FIG.1A). After 1 week, the serum insulin levels in mice on the western diet increased by 5-fold over that in mice on the control diet and remained increased for 12 weeks (WD over CD) (FIGs.1B and 5A). Miceon the western diet showed increase in glucose levels 20% over those in control mice, 2 weeks after its initiation, and remained hyperglycemic over 12 weeks (FIG.1C). Insulin sensitivity was measured using the glucose tolerance test (GTT) and a reduction in glucose tolerance began between 2 days and 1 week after western diet consumption and glucose tolerance was severely compromised by 2 weeks, coinciding with the start of hyperglycemia (FIG.1D). Glucose intolerance increased steadily over 6 weeks of the western diet (FIG. 1B) as demonstrated by the increase in peak glucose (30min after glucose stimulation) and adapted glucose levels (2 hours after glucose stimulation) of the GTT which remained increased by 55% and 46% respectively in mice on western diet over normal diet at the end of week 6 (FIGs.5B, 5C, and 5D). As previously reported, it was found that mice on the western diet developed hepatic lipidosis and steatosis and a significant increase in leptin levels (11-fold over mice on CD) within 4 weeks (FIGs.1E-1F and 1J)2,4. Altogether these data indicates that upon high fat diet, these mice developed insulin resistance and the onset of related metabolic pathologies.

[0124] It was next assayed whether PTEN expression was increased in insulin sensitive tissues (epididymal white adipose tissue (eWAT), muscle, and liver). PTEN expression was significantly increased in eWAT and liver tissues after 1 week of western diet and in muscle after 2 weeks (18-fold in eWAT, 1.5- fold in liver and 2.7-fold muscle) as demonstrated by immunoblotting and immunohistochemistry (FIGs.1G-1I and 5E). The increase in PTEN expression measured by immunohistochemistry in the liver was especially striking (FIG. 1I). Phosphorylated AKT T308 and S473 also increased significantly in eWAT and AKT T308 phosphorylation increased modestly in liver and muscle (FIG.1G). Thus, in mice on the western diet, insulin activates the expression of PTEN, a negative regulator of upstream elements of the pathway. By contrast, levels of insulin receptor and IRS1 increase in eWAT and are unchanged in the other two tissues (FIGs.1G and 5F-5G). In eWAT at 1 week, elevated insulin levels activate AKT T308 significantly, enough to maintain normoglycemia initially (FIG.1C).

[0125] These data show that induction of insulin resistance in animals on a Western diet is accompanied by an increase in weight, serum insulin levels, insulin resistance and PTEN expression in insulin target organs.Example 3: PTEN increases while AKT activity declines over 12 weeks in western diet

[0126] The kinetics of changes in PTEN expression and PI3K activity on the western diet was investigated. Epididymal white adipose (eWAT), muscle and liver tissue were collected from mice that were on western or control diet as a function of time. In eWAT, PTEN and pAKT T308 increase within a week of western diet (FIGs.1G, 2A, 2C-2D, and 6A) as does Insulin (FIG.1B). While PTEN is induced by 18-fold in eWAT in mice on western diet, pAKT T308 is induced by 16-fold (FIGs.2A and 2C-2D). Subsequently both decline and while PTEN remained upregulated to about 2 to 3-fold for 10 weeks, pAKT declined to approximately the same levels as the control animals in spite of the high insulin production. It is likely that the PTEN declined in response to the AKT decline but remained high enough to lower AKT signaling. In the muscle tissue of mice on western diet, PTEN induction started by 1 week and peaked at 2 weeks (induced by 2.7-fold over mice on control diet), remains increased by 2-fold above control tissues for almost 12 weeks (FIGs. 2B-2C and 6A). Phospho-AKT on the other hand, increased within 2 days after start of the western diet and continued to decline right after, its level being lower than the control tissue for the rest of 12 weeks (FIGs.2B, 2D, and 6A). In the liver of mice on western diet, PTEN increased within a week by 1.5-fold over mice on control diet, oscillated over the next 6 weeks but remained increased after that by approximately 1.5-fold until 10 weeks (FIGs.2C and 6A-6B). Phospho-AKT increased within 1 week of western diet consumption and subsequently remained reduced compared to control animals for approximately all of 10 weeks (FIGs.2D and 6A-6B).

