Poly-glutamine androgen receptor knock-in animal models, reagents and methods

Abstract

A recombinant cell or knock-in mammal comprising a recombinant androgen receptor (AR) cassette containing an exogenous polyglutamine (polyQ) tract encoding sequence in exon 1, wherein the polyQ tract encoding sequence is stably integrated into the genome of the cell.

Description

Title: POLY-GLUTAMINE ANDROGEN RECEPTOR KNOCK-IN ANIMAL MODELS, REAGENTS AND METHODS Field

[0001] This disclosure relates to reagents, screening assays and animals models and particularly to mouse models comprising a humanized androgen receptor with a polyglutamine (polyQ) tract as an animal model of various diseases including type 2, diabetes, metabolic syndrome and related liver disorders, prostatic and cardiovascular disorders as well as screening assays employing said animal models and reagents related thereto. Background The Androgen Receptor Protein and its Polyglutamine Tract

[0002] The X-linked androgen receptor (AR) protein is a member of the nuclear receptor superfamily. It is a ligand-inducible transcription factor containing a polymorphic N-terminal region, a central DNA-binding domain (DBD) and a C-terminal ligand-binding domain (LBD). The AR DBD contains two zinc fingers, each anchored by four cysteines, and two <semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>-helical segments. The AR DBD binds to a bipartite androgen response element (ARE) and contributes to the dimerization that underlies co-operative interaction between a pair of AR monomers and the ARE. The AR LBD has the typical nuclear receptor LBD structure of 12 <semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>-helices and small <semantics>β<annotation encoding="application / x-tex">\beta< / annotation>< / semantics>-sheets in a 3-layer "sandwich". In response to ligand binding, the LBD undergoes conformational changes, completing the assembly of an AF-2 domain and initiating coactivator recruitment.

[0003] The structurally flexible N-terminal domain (NTD), harbours a long polymorphic polyglutamine (polyQ) tract (n=11-33). Encoded by a genomic CAG repeat, it helps modulate AR transcriptional activity; ARs with shorter polyQ tracts are the most transcriptionally active.

[0004] Trinucleotide repeats are genomically very unstable, i.e., they can expand or contract in length in somatic cells. This exceeds, by many orders of magnitude, the comparative genetic stability of common nucleotide sequences. Using laser microdissection significant somatic AR CAG repeat instability in prostate and breast pathology including short AR CAG repeat lengths has been reported, including polyGln tract deletions.

[0005] An attempt to humanize the AR in mouse has been done previously; a knock-in mouse model was created by replacing all of mouse exon one with human exon one (with a 21Q tract) but had no phenotype (Lieberman AP, Robins DM. The androgen receptor's CAG / glutamine tract in mouse models of neurological disease and cancer. J Alzheimers Dis 2008;14:247-55). Vectors for making a humanized mouse model are described in Mousavi G. (Generating vectors for and its CAG repeat in human prostate cancer. Masters Thesis, Dept. of Human Genetics, McGill University. 2005). The Androgen Receptor and Intermediary Metabolism

[0006] The AR mechanism of action has classically been studied in target organs of the reproductive system. In prostate tissue the AR has very important effects on growth and maintenance of the organ by supporting intracellular growth pathways, but tied to enhancing the basic metabolic machinery in the cell. Thus androgens support glucose metabolism and do this with the direct involvement of mTOR. The effects of AR on intermediary metabolism are well documented within the liver, again supporting both glucose metabolism and protein metabolism. Metabolic Syndrome and Related Liver Disorders

[0007] Insulin resistance in target tissues, combined with a relative deficiency of insulin secretion are the major features found in both overt T2D and the associated metabolic syndrome (obesity, diabetes, hyperlipidemia, hypertension). Under physiological conditions, when insulin sensitivity decreases, insulin secretion increases to maintain normal glucose levels; when insulin secretion fails to compensate, (β-cell dysfunction / islet failure) abnormal glucose tolerance develops with further progression to overt T2D. Nonalcoholic fatty liver disease (NAFLD) caused by excessive uptake of lipids by the liver has now become a major complication of diabetes. In 30% of patients, chronic inflammation and fibrosis referred to as nonalcoholic steatohepatitis (NASH) ensues with a high risk of hepatocellular carcinoma (HCC). NASH cirrhosis is becoming one of the most common pathological conditions requiring liver transplantations. This progressive hepatic disease often accompanies obesity, and has a complex set of causes that include aforementioned insulin resistance, as well as signaling effects from adipose tissue, pancreatic islets, and skeletal muscle. NASH, NASH / cirrhosis and NASH / HCC is rapidly becoming an important health care concern with extensive morbidity and mortality and at the moment there is no assigned therapeutics.

[0008] Hepatic steatosis exemplified by nonalcoholic fatty liver disease (NAFLD) is the accumulation of fat in the liver that occurs up to 25% of individuals. NAFLD is identified as a serious risk factor for (30%) nonalcoholic steatohepatitis (NASH) an inflammatory hepatitis that can ultimately culminate in liver failure and cirrhosis. NAFLD is the most common liver disorder in developed countries. NAFLD and NASH are significant risk factors for hepatocellular carcinoma. In developed countries NASH cirrhosis is poised to be the most common disease accounting for liver transplantation.

[0009] In Type 2 Diabetes mellitus (T2D), the incidence of NAFLD is even more pronounced as the majority of diabetics (70%) have hepatic steatosis. Also in T2D NASH, NASH cirrhosis and NASH liver failure is even more aggressive. Currently close to 4 million have diabetes and another 5 million have prediabetes in Canada. It is estimated that as much as 5% will develop liver failure.

[0010] Over time clinicians and researchers have made substantial improvements in addressing other serious diseases associated with diabetes (hypertension cardiovascular diseases, diabetic kidney diseases) by introducing new therapeutics. For instance better hypertension medications with better control of hypertension, the use of powerful lipid lowering agents, and medications that help prevent kidney failure all have had a substantial beneficial effect on controlling complications. This is not the case for diabetes related liver disorders where there are no designated treatment regimens. Presently, close to 400 Canadians receive a liver transplant a year; at a cost of 50 million dollars for the transplant itself, excluding ancillary costs of medications nor costs associated with significant morbidities in individuals with ongoing liver disease. The costs to be incurred in all type II diabetes related liver disorders will become the largest expense overall of diabetic complications.

[0011] NAFLD and its progression to NASH with respect to other liver disorders is relatively newly recognized disorder and its morbidity and mortality effects are now being appreciated. For significant and effective therapies to be developed a better understanding of the pathophysiology is needed. To be successful animal models are critical not just for investigative studies but also to test new therapeutics in a convincing manner. Animal mouse models do exist but are far from representing the human condition; as they rely on genetic alterations (mostly through the leptin pathway, which patients do not have) or exaggerated diets are applied. Summary

[0012] An aspect is a construct comprising a) a nucleotide sequence comprising a 5' AR arm comprising a portion of exon 1 of non-human androgen receptor (AR), and b) an exogenous polyglutamime (polyQ) tract encoding sequence inserted into, and / or replacing the endogenous polyQ / H tract encoding sequence, and c) optionally a selectable marker

[0013] Another aspect of the disclosure includes a recombinant cell or knock-in mammal comprising a recombinant androgen receptor (AR) cassette containing an exogenous polyglutamine (polyQ) tract encoding sequence in exon 1, wherein the polyQ tract encoding sequence is stably integrated into the genome of the cell.

[0014] Other aspects include animal disease models, methods of making said animal disease model and screening assays as using said animal models.

[0015] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. Brief description of the drawings An embodiment of the present disclosure will now be described in relation to the drawings in which:

[0016] Fig. 1. Construction of the AR-19Q mouse. (A) Schematic representation of the human AR. The NTD harbors a polymorphic nucleotide repeat (CAG) (PolyQ) which regulates the transcriptional activity of the AR. (B) AR CAG (19Q) knock-in targeting vector. Fragments of the mouse AR gene (5' arm, 5' UTR and exon 1; 3' arm, start of intron 1); were used to create the pPGKneoNTRtkpA-plox 3' / 5' AR targeting vector. C. Genotyping of genomic DNA from AR- 19Q knock-in mice. Males positives for the AR-19Q allele were bred to heterozygous females to generate the AR-19Q males, AR males, and homozygous AR-19Q female mice. Positive AR- 19Q males were inbred to homozygous AR-19Q females to establish a stable lineage. The CAG tract was amplified by PCR using a set of primers flanking the CAG tract to give a PCR product of 256 bp, and positive clones selected by Bpml restriction digests (204bp + 52bp). D. Sequencing of the CAG tract of the AR-19Q knock-in mouse. The CAG repeat tract was amplified from genomic DNA. PCR products of interest were excised and sequenced. The sample from C57BL / 6 mouse shows the presence of a mixed glutamine / histidine (CAG / CAA / CAC) tract (SEQ ID NO: 10). The sample from a hemizygous AR-19Q male shows the presence of a homogeneous CAG tract (SEQ ID NO: 11).

[0017] Fig. 2. Sequencing of pPGKneoNTRtkpA-plox 3' / 5'AR plasmids. Confirmation of the generation of targeting vector containing different CAG or CAG / CAA tract lengths. (A) 0 CAG (SEQ ID NO: 12), (B) 39 CAG (SEQ ID NO: 13) and (C) mixed 40 CAG / CAA tract (SEQ ID NO: 14).

[0018] Fig. 3. CAG tract length activities. Relative luciferase activity in transfected COS-1 cells comparing transcriptional activity between wildtype AR and AR with various knock-in CAG repeats. * denotes significant p-value (<0.05) relative to human AR (hAR).

[0019] Fig. 4. Characterization of AR-19Q knock-in mouse. (A) Obese phenotype of the AR-19Q knock-in mouse. The picture depicts a 16 month old AR-19Q mouse compared with a 16 month C57BL / 6 (wild type) mouse. (B) Determination of body mass difference between C57BL / 6 mouse and the AR-19Q mouse. Mice were kept and fed with standard chow (18% protein rodent diet with 6% fat) and water ad libitum. Body mass started to increase in the AR- 19Q mouse early and by six months was quite significant. (C) Fat analysis of AR-19Q mice. White and brown adipose tissue (WAT and BAT, respectively) weight was analyzed between C57BL / 6 vs. AR-19Q mice, and found that AR-19Q mice had significantly (p<0.005, p<- / 05) more all types of white adipose tissue (inguinal, visceral, and subcutaneous). (D) Intraperitoneal Glucose Tolerance Test (IPGTT) of C57BL / 6 and AR-19Q mice: Mice were fasting for 4 hour prior to the test. C57BL / 6 and AR-19Q were injected intraperitoneally with a 20% glucose solution (2g / kg body mass). Blood glucose was measured by pricking the saphenous vein and using an Accu-Check® Glucometer (Roche). Samples were taken at t=0 (baseline glucose level), and then post injection at t=20, t=40 and t=60 minutes. (E) Blood Glucose determination of C57BL / 6 and AR-19Q mice: C57BL / 6 mice and AR-19Q mice at an age ≥ 18 months are kept fasting for two hours and then blood samples are taken using an Accu-Check® Glucometer. (F) Systolic Blood Pressure (SBP) of C57BL / 6 and AR-19Q mice. SBP was determined by the Tail Cuff method using the MC 4000 Blood Pressure System (Hatteras). C57BL / 6 (n=2) and AR-19Q (n=3) at an age ≥ 18 months were allowed to acclimatize to the system for 3 days after which the readings for the 4th and 5th day were recorded for analysis.

[0020] Fig. 5. Islet cell hyperplasia in AR-19Q knock-in mice. Histopathology of the pancreas from a 21 month old AR-19Q and a 20 month old C57BL / 6 mice was conducted using hematoxylin and eosin (H / E) stain. The analysis indicates that the AR-19Q mouse presents significant pancreatic islet hyperplasia as indicated by the arrows. AR-19Q mice also show robust expression of insulin and glucagon.

[0021] Fig. 6. AR-19Q Prostate Histopathology. (A) H / E histopathology analysis was made at different aged mice containing the humanized 19Q androgen receptor (AR-19Q). Benign prostatic hyperplasia (BPH) (II) and lymphocytic inflitration (IV) were observed in the AR- 19Q mice compared to control mice (I and III). (B). Immunostaining of mice prostate tissue. Prostate samples from wild-type (WT) and AR-19Q mice were immunostained for cell proliferation using Ki-67 antibody, detection of AR, and of the macrophage colony stimulating factor receptor (cFMS). (C) AR-19Q also more commonly display multi- nucleic / heterochromatic cells vs. WT mice (illustrated by arrows).

[0022] Fig. 7. Qiagen RT2 mouse gene expression array platform. Each array is designed to analyze the expression of 84 genes commonly involved in either prostate cancer development or each of the metabolic pathways selected. Besides containing specific genes to study, each array contains 5 housekeeping genes, 1 genomic DNA control, 3 reverse- transcription controls, and 3 positive PCR controls. Triplicate total RNA samples for all arrays were prepared and analyzed. Genes that showed a > + / - 1.5 fold change in AR-19Q vs. WT mice are included in the figure. Shown are results for: (A) 6, 12, and >18 month AR-19Q mouse prostate vs. WT time course on a prostate specific array. (B) Array profiling of >18 month AR- 19Q vs. WT mouse prostate, performed on arrays specially for Glucose metabolism, AMP- activated protein kinase (AMPK) Signaling, and Mitochondrial Energy Metabolism. In addition to the genes mentioned previously, most notable changes include genes involved in cycle regulation (CCNA1, CDKN1A, CDKN2A, RB1), Fatty acid and Glucose metabolism (ACACB, PFKFB3, ENO1,ENO2), and * indicate components of the electron transport chain and oxidative phosphorylation complexes; ATP6v0D2, a proton pump and marker for lysosomal biogenesis shows the highest expression. (C) Another array assessing 84 Inflammatory genes was used. Of the 84 genes, 80% of chemokines / cytokines (27 genes), 60% of interleukins (8 genes), and 50% of the associated receptors (15 genes) are changed in their expression in AR- 19Q vs. WT prostate samples.

[0023] Fig. 8. NAFLD progression. (A) Alanine amino transferase (ALT) measurement in AR-19Q vs. C57BL / 6 (wild-type) mice. A Biochemical Profile was done using a Comprehensive Profile from Abaxis. C57BL / 6 mice and AR-19Q mice at an age ≥ 18 months were used. Data indicate that AR-19Q mice present with abnormally high values for ALT indicative of hepatic damage. (B) H / E stainings of representative livers from C57BL / 6 (wild-type) vs. AR-19Q mice, at 6, 12, and 24 months. It is strikingly clear of the fatty liver phenotype of the AR-19Q as it ages, towards the development of NASH. The fatty liver NAFLD and NASH progression phenotype is observed in 100% of the mice.

[0024] Fig. 9. Characterization of liver phenotype of AR-19Q mice. (A) Liver Histopathology of C57BL / 6 vs. AR-19Q mice. B- NASH, steatosis; AR-19Q sample shows macrovesicular steatosis, confirming the hepatic damage. (B) Inflammation, confirmed from the infiltration of leukocytes. (C) Fibrosis. AR-19Q sample stained with Masson's trichrome stain presents with a high density of collagen fibers indicative of advanced liver damage. (D) Hepatocellular carcinoma (HCC). Positive, immunostaining with HepPar1 antibody for HCC in AR-19Q, illustrated by the strong, punctate staining, is indicative of HCC.

[0025] Fig. 10. Liver Gene Profiling of the AR knock-in mouse. Analysis was carried out using a RT2 Profiler PCR Array Mouse Fatty Liver platform from QIAGEN that allows for the analysis of 84 genes involved in the pathogenesis of fatty liver. Analysis performed on C57BL / 6 <semantics>(n=3)<annotation encoding="application / x-tex">(n=3)< / annotation>< / semantics>, AR-19Q <semantics>(n=8)<annotation encoding="application / x-tex">(n=8)< / annotation>< / semantics>.

[0026] Fig. 11. Assessment of AR-19Q mice under high fat diet. AR-19Q mice were placed on standard fat diet (SFD) or high fat diet (HFD) for a 3 month period. (A) HFD mice gained a considerable amount of weight in that period. (B) Livers also looked lighter an indication of higher fat connect. HFD AR-19Q mice also had higher blood glucose (C) and ALT levels (D) than SFD fed AR-19Q mice.

[0027] Fig. 12. Sequence of the exon 1 / 19Q plus junction / intron 1 in AR-19Q mice.

[0028] Fig. 13. Sequence of exon 1 region of mouse AR knocked-in with a 19Q tract. Detailed description of the Disclosure

[0029] In the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. . Definitions

[0030] The term "knock-in mammal" as used herein means a non-human mammal, for example a mouse, rat, hamster, rabbit having an exogenous nucleotide sequence, for example a polyQ tract encoding sequence, stably integrated in its genome. In the context of a polyQ knock-in non-human mammal, the non-human mammal can be any mammal lacking a pure endogenous polyQ tract encoding sequence in its AR gene that is amenable to gene knock-in technologies.

[0030] The term "androgen receptor" or "AR" (also known as NR3C4) as used herein refers to an AR polypeptide or nucleic acid encoding an AR, from any species and including naturally occurring variants. In humans and in mice the AR gene, is located at the long arm of X- chromosome and is composed of 8 exons. In humans, the AR comprises in exon 1 of its N- terminal domain a nucleotide sequence encoding a polyQ tract. The mouse AR shares over 90% homology with the human ortholog, but lacks the polyQ tract; instead the mouse possess a mixed CAG / CAA / CAC-glutamine / histidine tract in the N-terminal domain of exon 1. The mouse mixed CAG / CAA / CAC tract may comprise 18, 19 or 20 units.

[0031] The term "polyglutamine tract" or "polyQ tract" alternatively "polyGln" as used in the context of the knock-in non-human mammal (e.g. knock-in mouse) refers to an amino acid sequence consisting of a plurality of uninterrupted (contiguous) glutamine residues, for example 16, 17, 18, 19, 20, 21, 22 or 23 amino acid residues, or depending on the usage, the nucleotide sequence of repeating CAG or CAA units, optionally only CAG units, encoding said amino acid sequence.

[0032] The term "polyQ tract encoding sequence" as used herein means a nucleic acid sequence comprising CAG and / or CAA units, or optionally only CAG units that encodes a polyQ tract.

[0033] The term "recombinant AR cassette" as used herein means a nucleic acid encoding a recombinant AR polypeptide comprising an exogenous polyQ tract instead of or partially replacing an endogenous polyQ / H tract. Typically, only the polyQ tract is exogenous. For example, a recombinant AR cassette in a mouse comprises mouse AR sequence and a non- mouse polyQ tract encoding sequence.

[0034] The term "polyQ AR knock-in gene", "knock-in gene" (in the context of AR) or "humanized AR gene" as used herein are used interchangeably and mean a recombinant non- human AR gene comprising a polyQ tract encoding sequence.

[0035] The term "replaces all of an endogenous polyQ / H tract encoding sequence" in the context of an exogenous polyQ tract encoding sequence means that the entire endogenous polyQ / H tract encoding sequence in exon 1 of the AR is replaced by the exogenous polyQ tract encoding sequence such that no endogenous polyQ / H tract encoding sequence forms part of the recombinant AR cassette. For example, in the context of the mouse whose AR comprises 19 mixed Q / H tract, the exogenous polyQ tract encoding sequence includes 16, 17, 18, 19, 20, 21, 22 or 23 CAG / CAA units and replaces the endogenous 19 Q / H tract encoding sequence.

[0036] The term "replaces part of an endogenous polyQ / H tract encoding sequence" in the context of an exogenous polyQ tract encoding sequence means that some residues of the endogenous polyQ / H tract encoding sequence form part of the recombinant AR cassette. The part can be for example 11, 12, 13 up to 18 codons. For example, in the context of the mouse whose AR comprises 19 mixed Q / H tract, the exogenous polyQ tract encoding sequence can comprise 16, 17, 18, 19, 20, 21, 22, 23 CAG / CAA units and replace at least 16, at least 17, at least 18 of the endogenous polyQ / H tract encoding sequence. In some embodiment, 1, 2, 3 or up to 8 CAG / CAA / CAC units from the endogenous polyQ / H tract encoding sequence form part of the recombinant AR cassette. In the C57BL / 6 mouse, the polyQ / H tract consists of an initial run of 8 Q encoding codons. Accordingly, a person skilled in the art would understand that an exogenous polyQ tract of 11 Q encoding codons could be used to replace the sequence encoding endogenous polyQ / H at 9 to 19 providing an AR recombinant cassette that includes a total of 19 Q encoding codons. In some embodiments, the exogenous polyQ tract encoding sequence and endogenous polyQ / H tract encoding sequence encode a maximum of 19, 20, 21, 22 or 23 amino acids, optionally a maximum of 19, 20, 21, 22 or 23 contiguous Q amino acids.

