WO2020223674A2 - Methods and compositions for regulating adipogenesis - Google Patents

Abstract

Provided herein are methods and compositions for reducing or inhibiting adipogenesis of a cell, such as: correcting a mutation in Ovo like zinc finger 2 (Ovol2) gene present in the cell; increasing an amount of OVOL2 protein or a functional fragment thereof; enhancing an activity of OVOL2 protein; and/or administering a C/EBPα inhibitor that mimics OVOL2 binding to C/EBPα.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. Provisional Patent

Application No.62/841,557 filed May 1, 2019, incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. NIH R01 AI125581 awarded by the National Institute of Health; the Government has certain rights in the invention. FIELD

Provided herein are methods and compositions for regulating adipogenesis, in particular for reducing adiposity in subjects in need thereof. BACKGROUND

Adiposity, the degree of body fat accumulation (Bays et al., 2013), is chiefly determined by the number of adipocytes in the body and the quantity of lipid stored within them. Excessive adiposity is a risk factor for type 2 diabetes and many other diseases (Gesta et al., 2007; van der Klaauw and Farooqi, 2015). Adipose tissue mass increases by enlargement of existing adipocytes and differentiation of new adipocytes, which arise from undifferentiated mesenchymal precursors that also give rise to muscle, fibroblasts, bone, cartilage, and other tissues. While food consumed by an organism may cause hyperplasia along the adipocyte line and also stimulate adipocyte hypertrophy, basal control of adipogenesis (independent of the anabolic stimuli induced by caloric intake) is understood only in part.

Adipocyte differentiation is elicited by cascades of transcription factors, among which Peroxisome Proliferator-Activated Receptor g (PPARg) and CCAAT/enhancer-binding protein (C/EBP) family proteins are major effectors (Farmer, 2006). During the early stages of differentiation, C/EBPb and C/EBPd are induced to facilitate the expression of PPARg, which along with C/EBPb and C/EBPd, drives the later expression of C/EBPa. Then, C/EBPa and PPARg activate genes responsible for the terminal differentiation of adipocytes. Recent studies have revealed a more complex adipocyte differentiation network and more adipose lineage determination factors (Rosen and Spiegelman, 2014), increasing our understanding of remained incomplete.

Thus, a need exists for new methods and compositions for regulating adipogenesis, in particular for reducing adiposity in subjects in need thereof. SUMMARY

In one aspect, provided herein is a method for reducing or inhibiting adipogenesis of a cell, comprising one or more of:

(a) correcting a mutation in Ovo like zinc finger 2 (Ovol2) gene present in the cell; (b) increasing an amount of OVOL2 protein or a functional fragment thereof in the cell;

(c) enhancing an activity of OVOL2 protein in the cell; and

(d) administering to the cell a CCAAT enhancer-binding protein a (C/EBPa) inhibitor that mimics OVOL2 binding to C/EBPa.

In some embodiments, the cell can be a preadipocyte such as a pluripotent stem cell, a mesenchymal stem cell, a white adipocyte progenitor cell, a brown adipocyte progenitor cell, a white adipocyte or a brown adipocyte. In certain embodiments, the method can be used to reduce or inhibit adipogenesis of a plurality of cells, wherein the cells can include one or more of pluripotent stem cells, mesenchymal stem cells, white adipocyte progenitor cells, brown adipocyte progenitor cells, white adipocytes, and/or brown adipocytes.

In some embodiments, the mutation can be a substitution of the cysteine at position 120 in OVOL2 isoform A. In some embodiments, the correcting is via gene therapy such as gene editing. In some embodiments, the gene therapy can use a construct under the control of platelet-derived growth factor receptor (PdgfR) a (PdgfRa) or PdgfRb promoter.

In some embodiments, the functional fragment binds to C/EBPa, thereby increasing OVOL2 binding to C/EBPa. In some embodiments, the functional fragment comprises amino acids H118 to K274 of the OVOL2 protein.

In some embodiments, the increasing comprises expressing OVOL2 from an engineered nucleic acid encoding the OVOL2 protein or functional fragment thereof. In some embodiments, the engineered nucleic acid can include a PdgfRa or PdgfRb promoter. In some embodiments, the engineered nucleic acid is provided by gene therapy.

In some embodiments, the comprises introducing into the cell a recombinantly produced OVOL2 protein or functional fragment thereof. In some embodiments, the recombinantly produced OVOL2 protein or functional fragment thereof is engineered to be In some embodiments, the enhancing comprises blocking a phosphotransferase such as a kinase that phosphorylates OVOL2. In some embodiments, the C/EBPa inhibitor is a small molecule drug.