[0127] In summary, in eWAT, within 1 week of western diet consumption there was a significant increase in AKT phosphorylation and PTEN expression that coincided with increased insulin levels (FIGs.1B, 2A, and 2C-2D). Hyperglycemia and severe glucose intolerance begun after 2weeks of western diet (FIGs.1C-1D). By 10 weeks AKT phosphorylation had declined to basal levels whereas PTEN declined but remained elevated above control animal tissues. Over ten weeks, changes in serum insulin and PTEN expression in eWAT almost coincided. Thus, PTEN levels in eWAT may be regulated by insulin levels and, in turn, may decrease insulin sensitivity further. Thus, at some level PTEN induced feedback may prevent further activation of the pathway, thus leading to hyperglycemia. On the other hand, in muscle tissues, which are considered as the primarytissue of glucose uptake in response to insulin, PTEN starts increasing after a week and peaks at 2 weeks, coinciding with the onsets of hyperglycemia and severe glucose intolerance. At that time, pAKT had declined to levels 50% lower than muscle in control animals (FIGs.2B-2D). Thus, unlike PTEN levels in eWAT, the PTEN levels in muscle are correlated with the onset of hyperglycemia. In the liver, the PTEN and pAKT levels increased with insulin levels (at 1 week) and pAKT levels drop below those of control animals at 2 weeks, when glucose levels rose (FIGs.2C-2D). Example 4: Inhibiting PTEN activity prevents and reverses insulin resistance

[0128] Since PTEN levels increased in insulin sensitive tissues and the increase correlated with the onset of hyperinsulinemia, hyperglycemia and glucose intolerance along with the decline of AKT phosphorylation, it was determined whether inhibiting PTEN activity would affect these phenotypes. To that effect an inhibitor of PTEN phosphatase activity, VO-OHpic, a vanadyl compound complexed to hydroxypicolinic acid31-33was used. This complex is a non-competitive inhibitor of PTEN protein and its selectivity towards PTEN over other cysteine-based phosphatases (CBP), including protein tyrosine phosphatases, is based on exploitation of the differences in the catalytic pockets of the phosphatases. The catalytic pocket of PTEN (8Ang) is much larger than that of the other CBPs allowing this compound to bind PTEN with an IC50 of 35nM while it binds other CBPs in the uM range31. Its selectivity has been confirmed by its induction of AKT phosphorylation in cells with WT PTEN but not in PTEN null cells31. This compound has been tested in vivo in multiple pre-clinical models in which it has effectively inhibited PTEN without noticeable toxicity31,34-36. It was confirmed that VO-OHpic induced the PI3K / AKT pathway by treatment of PIK3CA mutant BT474 and MCF7 breast cancer cells with the drug (FIGs.7A-7B). Cancer cell lines with activating mutation in PIK3CA and wild type for PTEN have higher PTEN expression levels since oncogenic activation of PI3K leads to an increase in PTEN translation24. Upon treatment of these cells with the drug, phosphorylation of AKT, its substrate PRAS40, and mTOR substrates increased as a function of time, consistent with its inhibition of PTEN (FIGs.7A-7B). In 3T3L-1 adipose cells, VO-OHpic increased the duration of insulin induction of AKT, and S6K phosphorylation (FIG.7C).

[0129] Treatment of mice on the Western diet with 10mg / kg of VOOH-pic once daily led to complete prevention of weight gain over 6 weeks (FIGs.3A and 7I). No toxicity of the drug was observed. Treatment with VO-OHpic completely prevented hyperinsulinemia, hyperglycemia and insulin resistance as demonstrated by glucose tolerance tests done after 4 and 6 weeks of drug treatment (FIGs.3B-3D, 3I-3J, and 7G). It was confirmed that the mice on western diet with and without drug treatment were consuming approximately the same amount of food (FIG.7D). It was also confirmed that treatment with the inhibitor on control diet did not affect the weight or glucose levels of mice (FIGs.7E-7F). It was next determined whether inhibiting PTEN was able to reverse the phenotypes associated with insulin resistance. Mice were treated with VO-OHpic 2 weeks after initiation of the western diet, at which time hyperinsulinemia, hyperglycemia and glucose intolerance were well established (FIGs.1A-1D). VO-OHpic completely reversed weight gain and hyperinsulinemia within 1 week of PTEN inhibition (FIGs.3E-3F, 3I, and 7I). Hyperglycemia and glucose intolerance were reversed after 2 weeks of inhibitor treatment and insulin sensitivity persisted on therapy and the western diet for four weeks (FIGs.3G- 3H, 3J, and 7H). PTEN inhibition also prevented the development of leptin resistance and reversed the increase in leptin levels within 2 weeks of drug treatment (FIGs.3K and 3J). eWAT adipocytes are the primary source of leptin hormone and expand upon consumption of a high fat diet. It was found that the adipocyte area per cell increased upon western diet consumption, and this was both prevented and reversed by inhibition of PTEN (FIGs.3L, 3M, and 7K).