[0037] The term "metabolic syndrome" as used herein refers to a condition characterized by at least three of central obesity, elevated triglycerides, reduced high-density lipoprotein (HDL), hypertension, and hyperglycemia.

[0038] The term "metabolic syndrome related liver disorders" as used herein includes non- alcoholic fatty liver disease (NAFLD), chronic liver inflammation, non-alcoholic steatohepatitis (NASH), hepatitis, NASH fibrosis, NASH cirrhosis, hepatomegaly and / or hepatocellular carcinoma (HCC) that are subsequent to developing metabolic syndrome.

[0039] The term "normal diet" as used herein refers to a diet providing sufficient caloric intake for the subject without causing more than about a 5%, or optionally a 10%weight fluctuation. For example a normal diet for mice could include a mixture of glucose lipid and protein composite. Examples of normal diets include for example a standard fat diet, a standard glucose diet, a standard fructose diet, a standard carbohydrate diet and / or a standard protein diet.

[0040] The term "high fat diet" or "HFD" as used herein refers to a diet wherein at least 60%, of the animal's calorie intake is provided by fat (e.g. lard and / or soy bean oil).

[0041] The term "high glucose diet" as used herein refers to a diet wherein at least 60%, of the animal's calorie intake is provided by dextrose.

[0042] The term "high fructose diet" as used herein refers to a diet wherein at least 60%, at least 45% or at least 50% of the animal's calorie intake is provided by fructose.

[0043] The term "high carbohydrate diet" as used herein refers to a diet wherein at least 70% of the animal's calorie intake is provided by carbohydrate (e.g. sucrose).

[0044] The term "high protein diet" as used herein refers to a diet wherein at least 40%, at of the animal's calorie intake is provided by protein (e.g. casein).

[0045] The term "test compound" means any compound or composition including any small molecule, metabolite, synthetic polymer nanoparticle, aptamer or biologic such as a nucleic acid (e.g. siRNA), peptide, polypeptide including for example an antibody or binding fragment thereof, composite molecule and / or any composition such as an extract, or combination thereof. The test compound may be in a library or a compound selected from a druggable genome database.

[0046] The term "treating" or "treatment" as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of addiction, stabilized (i.e. not worsening) state of addiction, delay or slowing of addiction progression, amelioration, diminishment of the reoccurrence of the addiction, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treating" and "treatment" as used herein also include prophylactic treatment.

[0047] In understanding the scope of the present disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0048] The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives. Finally, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0049] In understanding the scope of the present disclosure, the term "consisting" and its derivatives, as used herein, are intended to be close ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0050] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural references unless the content clearly dictates otherwise. Thus for example, a composition containing "a compound" includes a mixture of two or more compounds. It should also be noted that the term "or" is generally employed in its sense including "and / or" unless the content clearly dictates otherwise.

[0051] The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.

[0052] The recitation of numerical ranges by endpoints, for example activity levels, blood pressure levels, enzyme levels, and the like herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about." Further, it is to be understood that "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. The term "about" means plus or minus 0.1 to 10%, 1-10%, or preferably 1-5%, of the number to which reference is being made.

[0053] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the present disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. II. Constructs

[0054] The present disclosure provides a nucleic acid construct for producing a recombinant non-human mammal that comprises a humanized androgen receptor (AR) gene.

[0055] In one embodiment, the construct is a targeting construct for making a recombinant non-human mammal that comprises a humanized AR gene or plasmid construct that includes a portion such as an arm (e.g. a 5' arm or 3' arm) for making the humanized AR targeting construct.

[0056] In one embodiment, the construct comprises a vector backbone and a 5' arm comprising all or part of a non-human AR exon1 and an exogenous polyQ tract encoding sequence. In all cases, the 5' arm includes i) non-human AR sequence upstream and downstream of the endogenous mixed poly glutamine / histidine tract encoding sequence and ii) the exogenous polyQ tract encoding sequence. The exogenous polyQ tract encoding sequence replaces all or part of the endogenous mixed poly glutamine / histidine tract.

[0057] The vector backbone comprising for example the 5' arm can be any suitable plasmid, for example pUC19 or plasmids described elsewhere. Suitable vector backbones for the targeting vector include for example the pPGKneoNTRtkpA-plox 3' / 5' targeting vector as was used herein.

[0058] Referring to Example 1 and Fig. 1B, fragments of the mouse AR gene (5' arm, 5' UTR and exon 1; 3' arm, start of intron 1) were used to create the pPGKneoNTRtkpA-plox 3' / 5' AR targeting vector.

[0059] Virus vectors for generating recombinant cultured cells can also be used. For example, recombinant adeno-associated viruses (rAAVs) have been adapted for targeting cells. Infectious rAAV stock can be produced comprising the targeting construct and used to prepare heterozygous and homozygous knock-in cells.

[0060] As shown in Fig. 1B, the 5' AR arm has a size of 4.6 kb, the 3' AR arm a size of 4.1 kb, and the assembled pPGKneoNTRtkpA-plox 3' / 5' AR targeting vector a size of about 16 kb. Depending on the available sequence other size arms can also be used.

[0061] Other constructs and other technologies such as CRISPR can be used to obtain a polyQ knock-in mammal.

[0062] For example, a method or construct can be any method or construct capable of inserting a polyQ tract encoding sequence and / or replacing the endogenous polyQ / H tract encoding sequence in an AR gene. As described herein, an AR Q19 knock-in mouse was made. The sequence of exon 1 / 19Q plus junction / intron 1 after Cre recombinase treatment is shown in Fig. 12 and SEQ ID NO: 8.

[0063] In an embodiment, the construct is one that produces a polyQ knock-in having the sequence as shown in SEQ ID NO: 8 or at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. For example the construct specific portions that are non-functional (e.g. pCN3 sequence) can be excluded or varied. The sequence can for example be more similar to SEQ ID NO: 9 which shows exon 1 region knocked-in with a 19Q tract (Fig. 13).

[0064] The length of the polyQ tract encoding sequence can for example encode 16 to 23 glutamine residues. The length of the polyQ tract can alternatively refer to the length of the exogenous polyQ tract or the polyQ tract that is partially endogenous (for example the mouse polyQ / H tract begins with a string of Q residues) and exogenous, such that all His encoding residues are replaced.

[0065] Referring to Fig. 3, in in vitro transactivation assays, it was shown that an AR construct comprising 19 CAG repeats had similar activity to the human androgen receptor whereas constructs comprising 0 CAG repeats or 39 CAG repeats had significantly different activity from human AR. The term "similar activity" as used in this context means a no greater than 40%, 30%, 20%, 10% or 5% difference in transactivation properties compared to human AR. Constructs having 16 to 23 CAG units may also show similar activity and would be expected to show a phenotype similar to the phenotype of the AR-19Q knock-in mouse described in the Examples.

[0066] In one embodiment, the polyQ tract encoding sequence comprises at least 16 contiguous CAG units. For example, the polyQ tract encoding sequence comprises 16, 17, 18, 19, 20, 21, 22 or 23 contiguous CAG units.

[0067] For example, the polyQ tract encoding sequence comprises 16 to 23 contiguous CAG units. For example, the polyQ tract encoding sequence comprises 18 to 21 contiguous CAG units, for example, the polyQ tract encoding sequence comprises 19 CAG units. For example, the polyQ tract encoding sequence consists of 19 CAG units.

[8000] In other embodiments, the polyQ tract encoding sequences comprises CAA units.

[0069] Referring to Example 1, the polyQ tract encoding sequence comprises 19 contiguous CAG repeats and replaces the endogenous poly glutamine / histidine encoding sequence.

[0070] The CAG units of the polyQ tract encoding sequence may fully or partially replace the native endogenous tract sequence. For example, in the context of native mouse AR, which natively comprises in exon 1 a mixed CAG / CAA / CAC-glutamine / histidine tract of 19 units, the polyQ tract encoding sequence may comprise 19 CAG units and fully replace the native endogenous tract sequence. For example, the exogenous polyQ tract encoding sequence can comprise fewer than 19 units, such as 16 CAG units. The 16 CAG units can fully or partially replace the endogenous tract. In an embodiment, it partially replaces the native CAG / CAA / CAC tract of mouse AR (e.g. the polyQ tract comprises 16 CAG units and 3 native units of mouse CAG / CAA / CAC tract remain).

[0071] If desired, the construct can be engineered to comprise selectable markers such as reporter genes. As used herein, a "selectable marker" refers to a genetic element that provides a selectable phenotype to a cell in which the selectable marker has been introduced. A selectable marker is generally a gene whose gene product provides resistance to an agent that inhibits cell growth or kills a cell. A variety of selectable markers can be used in the DNA constructs of the disclosure, including, for example, Neo, Hyg, hisD, Gpt, hsv-tk and Ble genes, as described, for example in Ausubel et al. (Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999)) and U.S. Pat. No. 5,981,830. Drugs useful for selecting for the presence of a selectable marker include, for example, G418 for Neo, hygromycin for Hyg, histidinol for hisD, xanthine for Gpt, and bleomycin for Ble (see Ausubel et al, supra, (1999); U.S. Pat. No. 5,981,830). DNA constructs can incorporate a positive selectable marker, a negative selectable marker, or both (see, for example, U.S. Pat. No. 5,981,830).

[0072] For example, as shown in Example 1, the neomycin resistance (neo) and herpes simplex virus-thymidine kinase (hsv-tk) selectable genes are included in the construct.

[0073] The constructs can be used for generating knock-in cells or knock-in non-human mammal models.

[0074] Also provided are reagents for making and / or confirming that the construct is stably integrated into genome of the knock-in non-human mammal.

[0075] As described in Example 1, specific primers flanking the CAG tract encoding sequence were used to amplify the CAG tract encoding sequence by PCR and to give a PCR product of 256 bp.

[0076] An aspect provided herein is a primer having the following nucleic acid sequence: FWD: 5'-GTTCCAGAGCGTGCGCGAAGT-3' (SEQ ID NO: 4).

[0077] Another aspect provided herein is a primer having the following nucleic acid sequence: REV: 5'-TGGGGCCTCTACGATGGGCTT-3' (SEQ ID NO: 5).

[0078] A further aspect includes an isolated nucleic acid sequence amplified using SEQ ID NO: 4 and 5,, or a portion of any of the foregoing. In an embodiment, the isolated nucleic acid comprises all or part of SEQ ID NO: 8 or a sequence with at least or at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. III. Cells and animals

[0079] The non-human mammal described herein comprise a humanized AR gene comprising endogenous AR sequence and an exogenous polyQ tract encoding sequence and can be made using for example a construct and methods described herein.

[0800] Targeting vectors described herein can be used to prepare humanized AR gene animal models and recombinant cells, the animal model or recombinant cell comprising a recombinant androgen receptor (AR) cassette, the recombinant AR cassette containing an exogenous polyQ tract encoding sequence in exon 1, wherein the polyQ tract encoding sequence is stably integrated into the genome of the knock-in non-human mammal.

[0081] In an embodiment, the non-human mammal is a mouse. The method of making the humanized AR mouse is described herein.

[0082] The construct can be integrated into the genome of a non-human mammal, for example a non-human mammal, by any method known to those skilled in the art. The construct be introduced into pluripotent cells, such as ES cells, by any suitable method that will permit replacing DNA in animal cells. Techniques that can be used include, but are not limited to, CRISPR technology, calcium phosphate / DNA co-precipitates, microinjection of DNA into the nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, and polycations, (e.g., polybrene, polyornithine, etc.). The DNA can be single or double stranded DNA, linear or circular. (See for example, Hogan et al. Manipulating the Mouse Embryo: A Laboratory Manual Cold Spring Harbor Laboratory (1986); Hogan et al. Manipulating the Mouse Embryo: A Laboratory Manual, second ed., Cold Spring Harbor Laboratory (1994), U.S. Pat. Nos. 5,602,299; 5,175,384; 6,066,778; 4,873,191 and 6,037,521; retrovirus mediated gene transfer into germ lines (Van der Putten et al. (1985)) Proc. Natl. Acad. Sci. U.S.A. 82, 6148- 6152; gene targeting in embryonic stem cells (Thompson et al. (1989) Cell 56, 313-321); electroporation of embryos (Lo (1983) Mol. Cell. Biol. 3, 1803-1814); and sperm-mediated gene transfer (Lavitrano et al. (1989) Cell 57, 717-723))

[0083] Referring to Fig. 1, mouse embryonic stem (ES) cells were electroporated with the construct (knock-in vector). ES cells identified as having homologous recombination of the polyQ AR knock-in gene were selected and then injected into mouse blastocysts which were implanted into female mice. Back-crossing of chimeras was carried out until pure homozygous AR-19Q knock-in mice were obtained (Fig. 1C / D).

[0084] It will be understood that embryonal cells at various developmental stages can also be used to introduce genes for the production of knock-in mammals. Also, different methods are used depending on the stage of development of the embryonal cell. For example, ES cells can be incorporated into a developing embryo through injection into the blastocyst cavity of a murine blastocyst-stage embryo, by injection into a morula-stage embryo, by co-culture of ES cells with a morula-stage embryo, or through fusion of the ES cell with an enucleated zygote. The resulting embryo is raised to sexual maturity and bred in order to obtain animals, whose cells (including germ cells) carry the polyQ AR knock-in gene. If the original ES cell was heterozygous for the insertion, several of these animals can be bred with each other in order to generate animals homozygous for the insertion. Upon making the knock-in non-human mammals having a polyQ AR knock-in gene, the animals may be screened using, for example, Southern blot analysis, Northern blot analysis, Western blot analysis or PCR techniques, to analyze animal tissues to verify the polyQ AR knock-in gene was been stably integrated in the genome of the animal.

[0085] Cells and animals comprising the polyQ AR knock-in gene are provided in the present application.

[0086] An aspect provided herein is a recombinant cell. The recombinant cell comprises a recombinant androgen receptor (AR) cassette, the recombinant AR cassette containing an exogenous polyglutamine (polyQ) tract encoding sequence in exon 1, wherein the polyQ tract encoding sequence is stably integrated into the genome of the cell.

[0087] Also provided is a recombinant cell expressing a recombinant AR polypeptide, the recombinant AR polypeptide comprising an exogenous polyQ tract, wherein the exogenous polyQ tract replaces all or part of the an endogenous AR polyQ / H tract.

[8800] In an embodiment, the polyQ tract comprises 16 to 23 glutamine residues.

[0089] For example, the polyQ tract comprises 18 to 21 glutamine residues.

[0090] In an embodiment, the polyQ tract comprises 19 glutamine residues.

[0091] In an embodiment, the recombinant cell is a non-prostate cell.

[0092] For example, the recombinant cell is a pancreatic cell, a kidney cell, a hepatic cell, a mammary cell or an ovarian cell.

[0093] In an embodiment, the recombinant cell is made using a targeting construct, for example a recombinant adeno-associated virus targeting construct.

[0094] In an embodiment, the recombinant cell is isolated.

[0095] In an embodiment, the recombinant cell is isolated from a recombinant non-human mammal comprising a humanized AR gene. For example, the mammal is a rodent. For example, the rodent is a mouse, rat or hamster. In other embodiments, the mammal is a rabbit.

[0096] In an embodiment, the recombinant cell is isolated from a mouse, optionally a AR- 19Q knock-in mouse.

[0097] In an embodiment, the cell is from a knock-in mouse and the polyQ tract encoding sequence fully or partially replaces the native mixed CAG / CAA / CAC-glutamine / histidine tract.

[0098] Also provided herein in another aspect is a polyQ AR knock-in non-human mammal comprising a recombinant androgen receptor (AR) cassette, the recombinant AR cassette containing an exogenous polyQ tract encoding sequence in exon 1, wherein the polyQ tract encoding sequence is stably integrated into the genome of the knock-in non-human mammal.

[0099] Also provided is a polyQ AR knock-in non-human mammal expressing a recombinant AR polypeptide, the recombinant AR polypeptide comprising an exogenous polyQ tract, wherein the polyQ tract replaces all or part of the an endogenous AR polyQ / H tract.

[00100] In an embodiment, the polyQ tract comprises 16 to 23 glutamine residues.

[00101] For example, the polyQ tract comprises 18 to 21 glutamine residues.

[00102] In an embodiment, the polyQ tract comprises 19 glutamine residues.

[00103] In an embodiment, the non-human mammal is a rodent. For example, the rodent is a mouse, rat or hamster. In an embodiment, the non-human mammal is a rabbit.

[00104] It will be understood that any suitable mouse strain in which an exogenous polyQ tract encoding sequence may be stably integrated into the genome of the mouse may be used (e.g. any strain used for genetic altered mouse models). For example, the mouse can be a C57BL / 6 mouse.

[00105] In an embodiment, the non-human mammal is a mouse, optionally an AR-19Q knock- in mouse.

[00106] In an embodiment, the polyQ AR knock-in mammal, optionally a polyQ AR knock-in mouse, is homozygous for the recombinant AR cassette, optionally wherein the recombinant AR cassette comprises a 19Q exogenous polyQ tract encoding sequence insert.

[00107] In another embodiment, the polyQ AR knock-in mammal, optionally a polyQ AR knock-in mouse, is heterozygous for the recombinant AR cassette, optionally wherein the recombinant AR cassette comprises a 19Q exogenous polyQ tract encoding sequence insert. Heterozygous females can be used for mating.

[00108] In some embodiments such as disease models and screening assays, mammals of a particular sex are desired. For example, in some embodiments the polyQ AR knock-in mammal (optionally a mouse) is male (e.g. a male mouse).

[00109] For example, the polyQ AR knock-in mammal (optionally a mouse) is female with altered androgen hormone levels, optionally wherein the hormone levels are altered by surgically removing the ovaries or by hormone replacement therapy resulting in for example increased testosterone levels. It is believed that higher estrogen levels in female mice may interfere and inhibit androgen receptor activity and that, for example, removing ovaries from the females or providing excess testosterone may alleviate androgen receptor inhibition.

[00110] In an embodiment, the animal is a knock-in mouse and the polyQ tract encoding sequence fully or partially replaces the native mixed CAG / CAA / CAC-glutamine / histidine tract encoding sequence.

[00111] The AR-19Q mice described in the Examples exhibit signs of type 2 diabetes, prostatic hyperplasia, a metabolic syndrome phenotype and related liver diseases, and cardiovascular abnormalities.

[00112] In an embodiment, the polyQ AR knock-in non-human mammal is a non-human knock-in mammal. For example, the mammal can be a rodent such as a mouse, rat or hamster. In another embodiment the mammal is a rabbit.

[00113] In an embodiment, the polyQ AR knock-in non-human mammal is a polyQ AR knock- in mouse made for example as described in the Examples. IV. Disease models and screening assays

[00114] The polyQ AR knock-in non-human mammals described herein comprising a recombinant AR cassette, for example the AR-19Q knock-in mouse described in Example 1, surprisingly exhibit phenotypes associated with several human diseases and based on the exhibited phenotype may be used as disease models, for example as a metabolic syndrome disease model, a type 2 diabetes mellitus (T2D) disease model, a prostate disorder model, a model for liver diseases associated with metabolic syndrome and a cardiovascular disease model. Animals for screening assays can be selected to have one or more of the features described herein. Metabolic syndrome and Type 2 diabetes mellitus

[00115] An aspect provided herein is a polyQ AR knock-in non-human mammal for use as a metabolic syndrome animal model. Such mammal models can be used for screening for new compounds, identifying useful biomarkers for disease monitoring, assessing disease progression in light of dietary and environmental manipulations. Exploring etiologies of disease initiation and progression

[00116] As detailed in the Examples, the AR-19Q knock-in mouse exhibits several disease features associated with metabolic syndrome in humans including obesity, elevated triglycerides, high-density lipoprotein (HDL), hypertension and hyperglycemia.