Also provided herein is a composition for use in the methods disclosed herein, such as gene editing compositions, recombinant nucleic acid compositions, recombinant protein compositions, and C/EBPa inhibitors.

In one specific aspect, provided herein is a composition for expressing OVOL2 in a cell, comprising an engineered nucleic acid encoding an amino acid sequence having at least 80%, at least 85%, at least 95% or at least 98% sequence identity to SEQ ID NO.: 2 or 3, or a functional fragment thereof. In some embodiments, the engineered nucleic acid encodes the amino acid sequence of SEQ ID NO.: 2 or 3. In some embodiments, expression of the engineered nucleic acid can be under the control of an adipocyte-specific promoter or is activated by a trans factor in adipocytes. In some embodiments, the engineered nucleic acid can include a PdgfRa or PdgfRb promoter. In some embodiments, the cell is a preadipocyte such as a pluripotent stem cell, a mesenchymal stem cell, a white adipocyte progenitor cell, or a brown adipocyte progenitor cell. In some embodiments, the cell is a white adipocyte or a brown adipocyte.

The compositions disclosed herein can be used in the manufacture of a medicament for the treatment of a metabolic disorder. In some embodiments, the metabolic disorder is one or more of obesity, type II diabetes, insulin resistance, hyperinsulinemia, hypertension, hyperlipidemia, hepatosteatosis, fatty liver, non-alcoholic fatty liver disease, hyperuricemia, polycystic ovarian syndrome, acanthosis nigricans, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Laurence-Moon syndrome, and Prader- Willi syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1X. The boh phenotype.

(Figure 1A) Photograph of a male boh homozygote (boh/boh) and WT (+/+) littermate at 24 wk of age.

(Figures 1B, 1C, 1D and 1E) Body weight (Figure 1B), fat to lean weight ratio (Figure 1C), fat weight (Figure 1D), and lean weight (Figure 1E) of 12-wk-old mice.

(Figure 1F) Representative photographs of eWAT, iWAT, iBAT with WAT, and iBAT from 16-wk-old male mice. tissues of 16-wk-old male mice. Scale bar: 100 ^m.

(Figures 1M and 1N) Serum glucose (Figure 1M) and insulin (Figure 1N) in 12-wk-old male mice after a 6-h fast.

(Figure 1O) Glucose tolerance test. Blood glucose was measured at indicated times after i.p. glucose injection in 12-wk-old male mice (n=4).

(Figure 1P) Insulin tolerance test. Blood glucose was measured at indicated times after i.p. insulin injection in 12-wk-old male mice (n=4).

(Figures 1Q, 1R and 1S) Serum leptin (Figure 1Q), cholesterol (Figure 1R), and triglyceride (Figure 1S) in 12-wk-old male mice after a 6-h fast.

(Figure 1T) Representative photographs of liver from 16-wk-old male mice.

(Figures 1U, 1V, 1W and 1X) Liver sections of 16-wk-old male mice stained with H&E (Figures 1U and 1V) and Oil Red O (Figures 1W and 1X). Scale bar: 100 ^m.

All data are presented as means ± SEM. * P £ 0.05; ** P £ 0.01; *** P £ 0.001; **** P £ 0.0001; ns, not significant with P > 0.05. See also Figure 5. Figures 2A-2O. Mutations in Ovol2 cause the boh phenotype.

(Figure 2A) Scaled body weight phenotypic data plotted vs. genotype at the Ovol2 mutation site. Each data point represents one mouse. Mean (m) and SD (s) are indicated.

(Figure 2B) Manhattan plot showing P values calculated using a recessive model of inheritance. The -log10 P values (y axis) are plotted vs. the chromosomal positions of 39 mutations (x axis) identified in the G1 founder of the pedigree. Horizontal lines represent thresholds of P = 0.05 with or without Bonferroni correction, respectively.

(Figure 2C) Protein domains of mouse OVOL2274-aa isoform A and 241-aa isoform B. The boh mutation is a cysteine to tyrosine substitution at position 120 (C120Y) in OVOL2- A and position 87 (C87Y) in the shorter OVOL2-B.

(Figure 2D) Immunoblots of lysates of 293T cells expressing HA-tagged WT OVOL2- A, OVOL2-B or OVOL2-A^boh, OVOL2-B^boh. Gapdh was used as a loading control.

(Figures 2E, 2F, 2G and 2H) Body weight (Figure 2E), fat weight (Figure 2F), lean weight (Figure 2G), and fat to lean ratio (Figure 2H) of 12-wk-old mice.