[0130] PTEN inhibition also prevented the increase in liver weight upon western diet and reduced the weight upon inhibitor treatment for 4 weeks after 2 weeks of the diet exposure (FIG.3N). Upon analysis of Oil red O (ORO) staining of liver sections, it was found that lipid accumulation was substantially prevented and reversed by inhibiting PTEN at the beginning or after 2 weeks of western diet (FIG.3L). Analysis of H&E sections, that were certified, and blind scored by a pathologist, revealed that VO-OHpic treatment completely prevented and reversed the development of macrovesicular steatosis (within 4 weeks of drug treatment for the latter) (FIG.3O). Microvesicular steatosis was reduced by preventive treatment of VO-OHpic and was reduced more by treating the animals with the drug after 2 weeks of western diet consumption (FIG.7L). Hepatocellular hypertrophy andlobular inflammation both were significantly reduced by preventive treatment while treatment at 2 weeks of the diet completely reversed these conditions (FIGs.7M-7N). The MASH scores that are an integration of the conditions of macro- and microvesicular steatosis, hepatocellular hypertrophy and lobular inflammation, averaged around 10 for the mice on western diet, 4 for VO-OHpic treatment at the beginning of the diet and 0.5 for mice that were treated with the inhibitor after 2 weeks on western diet for 4 weeks (FIG. 3P). Other markers of liver disease or function (AST, ALT, GGT, albumin) were normal in all groups of mice (data not shown) except cholesterol which was elevated in the range of hypercholesteremia in mice on the western diet. Treatment with VO-OHpic prevented or lowered the cholesterol levels within the normal range (FIG.3Q). PTEN inhibition for 4 weeks during western diet consumption in the eWAT and liver caused an increase in AKT T308 and S473 phosphorylation (80% increase in AKT S473 in eWAT and 105% in liver of mice treated with VO-OHpic over vehicle treated mice on western diet) (FIGs.3R-3S, 3U, 7O-7P, and 7R-7S). PTEN inhibition for as little as 2 weeks in muscle tissue caused an increase of 140% in pAKT S473 in muscle (FIGs.3T, 3U, 7Q, and 7T). Phosphorylated PRAS40 and mTORC1 activity were also increased in all three tissues.

[0131] Taken together these data show that inhibition of PTEN phosphatase activity is sufficient to reverse insulin resistance and its metabolic sequelae in mice on a western diet. Insulin induction of PTEN is therefore necessary for maintenance of the phenotype. Moreover, inhibition of PTEN activity in cells in which its expression has been induced by insulin prevents the development of insulin resistance. Accordingly, these results demonstrate that the methods disclosed herein are useful for preventing or treating Type 2 diabetes, reducing risk factors associated with Type 2 diabetes, and / or reducing the likelihood or severity of Type 2 diabetes in a subject in need thereof. Example 5: Inhibiting mTORC1 prevents and reverses obesity and insulin resistance

[0132] Regulation of mTOR activation by PI3K controls the 4E-BP1 dependent translation of PTEN protein. Further experiments were performed to confirm that the diet dependent increase in PTEN protein in mice is sensitive to mTORC1 inhibition. mTOR phosphorylates 4E-BP and causes it to dissociate from eIF4E, thus relieving its inhibition of formation of the eIF4E initiation complex37,38. Stimulation of translation of capped mRNAs including PTEN mRNA ensues. In MDA-MB-468 PTEN null cells, expression of PTENmRNA without the PTEN 5’UTR prevented the induction of PTEN expression after insulin stimulation and the duration of insulin stimulated AKT phosphorylation was increased (FIG.8A).