[00117] Referring to Fig. 4A to 4C, the AR-19Q mice exhibit weight gain commencing at 3 months, compared to wild-type mice. Both the AR-19Q and wild-type mice were fed the same diet.

[00118] In an embodiment, the polyQ AR knock-in non-human mammal has at least at least 30%, at least 40%, at least 50%, at least 60% or at least 70% greater weight compared to a control age-matched mammal lacking the recombinant AR cassette.

[00119] In Fig. 4F, it is shown that the systolic blood pressure of AR-19Q mice is elevated compared to wild-type mice.

[00120] In an embodiment, the polyQ AR knock-in non-human mammal exhibits systolic and / or diastolic hypertension.

[00121] In an embodiment, the polyQ AR knock-in non-human mammal has a systolic blood pressure of at least 10%, at least 20%, at least 30% or at least 40% greater than the systolic blood pressure of a control age-matched mammal lacking the recombinant AR cassette.

[00122] In an embodiment, the polyQ AR knock-in non-human mammal has a diastolic blood pressure of at least 10%, at least 20%, at least 30% or at least 40% greater than the systolic blood pressure of a control age-matched mammals lacking the recombinant AR cassette. Referring now to Fig. 4D and 4E, it was found that the AR-19Q mice had elevated blood glucose compared to wild-type mice.

[00123] In an embodiment, the polyQ AR knock-in non-human mammal exhibits hyperglycemia, optionally post-prandial and / or fasting hyperglycemia.

[00124] Referring now to Fig. 5, the AR-19Q knock-in mice presented pancreatic islet hyperplasia as well as a robust expression of insulin and glucagon.

[00125] In another aspect, the polyQ AR knock-in non-human mammal is used as a type 2 diabetes mellitus (T2D) model.

[00126] For example the mammal displays hyperglycemia, pancreatic hyperplasia, beta islet cell hyperplasia and / or insulin resistance.

[00127] For example, the mammal develops diabetic kidney disease and may eventually develop kidney failure. For example, the mammal displays renal function, optionally as measured by albumin to creatinine ratio in the urine.

[00128] In an embodiment, the mammal is a male mammal, optionally a male mouse.

[00129] Referring now to Fig. 11, it was shown that placing the AR-19Q mice on a high fat diet may accelerate the appearance of the metabolic syndrome and T2D phenotype. It was shown that AR-19Q mice on a high fat diet (HFD) gained more weight (e.g. at least 20%, at least 30%, at least 40% or at least 50% more weight) compared to AR-19Q mice on a standard fat diet (SFT). The HFD fed mice also presented fatty liver, higher blood glucose and increased ALT levels.

[00130] In further embodiments, the AR-19Q mice may be fed on special diets, for example high fat diets (HFD), to accelerate the appearance of disease phenotypes. For example, the AR- 19Q mouse fed on a HFD may develop hepatocellular carcinoma (HCC).

[00131] For example, the polyQ AR knock-in non-human mammal is fed on I) a high glucose diet, II) a high fat diet, III) a high fructose diet, IV) a high carbohydrate diet and / or V) a high protein diet.

[00132] PolyQ AR knock-in mammals can be selected that have modulations in some or all of the genes or markers, or particular levels of modulation for example as may be exhibited at different ages. The mammals can be used in screening assays described herein. Metabolic syndrome related liver disease

[00133] Another aspect provided herein is a knock-in non-human mammal for use as a liver disease model. Liver disease can develop subsequent to developing metabolic syndrome and as described in the Examples, the AR19Q mouse exhibits manifestations of liver disease.

[00134] In an embodiment, the polyQ AR knock-on non-human displays one or more of non- alcoholic fatty liver disease (NAFLD), chronic liver inflammation, non-alcoholic steatohepatitis (NASH), hepatitis, NASH fibrosis, NASH cirrhosis, hepatomegaly and / or hepatocellular carcinoma (HCC).

[00135] As described in Fig. 8, AR-19Q mice had increased alanine amino transferase (ALT) levels compared to wild type mice, which is indicated of hepatic damage. Also, AR-19Q mice all presented the fatty liver phenotype which progressed to nonalcoholic steatohepatitis (NASH) in older AR-19Q mice. Referring now to Fig. 9, AR-19Q mice presented other liver disease phenotypes such as NASH, macrovesicular steatosis, inflammation, and hepatocellular carcinoma (HCC).

[00136] In an embodiment, the polyQ AR knock-in non-human mammal has an increased level of ALT compared to a control age-matched mammal lacking the recombinant AR cassette, by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or greater than 100%.

[00137] In Fig. 10, gene expression analysis was conducted to assess genes involved in the pathogenesis of fatty liver. At least one of the following genes involved in lipid / fatty acid metabolism were upregulated in AR-19Q mice: Abcg1, Cd36, Fabp5, G6pdx, Lepr, Lpl, Mtor, Pparg, Srebf2 and / or Xbp1 and at least one of the following were down regulated: Abca1, Acaca, Acly, Acox1, Acsl5, Acsm3, Apoe, Cpt2, Cyp7a1, Fabp1, Fabp3, Fasn, Irs1, Ldlr, Nr1h2, Nr1h4, Ppara.

[00138] Table 1 and 2 below show respectively the up-regulated and down-regulated genes in fatty liver in AR-19Q mice. Table 1: Up-regulated genes in fatty liver in AR-19Q mice [Image disponible dans le document PDF, Image available in the PDF document] . ÷. 5 Table 2: Down-regulated genes in fatty liver in AR-19Q mice. [Image disponible dans le document PDF, Image available in the PDF document] · ·

[00139] At least one of the following genes involved in glucose metabolism were upregulated in AR-19Q mice: Adipor1, Akt1, Pck2 and / or Pik3ca and at least one of the following were down regulated: Adipor2, Foxa2, G6pc, Gck, Gsk3b, Gyk, Igf1, Pdk4, Pklr, Slc2a2, Slc2a4.

[00140] At least one of the following genes involved in inflammation were upregulated in AR- 19Q mice: II10, II6, Serpine 1 and / or Tnf.

[00141] At least one of the following genes involved in hepatocyte function were upregulated in AR-19Q mice: Ifng and / or Ptpn1.

[00142] At least one of the following genes also involved in liver disease were upregulated in AR-19Q mice: Cnbp, Ndufb6, Nfkb1 and / or Socs3 and at least one of the following were down regulated: Cyp2e1, Mlxipl, Ppargc1a, Rxra.

[00143] In an embodiment, the polyQ AR knock-in non-human mammal exhibits an up- regulation by at least 0.5 fold (e.g. a 50% increase), at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold or at least 14 fold, compared to a control age-matched mammal lacking the recombinant AR cassette, of at least one of the following genes: Abcg 1, Cd36, Fabp5, G6pdx, Lepr, Lpl, Mtor, Pparg, Srebf2, Xbp1, Adipor1, Akt1, Pck2, Pik3ca, II10, Il6, Serpine 1, Tnf, Ifng, Ptpn1, Cnbp, Ndufb6, Nfkb1 and / or Socs3.

[00144] In an embodiment, the polyQ AR knock-in non-human mammal exhibits a down- regulation by at least 0.5 fold, at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4, at least 5, at least 6 or at least 7 fold, compared to a control age-matched mammal lacking the recombinant AR cassette, of at least one of the following genes: Abca1, Acaca, Acly, Acox1, Acsl5, Acsm3, Apoe, Cpt2, Cyp7a1, Fabp1, Fabp3, Fasn, Irs1, LdIr, Nr1h2, Nr1h4, Ppara, Adipor2, Foxa2, G6pc, Gck, Gsk3b, Gyk, Igf1, Pdk4, Pklr, Slc2a2, Slc2a4, Cyp2e1, Mlxipl, Ppargc1a and / or Rxra.

[00145] In an embodiment, the mammal is a male mammal, optionally a male mouse.

[00146] PolyQ AR knock-in mammals can be selected that have modulations in some or all of the genes or markers, or particular levels of modulation for example as may be exhibited at different ages. The mammals can be used in screening assays described herein. Prostate disorder

[00147] In a further embodiment, the polyQ AR knock-in non-human mammal is a prostate disorder mouse model.

[00148] In an embodiment, the mammal is a male mammal, optionally a male mouse.

[00149] Referring now to Fig. 6, it was shown that AR-19Q mice presented benign as well as prostatic hyperplasia lymphocytic infiltration, compared to wild-type mice of similar age.

[00150] For example, the polyQ AR knock-in non-human mammal displays prostate abnormalities, for example prostatic hyperplasia or hypertrophy, which can be precursor disorders to prostate cancer.

[00151] As described in Fig. 7, certain genes commonly involved in prostate cancer are up- regulated or down-regulated in AR-19Q mice.

[00152] Table 3 and 4 below show respectively the up-regulated and down-regulated genes in prostate tissue in AR-19Q mice. Table 3: Up-regulated genes in prostate tissue in AR-19Q mice [Image disponible dans le document PDF, Image available in the PDF document] [Image disponible dans le document PDF, Image available in the PDF document] [Image disponible dans le document PDF, Image available in the PDF document] [Image disponible dans le document PDF, Image available in the PDF document] Table 4: Down-regulated genes in prostate tissue in AR-19Q mice [Image disponible dans le document PDF, Image available in the PDF document] [Image disponible dans le document PDF, Image available in the PDF document] --------------------------------------

[00153] In an embodiment, the polyQ AR knock-in non-human mammal exhibits an up- regulation by at least 0.5 fold, at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, compared to a corresponding wild type non-human mammal at least one of the following genes: Ccna1, Gnrh1, Ptgs1, Abcb1b, Cdkn2a, Dlc1, Egr3, Gpx3, II6, Lgals4, Loxi1, Sfrp1, Slc5a8, Socs3, Bcl2, Cdkn1a, Cdkn2a, Dab2ip, Dkk3, Egr3, Erg, Gpx3, Loxl1, Msx1, Ptgs1, Rbp1, Sfrp1, Socs3, Sox4 and / or Tfpi2.

[00154] In an embodiment, the polyQ AR knock-in non-human mammal exhibits a down- regulation by at least 0.5 fold, at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold or at least 4 fold compared to a corresponding wild type non-human mammal to at least one of the following genes: Ect2, Hal, Slc5a8, Arntl, Klkb1, Ptgs1, Abcb1a, Apc, Casp3, Ccna1, Cdh1, Gnrh1, Hmgcr, Ints6, Lgals4, Mapk1, Mto1, Nrip1, Pten, Rbm39, Sept7, Shbg and / or Vegfa,

[00155] For example, when the polyQ AR knock-in non-human mammal is an AR-19Q mouse, at least one of the following genes is up-regulated in mice having about 6 months: Ccna1, Gnrh1 and / or Ptgs1.

[00156] For example, when the polyQ AR knock-in non-human mammal is an AR-19Q mouse, at least one of the following genes is down-regulated in mice having about 6 months: Ect2, Hal and / or Slc5a8.

[00157] For example, when the polyQ AR knock-in non-human mammal is an AR-19Q mouse, at least one of the following genes is up-regulated in mice having about 12 months: Abcb1b, Cdkn2a, Dlc1, Egr3, Gpx3, ll6, Lgals4, Loxl1, Sfrp1, Slc5a8 and / or Socs.

[00158] For example, when the polyQ AR knock-in non-human mammal is an AR-19Q mouse, at least one of the following genes is down-regulated in mice having about 12 months: Arntl, Klkb1 and / or Ptgs1.

[00159] For example, when the polyQ AR knock-in non-human mammal is an AR-19Q mouse, at least one of the following genes is up-regulated in mice having greater than 18 months: Bcl2, Cdkn1a, Cdkn2a, Dab2ip, Dkk3, Egr3, Erg, Gpx3, Loxl1, Msx1, Ptgs1, Rbp1, Sfrp1, Socs3, Sox4 and / or Tfpi2.

[00160] For example, when the polyQ AR knock-in non-human mammal is an AR-19Q mouse, at least one of the following genes is down-regulated in mice having greater than 18 months: Abcb1a, Apc, Casp3, Ccna1, Cdh1, Gnrh1, Hmgcr, Ints6, Lgals4, Mapk1, Mto1, Nrip1, Pten, Rbm39, Sept7, Shbg and / or Vegfa.

[00161] In an embodiment, one or more genes shown in Fig. 7 are measured to assess prostatic changes in the polyQ AR knock0in non-human mammal.

[00162] PolyQ AR knock-in mammals can be selected that have modulations in some or all of the genes or markers, or particular levels of modulation for example as may be exhibited at different ages. The mammals can be used in screening assays described herein.

[00163] As the AR-19Q mice develop a prostatic phenotype including hyperplasia, the AR- 19Q mice may be manipulated, for example genetically by crossing to other mutant mice, introducing cancer promoting genes optionally dominant active forms using for example a modified adenovirus system for long-term expression or optionally by diet hormone replacement therapy or other treatment, to illicit prostate cancer. Such manipulated mice may also be used in screening assays described herein. Cardiovascular disease

[00164] In yet another embodiment, the polyQ AR knock-in non-human mammal is a cardiovascular disease model. For example, as described above, the mammal displays hypertension, obesity, diabetes and / or hyperlipidemia. For example, the mammal has elevated triglyceride and LDL cholesterol levels.

[00165] In an embodiment, the mammal is a male mammal, optionally a male mouse.

[00166] PolyQ AR knock-in mammals can be selected that have modulations in some or all of the genes or markers, or particular levels of modulation for example as may be exhibited at different ages. The mammals can be used in screening assays described herein. Glucose metabolism AMPK signaling, mitochondria energy metabolism and inflammatory processes

[00167] In addition, as described in Fig. 7, genes involved in glucose metabolism, AMPK signaling, mitochondria energy metabolism and inflammatory processes are altered. These genes may be relevant for each of the models described herein.

[00168] At least one of the following genes involved in glucose metabolism are up-regulated in AR-19Q mice: Aldoc, Bpgm, Eno1, Eno2, Hk2, Hk3, Pdk4, Pfkl, Pklr, Prps2 and / or Pygl; and at least one of the following genes involved in glucose metabolism are down-regulated: Acly, Aldob, Fbp1, Fh1, G6pc, Gck, Gys2, Mdh1b, Pck2 and / or Pgk2.

[00169] At least one of the following genes involved in AMPK signaling are up-regulated in AR-19Q mice: Acacb, Camkk1, Chrnb1, Cpt1b, Gpat2, Lepr, Pfkfb3, Ppp2r2b, Prkaa2 and / or Prkar1b; and at least one of the following genes involved in AMPK signaling are down- regulated: Acaca, Adra2a, Akt3, Chrna1, Gys2, Hnf4a and / or Prkag3.

[00170] At least one of the following genes involved in mitochondria energy metabolism are up-regulated in AR-19Q mice: Atp4a, Atp6v0d2, Atp6v1g3, Cox6a2 and / or Cox6b2; and at least one of the following genes involved in mitochondria energy metabolism are down-regulated Atp12a, Atp4b, Atp6v1c2, Atp6v1e2, Ndufa1, Ndufb2, Ndufb4, Ndufs6 and / or Uqcrq.

[00171] At least one of the following genes involved in inflammatory processes are up- regulated in AR-19Q mice: Bmp2, Ccl11, Ccl12, Ccl17, Ccl19, Ccl2, Ccl20, Ccl22, Ccl24, Ccl3, Ccl4, Ccl5, Ccl6, Ccl7, Ccl8, Ccl9, Ccr1, Ccr10, Ccr2, Ccr3, Ccr5, Ccr6, Csf1, Csf3, Cx3cl1, Cxcl1, Cxcl10, Cxcl13, Cxcl15, Cxcl5, Cxcl9, Cxcr3, Cxcr5, Fasl, II10ra, II10rb, II11, II13, II16, II17b, II1b, II1rn, II2rb, II2rg, II33, II4, II6ra, II6st, Ltb, Pf4, Spp1, Tnfrsf11b, Tnfsf11 and / or Tnfsf13b; and at least one of the following genes involved in inflammatory processes are down- regulated: Aimp1, Csf2, II15, IL17a, II21, II3 and / or II5ra.

[00172] By understanding the changes exhibited by polyQ AR knock-in mammals and recombinant cells, it is possible to interrogate these mammals and cells with test compounds, treatment regimens, diet changes etc that lead to disease amelioration and to identify factors which can negatively impact disease progression. Screening assays

[00173] Any of the described expression products, or changes described can be assessed in screening assays.

[00174] An aspect provided herein is a method of screening for putative treatment for treating metabolic syndrome, metabolic syndrome related liver disorder, type 2 diabetes mellitus and / or a prostate disorder comprising: a. i) contacting a test compound with a recombinant cell described herein or ii) administering one or more doses of the test compound to a knock-in non- human mammal described herein, and b. determining any effect of the test compound on the recombinant cell or knock-in mammal compared to suitable control, optionally untreated control cells or untreated age-matched control animals.

[00175] In an embodiment, untreated control cells are vehicle treated recombinant cells.

[00176] In an embodiment, untreated age-matched control animals are vehicle treated knock- in non-human mammal.

[00177] In an embodiment, the cell is an AR-19Q mouse cell and the knock-in non-human mammal is an AR-19Q mouse.

[00178] In an embodiment, the test compound or diet is for weight loss and an effect measured in the mammal is a decrease in weight, optionally a decrease in volume of fat pads, optionally epididymal or subcutaneous fat pads.

[00179] In an embodiment, the test compound is for treating hypertension and an effect measured in the mammal is a decrease in blood pressure, optionally systolic or diastolic blood pressure.

[00180] In an embodiment, the test compound is for treating T2D and an effect measured in the mammal is a decrease in glycemia, optionally post-prandial hyperglycemia and / or fasting hyperglycemia, and / or a decrease in insulin resistance, optionally measured by a decrease in glucose tolerance test (GTT).

[00181] In an embodiment, the test compound for treating T2D and an effect measured in the mammal is no change (e.g. no increase) in pancreatic volume, optionally as measured by histopathology, or an increase in insulin content in the pancreas and / or increase in glucagon in the pancreas.

[00182] In an embodiment, the test compound is for treating hypertriglyceridemia in the mammal and an effect measured is a decrease in triglyceride levels, optionally a decrease in lipoprotein lipase (LPL) levels.

[00183] In an embodiment the test compound is for treating liver disease related to metabolic syndrome in the mammal and an effect measured is improved liver function, optionally as determined by liver enzyme activity, optionally alanine aminotransferase (ALT) AST, ALP and / or GGT levels, and / or a decrease in liver volume and content, optionally as measured by histopathology or ultrasound.

[00184] In an embodiment the test compound is for treating prostatic disease, optionally prostatic hyperplasia, prostatic hypertrophy, prostatic neoplasia, optionally prostatic interepithelial neoplasia or prostatic interepithelial neoplasia, prostatic neoplasia in situ, prostatic hyperplasia lymphocytic infiltration, and the effect measured is a change in prostate morphology and / or an increase in volume, optionally as measured by microscopy.

[00185] In an embodiment, the method comprises administering the test compound to the polyQ AR knock-in cell or mammal and measuring the levels of gene expression as described above, for example by conducting gene expression analysis as described in Fig. 7 and 10. For example, in genes that are up-regulated in polyQ AR knock-in cells or mammals, the effect measured for a test compound is a decrease in expression of said genes compared to an untreated control. In genes that are down-regulated in polyQ AR knock-in cells or mammals, the effect measured for a test compound is an increase in expression of said genes compared to an untreated control.