[0133] It was assayed whether inhibition of TORC1 kinase prevented the increase in PTEN protein by insulin stimulation. RMC-6272 is a selective inhibitor of TORC1 kinase (IC50 for mTORC1 inhibition is 0.44 nM for p4E-BP, IC50 for TORC2 inhibition of pS473 AKT is 12nM)39. At TORC1 selective doses this drug inhibits 4E-BP1 phosphorylation but not AKT phosphorylation, which is TORC2 dependent. RMC-6272 prevented the induction of PTEN after insulin stimulation in 3T3L1 adipocytes (FIG.8B).

[0134] It was tested whether RMC-6272 inhibited the increase in PTEN expression that occurs in mice on the western diet and, in doing so, prevents its induction of obesity, insulin resistance and MASH. RMC-6272 (3m / kg) was administered to mice once per week when the western diet was initiated and then once weekly. Treatment of RMC-6272 at the beginning of the diet completely prevented weight gain, reduced insulin levels after 4 weeks and glucose levels after 2 weeks to normal levels, and prevented the development of insulin resistance (FIGs.4A-4D, 4I-4J, and 8C). It was confirmed that the mice in different groups approximately ate the same amount of food (FIG.8D) and the inhibitor alone did not induce major changes in weight or glucose levels in animals (FIGs.8E-8F). It was found that weight gain, hyperinsulinemia, hyperglycemia and the development of insulin resistance were completely reversed when the mice were treated with the drug two weeks after the diet was given to them (FIGs.4E-4H, 4I-4J, and 8C). mTORC1 inhibition also prevented and reversed increases in leptin levels and adipocyte cell area in eWAT tissues (FIGs.4K-4N and 8G).

[0135] By contrast, although PTEN inhibition prevented and reversed the MASH phenotypes in mice fed on the western diet, mTORC1 inhibition did not. Liver weight, lipidosis, steatosis and overall MASH scores were not decreased by RMC-6272 (FIGs.4O- 4P). This result was confirmed by H&E and ORO staining of liver sections that showed no reduction in the lipid accumulation had occurred on the western diet (FIG.8H). Neither was the diet-induced hypercholesterolemia reduced by mTORC1 inhibition (FIG.8I) whereas serum liver enzymes such as serum AST, ALT, GGT as well as albumin levels were normal in all groups of mice (data not shown). To confirm that the failure to inhibitthe MASH phenotype was not specific to RMC-6272, a different mTORC1 inhibitor, Rapamycin40, was used. Rapamycin inhibited weight gain in mice on western diet and quite effectively reduced hyperinsulinemia and hyperglycemia (FIGs.8J-8L). However, H&E sections of the liver treated with Rapamycin revealed the presence of liver lipidosis (FIG. 8M).

[0136] Lastly, pAKT / mTORC1 activity and PTEN protein levels in the eWAT, liver and muscle tissues upon treatment with RMC-6272 or Rapamycin was analyzed. Treatment of RMC-6272 or Rapamycin along with consumption of western diet for 4 weeks in the eWAT or 2 weeks in the muscle, led to a decrease in p4E-BP1 T47 / 46 and S65 sites and there was a significant decrease in PTEN expression accompanied by increase in pAKT and its substrate pPRAS40 (FIGs.4Q-4R and 4N). In contrast, in the liver, neither RMC-6272 nor Rapamycin treatment for 4 weeks inhibited mTORC1 activity when the mice were fed with western diet, and PTEN levels were almost unchanged (FIGs.8Q-8R and 8N). Consistent with persistent PTEN overexpression, pAKT T308 phosphorylation and that of the AKT substrate remained low.