[00186] For example, the test compound is for treating prostatic disease and an effect measured effect is a decrease in expression of by at least 0.5 fold, at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold or at least 12 fold of at least one of the following genes: Ccna1, Gnrh1, Ptgs1, Abcb1b, Cdkn2a, Dlc1, Egr3, Gpx3, Il6, Lgals4, Loxl1, Sfrp1, Slc5a8, Socs, Bcl2, Cdkn1a, Cdkn2a, Dab2ip, Dkk3, Egr3, Erg, Gpx3, Loxl1, Msx1, Ptgs1, Rbp1, Sfrp1, Socs3, Sox4, Tfpi2, Aldoc, Bpgm, Eno1, Eno2, Hk2, Hk3, Pdk4, Pfkl, Pklr, Prps2, Pygl, Acacb, Camkk1, Chrnb1, Cpt1b, Gpat2, Lepr, Pfkfb3, Ppp2r2b, Prkaa2, Prkar1b, Atp4a, Atp6v0d2, Atp6v1g3, Cox6a2, Cox6b2, Bmp2, Ccl11, Ccl12, Ccl17, Ccl19, Ccl2, Ccl20, Ccl22, Ccl24, Ccl3, Ccl4, Ccl5, Ccl6, Ccl7, Ccl8, Ccl9, Ccr1, Ccr10, Ccr2, Ccr3, Ccr5, Ccr6, Csf1, Csf3, Cx3cl1, Cxcl10, Cxcl13, Cxcl15, Cxcl5, Cxcl9, Cxcr3, Cxcr5, Fasi, Il10ra ,Il10rb, Il11, Il13, Il16, Il17b, Il1b, Il1rn, Il2rb, Il2rg, Il33, Il4, Il6ra, Il6st, Ltb, Pf4, Spp1, Tnfrsf11b, Tnfsf11 and / or Tnfsf13b, compared to an untreated control.

[00187] For example, the test compound is for treating prostatic disease and an effect measured is an increase in expression by at least 0.5 fold, at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold or at least 4 fold to at least one of the following genes: Ect2, Hal, Slc5a8, Arntl, Klkb1, Ptgs1, Abcb1a, Apc, Casp3, Ccna1, Cdh1, Gnrh1, Hmgcr, Ints6, Lgals4, Mapk1, Mto1, Nrip1, Pten, Rbm39, Sept7, Shbg, Vegfa, Acly, Aldob, Fbp1, Fh1, G6pc, Gck, Gys2, Mdh1b, Pck2, Pgk2, Acaca, Adra2a, Akt3, Chrna1, Gys2, Hnf4a, Prkag3, Atp12a, Atp4b, Atp6v1c2, Atp6v1e2, Ndufa1, Ndufb2, Ndufb4, Ndufs6, Uqcrq, Aimp1, Csf2, II15, IL17a, II21, II3 and / or II5ra, compared to an untreated control.

[00188] For example, the test compound is for treating liver disease and an effect measured is a decrease by at least 0.5 fold, at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold or at least 14 fold, to at least one of the following genes: Abcg 1, Cd36, Fabp5, G6pdx, Lepr, Lpl, Mtor, Pparg, Srebf2, Xbp1, Adipor1, Akt1, Pck2, Pik3ca, II10, II6, Serpine 1, Tnf, Ifng, Ptpn1, Cnbp, Ndufb6, Nfkb1 and / or Socs3, compared to an untreated control.

[00189] For example, the test compound is for treating liver disease and an effect measured is an increase by at least 0.5 fold, at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4, at least 5, at least 6 or at least 7 fold, to at least one of the following genes: Abca1, Acaca, Acly, Acox1, Acsl5, Acsm3, Apoe, Cpt2, Cyp7a1, Fabp1, Fabp3, Fasn, Irs1, Ldlr, Nr1h2, Nr1h4, Ppara, Adipor2, Foxa2, G6pc, Gck, Gsk3b, Gyk, Igf1, Pdk4, Pklr, Slc2a2, Slc2a4, Cyp2e1, Mlxipl, Ppargc1a and / or Rxra, compared to an untreated control.

[00190] For example, the mammal or cell is an AR-19Q mouse or cell and the control is an AR-19Q mouse or cell that was not treated / contacted with said putative treatment. For example, if liver disease related genes, normally up-regulated in AR-19Q mice, exhibit a decrease in expression following administering of a test compound (e.g. a decrease compared to an untreated AR-19Q mouse), then the compound is considered to be effective for treating liver disease.

[00191] In other embodiments, the effect measured of the test compound is a similarity of physiological / functional parameters, parameters, gene expression levels, etc. of the polyQ AR knock-in non-human mammal compared to a wild type age controlled non-human mammal. For example, if following treatment with a test compound for treating obesity, the weight of the AR- 19Q mouse decreases such that it is similar to the weight of a wild type age controlled mouse of the same strain, then the compound is considered to be effective for treating obesity.

[00192] In an embodiment, the test compound is tested in combination with an existing therapeutic.

[00193] In another embodiment, the test compound is an existing therapeutic.

[00194] In an embodiment, the effect of the test compound is determined after 1, 7, 14, 21, 28 days and / or 1, 2, 3, 6, and / or 9 months of treatment.

[00195] As described herein the polyQ non-human mammal models described herein may be validated as disease models, for example to confirm that the models correlate with clinical trials.

[00196] For example, the AR-19Q mice are treated with therapeutic compounds, for example elafibranor, MSCD-0602L or obeticholic acid, to assess the AR-19Q mouse disease model correlation with clinical trials. AR-19Q mice exhibiting a NAFLD Activity Score of greater than 4 are determined suitable for treatment.

[00197] In an embodiment, the polyQ non-human mammal model is used as a pre-clinical model, for example for in-vivo testing of elafibranor, MSCD-0602L or obeticholic acid, or other therapeutic compounds and / or diets for treating metabolic syndrome, type 2 diabetes mellitus, metabolic syndrome related liver disease, prostatic disease and carbvascular disease. For example, the models may be used to identify biomarkers involved in the disease disclosed herein.

[00198] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[00199] The following non-limiting examples are illustrative of the present disclosure: Examples Example 1: AR-19Q MOUSE The Androgen Receptor Protein and its Polyglutamine Tract

[00200] The X-linked androgen receptor (AR) protein is a member of the nuclear receptor superfamily14,15. It is a ligand-inducible transcription factor containing a polymorphic N-terminal region, a central DNA-binding domain (DBD) and a C-terminal ligand-binding domain (LBD)16-18 (Figure 1A).

[00201] The structurally flexible N-terminal domain (NTD)29,30, harbours a long polymorphic polyglutamine (polyQ) tract (n=11-33). Encoded by a genomic CAG repeat, it helps modulate AR transcriptional activity; ARs with shorter polyQ tracts are the most transcriptionally active31- 34. This functional polymorphism has now been well documented to participate in all facets of androgen physiology35 and is an independent factor affecting almost every aspect of both AR reproductive and non-reproductive biology both in males and females (PubMed >1000 references). Much emphasis has been placed upon the polyQ tract as risk factor for prostatic hypertrophy36-40 and carcinoma41,42.

[00202] Trinucleotide repeats are genomically very unstable, i.e., they can easily expand or contract in length in somatic cells43,44. This exceeds, by many orders of magnitude, the comparative genetic stability of common nucleotide sequences. Using laser microdissection significant somatic AR CAG repeat instability in prostate 45,46 and breast 47 pathology including short AR CAG repeat lengths were documented, and polyGln tract deletions were found, which would result in a superactive AR.

[00203] Using proteomics coupled to a systems biology approach the first AR interactome was constructed. In the process new and novel functional properties of the AR were redefined and validated, beyond its well-recognized role as classical ligand dependent transcription factor. It is clear from this data that the AR contributes to cell biology by directly affecting RNA metabolism (initiating RNA splicing events and modulating RNA translation by ribosomes), DNA methylation and proteasomal interaction48,49. AR-19Q KNOCK-IN MOUSE: A METABOLIC SYNDROME MOUSE

[00204] The mouse AR shares over 90% homology with the human ortholog, but interestingly lacks the CAG encoded-polyglutamine tract; instead mice possess a mixed CAG / CAA / CAC-glutamine / histidine tract. To study AR functionality in a more structured fashion, including the newly identified AR properties, a knock-in mouse model that would have the equivalent of human polyQ tract was created. This would allow to study AR functions in more controlled experimental conditions and at the same time assess the impact of polyQ in overall reproductive physiology. A similar attempt to humanize the AR in mouse has been done previously; a knock-in mouse model was created by replacing all of mouse exon one with human exon one 60, a rather large and drastic change considering both exon ones have more than 400 amino acids. This particular whole exon 1 knock-in mouse model (21Q tract) does not have an obvious phenotype, but when crossed with the TRAMP prostate mouse model it showed that the presence of a CAG tract accelerates prostate neoplasia but minimally so 61,62. A more precise humanized mouse AR model was made by replacing only the mouse 19- glutamine / histidine tract with the human pure 19-glutamine tract by knock-in methodology. Generating the Targeting vector for transfection of ES cells. To construct the targeting vector, sequences from the ploxPneo-1 plasmid and pPGKneo-NTR- tkpA plasmid were used. The pPGKneo-NTR-tkpA plasmid encodes the neomycin resistance (neor) and herpes simplex virus-thymidine kinase (hsv-tk) selectable genes with the 5' nontranslated region (NTR) of encephalomyocarditis virus inserted in between. To generate the targeting vector, the PGK-neo cassette in ploxPneo-1 was replaced with the neo-NTR-hsv-tk sequences from pPGKneoNTR-tkpA as follows: Five micrograms of ploxPneo-1 plasmid were partially digested with Xba I (37°C / 16 h). Xba I was heat inactivated for 20 min at 65°C and the ends were blunted using Klenow polymerase (37°C / 10 min) The DNA was then completely digested with Blp I (37°C / 16 h). Five micrograms of pPGKneoNTR-tkpA plasmid was digested with Nsi I (37°C / 16 h). Enzyme was inactivated by heating at 65° for 20 min and the ends were blunted with T4 polymerase (RT / 5 min). The DNA was then digested with Blp I (37°C / 16 h). The Nsi I – Blp I fragment was ligated to the Xba I – Blp I vector backbone using T4 DNA ligase (RT / 4 h), such that the neo-hsv-tk fragment removed from pPGKneoNTR-tkpA was introduced into the ploxPneo-1 vector, flanked by two loxP sites. Ligation was confirmed by restriction enzyme digestion. Constructing 3' and 5' mouse AR genomic DNA clones. Mouse AR mRNA (NM_013476) was used to search Genbank using BLAST. A 17202 base pair long Sanger assembly f162083 was found. f162083 is the contig of a number of different BAC clones, including RPCI-23-191K6 (191K6 BAC clone) and RPCI-23-259K23 (259K23 BAC clone), which contain different mouse AR genomic DNA fragments. 191K6 BAC clone contains the 5' untranslated region and its sequenced region starts at bp 1311 of f162083 and ends at bp 1775. 259K23 BAC clone starts at exon 1 and continues up to exon eight of the mouse AR gene. Its sequence region starts at bp 4470 of f162083. The ORF of the AR starts at bp 4470 of f162083. Verification of the clones for AR sequences was carried out by PCR. 191K6 or 259K23 were used as DNA template and a set of specific primers used: TR12404A (FWD: 5'-ACGAGGCCGGCACCATGCAACTTCTC-3') (SEQ ID NO: 1) and TR12404B (REV: 5'-CTTCGTCCAGGGGAAGACCTTT-3') (SEQ ID NO: 2). PCR was done using Vent DNA polymerase in a total reaction volume of 50 µl. After initial denaturation at 94°C / 45 s, 36 cycles (94°C / 45 s; 55°C / 45 s; 72°C / 1 min) were carried out. The product of PCR was predicted to be a 317 bp fragment. The 191K6 BAC clone contains 4 Bgl II sites according to the f162083 assembly, located at bp 891, bp 1651, bp 6310, and at bp 12601. The interval from 1651 to 6310 (4.6 kb) includes the 5' UTR, exon 1 and 300 bp downstream of exon 1 of the mouse AR gene. The interval from 6310 to 12601 (6.2 kb) includes intron 1 sequence. The 191K6 BAC clone was digested with Bgl II (37°C / 16 h) and run in a 1% agarose gel. The 4.6 kb and 6.2 kb bands were excised and the DNA extracted. Confirmation that the right bands had been excised was done by Southern blot using the PCR fragments of 191K6 and 259K23 clones as probes. The 4.6 kb piece is referred to as the 5'-end AR sequence. The 6.2 kb piece is referred to as the 3'-end AR sequence. Both pieces, 4.6 kb and 6.2 kb, were subcloned into the pcDNA3 vector. pcDNA3 was opened by digesting it with Bam HI, dephosphorylated with Calf Intestinal Alkaline Phosphatase to remove the 5'-phosphate group. The product of Bam HI digestion was run in agarose gel containing guanosine. Band was excised and the DNA extracted. The Bgl II – Bgl II 5'-end was then ligated into pcDNA3. Clones were screened by Southern blot using the 191K6 PCR product as probe. Southern blot results were confirmed by restriction enzyme digestion. Similar procedure was used to subclone the 3'-end into the pcDNA3 vector. To confirm that the 3'-end and 5'-end AR fragments were intact in pcDNA3, positive clones were sequenced using the Thermo Sequenase Radiolabeled Terminator Cycle sequencing kit. The Sp6 primer (REV: 5'-AGCATTTAGGTGACACTATAGAATAG-3') (SEQ ID NO: 3), downstream of Bam HI (pcDNA3 cloning site) was used for sequencing. Creating the 19 CAG tract A pSVhAR.BHEX plasmid containing 19 CAG repeats and 1 CAA, which had been created in the laboratory, was used as template for PCR to amplify the CAG repeat. Specific primers flanking the CAG tract were used: Primer 1.15A (FWD: 5'-GTTCCAGAGCGTGCGCGAAGT-3') (SEQ ID NO: 4). Primer 1.15B (REV: 5'-TGGGGCCTCTACGATGGGCTT-3') (SEQ ID NO: 5). The PCR reaction consisted of an initial denaturation step at 94°C / 45 s followed by 36 cycles (94°C / 45 s; 55°C / 45 s; 72°C / 1 min). The PCR product is loaded into a 2.3% agarose gel and the sample is fractionated. The right product is approximately 230 bp. The band is excised and the DNA extracted and then digested with Alw261 (37°C / 16 h), which site is downstream of the CAG repeat. Klenow polymerase was used to fill in using only dATP / dGTP. The end was blunted using Mung Bean Nuclease (30°C / 30 min) The DNA was the digested with HpyCH4V (37°C / 16 h), upstream of the human CAG repeat. These procedures produced a blunt ended fragment containing only 19 CAG repeats, without flanking sequences. Changes in the CAG repeat length due to PCR artifacts gave as results fragments with 18 CAG repeats, which upon ligation with the 19 CAG repeat fragment produced an insert with 37 CAG repeats. In order to subclone different CAG repeat lengths into the 5'-AR, the 5'-end AR 0 CAG cloned in pUC19 was opened at the Xho I site (37°C / 16 h), filled in using Klenow polymerase with dTTP / dCTP only (37°C / 15 min), and then blunted using Mung Bean nuclease (30°C / 30 min) in order to keep the ORF. Subsequently, different length CAG fragments were cloned into the opened vector. Resulting clones were screened by restriction enzyme digestion. Results indicated that there were two clones containing 19 CAG repeats, a single clone containing 18 CAGs, and a single clone containing 37 CAGs in the correct orientation and reading frame. Introducing the 3'-end AR fragment into pPGKneoNTR-tkpA-plox The 3'-end of the AR was obtained by digesting the pcDNA3-3'end clone with Kpn I (37°C / 16 h) restriction site in pcDNA3 and SnaB I (37°C / 16 h)site in the AR. After digestion the Kpn I site was blunted with T4 DNA polymerase (RT / 15 min). The pPGKneoNTR-tkpA-plox vector was opened with Xho I (37°C / 16 h) and then blunted. The 3'-end AR released from pcDNA3 is then subcloned into the pPGKneoNTR-tkpA-plox to produce a new construct, pPGKneoNTR-tkpA-plox-3'- AR, of approximately 11 kb. (Figure 1B) Mouse 129 / SV embryonic stem (ES) cells were electroporated with the AR 19 CAG knock-in vector. PCR was used to confirm homologous recombination had taken place in certain ES clones. Specific 19 CAG ES clones were electroporated with Cre recombinase. Following PCR screening a number of ES clones in which the neo-tk cassette had been correctly excised were identified. Positive ES cells were used for blastocyst injections and chimeric mice obtained. 25 chimeras from four 19-CAG clones were obtained. Only males with ≥80% chimerism were mated with C57BL / 6 females. Females of the F1 generation were genotyped and positive females were back-crossed with C57BL / 6 males. Back-crossing continued until the appearance of positive males for the AR-19Q allele in the F7 generation (Figure 1C / 1D). Males positive for the AR-19Q allele were mated with heterozygous females and their litters genotyped. Homozygous females were obtained in F10. Since then it has been possible to breed pure homozygous AR-19Q knock-in mice. Constructing the 0 CAG repeat In order to make 0 CAG in exon 1 of the mouse AR gene, PCR methodology was used. Two specific primers were made: Primer #1 (FWD: 5'-ACGAGGCCGGCACCATGCAACTTCTCGAGGTAATCTCCGAAGGCAGC- 3') (SEQ ID NO: 6) which starts 26 bp upstream of CAG / CAA / CAC repeat, skips over the CAG / CAA / CAC repeat and continues 21 bp downstream of it. One single T bp was change to C to create an Xho I site for future manipulations. Primer #2 (REV: 5'-CTTCGTCCAGGGGAAGACCTTT-3') (SEQ ID NO: 2) 191K6 or / and 259K23 BAC clones were used as DNA templates. PCR reaction consisted of an initial denaturation at 94°C / 2 min, followed by 35 cycles (96°C / 45 s, 60°C / 30 s, 72°C / 1 min). PCR product lacked the mouse CAG / CAA / CAC repeat. Introducing 0 CAG fragment into the 5'-end AR fragment The 5'-end fragment (4.6 kb) was taken out from the pcDNA3 plasmid and introduced into the pUC19 cloning vector. Ten micrograms of pcDNA3 were digested with Kpn I and Not I (37°C / 16 h), and blunted with T4 DNA polymerase (RT / 5 min). pUC19 was opened with Sma I (25°C / 16 h) and then dephosphorylated with CIP (37°C / 15 min). The Kpn I / Not I fragment from pcDNA3 was ligated into the pUC19 vector using T4 DNA ligase (RT / 16 h). The pUC19 vector containing the 5'-end fragment was the digested with Bsm I (sticky ends) (65°C / 5 h) and partially with Nae I (blunt) (37°C / 2 h). The PCR fragment lacking the CAG / CAA / CAC repeat (0 CAG) was also digested with Bsm I and Nae I, and then ligated into the 5'-end fragment in pUC19 using T4 DNA ligase (RT / 16h). A positive clone was sequenced using the TR12404B primer (REV: 5'- CTTCGTCCAGGGGAAGACCTTT-3') (SEQ ID NO: 2) and also a second primer called preCAG (FWD: 5'-CCAGGCTTAAGCAGCTGCTCCG-3') (SEQ ID NO: 7), which anneals upstream of the Xho I site that was created. Sequencing results indicated that the positive clone had three Bsm I – Nae I insert, that correspond to one 0 CAG and two multiple CAG repeats. In order to eliminate the two multiple CAG repeats, the clone was digested with Avr II (37°C / 16h), giving as a result the obtention of four pieces. Two small bands (<500 bp) corresponding to the extra inserts, which had to be eliminated. The two largest bands corresponds to the vector itself (6.3 kb) and to the correct insert (1.6 kb), and they were excised, the DNA extracted, dephosphorylated with CIP (37°C / 15 min) and religated to each other using T4 ligase (RT / 16 h). To confirm that the ligation worked properly, resulting clones were sequenced using the primer preCAG (FWD: 5'-CCAGGCTTAAGCAGCTGCTCCG-3') (SEQ ID NO: 7), which is upstream of the Xho I site. Results indicated the presence of a unique Xho I site and lacking of the CAG / CAA / CAC repeat. (Figure 2A) Creating various lengths of CAG tract Introducing different lengths of CAGs into the targeting vector The 5'-end AR clones in pUC19, which included the different length CAG repeats, were digested with EcoRV (37°C / 16 h), run in a 2% agarose gel and then extracted. The linearized vector was then digested with Sac I (37°C / 16 h), and then the ends blunted with T4 polymerase (RT / 5 min). The product was run in a 2% gel and two bands were observed. The upper band (4.6 kb) that corresponded to the 5'-end AR fragment containing the CAG repeat was extracted. The extracted fragment, either containing 0, 19 or 37 CAG or the mixed repeats, 19 CAG / CAC or 40 CAG / CAC, was subcloned into the pPGKneoNTR-tkpA-plox-3'-end AR vector that had been digested with BamHI and blunted with Klenow polymerase. Ligation was done using T4 DNA ligase (RT / 16 h) Clones were screened by restriction enzyme digestion. Confirmation of the right CAG tract length was done by sequencing.

[00205] The 0Q tract was lethal in male and female mice and only females heterozygous for the expanded 39Q tract survived.

[00206] To assess the impact of introducing the 19Q tract into the mouse AR (mAR), a 19Q mAR construct was used in transactivation assays; the mAR-19Q construct demonstrated slightly higher transactivation properties than the wild-type mAR vector at identical androgen ligand concentrations (Figure 3).

[00207] The stability of the CAG repeat of the mouse has been analyzed and shown to be stable. 19Q Phenotype

[00208] The 19Q mice phenotype can be best described as a mice counterpart to the Metabolic Syndrome or MetS, reflecting the dependence on androgens as well as the AR 19Q tract; to date castrated mice have not expressed the phenotype. Females have not yet been challenged with estrogen depletion (castration) or androgen supplementation. No reproducible phenotype has been observed in females. Penetrance of the phenotype in males is very high (>95%). All mice are fed completely normal chow. The first obvious characteristic is the appreciable gain of weight, commencing at 3 months (Figure. 4A). The extent of obesity and weight gain is significant, with the 19Q mice displaying 40% greater weight vs. C57BL5 mice (Figure 4B). To assess obesity status we weighed fat pads, epididymal, subcutaneous, and brown adipose tissue vs. age-matched controls (Figure 4C)The 19Q mice first appear to have post-prandial hyperglycemia and eventually develop moderate fasting hyperglycemia, as seen from glucose tolerance tests and elevated blood glucose levels (Figures 4D / 4E). Moreover, abdominal ultrasound reveals hepatomegaly and features of a fatty liver, without ascites fluid accumulation. The mice soon after have documented systolic hypertension that persists throughout their lifespan (Figure 4F). Example 2: Metabolic Syndrome Mouse Model and Type 2 Diabetes (T2D) and islet β-cell failure Model Pioneer studies1 clearly demonstrated that T2D is a "in continuum" disease entity that primarily starts with peripheral insulin resistance with normal serum glucose dynamics, although at the expense of higher circulating insulin levels. This demand is reflected by higher insulin production and by the sine qua non finding of islet hyperplasia. There are very few pathological sequelae at this stage and it is only when insulin demands are not met that postprandial hyperglycemia, and eventually frank hyperglycemia, is seen. Late stage disease is marked by significant loss of insulin production and islet accumulation of amyloid material. The critical element responsible for initiation of disease entities associated with diabetes is islet β-cell failure. Thus, insulin resistance in target tissues, combined with a relative deficiency of insulin secretion from pancreatic islet β-cells, are the major features of T2D2. Under physiological conditions, when insulin sensitivity decreases, insulin secretion increases to maintain normal glucose levels; when insulin secretion fails to compensate, abnormal glucose tolerance develops and further progression to overt T2D is characterized by severe β-cell dysfunction3-5. Islet cell dysfunction remains the pivotal event in T2D. The molecular mechanisms responsible for islet β-cell failure remain elusive and few models offer the opportunity to make progress towards unraveling its pathophysiology. The mouse model described in Example 1 is relevant to T2D, is more physiological (lacks significant genetic alterations, no dietary manipulation is needed, no hyperphagia is seen) and is a clear departure from other models59. It is proposed that glucose metabolism in the β islets is accelerated, provoked by either insulin resistance and / or hyperglycemia itself, leading to excessive mitochondrial ROS, cell stress, metabolic alterations and subsequent cellular damage, most notably DNA damage that eventually leaves cells incapable of normal function. Such an analysis in a comparative and chronological fashion will be pursued so as to define molecular underpinnings at various stages of islet β-cell function: from the earliest stage prior to the establishment of islet cell hyperplasia, to hyperplasia with normal glycemia, and finally to dysfunctional hyperplasia with hyperglycemia. Four groups of mice will be used: Wild-type C57BL / 6 on normal or high glucose diets; and 19Q on normal or high glucose diets. Dietary manipulation will also allow us to identify both the islet β-cell molecular responses (or the lack thereof in 19Q mice) to heightened glucose loads (glucose stress), and the molecular mechanisms associated with possible long- term decline in islet β-cell functionality, especially in the 19Q mice. The studies will entail in situ islet cells immunohistochemistry, and isolating islets to perform expression experiments, mass spectroscopy metabolomics and ultra-deep DNA sequencing. Islets will be isolated using a classical approach at 3, 6, and 18 months. All mice will have routine blood sugars drawn and GTTs (oral and intraperitoneal) performed with concomitant systemic insulin levels determined at the same time points. Results The mouse model described in Example 1 was used to investigate the AR-19Q mice as a model for T2D. As described herein, that 19-Q mouse provides a suitable model for T2D. As mentioned in Example 1, the 19Q mice first appear to have post-prandial hyperglycemia and eventually develop moderate fasting hyperglycemia, as seen from glucose tolerance tests and elevated blood glucose levels Equally important are the pancreases of middle-aged 19Q mice, which have significant islet hyperplasia (Figure 5), suggesting peripheral insulin resistance. Blood glucose levels are monitored on a biweekly basis on all 19Q mice with age matched controls, as well as circulating triglycerides and non-esterified fatty acids levels (monthly). Insulin resistance is formally established by analyzing in vivo insulin-induced phosphorylation of insulin receptor, IRS1 and Akt (T308 and S473) in liver, skeletal muscle and adipose tissue homogenates by western blot using corresponding commercially available antibodies (monthly). Furthermore, to assess obesity status mice fat pads, both epididymal and subcutaneous will be weighed, as well as brown adipose tissue vs. age-matched controls (monthly). In addition, liver and pancreas will be weighed and pancreas analysis for insulin and glucagon content by immunofluorescence and quantitatively using Metabolic Syndrome and Assay (MSA) kits (months 3, 6 12, 18, 24); white adipose tissue will be assessed for leptin and adiponecting contents (MSA) (months 3, 6 12, 18, 24). Circulating insulin leptin and adiponectin levels will be assessed at different ages using MSA kits while triglycerides and non-esterified fatty acids (NEFAs) will be determined (months 3, 6 12, 18, 24). Immunohistochemistry in situ Islet cell quantitative immunochemistry techniques will be employed in situ in the 4 groups (C57BL / 6 or 19Q mice, on normal or high glucose diets). Insulin, glucagon and glucokinase content will be analyzed in conjunction with proteins supporting insulin promoter activity (PDX1, MafA)13. Other assays will include: i) oxidative damage analysis [cellular ROS detection kit e.g. MitoSOX staining of mitochondrial ROS]; ii) assessment of antioxidant / oxidative stress response [SOD1, Ape1 / Ref-1 (or NM23-H1 and MPG), thioredoxin, thioredoxin reductase and protein disulfide isomerase]; iii) ER stress [ER stress antibody sampler kit]; iv) oxidative DNA damage markers (8-OxoG, γ-H2AX and protein carbonyls); vi) induction of DNA transcriptional repair (Xbp Xbd); and vii) standard apoptosis (annexin v / propidium iodide). Thus baseline immunohistochemical data (normal islets, normal insulin / glucagon content of C57BL / 6 animals on a normal diet) will be established. With the C57BL / 6 animals on a high glucose diet it is expected to observe potentially enhanced insulin content, but not any excessive oxidative damage nor DNA damage over time (1-24 months). On the other hand, it is expected that very young19Q mice will have features similar to their C57BL / 6 counterparts, but rapidly develop islet cell hyperplasia with exaggerated insulin production, and later on, oxidative stress, ER stress and DNA damage that may be accelerated in 19Q mice on a high glucose diet; in both latter cases, islet <semantics>β<annotation encoding="application / x-tex">\beta< / annotation>< / semantics>-cell failure will be defined as inadequate insulin production. Additional immunohistochemistry studies will be prompted by ancillary data obtained from the expression studies and / or metabolomic studies described below. Pancreatic Islet Studies a) Insulin Production. Pancreatic islets will be isolated from all 4 groups (C57BL / 6 or 19Q mice, on normal or high glucose diet) at time points 3, 6, and 18 months. After 2 days in culture, islets will be subjected to glucose-induced insulin secretion assays, using 5-16.7 mM glucose for 1h. These experiments will be repeated in the presence of GLP-1 (10 nM). Insulin released in the medium will be normalized according to insulin content and both quantified using the appropriate MSA kit. b) Gene Expression Studies. Perusing the NCBI database hundreds of gene expression (GEO) datasets pertinent to diabetes have been identified, but fewer derived from pancreatic islets themselves. Though the latter are informative, many limitations were found with regards to experimental approaches in characterizing gene expression profiles: most or all are cross sectional analysis, many are sporadic humans cases (mostly cadavers after trauma), or from islet cell lines. In T2D rodent models there is a scarcity of expression data obtained in a direct comparative fashion (wild-type vs. rodent model), with alternative diets, coupled with time frames reflecting islet developmental / maturation studies, hyperplasia and eventual islet dysfunction. By combining studies of islet gene expression, further information will be known on genes and pathways that are responsible for islet hyperplasia and, more importantly, for islet failure during T2D development. Thus, full gene expression data assessment will be performed on freshly isolated pancreatic islets from the 4 mouse groups [(normal or high glucose diet) at time points 3, 6, and 18 months], using the Affymetrix Mouse 2.0 ST microarray platform. This microarray platform will allow to assess over 26,000 annotated transcripts, including almost 6,000 non-coding RNAs (miRNAs and IncRNAs). Simultaneously, this array platform will also allow to study alternative RNA splicing. All 4 groups of animals will be assessed, but only 3 time points (3, 6, and 18 months) will be chosen to carry out the analysis. Approximately 3-5 million islet cells per mouse group and time point are expected to be isolated, which would be adequate to obtain sufficient input RNA for analysis. After assessment of microarray studies, islet cell expression data will be supplemented with RNA Sequencing experiments of the same samples, but at selected time points (3, 6, and 18 months). 15 References for Examples 1 and 2 1. DeFronzo RA, Bonadonna RC, Ferrannini E. Pathogenesis of NIDDM. A balanced overview. Diabetes care 1992;15:318-68. 2. Hameed I, Masoodi SR, Mir SA, Nabi M, Ghazanfar K, Ganai BA. Type 2 diabetes mellitus: From a metabolic disorder to an inflammatory condition. World journal of diabetes 2015;6:598-612. 3. Fonseca VA. Defining and characterizing the progression of type 2 diabetes. Diabetes care 2009;32 Suppl 2:S151-6. 4. Kahn SE, Cooper ME, Del Prato S. 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The CAG and GGC microsatellites of the androgen receptor gene are in linkage disequilibrium in men with prostate cancer. Cancer research 1995;55:1937- 40. 40. Stanford JL, Just JJ, Gibbs M, et al. Polymorphic repeats in the androgen receptor gene: molecular markers of prostate cancer risk. Cancer research 1997;57:1194-8. 41. Gottlieb B, Beitel LK, Wu JH, Trifiro M. The androgen receptor gene mutations database (ARDB): 2004 update. Human mutation 2004;23:527-33. 42. Hardy DO, Scher HI, Bogenreider T, et al. Androgen receptor CAG repeat lengths in prostate cancer: correlation with age of onset. J Clin Endocrinol Metab 1996;81:4400-5. 43. Ferro P, Catalano MG, Raineri M, et al. Somatic alterations of the androgen receptor CAG repeat in human colon cancer delineate a novel mutation pathway independent of microsatellite instability. Cancer genetics and cytogenetics 2000;123:35-40. 44. Zhang L, Leeflang EP, Yu J, Arnheim N. Studying human mutations by sperm typing: instability of CAG trinucleotide repeats in the human androgen receptor gene. Nature genetics 1994;7:531-5. 45. Alvarado C, Beitel LK, Sircar K, Aprikian A, Trifiro M, Gottlieb B. Somatic mosaicism and cancer: a micro-genetic examination into the role of the androgen receptor gene in prostate cancer. Cancer research 2005;65:8514-8. 46. Sircar K, Gottlieb B, Alvarado C, et al. Androgen receptor CAG repeat length contraction in diseased and non-diseased prostatic tissues. Prostate Cancer Prostatic Dis 2007;10:360-8. 47. Gottlieb B, Alvarado C, Wang C, et al. Making sense of intratumor genetic heterogeneity: altered frequency of androgen receptor CAG repeat length variants in breast cancer tissues. Human mutation 2013;34:610-8. 48. Paliouras M, Zaman N, Lumbroso R, et al. Dynamic rewiring of the androgen receptor protein interaction network correlates with prostate cancer clinical outcomes. Integr Biol 2011;10:1020-32. 49. Zaman N, Giannopoulos PN, Chowdhury S, et al. Proteomic-coupled-network analysis of T877A- androgen receptor interactomes can predict clinical prostate cancer outcomes between White (non- Hispanic) and African-American groups. PLoS One 2014;9:e113190. 50. Hiort O. The differential role of androgens in early human sex development. BMC medicine 2013;11:152. 51. Tsouko E, Khan AS, White MA, et al. Regulation of the pentose phosphate pathway by an androgen receptor-mTOR-mediated mechanism and its role in prostate cancer cell growth. Oncogenesis 2014;3:e103. 52. Wu Y, Chhipa RR, Cheng J, Zhang H, Mohler JL, Ip C. Androgen receptor-mTOR crosstalk is regulated by testosterone availability: implication for prostate cancer cell survival. Anticancer Res 2010;30:3895-901. 53. Kelly DM, Jones TH. Testosterone: a metabolic hormone in health and disease. The Journal of endocrinology 2013;217:R25-45. 54. Navarro G, Allard C, Xu W, Mauvais-Jarvis F. The role of androgens in metabolism, obesity, and diabetes in males and females. Obesity (Silver Spring) 2015;23:713-9. 55. Stanworth RD, Jones TH. Testosterone in obesity, metabolic syndrome and type 2 diabetes. Frontiers of hormone research 2009;37:74-90. 56. Kang HY. Beyond the male sex hormone: deciphering the metabolic and vascular actions of testosterone. The Journal of endocrinology 2013;217:C1-3. 57. Mohlig M, Arafat AM, Osterhoff MA, et al. Androgen receptor CAG repeat length polymorphism modifies the impact of testosterone on insulin sensitivity in men. European journal of endocrinology / European Federation of Endocrine Societies 2011;164:1013-8. 58. Li RJ, Qiu SD, Wang HX, Tian H, Wang LR, Huo YW. Androgen receptor: a new player associated with apoptosis and proliferation of pancreatic beta-cell in type 1 diabetes mellitus. Apoptosis: an international journal on programmed cell death 2008;13:959-71. 59. Kennedy AJ, Ellacott KL, King VL, Hasty AH. Mouse models of the metabolic syndrome. Dis Model Mech 2010;3:156-66. 60. Lieberman AP, Robins DM. The androgen receptor's CAG / glutamine tract in mouse models of neurological disease and cancer. J Alzheimers Dis 2008;14:247-55. 61. Albertelli MA, O'Mahony OA, Brogley M, et al. Glutamine tract length of human androgen receptors affects hormone-dependent and -independent prostate cancer in mice. Hum Mol Genet 2008;17:98-110. 62. Albertelli MA, Scheller A, Brogley M, Robins DM. Replacing the mouse androgen receptor with human alleles demonstrates glutamine tract length-dependent effects on physiology and tumorigenesis in mice. Molecular endocrinology (Baltimore, Md 2006;20:1248-60. 63. Mousavi G. Generating vectors for production of transgenic mouse models to investigate the role of the androgen receptor and its CAG repeat in human prostate cancer. Masters Thesis, Dept. of Human Genetics, McGill University. 2005. 64. Abdullah A, Trifiro MA, Panet-Raymond V, et al. 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Experimental and toxicologic pathology: official journal of the Gesellschaft fur Toxikologische Pathologie 2005;57 Suppl 1:205-12. 82. Poovathingal SK, Gruber J, Ng LF, Halliwell B, Gunawan R. Maximizing signal-to-noise ratio in the random mutation capture assay. Nucleic acids research 2012;40:e35. 83. Cui J, Yin Y, Ma Q, et al. Comprehensive characterization of the genomic alterations in human gastric cancer. International journal of cancer Journal international du cancer 2015;137:86-95. Example 3: Prostate PreCancer / Cancer Mouse Model and Mutator Phenotype Mouse Model The Mutator Phenotype Advanced sequencing methodology, allowing for whole genome analysis, has clearly redefined cancer genetics. It has demonstrated the enormous extent of DNA somatic alterations underlying the high degree of tumoral genetic heterogeneity (inter-tumoral and intra-tumoral) and provides evidence that metastasis and tumor evolution is provoked by even a higher burden of DNA alterations 1-4. Cancers thus rely on inherent genomic instability to create a constant source of new mutations that ultimately lend to cancer evolution (mutator phenotype)<semantics>5−122<annotation encoding="application / x-tex">\frac{5-12}{2}< / annotation>< / semantics>. Although great effort has been made to catalogue specific DNA sequence alterations ("mutational landscape profiling of tumors"), relatively very little is known about the mechanisms neoplasms employ to develop the mutator phenotype. In prostate cancer (CaP), years of investigative studies, supplemented by many recent and comprehensive genomic analyses 13-15, have helped enormously to garner a better understanding of CaP genetics. Although a fair number of CaP mutational landscape analyses have been disclosed, a more recent comprehensive analysis has become the new reference for CaP genetics 16. This includes the analysis (whole exome, matched germline, transcriptome characterization) of 150 metastatic CaPs (localized, bone and soft tissue). The vast majority of previous CaP genetic analyses support the androgen receptor (AR) as a pivotal role-player in prostate cell tumor biology<semantics>17−191<annotation encoding="application / x-tex">\frac{17-19}{1}< / annotation>< / semantics>, but this recent analysis reports an even higher incidence of AR alterations in metastatic CaP (62.7%). Thus, the AR and its alterations play a fundamental role in both prostate cell and CaP biology, and therefore it is not unexpected that AR functionality is a real determinant of CaP progression and may play a crucial role in participating in the mutator phenotype. A proper investigation of the mutator phenotype requires assessment of ongoing pathological status, (DNA alterations, specific gene analysis, and metabolic studies) in a continuum, with special emphasis on early histological abnormalities prior to established CaP, which is simply not feasible with clinical specimens (typically only cross sectional in nature), but feasible in an appropriate animal model. Thus based various investigations, CaP displays very abnormal glucose metabolism, well beyond that expected to meet energy demands and building block synthesis 41,51-60. It is thought that glucose hyper-metabolism via either glycolysis or oxidative phosphorylation creates excessive ROS and is accelerated at more advanced stages that dramatically changes the rate of DNA mutations (mutator phenotype). The continued supply of DNA mutations allows powerful selection processes to ensue, ensuring neoplastic evolution; early stage hypermetabolism may initiate mutator pathways eventually leading to neoplatic transformation. The mouse AR shares over 90% homology with the human ortholog, but, interestingly, lacks the NTD CAG / polyQ tract; instead mice possess a mixed CAG / CAC-glutamine / histidine tract. Mice normally do not develop any element of prostatic hypertrophy or hyperplasia, and mouse models displaying prostate pathology are oncogenic transgenics where pathological time frames are not in keeping with humans68. As well, the transgenic oncogenes do not always represent the human condition, as many human CaPs do not have the same oncogenic expression <semantics>16,69,7010<annotation encoding="application / x-tex">\frac{16,69,70}{10}< / annotation>< / semantics>. AR-19Q Phenotype A "humanized" knock-in mouse model containing the equivalent of the human polyQ tract was created, as described in Example 1. The AR-19Q mice phenotype can be best described as a mice counterpart to the Metabolic Syndrome X seen in humans. Metabolic syndrome is a well- recognized association and possible risk factor for CaP74, / 5. AR-19Q Spontaneous Prostate Phenotype Histopathological studies have been performed on prostate tissue from AR-19Q mice at different times (6, 12, 18, and 24 months). AR-19Q mice have shown a phenotype that presents high degree of benign prostatic hyperplasia / hypertrophy with lymphocytic infiltration indicating an inflammatory process (Figure 6); to date castrated mice have not expressed any phenotype. Inflammation is a key component in the initiation and development of CaP<semantics>′′<annotation encoding="application / x-tex">^{\prime\prime}< / annotation>< / semantics>. Immunohistochemical analyses have been done using antibodies against AR, cellular proliferation (Ki-67) and mouse macrophages (CFm-C20), and an increase in expression of these proteins in AR-19Q mice was found. In AR-19Q mice prostatic tissue, multi- nuclear / heterochromatic cells have been seen, which are commonly observed in low-grade prostatic intraepithelial neoplasia (PIN). To analyze gene expression in the prostate samples, Qiagen RT2 array platforms were employed, allowing to use real time RT-PCR to assess the gene expression profiles related to several pathways involved in CaP development at three different time points (18 months) [Platforms; Prostate Cancer, Metabolism, Mitochondrial Function / Energy Metabolism, AMPK Signaling, and Inflammation (Figure 7-Inflammation pathway data not shown)]. The genes that show changes in expression between the AR-19Q mice vs. wild-type (WT) including up- regulation of BCL278, EGR379, ERG80,81, HK2 and HK382-85, CPT1B86, and cytokines and interleukins77. Similarly, the down-regulation of PTEN87-89, APC90-92, and GYS293 was observed. Gene expression abnormalities developing in a time dependent fashion, by analysis 6 vs. 12 vs. >18 prostate samples were also observed. Early up-regulation of cell cycle genes (CCNA1 and CDNK2A) at 6 and 12 months, expression of EGR3 and GPX3 at 12 months and continuing through to 18 months can be seen. Another group of genes found to be upregulated in the analysis included, H+-ATPase synthases, major components in ROS production in cells 94,95. Proteomic analysis also found the expression of ATP synthases were able to differentiate between BPH and CaP 96. Therefore, these changes in gene expression patterns in the AR- 19Q-knock-in mouse are parallel observations that have been very well characterized in CaP, but here the observation of these gene expression patterns suggests initiation events occur prior to the full neoplasia phenotype. EXPERIMENTS The focus on will be primarily assessing metabolic profiles, ROS generation and quantify extent of DNA damage in prostates of 19Q mice vs WT mice. An assay will be introduced to determine the DNA mutation rates of pertinent genes in order to gauge the mutator phenotype. Five mice of each group (WT and AR-19Q) will be used at 5 different time points (2, 6, 12, 18, and 24 months) for the investigations described below. RNA sequencing - Gene expression profiling RNA sequencing will be used to define overall gene expression and transcript splicing profiles of prostate tissue isolated from each group. Whenever possible, if the prostates contain distinct areas of pathology and inflammation, these areas will be laser micro-dissected and analyzed independently. It is expected that the AR-19Q mice will have unique expression profiles associated with alterations in metabolic pathways, DNA repair and oxidative stress. 5 Immunohistochemistry for Protein Expression, ROS, and DNA Damage Specific protein quantification by immunohistochemistry will be completed: androgen receptor, glucose transporters, and hexokinases including glucokinase will be assessed. Mitochondrial ROS levels will be quantified and the antioxidant / oxidative stress response [SOD1, Ape1 / Ref-1, thioredoxin (TRX), thioredoxin reductase (TRXR), and protein disulfide isomerase (PDI)], and induction of DNA repair proteins (Ape1 / Ref-1, NM23-H1 and MPG), and oxidative damage markers (8-OxoG, γ-H2AX and protein carbonyls) will be assessed. Other analyses will be sought which will be dependent on data generated by metabolomics studies described herein. It is expected that there will be differences in a number of markers in 19Q mice vs. their WT counterparts, and within the 19Q mice themselves in areas of prostate hyperplasia. Metabolomic Tumor Profiling The association of altered metabolism with the evolution of prostate pathology will be assessed by repeating the analysis at different time points: 2, 6, 12, 18, and 24 months in the 19Q mice. The metabolomics analysis will assess metabolic fluxes through several important pathways, namely the glycolytic pathway, the pentose phosphate pathway, and oxidative phosphorylation. All of the major metabolites can be ascertained; glucose-6-P, glucose-1-P, 6- phosphogluconolactone, pyruvate, lactate, oxaloacetate, fructose-6-P, glyceraldehyde 3-P, ribose 5-P and many others. Glutathione levels ratios will be done to assess antioxidant status 106. Whenever possible, if the prostates contain distinct areas of pathology and inflammation, these areas will be laser micro-dissected and analyzed independently. It will be determined whether the 19Q mice harbor abnormal glucose metabolism profiles and it is expected that the full metabolomics studies will give insight into the intracellular molecular abnormalities accounting for the glucose hypermetabolism phenotype. There exist many signature metabolomics profiles that are well characterized and cataloged, defined by key regulators of metabolism, e.g., PI3K, TP53, mTOR, AMPK 107-109. It is expected to see a time- dependent evolution of altered metabolomics, in hand with time-dependent prostatic pathology evolution. Rates of DNA Mutation The current description of DNA mutation rates accompanying mutational landscape investigation is a misnomer, as the analysis is cross-sectional in nature, and determined at one time point. Commonly available next generation whole genome DNA sequencing methods have extremely limited depth coverage and can easily miss 15% of cells harboring DNA mutations. Assays are now in place to assess with much more sensitivity somatic DNA sequence rate changes and include modern versions of the HPRT assay110, random mutation capture assay <semantics>(RMC)111<annotation encoding="application / x-tex">(RMC)^{111}< / annotation>< / semantics> and extensive duplex sequencing <semantics>1122<annotation encoding="application / x-tex">\frac{112}{2}< / annotation>< / semantics>. An abridged version of quantifying DNA sequence alterations using ultra-deep sequencing methodology that determines the exact percentage representation of a mutation in a group of cells by over sequencing (50,000 reads) the gene in question has been developed. Thus low- level mutations are exactly quantitated 113. Ultra-deep massively parallel sequencing of the genes found to be most frequently mutated in clinical specimens of CaP16 will be assessed. Candidates include: AR (AR CAG tract contractions and AR splicing variants); TP53; PTEN; AR-associated proteins (ETS fusions, FOXA1, ZBTB16, NCOR1, NCOR2); PIK3 pathway (PIK3CA, PIK3CB, PIK3R1); DNA repair (APC, BRCA2); cell cycle (RB1); chromatin modifiers (KMT2C, CHD1); and others (SPOP, ZFHX3 GNAS). PTEN, ETS fusions, ZBTB16, PIK3R1, CHD1, APC, and RB1 predominantly have gene fusions or loss of genomic copies as their primary mutation, and thus are not amendable to the methodology. However, AR (CAG tract contractions, amplifications, and missense mutations) FOXA1, PIK3CA KMT2C, SPOP and GNAS typically have predominant mutational hot spots, for which the sequencing approach is feasible. Six gene analysis is sufficient to assess DNA mutation rates; housekeeping gene will be also used to assess background DNA mutation rates in order to establish "hot" mutational rate within the chosen genes. Full exome sequencing of diseased tissue can also be used to assess total mutation rates over the development period of the prostatic phenotype. REFERENCES 1. Loeb KR, Loeb LA. Significance of multiple mutations in cancer. Carcinogenesis 2000;21:379-85. 2. Horne SD, Pollick SA, Heng HH. Evolutionary mechanism unifies the hallmarks of cancer. International journal of cancer Journal international du cancer 2015;136:2012-21. 5 3. Knijnenburg TA, Bismeijer T, Wessels LF, Shmulevich I. A multilevel pan-cancer map links gene mutations to cancer hallmarks. Chinese journal of cancer 2015;34:48. 4. Kandoth C, McLellan MD, Vandin F, et al. Mutational landscape and significance across 12 major cancer types. Nature 2013;502:333-9. 5. Fox EJ, Prindle MJ, Loeb LA. 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Example 4: Non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and hepatocellular carcinoma Mouse Model The mouse model described in Example 1 was used to investigate the AR-19Q mice as a model for alcoholic fatty liver disease, non-alcoholic steatohepatitis and hepatocellular carcinoma and was found to be a suitable model for these diseases. Blood tests showed a significant increase in alanine aminotransferase (ALT) levels, indicative of liver damage (Figure 8A), and an intense fatty infiltration that with time develops into classical nonalcoholic steatohepatitis (NASH), hepatitis and subsequent NASH fibrosis (Figure 8B). Nonalcoholic fatty liver disease (NAFLD) is caused by excessive uptake of lipids by the liver and, if left untreated, can result in chronic inflammation and eventually NASH (Figure 9). Of AR-19Q mice, approximately 20% develop hepatocellular carcinoma (HCC). This progressive hepatic disease often accompanies obesity, and has a complex set of causes that include insulin resistance, as well as signaling effects from adipose tissue, pancreatic islets, and skeletal muscle. Preliminary Mouse Liver RT2 Profiler PCR Arrays were completed for AR-19Q livers (Figure 10) (profiles the expression of 84 key genes involved in the mechanisms of NAFLD / NASH and hepatic insulin resistance). We observed an upregulation of inflammatory / anti-inflammatory genes, II-6, II-10; tumor necrosis factor (Tnf); lipid / fatty acid metabolism notably, lipoprotein lipase (Lpl) leptin receptor, adiponectin receptor, PPAR gamma; and glucose metabolism, Akt, Mtor, PIK3a genes 16; Xbp1 mediator of cellular ER stress: Ptpn1 regulates phosphorylation of insulin receptor kinase and negatively regulates insulin signaling. Notable down-regulated genes include, Abca1, fatty acid synthase and glucose-6- phosphatase77, as well as decreased expression of important glucose transporter genes, Slc2a2 and Slc2a4 encoding GLUT2 and GLUT4 respectively. Mlxipl, a basic HLH leucine zipper transcription factor that activates carbohydrate response element motifs in the promoters of triglyceride synthesis genes, is also decreased. Finally, Pdk4 is a kinase a regulator pyruvate dehydrogenase activity (pyruvate to acetyl-CoA)78-80. Decreased expression of Pdk4 will allow for significant increases in mitochondrial-mediated oxidative phosphorylation of glucose metabolites and ROS generation81 also found in limited clinical specimens82. Pdk4 also has been linked to HCC. PPAR-y-coactivator 1a is a transcription factor that coordinates genes required for healthy mitochondria biogenesis and is known to be deficient in NASH83,84. At least half of these are novel genes associated with NAFLD / NASH. The androgen receptor (AR) Q19 mouse model described in Example 1, manifesting metabolic syndrome accompanied by the complete gamut of progressive liver diseases, (alcoholic fatty liver disease (NAFLD), non- alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) alongside type 2 diabetes (T2D) can be used in the following studies to garner a better understanding of the molecular mechanisms responsible for liver diseases associated with metabolic syndrome and, as well, to serve as a therapeutic platform to assess treatment regimens. 