[0137] In summary, inhibiting PTEN activity in the insulin sensitive tissues inhibited the obesity, insulin resistance and MASH phenotypes whereas inhibiting mTORC1, suppressed PTEN and its metabolic effects in eWAT and muscle but not liver. Accordingly, these results demonstrate that the methods disclosed herein are useful for preventing or treating Type 2 diabetes, reducing risk factors associated with Type 2 diabetes, and / or reducing the likelihood or severity of Type 2 diabetes in a subject in need thereof. EQUIVALENTS

[0138] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications andvariations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0139] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0140] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non- limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0141] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. REFERENCES 1 Westman, E. C. Type 2 Diabetes Mellitus: A Pathophysiologic Perspective. Front Nutr 8, 707371 (2021). https: / / doi.org:10.3389 / fnut.2021.7073712 Lee, S. H., Park, S. Y. & Choi, C. S. Insulin Resistance: From Mechanisms to Therapeutic Strategies. Diabetes Metab J 46, 15-37 (2022). https: / / doi.org:10.4093 / dmj.2021.0280 3 Di Pino, A. & DeFronzo, R. A. Insulin Resistance and Atherosclerosis: Implications for Insulin-Sensitizing Agents. Endocr Rev 40, 1447-1467 (2019). https: / / doi.org:10.1210 / er.2018-00141 4 Nakamura, A. & Terauchi, Y. Lessons from mouse models of high-fat diet-induced NAFLD. Int J Mol Sci 14, 21240-21257 (2013). https: / / doi.org:10.3390 / ijms141121240 5 Wondmkun, Y. T. Obesity, Insulin Resistance, and Type 2 Diabetes: Associations and Therapeutic Implications. Diabetes Metab Syndr Obes 13, 3611-3616 (2020). https: / / doi.org:10.2147 / DMSO.S275898 6 Saini, V. Molecular mechanisms of insulin resistance in type 2 diabetes mellitus. World J Diabetes 1, 68-75 (2010). https: / / doi.org:10.4239 / wjd.v1.i3.68 7 Sears, B. & Perry, M. The role of fatty acids in insulin resistance. Lipids Health Dis 14, 121 (2015). https: / / doi.org:10.1186 / s12944-015-0123-1 8 Petersen, M. C. & Shulman, G. I. Mechanisms of Insulin Action and Insulin Resistance. Physiol Rev 98, 2133-2223 (2018). https: / / doi.org:10.1152 / physrev.00063.2017 9 Auger, K. R., Serunian, L. A., Soltoff, S. P., Libby, P. & Cantley, L. C. PDGF- dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell 57, 167-175 (1989). 10 Vanhaesebroeck, B., Stephens, L. & Hawkins, P. PI3K signalling: the path to discovery and understanding. Nature reviews. Molecular cell biology 13, 195-203 (2012). https: / / doi.org:10.1038 / nrm3290 11 Ruderman, N. B., Kapeller, R., White, M. F. & Cantley, L. C. Activation of phosphatidylinositol 3-kinase by insulin. Proceedings of the National Academy of Sciences of the United States of America 87, 1411-1415 (1990). 12 Metz, H. E. & Houghton, A. M. Insulin receptor substrate regulation of phosphoinositide 3-kinase. Clinical cancer research : an official journal of the American Association for Cancer Research 17, 206-211 (2011). https: / / doi.org:10.1158 / 1078- 0432.CCR-10-0434 13 Alessi, D. R. et al. Mechanism of activation of protein kinase B by insulin and IGF- 1. The EMBO journal 15, 6541-6551 (1996). 14 Samuel, V. T. & Shulman, G. I. The pathogenesis of insulin resistance: integrating signaling pathways and substrate flux. J Clin Invest 126, 12-22 (2016). https: / / doi.org:10.1172 / JCI77812 15 Manning, B. D. & Toker, A. AKT / PKB Signaling: Navigating the Network. Cell 169, 381- 405 (2017). https: / / doi.org:10.1016 / j.cell.2017.04.001 16 Williams, M. R. et al. The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells. Current biology : CB 10, 439-448 (2000). https: / / doi.org:10.1016 / s0960-9822(00)00441-317 Bayascas, J. R. et al. Mutation of the PDK1 PH domain inhibits protein kinase B / Akt, leading to small size and insulin resistance. Molecular and cellular biology 28, 3258-3272 (2008). https: / / doi.org:10.1128 / MCB.02032-07 18 Alessi, D. R. et al. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Current biology : CB 7, 261- 269 (1997). https: / / doi.org:10.1016 / s0960-9822(06)00122-9 19 Ma, X. M. & Blenis, J. Molecular mechanisms of mTOR-mediated translational control. Nature reviews. Molecular cell biology 10, 307-318 (2009). https: / / doi.org:10.1038 / nrm2672 20 Dibble, C. C. & Cantley, L. C. Regulation of mTORC1 by PI3K signaling. Trends in cell biology 25, 545-555 (2015). https: / / doi.org:10.1016 / j.tcb.2015.06.002 21 Stokoe, D. et al. Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science 277, 567-570 (1997). https: / / doi.org:10.1126 / science.277.5325.567 22 Zhang, H. H. et al. Insulin stimulates adipogenesis through the Akt-TSC2-mTORC1 pathway. PloS one 4, e6189 (2009). https: / / doi.org:10.1371 / journal.pone.0006189 23 Stambolic, V. et al. Negative regulation of PKB / Akt-dependent cell survival by the tumor suppressor PTEN. Cell 95, 29-39 (1998). https: / / doi.org:10.1016 / s0092- 8674(00)81780-8 24 Mukherjee, R. et al. Regulation of PTEN translation by PI3K signaling maintains pathway homeostasis. Mol Cell 81, 708-723 e705 (2021). https: / / doi.org:10.1016 / j.molcel.2021.01.033 25 de Moura, E. D. M. et al. Diet-induced obesity in animal models: points to consider and influence on metabolic markers. Diabetol Metab Syndr 13, 32 (2021). https: / / doi.org:10.1186 / s13098-021-00647-2 26 Preguica, I. et al. Diet-induced rodent models of obesity-related metabolic disorders- A guide to a translational perspective. Obes Rev 21, e13081 (2020). https: / / doi.org:10.1111 / obr.13081 27 Martins, T. et al. Murine models of obesity. 2, 127-147 (2022). 28 Clemente-Suarez, V. J., Beltran-Velasco, A. I., Redondo-Florez, L., Martin- Rodriguez, A. & Tornero-Aguilera, J. F. Global Impacts of Western Diet and Its Effects on Metabolism and Health: A Narrative Review. Nutrients 15 (2023). https: / / doi.org:10.3390 / nu15122749 29 McDonald, S. D., Pesarchuk, E., Don-Wauchope, A., El Zimaity, H. & Holloway, A. C. Adverse metabolic effects of a hypercaloric, high-fat diet in rodents precede observable changes in body weight. Nutr Res 31, 707-714 (2011). https: / / doi.org:10.1016 / j.nutres.2011.08.009 30 Fang, L. Z., Lily Vidal, J. A., Hawlader, O. & Hirasawa, M. High-fat diet-induced elevation of body weight set point in male mice. Obesity (Silver Spring) 31, 1000-1010 (2023). https: / / doi.org:10.1002 / oby.2365031 Rosivatz, E. et al. A small molecule inhibitor for phosphatase and tensin homologue deleted on chromosome 10 (PTEN). ACS Chem Biol 1, 780-790 (2006). https: / / doi.org:10.1021 / cb600352f 32 Mak, L. H., Vilar, R. & Woscholski, R. Characterisation of the PTEN inhibitor VO- OHpic. J Chem Biol 3, 157-163 (2010). https: / / doi.org:10.1007 / s12154-010-0041-7 33 Mak, L. H. & Woscholski, R. Targeting PTEN using small molecule inhibitors. Methods 77- 78, 63-68 (2015). https: / / doi.org:10.1016 / j.ymeth.2015.02.007 34 Du, J. et al. A small-molecule cocktail promotes mammalian cardiomyocyte proliferation and heart regeneration. Cell Stem Cell 29, 545-558 e513 (2022). https: / / doi.org:10.1016 / j.stem.2022.03.009 35 Chen, C. et al. Resveratrol as a new inhibitor of immunoproteasome prevents PTEN degradation and attenuates cardiac hypertrophy after pressure overload. Redox Biol 20, 390-401 (2019). https: / / doi.org:10.1016 / j.redox.2018.10.021 36 Wang, X. Q., Zhang, L., Xia, Z. Y., Chen, J. Y., Fang, Y. & Ding, Y. Q. PTEN in prefrontal cortex is essential in regulating depression-like behaviors in mice. Transl Psychiatry 11, 185 (2021). https: / / doi.org:10.1038 / s41398-021-01312-y 37 Gingras, A. C. et al. Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. Genes Dev 13, 1422-1437 (1999). https: / / doi.org:10.1101 / gad.13.11.1422 38 Sonenberg, N. & Hinnebusch, A. G. Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136, 731-745 (2009). https: / / doi.org:10.1016 / j.cell.2009.01.042 39 Burnett, G. L. et al. Discovery of RMC-5552, a Selective Bi-Steric Inhibitor of mTORC1, for the Treatment of mTORC1-Activated Tumors. J Med Chem 66, 149-169 (2023). https: / / doi.org:10.1021 / acs.jmedchem.2c01658 40 Dumont, F. J. & Su, Q. Mechanism of action of the immunosuppressant rapamycin. Life Sci 58, 373-395 (1996). https: / / doi.org:10.1016 / 0024-3205(95)02233-3 41 James, D. E., Stockli, J. & Birnbaum, M. J. The aetiology and molecular landscape of insulin resistance. Nat Rev Mol Cell Biol 22, 751-771 (2021). https: / / doi.org:10.1038 / s41580-021-00390-6 42 Li, M., Chi, X., Wang, Y., Setrerrahmane, S., Xie, W. & Xu, H. Trends in insulin resistance: insights into mechanisms and therapeutic strategy. Signal Transduct Target Ther 7, 216 (2022). https: / / doi.org:10.1038 / s41392-022-01073-0 43 George, S. et al. A family with severe insulin resistance and diabetes due to a mutation in AKT2. Science 304, 1325-1328 (2004). https: / / doi.org:10.1126 / science.1096706 44 Shah, O. J. & Hunter, T. Tuberous sclerosis and insulin resistance. Unlikely bedfellows reveal a TORrid affair. Cell Cycle 4, 46-51 (2005). https: / / doi.org:10.4161 / cc.4.1.1343 45 Pal, A. et al. PTEN mutations as a cause of constitutive insulin sensitivity and obesity. N Engl J Med 367, 1002-1011 (2012). https: / / doi.org:10.1056 / NEJMoa111396646 Li, Y. Z., Di Cristofano, A. & Woo, M. Metabolic Role of PTEN in Insulin Signaling and Resistance. Cold Spring Harb Perspect Med 10 (2020). https: / / doi.org:10.1101 / cshperspect.a036137 47 Johnson, M. L. et al. Mechanism by Which Caloric Restriction Improves Insulin Sensitivity in Sedentary Obese Adults. Diabetes 65, 74-84 (2016). https: / / doi.org:10.2337 / db15-0675 48 Ishihara, H. et al. Association of the polymorphisms in the 5'-untranslated region of PTEN gene with type 2 diabetes in a Japanese population. FEBS Lett 554, 450-454 (2003). https: / / doi.org:10.1016 / s0014-5793(03)01225-0 49 Le Bacquer, O. et al. Elevated sensitivity to diet-induced obesity and insulin resistance in mice lacking 4E-BP1 and 4E-BP2. J Clin Invest 117, 387-396 (2007). https: / / doi.org:10.1172 / JCI29528 50 Tsai, S. Y. et al. Increased 4E-BP1 Expression Protects against Diet-Induced Obesity and Insulin Resistance in Male Mice. Cell Rep 16, 1903-1914 (2016). https: / / doi.org:10.1016 / j.celrep.2016.07.029 51 Shi, Y. et al. PTEN is a protein tyrosine phosphatase for IRS1. Nat Struct Mol Biol 21, 522- 527 (2014). https: / / doi.org:10.1038 / nsmb.2828 52 Ueno, M. et al. Regulation of insulin signalling by hyperinsulinaemia: role of IRS- 1 / 2 serine phosphorylation and the mTOR / p70 S6K pathway. Diabetologia 48, 506-518 (2005). https: / / doi.org:10.1007 / s00125-004-1662-6 53 Um, S. H. et al. Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity. Nature 431, 200-205 (2004). https: / / doi.org:10.1038 / nature02866 54 Barbour, L. A., McCurdy, C. E., Hernandez, T. L. & Friedman, J. E. Chronically increased S6K1 is associated with impaired IRS1 signaling in skeletal muscle of GDM women with impaired glucose tolerance postpartum. J Clin Endocrinol Metab 96, 1431- 1441 (2011). https: / / doi.org:10.1210 / jc.2010-2116 55 Blagosklonny, M. V. Fasting and rapamycin: diabetes versus benevolent glucose intolerance. Cell Death Dis 10, 607 (2019). https: / / doi.org:10.1038 / s41419-019-1822-8 56 Liang, W. et al. Establishment of a general NAFLD scoring system for rodent models and comparison to human liver pathology. PLoS One 9, e115922 (2014). https: / / doi.org:10.1371 / journal.pone.0115922 57 Xue, W. et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514, 380-384 (2014). https: / / doi.org:10.1038 / nature13589 58 Galarraga, M. et al. Adiposoft: automated software for the analysis of white adipose tissue cellularity in histological sections. J Lipid Res 53, 2791-2796 (2012). https: / / doi.org:10.1194 / jlr.D023788