1) Histopathology, immunochemistry studies of livers to assess metabolic stress and oxidative damage in the early spectrum of NAFLD to NASH to HCC. 2) gene expression analysis of the livers using RNA Seq to evaluate disease associated genes and pathways again at early stages of tissue pathology, coupled with Mass spectroscopy metabolomic profiling of livers to assess dysfunctional metabolism lending to NAFLD, NASH, and NASH cirrhosis. 3) Using recently acquired human clinical data, effectiveness of treatment regimens that are currently undergoing clinical trial using the AR-Q19 mouse as a pre-clinical test platform will be tested. Animal Models Animal models are key to garner more information especially relevant to the early etiologies of HCC. Mouse models have been employed for HCC studies and include xenograft and orthotropic models, chemical models and allograft models. Genetically engineered models include transgenes of Hepatitis B (or C), c-MYC, TGFα or SV40-T Antigen82 and a very recent mitochondrial prohibitin gene knockout mouse (MITO-mouse)83. While these mouse models are appropriate in their respective areas they are not representative of HCC in the background of common metabolic syndrome liver diseases and are not "best-fit models". As a result, a complete understanding of the molecular pathological mechanisms responsible for NASH and NASH related diseases including HCC have been severely hampered by inadequate mouse models84, where either a significant gene alterations or highly manipulated diets are required to capitulate phenotypes that are not in-keeping with the manifestation of the human condition85,86. The AR Q19 mouse model offers a unique opportunity to investigate the molecular pathological etiologies of several important livers diseases associated with metabolic syndrome and T2D. Thus investigations can be had in a very systematic fashion and will give new insight on the pathophysiology of each related disease entity and potential future drug targets. Omics are very inclusive in nature and are excellent choices to pursue systematic studies. The mouse model is equally attractive to be employed as a therapeutic platform and is an important as the investigative model. Here recent data will be used to pursue new and novel therapeutic options aimed at early disease NAFLD which if successful should prevent evolution to NASH, cirrhosis and HCC. The anatomy and histology of the fatty acid deposition in the AR Q19 mouse appears to be quite identical to the human counterpart (macro vs micro steatohepatomegaly). High fat diet Most recently we have fed the 19Q mouse a high fat diet (HFD) that resulted in an accelerated weight gain vs. the normal chow 19Q mice in 3 months (Figure 11). Ultrasound showed significantly larger livers, as well as fatty liver. Higher glucose and liver ALT enzyme were also observed in HFD AR-19Q fed mice. The AR-19Q model developed in is physiologically relevant to metabolic syndrome and related liver disorders, displaying liver pathologies in continuum from early fatty liver NAFLD to NASH to HCC, without significant genetic alterations or dietary manipulations. Thus it is unique and is a clear departure from other models 102 mimicking the human condition and represents a major advancement in available working models for HCC. This model will allow us to assess ongoing genetic and metabolic changes in the evolution of liver disorders from NASH to HCC in a chronological fashion and will certainly help establish initial pathways and events related to the initiation of several stages of liver diseases. Critically the mouse model is equally qualified to be used as a competent therapeutic platform allowing the assessment of treatment regimens to defer disease pathologies 103,104. Preliminary data strongly suggests that intermediary metabolism, provoked by either insulin resistance and / or hyperglycemia itself with inflammation initiates liver disorders commonly seen in metabolic syndrome 105,106. It is believed that heightened metabolic fluxes leads to excessive mitochondrial ROS and lipid synthesis, cell stress, and subsequent cellular damage with associated inflammatory involvement; excessive oxidative damage lends to continuing DNA alterations eventually allowing for a neoplastic HCC transformation. To investigate the link between abnormal metabolism, oxidative damage and inflammatory alterations studies in a comparative and temporal fashion, to define the molecular underpinnings at various stages of disease progression, from normal liver to fatty liver to hepatitis to cirrhosis and HCC will be pursued. These studies will be done under different conditions that already have been shown clearly to have an important effect on the phenotype i.e diet and hormonal status. For Specific Aims 1-3 the cohorts that will be studied are as follows: AR Q19 and WT mice normal diet (SFD), AR Q19 and WT mice fed high fat diet (HFD), and AR Q19 castrated and wt mice castrated normal diet (SFD). The former studies help delineate and define the critical molecular pathological mechanisms underlying the several stages of liver related diseases associated with metabolic syndrome / T2D and the latter castrated mice studies will help discover the molecular pathways by which the AR Q19 AR vs WT AR utilize to confer disease etiology. With respect to mice harboring HCC tumors, adjacent non-HCC-matched tissue will be included. The studies will be comparative and sequential in nature; normal vs. NASH vs. HCC. For Specific Aim 3 (therapeutic intervention) only AR Q19 vs WT mice normal diet will be used. Early diseased NAFLD mice will be chosen, as successful treatment of NAFLD can be considered to be a preventative measure avoiding later disease pathologies. 1. Metabolic Syndrome (10 mice / cohort). Glucose tolerance tests performed with concomitant systemic insulin levels measurement and assessment of insulin resistance using as readout in vivo insulin-induced phosphorylation of the insulin receptor, IRS1 and Akt (T308 and S473) in liver, skeletal muscle and adipose tissue homogenates by western blot using corresponding commercially available antibodies (monthly). In addition, a liver Metabolic Syndrome and Assay (MSA) kits (months 3, 6 12, 18, 24) will be used and changes in leptin and adiponectin contents (MSA) in white adipose tissue (months 3, 6 12, 18, 24) will be assessed. Circulating insulin leptin and adiponectin levels will be assessed at different ages using MSA kits while triglycerides, cholesterol profiles and non-esterified fatty acids (NEFAs) will be determined (months 3, 6 12, 18, 24). Finally, a group of control and 19Q mice assessed for their metabolic activity and food intake in metabolic cages. For liver immunohistochemistry (months 3, 6 12, 18, 24), assays will include: i) oxidative damage analysis [cellular ROS detection kit e.g. MitoSOX staining of mitochondrial ROS]; ii) assessment of antioxidant / oxidative stress response [SOD1, Ape1 / Ref-1 (or NM23-H1 and MPG), thioredoxin, thioredoxin reductase and protein disulfide isomerase]; iii) ER stress [ER stress antibody sampler kit]; iv) oxidative DNA damage markers (8-OxoG, γ-H2AX and protein carbonyls); vi) induction of DNA transcriptional repair systems (XbpXbd); and vii) standard apoptosis (annexin v / propidium iodide). 2. Liver Studies: OMIC Studies. (5 mice / cohort). The AR Q19 Mouse model offers a unique opportunity to study the molecular mechanism and evolution of significant liver diseases associated with the metabolic syndrome. The preliminary RT2 data, though providing limited profiling, suggests significant alterations in carbohydrate, lipid and mitochondrial oxidative phosphorylation; a full expression study is warranted and will be pursued with emphasis placed on metabolism and inflammation pathways. Altered metabolism is clearly one the major properties of early to late stages of liver diseases NAFLD and NASH. Most cancers also have clear departures in metabolism that plays a pivotal role in cancer evolution. As such the AR Q19 mouse will allow us to characterize metabolic properties of each in a systematic fashion. AR Q19 castrated vs WT mice castrated normal diet cohort is a critical group as it will help delineate and define the critical molecular pathological mechanisms by which the AR Q19 AR vs WT AR utilize to confer disease etiology. OMIC Studies: Gene Expression RNA Seq. RNA Sequencing has a greater ability, and outperforms microarrays, in quantifying and identifying expressed genes (plus alternative RNA) splicing), RNAs of low abundance and a greater number of non-coding RNAs including miRNAs. miRNAs are critical non coding RNAs that can master-regulate multiple expression of many genes simultaneously. The preliminary RT2 data, though providing limited profiling, suggests significant alterations in carbohydrate, lipid and mitochondrial oxidative phosphorylation; a full expression study is warranted with emphasis placed on metabolism and inflammation pathways. Analysis NAFLD, NASH, adjacent HCC and HCC will be performed at months 3, 6 12, 18. RNA Seq will be completed at facilities within the McGill Genome Center. OMIC Studies: Metabolomics (fasting vs 1hr post prandial). The Metabolomics Center ultra- high-pressure liquid chromatography quadrupole time of fight mass spectrometry to conduct metabolomics. Liquid chromatography is preferred as it gives greater metabolite coverage and higher mass metabolites. The chemical analysis will be conducted in different ways, to provide good coverage across the metabolome: C18 reverse phase for non-polar components, and hydrophobic interaction liquid chromatography (HILIC) for polar components. A total of 4 orthogonal data sets will be obtained. In addition, MS / MS analysis will also be performed on each respective mode for subsequent metabolite identification and matching with known databases such as the Human Metabolite Database. The data sets will then be processed via Agilent qualitative analysis and mass profiler professional for peak detection and alignment for peak table generation for the identification of dysregulated metabolites and pathways in the various stages of liver pathologies. Fresh liver cell preparations from the 4 mouse groups, isolated time points, from fasting and 1 hr post prandial mice will be used. The tissue will be collected and flash frozen, awaiting cell extraction using an acetonitrile extraction protocol (10 replicates) <semantics>382<annotation encoding="application / x-tex">\frac{38}{2}< / annotation>< / semantics>. The metabolomics analysis will assess metabolic fluxes through several important pathways, namely the glycolytic and lipid pathways, the pentose phosphate pathway, and oxidative phosphorylation. All the major metabolites can be ascertained: glucose-6-P, glucose-1-P, 6-phosphogluconolactone, pyruvate, lactate, oxaloacetate, fructose-6-P, glyceraldehyde 3-P, ribose 5-P and many others. Glutathione levels and NADP / NADPH ratios will be done to assess antioxidant status39. Only a few metabolomic studies 40,41 have been completed but are of limited scope: cross sectional pathologies including HCC. 3. Therapeutic Intervention Studies Therapeutic Intervention Study: Metabolism. (5 mice / cohort) Human NAFLD liver samples were obtained and used to perform extensive omics, histochemistry, immunohistochemistry and spatially resolved mass spectroscopy imaging (MALDI). Macro steatosis contained higher proportions of saturated triglycerides compared to micro steatosis. Laser capture microdissection transcriptomics illustrated several genes (EIF2S1, MBTPS1 ATF6) higher in macro vs micro steatosis. Thus macro steatosis correlates with lipogenesis, increased ER stress and CLDs containing higher saturated lipids that can be converted to unsaturated lipids augmenting ER stress. Overall fatty acid production results in the generation of reactive oxygen species (ROS) damaging components involved in the mitochondrial electron transport chain, accruing even more ROS production. ROS also creates ER stress directly by forming adducts with both lipids and proteins. This triggers several stress signaling pathways including the UPR and autophagy processes. It will be first validated that the AR Q19 mice follow a similar hepatic pattern of increased ER stress (e.g. EIF2S1 and MBTPS1), lipogenesis (i.e. nuclear localization of SREBP-1c and SREBP-2 by immunohistochemistry) and macro steatosis (as determined by histochemistry) as outlined by human samples. These studies will be used together with histological features as endpoints of early disease and disease progression in drug intervention studies (see below). During these studies, AR Q19 mice will be followed beyond NAFLD and ER stress progression and correlate with NAFLD, NASH, cirrhosis and HCC (months 3, 6, 9, 18) will be examined. The time schedule was chosen as an ideal time period allowing for early intervention and possible prevention of later disease phenotype. Dietary manipulations will first include palmitic acid 108 (30 µmoles / mouse in 50ul castor oil intraperitoneal (IP) injection daily vs vehicle alone starting at 6 months for 3 months), a saturated fatty acid known to increase ER stress and therefore should provoke macro steatosis and disease progression; The second dietary manipulation will include an inhibitor of stearoyl- CoA deasturase-1 (N-(2-hydroxy-2-phenylethyl)-6-[4-(2-methylbenzoyl)piperidin-1-yl]pyridazine- 3-carboxamide) 109 already proven to be effective in inhibiting the conversion saturated fats to unsaturated fats ( 30mg / kg / d oral administration starting at 6 months for 3 months duration). Again it will be examined whether this ameliorates disease progression (months 3, 6, 9, 18) as described above. Removal of ROS and other reactive species (e.g. NOS) through scavengers should mitigate damage caused on organelles, proteins, lipids and alleviate ER stress. Thus AR Q19 mice will be treated with MnTBAP (a mimic of superoxide dismutase converting ROS to hydrogen peroxide) 110 20 mg / kg in PBS IP daily vs vehicle starting at 6 months for 3 months). To assess ROS, thiobrabituric acid-reactive substances and mitochondrial glutathione levels will be monitored (months 3 6, 9, 18). Again treatments will be correlated with disease status by routine histochemistry of liver sections and endpoints described above and assess roles in liver pathologies beyond NALFD ie NASH, cirrhosis and HCC. (months 3, 6, 9, 18). Therapeutic Intervention Study: Autophagy (5 mice / cohort) Classical autophagy uses a double membrane phagophore that encloses the damaged membrane fusing with lysosomes for degradation. A master controller of autophagy is MTORC1 111,112, a kinase protein complex involved in regulation of both anabolic and catabolic cellular processes; when active autophagy is inhibited through its action on ATG proteins allowing damaged organelles to accumulate and lend to cellular dysfunction. Thus promoting autophagy or the replacement of damaged organelles should also slow or halt NAFLD disease progression. By constructing human liver organellar proteomes from human samples, we have now assigned proteins TPD52 and autophagic regulator protein SH3GLB1 and ATG2 involved in autophagy specific to CLDs and also to be involved in a form of autophagy involving direct interactions between CLDs and lysosomes (lipohagy). Gene therapy has made great strides in recent years especially in cancer therapy where a protein of interest needs to be introduced (or reduced by RNA silencing) into malignant cells. P53 cancer gene therapy is an excellent example of the progress that has been made 113. The platforms can be also used for non-cancer therapies especially applicable to serious chronic disorders like metabolic syndrome associated liver disorders and are extremely promising therapeutics soon to be introduced. The activity of MTORC1 will be inhibited using rapamycin (Sirolimus) 114,115. This will be achieved through IP injection 4mg / kg / every other day starting at 3 months for 6 months duration114. Degree of hepatic autophagy will be monitored in Epon-embedded plastic sections of frozen livers and transmission electron microscopy. Again, this will be correlated with a predicted reduction in NAFLD disease progression and other liver disease progression. Treatments will be correlated with disease status by routine histochemistry of liver sections (months 3, 6, 12, 18). The efficacy of introducing TPD52 and SH3GLB1 including dominant active forms using a modified adenovirus system for long-term expression based on the system developed by Kim et al. 116 will be tested, where the original CMV promoter has been replaced with the liver-specific albumin (Alb) promoter (to ensure liver specificity). To achieve silencing, RNAi technique (Invivofectamine 2.0; Life Technologies) will be used. This in vivo method achieves >80% knockdown in the liver 48 h post-injection and lasts for 36 days. Overexpression and depletion of both proteins in the liver will be determined by Western blot analysis and routine histochemistry of livers will be completed. 5 Therapeutic Intervention Study. Rationale: Historically, molecules proven to be the most effective against NASH have been thiazolidinediones (TZDs), particularly Pioglitazone, a PPARy agonist having demonstrated significant results in clinical studies (NCT00062764) with a 30mg / day doses. Unfortunately, at this dosage, side effects of pioglitazone are noteworthy: weight gain, edema, bone fractures and increased risk of bladder cancer. For this reason the PPARy agonists could not be considered as a therapeutic option for the treatment of NASH. Therefore, other compounds are being pursued and upon a review of the literature and currently ongoing clinical trials, two compounds to test with the AR19Q mouse were selected and ranked. Elafibranor / GFT505 (Genfit). In November 2015, GENFIT announced the design of the global Phase 3 clinical trial RESOLVE-IT (NCT02704403) to evaluate the benefits of Elafibranor treatment on NASH patients. RESOLVE-IT is a randomized pivotal trial, double-blind, placebo- controlled (2:1), conducted in approximately 2000 patients, at 250 centers worldwide. The patient population are NASH patients with (NAS≥4) with F2 or F3 fibrosis given Elafibranor 120 mg or placebo, administered once daily. Elafibranor, initially developed to target T2D, is a new dual activator of the nuclear receptors PPARα / δ having multiple properties: modulates plasma glucose and lipid levels, have prominent anti-fibrotic and anti-inflammatory affects. From their Phase IIb trials (NCT01694849), the following claims were made: 1) Improvement in markers of hepatic dysfunction, including liver enzymes: ALT, AST, GGT, and ALP 2) Improved insulin sensitivity and glucose homeostasis 3) Beneficial effects on plasma lipids, with a reduction in triglyceride and LDL-C and increased HDL-C levels 4) Anti-inflammatory effects 5) Beneficial effects on histological parameters NASH (steatosis, inflammation, fibrosis) in animal models (anti-fibrotic activities) 6) A good safety profile with no unwanted side effects in clinical trials (reinforced profile in toxicology studies with up to two-year carcinogenicity studies). The study included patients with NASH / NAFLD scores of 3 to 8 inclusive. This inclusion, which had been validated by the FDA, represented a small group of patients (~15%). The study advocated to the patients a healthy eating and exercise lifestyle. Thus the statistical impact of patients with a NAS score <4, prevented Elafibranor to have a statistically significant results across the entire study population. The mere withdrawal of NAS < 4 patients immediately altered the results of study:t a statistically acceptable positive result (22.4% vs 12.7%; Elafibranor vs placebo, <semantics>p=0.046<annotation encoding="application / x-tex">p = 0.046< / annotation>< / semantics>, <semantics>RR=1.9<annotation encoding="application / x-tex">RR = 1.9< / annotation>< / semantics>). MSDC-0602K (Octeta). To target NASH, Ocetata Therapeutics have developed a different strategy by targeting mTOT (a mitochondrial target of thiazolidinediones), which would defer the activation of PPARy. By modulating mTOT one can increase insulin sensitivity significantly without side effects PPARy agonists. Regulating NASH via insulin sensitizer has been a promising pathway. A clinical Phase IIa was conducted for 28 days weeks comparing the effect of the MSDC-0602K (100 mg, 250 mg) to the Pioglitazone (45 mg). Their results show MSCD-0602K was equivalent to Pioglitazone on overall glucose control, adiponectin levels, and extrapolated a potential positive effect of MSCD-0602K on NASH prompting a 12 month Phase Ilb trial (NCT02784444) which is currently underway, assessing tolerability and efficacy, with the goal of hepatic histological improvement in NASH with a NAS decrease in at least 2 points with no concurrent worsening of fibrosis. Also the reduction in NAS must include either a 1 point reduction in ballooning or inflammation. Obeticholic Acid / Ocaliva (Intercept). Ocaliva is approved for primary biliary cirrhosis. Intercept is currently recruiting NASH patients for a phase 3 trial: "Randomized Global Phase 3 Study to Evaluate the Impact on NASH With Fibrosis of Obeticholic Acid Treatment (REGENERATE)": NCT02548351. In a Phase 2 trial, NCT01265498 (Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multi- centered, randomized, placebo-controlled trial results), 141 patients were randomly assigned to receive obeticholic acid and 142 to placebo. 50 (45%) of 110 patients in the obeticholic acid group who were meant to have biopsies at baseline and 72 weeks had improved liver histology compared with 23 (21%) of 109 such patients in the placebo group (relative risk 2.2, 95% CI 1.4) to 3.3; p=0.0002) <semantics>952<annotation encoding="application / x-tex">\frac{95}{2}< / annotation>< / semantics>. Drug Intervention Studies: AR-19Q animal studies with Elafibranor, MSDC-0602K and Obeticholic acid. AR Q19 mice on normal diet or high fat diet will receive either Elafibranor or MSDC- 0602K96 (30 mg / kg / day and 50 mg / kg / day orally) or (10 mg / kg / day and 20 mg / kg / day orally) obeticholic acid vs. a vehicle control (1% methyl cellulose), for 6 and 12 weeks. The study will begin at 9 months where it is believed that mice will be displaying NAS> 4 an appropriate stage for treatment. The mice will be evaluated for the following criteria and expected improve outcomes for each compound. For these experiments 10 mice / cohort will be used Metabolic Studies. Blood glucose will be monitored weekly, with liver enzyme profiling (Abaxis - Mammalian Liver Profile) and liver ultrasounds to assess size and content will performed every two weeks. Weights are performed weekly. Histopathology and Immunohistochemistry. Following the 6 week period, mice will be sacrificed and dissected, with histopathology performed on the livers. 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In prostate tissue the AR has very important effects on growth and maintenance of the organ by supporting intracellular growth pathways, but tied to enhancing the basic metabolic machinery in the cell57. Thus androgens support glucose metabolism and do this with the direct involvement of mTOR57,58. The effects of AR on intermediary metabolism are also well documented within the liver, again supporting both glucose metabolism and protein metabolism59,60. More recent investigations have shown that androgens help establish peripheral insulin sensitivity / insulin resistance parameters 61-63. With respect to islet β-cell functions, androgens have been shown to increase ROS activity and, in mice with streptozotocin-induced diabetes, pancreatic β-cells are more prone to apoptosis when exposed to androgens64. In the liver androgens, increase insulin receptor, glycogen synthesis, cholesterol uptake and synthesis, decrease lipogenesis, glucose uptake and cholesterol removal; estrogens have the opposite effect on metabolic parameters65. Generally NAFLD occurs most often in men than women with a ratio 0f 1.75 <semantics>66,67<annotation encoding="application / x-tex">\frac{66,67}{}< / annotation>< / semantics>. The AR CAG tract polymorphism has now been recognized as a significant risk factor in HCC. Individuals with <23 CAG AR tract lengths had a 20% increase in HCC risk68,69 and individuals with higher testosterone and <20 CAG AR tract lengths had a 4-fold increase in <semantics>HCC70<annotation encoding="application / x-tex">HCC^{70}< / annotation>< / semantics>. AR is known to interact with the WNT / β-catenin pathway that is aberrantly activated in >90% of HCC. AR can also activate β-catenin through the transcriptional modulation of CCRK a novel cyclin dependent kinase-activating kinase an important regulator of tumorigenicity in many malignancies 71-73. Wnt signaling also regulates mitochondria physiology contributing to mitochondrial biogenesis, ROS generation and cellular senescence74. Thus AR has important implications in hepatocarcinogenesis 75. Preliminary data strongly suggests that intermediary metabolism, provoked by either insulin resistance and / or hyperglycemia itself with inflammation initiates liver disorders commonly seen in metabolic syndrome88,89. It is believed that heightened metabolic fluxes leads to excessive mitochondrial ROS and lipid synthesis, cell stress, and subsequent cellular damage with associated inflammatory involvement. To investigate the link between abnormal metabolism, oxidative damage and inflammatory alterations relevant studies in a comparative and temporal fashion will be pursued, to define the molecular underpinnings at early stages of disease progression, from normal liver to fatty liver to hepatitis and possible cirrhosis (animals 1 year old and less). These studies will be done under different conditions that already have been shown clearly to have important effects on the phenotype i.e diet and hormonal status. The cohorts that will be studied are as follows: AR-Q19 and WT mice, on standard (SFD) and high fat diet (HFD). For Specific Aim 2 (therapeutic intervention) only AR-Q19 vs WT mice, on SFD and HFD. Characterization of AR Q19 Mouse Metabolic Syndrome. Metabolic Studies. Blood glucose levels will be done on a weekly basis on all AR-19Q mice with age matched controls cohorts. Standard blood analysis [Comprehensive Diagnostic Profile (#500-0038) Abaxis] and liver enzymes AST, ALT, ALP and GGT [Mammalian Liver Profile (#500-0040) Abaxis] will be done at months 6, 9, 12, and 15 as well as circulating triglycerides, cholesterol profile and non-esterified fatty acids levels. Liver ultrasounds to assess size and content will also be done at the same time schedule. Weights are performed weekly. Glucose tolerance tests performed with concomitant systemic insulin levels measurement will be completed (months 6, 9, 12, and 15). Insulin resistance will be formally established by analyzing in vivo insulin-induced phosphorylation of insulin receptor, IRS1 and Akt (T308 and S473) in liver, skeletal muscle and adipose tissue homogenates by western blot methods using corresponding commercially available antibodies (months 6, 9, 12, and 15). Furthermore, to assess obesity status mice fat pads, both epididymal and subcutaneous will be weighed, as well as brown adipose tissue vs. age-matched controls (months 6, 9, 12, and 15). In addition, liver and pancreas (islets) will be weighed and pancreas analysis for insulin and glucagon content by immunofluorescence and quantitatively (months 6, 9, 12, and 15); white adipose tissue will be assessed for leptin and adiponectin contents (months 6, 9, 12, and 15) with concomitant circulating insulin, leptin and adiponectin levels [using Meso Scale kits (cat#K15124C-1 cat#K152BXC-1). Hypertension studies. Real time intravascular pressure analysis remains the gold standard in blood pressure measurements. Data Sciences International pioneered small animal blood pressure telemetry and their hardware and software will be employed to complete precise blood pressure measurements. Mice will have precise blood pressure assessments using vascular implanted pressure transducers (aorta) wired to an abdominal transmitter (DSI PhysioTel transmitter PA-c10). Blood pressure is recorded by telemetry to DSI PhysioTel Receiver (RPC-1). Dataquest ART software and DSI Data Exchange Matrix are employed to interpret the accrued measurements. Typical real time data acquisition is for a period of seven days to ten days (months 6, 9, 12, and 15). Metabolic Cage Studies. A group of control and 19Q mice will be followed to assess their metabolic activity and food intake in metabolic cages. The LabMaster / PhenoMaster system (TSE Germany) comprises three individual components: metabolic chambers, telemetry based monitoring and an Echo / MRI body composition analysis. For metabolic analysis, indirect calorimetry is used to assess O2 consumption, CO2 production, respiratory exchange rate and energy expenditure; food and water intake; locomotor activity (via infrared breaking), including total activity, ambulatory fine movements and rearing. The telemetry platform assesses locomotor activity, heart rate, body temperature and food and water intake. Echo / MRI is a precise system that measures total body fat, lean mass, free water and total body water in live mice. Urinalysis will also be performed to assess renal function, with proteinuria measured. Histochemistry and Immunohistochemistry. Tissue Microarrays (TMA) will be prepared for liver analysis from the most representative areas of paraffin embedded livers. Liver histochemistry will comprise of standard liver histochemistry, which will include assessment of steatosis (micro vs macro-vesicular), cellular ballooning, hepatocytolysis, later stage perisinusoidal fibrosis to periportal fibrosis, Mallory bodies and important inflammatory (neutrophils, eosinophils, lymphocytes, Kupffer cells) infiltrates (intralobular and periportal). Attention will be paid to pathological findings according to zonal liver architecture. Standard H&E staining and Masson's trichrome (fibrosis) will be completed supplemented by Sirius red (collagen proportional area) using digital imaging 90,91. The NASH Clinical Research Network criteria will be used as much as possible in staging disease 92-94. For liver immunohistochemistry, adiponectin (stimulates anti-inflammatory cytokines), insulin receptor, TNFα will be assessed, as well as a panel of extensive CD markers to identify and characterize classes and subclasses of inflammatory cellular infiltrates [B lymphocytes, T lymphocytes, macrophages, To assess more specifically cellular stress and oxidative damage (months 6, 9, 12, and 15), assays will include: 1) oxidative damage analysis [cellular ROS detection kit e.g. MitoSOX staining of mitochondrial ROS] and iNOS (nitric oxide synthase associated with insulin resistance inflammatory mediators) 2) assessment of antioxidant / oxidative stress response [SOD1, Ape1 / Ref-1 (or NM23-H1 and MPG), thioredoxin, thioredoxin reductase and protein disulfide isomerase]; 3) ER stress [ER stress antibody sampler kit]; 4) oxidative DNA damage markers (8-OxoG, γ-H2AX and protein carbonyls); 5) induction of DNA transcriptional repair systems (XbpXbd); and 6) standard apoptosis (annexin v / propidium iodide). 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[00209] While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Claims

1. A construct comprising a) a nucleotide sequence comprising a 5' AR arm comprising a portion of exon 1 of mouse androgen receptor (AR), and b) an exogenous polyglutamine (polyQ) tract encoding sequence, wherein the exogenous polyQ tract encoding sequence replaces an endogenous polyglutamine / histidine (polyQ / H) tract encoding sequence corresponding to at least amino acids 9 to 19 of the endogenous polyglutamine / histidine (polyQ / H) tract and up to all of the polyQ / H tract in the portion of exon 1 of mouse AR gene, wherein only the endogenous poly Q / H encoding tract is replaced and wherein the exogenous polyQ tract encoding sequence encodes 16to 23 glutamine residues.

2. The construct of claim 1, further comprising c) a selectable marker.

3. The construct of claim 1 or 2 wherein the exogenous polyQ tract encoding sequence encodes 18 to 21 glutamine residues.

4. The construct of any one of claims 1-3, wherein the exogenous polyQ tract encoding sequence encodes 19 glutamine residues.

5. The construct of any one of claims 1-4, wherein the exogenous polyQ tract is contiguous CAG repeats.

6. A recombinant cell comprising a recombinant androgen receptor (AR) cassette, the recombinant AR cassette containing an exogenous polyglutamine (polyQ) tract encoding sequence in mouse exon 1 of said cassette, wherein the exogenous polyQ tract encoding sequence is stably integrated into the genome of the cell and replaces an endogenous AR polyglutamine / histidine (polyQ / H) tract encoding sequence corresponding to at least amino acids 9 to 19 of the endogenous polyQ / H tract in exon 1 of mouse AR gene and expressing a recombinant AR polypeptide, wherein only the endogenous poly Q / H encoding tract is replaced and wherein the exogenous polyQ tract encoding sequence encodes 16 to 23 glutamine residues.