Claims

WHAT IS CLAIMED IS 1. A method for treating or preventing Type 2 diabetes in a subject in need thereof comprising administering to the subject an effective amount of an agent that inhibits PTEN expression and / or activity.

2. The method of claim 1, wherein administration of the agent prevents or reverses insulin resistance, hyperglycemia, and / or hyperinsulinemia in the subject.

3. The method of claim 1 or 2, wherein administration of the agent prevents or reverses liver lipidosis, dyslipidemia, and / or leptin resistance.

4. The method of any one of claims 1-3, wherein administration of the agent prevents or reverses obesity in the subject.

5. The method of any one of claims 1-4, wherein the Type 2 diabetes is diet- induced.

6. The method of any one of claims 1-5, wherein the agent is a small molecule PTEN inhibitor, or a PTEN-specific inhibitory nucleic acid, optionally wherein the PTEN- specific inhibitory nucleic acid is a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA.

7. The method of claim 6, wherein the small molecule PTEN inhibitor is hydroxyl(oxo)vanadium 3-hydroxypiridine-2-carboxylic acid (VO-OHpic), bisperoxovanadium 1,10-phenantroline (bpV(phen)), bisperoxovanadium 5-hydroxipyridine (bpV(pic)), bisperoxovanadium 5-hydroxipyridine-2-carboxylic acid bpV(HOpic), bisperoxovanadium pyridin-2-squaramide (bpV(pis)), or N-(9,10-dioxo-9,10- dihydrophenanthren-2-yl) pivalamide (SF1670).

8. The method of any one of claims 1-5, wherein the agent is a small molecule mTORC1 inhibitor, or a mTORC1-specific inhibitory nucleic acid, optionally wherein the mTORC1-specific inhibitory nucleic acid is a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA.

9. The method of claim 8, wherein the small molecule mTORC1 inhibitor is rapamycin, everolimus, temsirolimus, INK128, OSI027, PP242, RapaLinks, Ku-0063794,PP30, XL 388, WYE-354, WAY-600, WYE-687, Torin-1, Torin-2, RMC-6272, or AZD8055.

10. The method of any one of claims 1-9, wherein the agent is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, iontophoretically, transmucosally, or intramuscularly.

11. The method of any one of claims 1-10, wherein administration of the agent results in a decrease in epididymal white adipose fat, liver or muscle PTEN polypeptide levels in the subject compared to that observed prior to administration.

12. The method of any one of claims 1-11, wherein the agent is administered for 4 weeks or more.

13. The method of any one of claims 1-12, further comprising separately, sequentially or simultaneously administering one or more additional therapeutic agents to the subject.

14. The method of claim 13, wherein the one or more additional therapeutic agents are selected from among insulin sensitizers (e.g., biguanides (e.g., metformin) and glitazones (e.g., rosiglitazone and pioglitazone)), DPP-4 inhibitors (e.g., sitagliptin, saxagliptin, and linagliptin), GLP-1 receptor agonists (e.g., exenatide, liraglutide and semaglutide), and SGLT2 inhibitors (e.g., canagliflozin, dapagliflozin and empagliflozin).

15. A method for selecting a patient suffering from or at risk for Type 2 diabetes for treatment comprising (a) detecting PTEN polypeptide expression and / or activity levels in epididymal white adipose fat, liver and / or muscle of the subject that are elevated compared to a control subject or a predetermined threshold; and (b) administering to the patient an effective amount of an agent that inhibits PTEN expression and / or activity.

Images