7. The recombinant cell of claim 6, wherein the exogenous polyQ tract encoding sequence encodes 18to 21 glutamine residues.

8. The recombinant cell of claim 6 or 7, wherein the exogenous polyQ tract encoding sequence encodes 19 glutamine residues.

9. The recombinant cell of any one of claims 6-8, wherein the recombinant cell is a pancreatic cell, a kidney cell, a hepatic cell, a mammary cell or an ovarian cell.

10. The recombinant cell of any one of claims 6-9, wherein the recombinant cell is an isolated recombinant cell.

11. The isolated recombinant cell of claim 10, wherein the cell is isolated from a knock- in mouse.

12. The isolated recombinant cell of claim 11, wherein the knock-in mouse is an AR- 19Q knock-in mouse, wherein the exogenous polyQ tract encoding sequence encodes 19 glutamine residues.

13. A method of making a polyQ AR knock-in mouse or a recombinant cell, comprising converting an endogenous androgen receptor (AR) polyglutamine / histidine (polyQ / H) tract encoding sequence to a polyglutamine (polyQ) tract encoding sequence, wherein the polyQ tract encoding sequence is stably integrated into the genome of the knock-in mouse or recombinant cell, and replaces the endogenous AR polyQ / H tract encoding sequence corresponding to at least amino acids 9 to 19 of the endogenous polyQ / H tract in exon 1 of mouse AR gene, wherein only the endogenous poly Q / H encoding tract is replaced and wherein the polyQ tract encoding sequence encodes 16 to 23 glutamine residues.

14. The method of claim 13, wherein converting the endogenous AR polyQ / H tract encoding sequence to the polyQ tract encoding sequence comprises use of the construct of any one of claims 1-5.

15. The method of claim 13 or 14, wherein androgen hormone levels of the knock-in mouse are altered by hormone therapy or by diet.

16. The method of any one of claims 13-15, wherein the knock-in mouse is a male mouse.

17. The method of any one of claims 13-16, wherein the knock-in mouse is a mouse model for metabolic syndrome, or metabolic syndrome related liver disorder.

18. The method of any one of claims 13-16, wherein the knock-in mouse is a mouse model for one or more of obesity, hypertension, Type 2 diabetes mellitus (T2D), prostate disorder, breast cancer, or polycystic ovarian syndrome.

19. The method of claim 17 or 18, wherein the knock-in mouse is a mouse model for obesity.

20. The method of claim 17 or 18, wherein the knock-in mouse is a mouse model for hypertension.

21. The method of claim 17 or 18, wherein the knock-in mouse is a mouse model for T2D.

22. The method of claim 17 or 18, wherein the knock-in mouse exhibits hypertriglyceridemia and / or elevated LDL cholesterol levels compared to control age matched mice.

23. The method of claim 17 or 18, wherein the knock-in mouse exhibits one or more of pancreatic hyperplasia, beta islet cell hyperplasia or insulin resistance.

24. The method of claim 17 or 18, wherein the knock-in mouse exhibits diabetic kidney disease.

25. The method of claim 17 or 18, wherein the knock-in mouse is a mouse model for metabolic syndrome related liver disorders.

26. The method of claim 25, wherein the mouse has non-alcoholic fatty liver disease (NAFLD).

27. The method of claim 25, wherein the mouse has chronic liver inflammation.

28. The method of claim 25, wherein the mouse has non-alcoholic steatohepatitis (NASH).

29. The method of claim 25, wherein the mouse has hepatitis.

30. The method of claim 25, wherein the mouse has NASH fibrosis.

31. The method of claim 25, wherein the mouse has cirrhosis.

32. The method of claim 25, wherein the mouse has hepatomegaly.

33. The method of claim 25, wherein the mouse has hepatocellular carcinoma (HCC).

34. The method of any one of claims 25-32, wherein the knock-in mouse exhibits one or more of increased alanine aminotransferase (ALT), aspartate transaminase (AST), gamma- glutamyl transferase (GGT), or alkaline phosphatase (ALP) levels.

35. The method of any one of claims 13-33, wherein the knock-in mouse exhibits a Nonalcoholic Fatty Liver Disease (NAFLD) Activity Score of greater than 4.

36. The method of claim 17 or 18, wherein the knock-in mouse is a mouse model for a prostate disorder, and wherein the mouse is a male mouse.

37. The method of claim 36, wherein the prostate disorder is prostatic hyperplasia, prostatic neoplasia, or prostatic hyperplasia lymphocytic infiltration.

38. The method of claim 37, wherein the prostatic neoplasia is prostatic interepithelial neoplasia, prostatic intraepithelial neoplasia, or prostatic neoplasia in situ.

39. The recombinant cell of any one of claims 6-12 or the method of any one of claims 13- 38, wherein the recombinant cell or the knock-in mouse is used in identifying dietary effects, genetic alterations, macromolecules, metabolites or pathways involved in metabolic syndrome, metabolic syndrome-related liver disorder, or prostate disorder.

40. The recombinant cell of any one of claims 6-12 or the method of any one of claims 13- 38, wherein the recombinant cell or knock-in mouse is used in a screening assay.

41. A method of screening for putative treatment for treating one or more of metabolic syndrome, metabolic syndrome related liver disorder, type 2 diabetes mellitus or a prostate disorder comprising: a. i) contacting a test compound with the recombinant cell of any one of claims 6-12, or ii) administering one or more doses of the test compound to the knock-in mouse as defined in any one of claims 13-38, and b) determining any effect of the test compound on the recombinant cell or knock-in mouse compared to suitable control.

42. The method of claim 41, wherein the suitable control is untreated control cells or untreated age-matched control mouse.

43. The method of any one of claim 41-42, comprising administering the test compound to the knock-in mouse.

44. The method of claim 43, wherein the knock-in mouse has obesity and the mouse is administered an agent or diet for weight loss and the effect measured is a decrease in weight and / or a decrease in volume of fat pads.

45. The method of claim 44, wherein the fat pads are epididymal or subcutaneous fat pads.

46. The method of claim 43, wherein the knock-in mouse has hypertension and the effect measured is a decrease in blood pressure.

47. The method of claim 46, wherein the blood pressure is systolic or diastolic blood pressure.

48. The method of claim 43, wherein the knock-in mouse has T2D and the effect measured is a decrease in glycemia and / or a decrease in insulin resistance.

49. The method of claim 48, wherein the decrease in glycemia is a decrease in post-prandial hyperglycemia and / or fasting hyperglycemia.

50. The method of claim 43, wherein the knock-in mouse has diabetic kidney disease and the effect determined is an improvement in renal function.

51. The method of claim 43, wherein the knock-in mouse has pancreatic hyperplasia, and the effect measured is (i) no change in pancreatic volume, or (ii) an increase in insulin content in the pancreas and / or increase in glucagon in the pancreas.

52. The method of claim 51, wherein the pancreatic hyperplasia is beta islet cell hyperplasia.

53. The method of claim 50, wherein the knock-in mouse has not developed beta islet cell hyperplasia, has developed beta islet cell hyperplasia with normal glycemia or has developed beta islet cell hyperplasia with hyperglycemia.

54. The method of any one of claims 48-53, wherein the method is for screening for therapeutically effective compounds involved in beta islet cell regeneration.

55. The method of claim 43, wherein the knock-in mouse has hypertriglyceridemia and the effect determined is a decrease in triglyceride levels.

56. The method of claim 55, wherein the decrease in triglyceride levels is a decrease in lipoprotein lipase (LPL) levels.

57. The method of claim 43, wherein the knock-in mouse has a metabolic syndrome related liver disorder and the effect measured is (i) improved liver function, and / or (ii) a decrease in liver volume and content.

58. The method of claim 57, wherein the metabolic syndrome related liver disorder is one or more of non-alcoholic fatty liver disease (NAFLD), chronic liver inflammation, non-alcoholic steatohepatitis (NASH), hepatitis, NASH fibrosis, cirrhosis, hepatomegaly or hepatocellular carcinoma (HCC).

59. The method of claim 57 or 658, wherein the improved liver function is determined by one or more of liver enzyme activity levels.

60. The method of claim 59, wherein the liver enzyme is alanine aminotransferase (ALT), aspartate transaminase (AST), gamma-glutamyl transferase (GGT), or alkaline phosphatase (ALP).

61. The method of claim 41 or 42, wherein the method is for screening for test compounds for treating prostate disorder.

62. The method of claim 43, wherein the knock-in mouse has prostatic hyperplasia, prostatic hypertrophy, prostatic neoplasia, prostatic neoplasia in situ, or prostatic hyperplasia lymphocytic infiltration, and the effect measured is a change in prostate morphology and / or an increase in volume.

63. The method of claim 62, wherein the prostatic neoplasia is prostatic interepithelial neoplasia or prostatic intraepithelial neoplasia.

64. The method of claim 41 or 42, wherein in methods comprising contacting the test compound with the recombinant cell, the method further comprises measuring expression levels of genes.

65. The method of claim 64, wherein the genes comprise genes involved in NAFLD / NASH or hepatic insulin resistance; lipid / fatty acid metabolism; glucose metabolism; or prostatic disease.

66. The method of claim 65, wherein the genes involved in NAFLD / NASH or hepatic insulin resistance comprise inflammatory / anti-inflammatory genes.

67. The method of claim 66, wherein the inflammatory / anti-inflammatory genes comprise one or more of IL-6, IL-10 or TNF.

68. The method of claim 65, wherein the genes involved in lipid / fatty acid metabolism comprise one or more of lipoprotein lipase (Lpl) leptin receptor, adiponectin receptor, or PPAR gamma.

69. The method of claim 65, wherein the genes involved in glucose metabolism comprise one or more of Akt, Mtor, PIK3a, Xbp1 mediator of cellular ER stress, Ptpn1, Abca1, fatty acid synthase, glucose-6-phosphatase, pyruvate dehydrogenase kinase, Slc2a2, Slc2a4, Mlxipl or Pdk4.

70. The method of claim 65, wherein the genes involved in prostatic disease are involved in one or more of mitochondrial ROS levels, antioxidant / oxidative stress response, or induction of DNA repair.

71. The method of claim 70, wherein the genes involved in antioxidant / oxidative stress response comprise one or more of SOD1, Ape1 / Ref-1, thioredoxin (TRX), thioredoxin reductase (TRXR), or protein disulfide isomerase (POI).

72. The method of claim 70, wherein the genes involved in induction of DNA repair comprise one or more of Ape1 / Ref-1, NM23-H1 or MPG.

73. The method of claim 70, wherein measuring expression levels of genes involved in prostatic disease comprises measuring oxidative damage markers.

74. The method of claim 73, wherein the oxidative damage markers comprise one or more of 8-0xoG, y-H2AX or protein carbonyls.

75. The method of claim 41 or 42, wherein the effect measured is a decrease in expression by at least 0.5 fold, at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold or at least 12 fold of at least one of the following genes: Ccna1, Gnrh1, Ptgs1, Abcb1b, Cdkn2a, Dlc1, Egr3, Gpx3, 116, Lgals4, Loxl1, Sfrp1, Slc5a8, Socs, Bcl2, Cdkn1a, Cdkn2a, Dab2ip, Dkk3, Egr3, Erg, Gpx3, LoxI1, Msx1, Ptgs1, Rbp1, Sfrp1, Socs3, Sox4, Tfpi2, Aldoc, Bpgm, Eno1, Eno2, Hk2, Hk3, Pdk4, Pfkl, Pklr, Prps2, Pygl, Acacb, Camkk1, Chrnb1, Cpt1b, Gpat2, Lepr, Pfkfb3, Ppp2r2b, Prkaa2, Prkar1b, Atp4a, Atp6v0d2, Atp6v1g3, Cox6a2, Cox6b2, Bmp2, Ccl11, Ccl12, Ccl17, Ccl19, Ccl2, Ccl20, Ccl22, Ccl24, Ccl3, Ccl4, Ccl5, Ccl6, Ccl7, Ccl8, Ccl9, Ccr1, Ccr10, Ccr2, Ccr3, Ccr5, Ccr6, Csf1, Csf3, Cx3cl1, Cxcl1, Cxcl10, Cxcl13, Cxcl15, Cxcl5, Cxcl9, Cxcr3, Cxcr5, Fasl, IL10ra, IL10rb, IL11, IL13, IL16, IL17b, IL1b, IL1rn, IL2rb, IL2rg, IL33, IL4, IL6ra, IL6st, Ltb, Pf4, Spp1, Tnfrsf11b, Tnfsf11 and / or Tnfsf13b, compared to an untreated control.

76. The method of claim 41 or 42, wherein the effect measured is an increase in expression by at least 0.5 fold, at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold or at least 4 fold to at least one of the following genes: Ect2, Hal, Slc5a8, Arntl, Klkb1, Ptgs1, Abcb1a, Ape, Casp3, Ccna1, Cdh1, Gnrh1, Hmgcr, Ints6, Lgals4, Mapk1, Mto1, Nrip1, Pten, Rbm39, Sept7, Shbg, Vegfa, Acly, Aldob, Fbp1, Fh1, G6pc, Gck, Gys2, Mdh1b, Pck2, Pgk2, Acaca, Adra2a, Akt3, Chrna1, Gys2, Hnf4a, Prkag3, Atp12a, Atp4b, Atp6v1c2, Atp6v1e2, Ndufa1, Ndufb2, Ndufb4, Ndufs6, Uqcrq, Aimp1, Csf2, IL15, IL17a, IL21, IL3 and / or IL5ra, compared to an untreated control.

77. The method of claim 41 or 42, wherein the test compound is for treating liver disease and the effect measured is a decrease by at least 0.5 fold, at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold or at least 14 fold, to at least one of the following genes: Abcg 1, Cd36, Fabp5, G6pdx, Lepr, Lpl, Mtor, Pparg, Srebf2, Xbp1, Adipor1, Akt1, Pck2, Pik3ca, IL10, IL6, Serpine 1, Tnf, Ifng, Ptpn1, Cnbp, Ndufb6, Nfkb1 and / or Socs3, compared to an untreated control.

78. The method of claim 41 or 42, wherein the test compound is for treating liver disease and the effect measured is a decrease by at least 0.5 fold, at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4, at least 5, at least 6 or at least 7 fold, to at least one of the following genes: Abca1, Acaca, Acly, Acox1, Acsl5, Acsm3, Apoe, Cpt2, Cyp7a1, Fabp1, Fabp3, Fasn, Irs1, Ldlr, Nr1h2, Nr1h4, Ppara, Adipor2, Foxa2, G6pc, Gck, Gsk3b, Gyk, lgf1, Pdk4, Pklr, Slc2a2, Slc2a4, Cyp2e1, Mlxipl, Ppargc1a and / or Rxra, compared to an untreated control.

79. The method of any one of claims 41-78, wherein the knock-in mouse is fed (i) a normal diet; or (ii) I) a high glucose diet, II) a high fat diet, III) a high fructose diet, IV) a high carbohydrate diet and / or V) a high protein diet.

80. The method of claim 43, further comprises performing one or more metabolic tests; hypertension tests; histochemistry or immunohistochemistry tests; metabolic cage tests; or metabolomics tests.

81. The method of claim 80, wherein the one or more metabolic tests comprise measuring blood glucose levels, glucagon levels, liver enzyme levels or activity, circulating triglyceride levels, cholesterol profile, non-esterified fatty acids levels, leptin level, adiponectin level, insulin-induced phosphorylation of insulin receptor IRS1 or Akt, or insulin levels.

82. The method of claim 43, wherein the knock-in mouse has a NAFLD activity score (NAS) of greater than 4, and the method further comprises measuring changes in NAS.

83. The method of claim 82, wherein the knock-in mouse has F2 or F3 fibrosis.

84. The method of claim 43, wherein the recombinant cell is a recombinant pancreatic cell, a recombinant hepatic cell, a recombinant kidney cell or a recombinant prostate cell.

85. The method of claim 84, wherein the recombinant pancreatic cell is a recombinant beta islet cell.

86. The method of claim 43, wherein the test compound is chosen from elafibranor, MSCD- 0602L and obeticholic acid.

87. The method of claim 43, wherein the test compound is a putative treatment for one or more of a metabolic syndrome disease model, a type 2 diabetes mellitus (T2D) disease model, a prostate disorder model, a model for liver diseases associated with metabolic syndrome, a cardiovascular disease model, or an ovarian disease with accompanying infertility model.

88. The method of claim 43, wherein determining the effect comprises measuring changes in one or more of metabolic profile, ROS generation, antioxidant / oxidative stress response, oxidative damage markers, extent of DNA damage, mutation rate, or gene expression.

89. The method of claim 88, wherein the gene expression is the expression of one or more of BCL2, EGR3, ERG, HK2, HK3, CPTIB, an ATPase proton transporter, a cytokine, an interleukin, or a cell cycle gene.

90. The method of claim 89, wherein the cell cycle gene is CCNA1, CDNK2A, PTEN, APC, or GYS2.

91. The method of any one of claims 41 to 90, wherein the test compound is tested in combination with an existing therapeutic.

92. The method of claim 41 or 42, wherein the effect comprises measuring changes in one or more genes selected from Abca1, Abcb1b, Abcg1, Acaca, Acacb, Acly, Acly, Acox1, Acsl5, Acsm3, Adipor1, Adipor2, Adra2a, Aimp1, Akt1, Akt3, Aldob, Aldoc, Ape, Apoe, Atp12a, Atp4a, Atp4b, Atp6v0d2, Atp6v1c2, Atp6v1e2, Atp6v1g3, Bcl2, Bmp2, Bpgm, Camkk1, Casp3, Ccl11, Ccl12, Ccl17, Ccl19, Ccl2, Ccl20, Ccl22, Ccl24, Ccl3, Ccl4, Ccl5, Ccl6, Ccl7, Ccl8, Ccl9, Ccna1, Ccr1, Ccr10, Ccr2, Ccr3, Ccr5, Ccr6, Cd36, Cdkn1a, Cdkn2a, Chrna1, Chrnb1, Cnbp, Cox6a2, Cox6b2, Cpt1b, Cpt2, Csf1, Csf2, Csf3, Cx3cl1, Cxcl1, Cxcl10, Cxcl13, Cxcl15, Cxcl5, Cxcl9, Cxcr3, Cxcr5, Cyp2e1, Cyp7a1, Dab2ip, Dkk3, Egr3, Eno1, Eno2, Erg, Fabp1, Fabp3, Fabp5, Fasl, Fasn, Fbp1, Fh1, Foxa2, G6pc, G6pdx, Gck, Gpat2, Gpx3, Gsk3b, Gyk, Gys2, Hk2, Hk3, Hmgcr, Hnf4a, Ifng, Igf1, IL10, IL 10ra, IL 10rb, IL 11, IL 13, IL 15, IL 16, IL 17a, IL 17b, IL 1b, IL 1rn, IL21, IL 2rb, IL2rg, IL3, IL 33, IL4, IL5ra, IL6, IL6ra, IL6st, Ints6, Irs1, LdIr, Lepr, Lgals4, Lpl, Mapk1, Mdh1b, Mlxipl, Msx1, Mto1, Mtor, Ndufa1, Ndufb2, Ndufb4, Ndufb6, Ndufs6, Nfkb1, Nr1h2, Nr1h4, Nrip1, Pck2, Pdk4, Pf4, Pfkfb3, Pfkl, Pgk2, Pik3ca, Pklr, Ppara, Pparg, Ppargc1a, Ppp2r2b, Prkaa2, Prkag3, Prkar1b, Prps2, Pten, Ptgs1, Ptpn1, Pygl, Rbm39, Rbp1, Rxra, Sept7, Serpine1, Sfrp1, Shbg, Slc2a2, Slc2a4, Slc5a8, Socs3, Sox4, Spp1, Srebf1, Srebf2, Tfpi2, Tgfb1i1, Tnf, Tnfrsf11b, Tnfsf11, Tnfsf13b, Uqcrq, Vegfa, or Xbp1.