(Figures 2I and 2J) Serum glucose (Figure 2I) and insulin (Figure 2J) in 12-wk-old male mice after a 6-h fast.

(Figure 2K) Glucose tolerance test. Blood glucose was measured at indicated times after i.p. glucose injection in 12-wk-old male mice (n=4). i.p. insulin injection in 12-wk-old male mice (n=4).

(Figures 2M, 2N and 2O) Serum leptin (Figure 2M), cholesterol (Figure 2N), and triglyceride (Figure 2O) in 12-wk-old male mice after a 6-h fast.

All data are presented as means ± SEM. * P £ 0.05; ** P £ 0.01; *** P £ 0.001; **** P £ 0.0001; ns, not significant with P > 0.05. See also Figures 6 and 7. Figures 3A, 3B, 3C, 3D, 3E and 3F. Identification of C/EBPa as an OVOL2 interacting protein.

(Figure 3A) Immunoblot analysis of immunoprecipitates (Top and Middle) or lysates (Bottom) of 293T cells expressing HA-tagged C/EBPa and 3xFLAG-tagged OVOL2.

(Figure 3B) Purified HIS-tagged OVOL2 was incubated with GST or GST-tagged C/EBPa. After GST pull-down, bound protein was analyzed by HIS immunoblot (Top). The amounts of GST and GST-C/EBPa were visualized by Coomassie blue staining (Bottom).

(Figure 3C) Protein domains of mouse OVOL2, C/EBPa and truncated forms for mapping the protein interaction region.

(Figure 3D) Immunoblot analysis of immunoprecipitates (Top and Middle) or lysates (Bottom) of 293T cells expressing HA-tagged C/EBPa and 3xFLAG-tagged full-length or truncated OVOL2.

(Figure 3E) Immunoblot analysis of immunoprecipitates (Top and Middle) or lysates (Bottom) of 293T cells expressing HA-tagged OVOL2 and 3xFLAG-tagged full-length or truncated C/EBPa.

(Figure 3F) Immunoblot analysis of immunoprecipitates (Top and Middle) or lysates (Bottom) of 293T cells expressing HA-tagged C/EBPa and 3xFLAG-tagged WT or boh mutant (C120Y) OVOL2. Figures 4A, 4B, 4C and 4D. OVOL2 represses the transcriptional activity of C/EBPa to regulate adipogenesis.

(Figure 4A) Oil Red O staining of fully differentiated Ovol2 KO 3T3-L1 adipocytes expressing WT or boh mutant OVOL2.

(Figure 4B) Quantification of Oil Red O staining by measurement of OD at 492nm (n=3).

(Figure 4C) Immunoblots of lysates of Ovol2 KO 3T3-L1 cells expressing different OVOL2 proteins before (day 0) or after (day 10) differentiation. C/EBPa in the presence of different dosages of His-OVOL2 protein. DNA probe was labeled with biotin for streptavidin-HRP visualization. EMSA samples were also analyzed by Western Blot (WB) to detect Ovol2 protein (Middle) and C/EBPa protein (Bottom). Figures 5A-5G. The body weight, food intake, adipocyte number, and lipid accumulation in livers of boh mice.

(Figure 5A) Growth curve of male homozygous boh mice (n=4) and WT littermates (n=4) from 6 wk to 13 wk of age.

(Figures 5B and 5C) Food intake of male homozygous boh mice (n=4) and WT littermates (n=4) was monitored from 6 wk to 13 wk of age. Average food intake per mouse per day (g/day) (Figure 5B) or average food intake per body weight per day (g food/g body weight/day) (Figure 5C).

(Figures 5D and 5E) Cell number quantification (Figure 5D) and total DNA amount (Figure 5E) of isolated fat cells from iBAT, eWAT, and iWAT fat pads of 16-wk-old male homozygous boh mice (n=3) and WT littermates (n=3).

(Figures 5F and 5G) Representative TEM images of liver sections from an18-wk-old female homozygous boh mouse (Figure 5F) and a WT littermate (Figure 5G). The boundary of a single liver cell is highlighted. Scale bar: 10 ^m.

All data are presented as means ± SEM. * P £ 0.05; ** P £ 0.01; *** P £ 0.001; **** P £ 0.0001; ns, not significant with P > 0.05. Figures 6A-6P. The boh phenotype is caused by a conserved mutation in Ovol2. (Figure 6A) Boh mutation is highly conserved among different species.

(Figure 6B) Representative photographs of eWAT, iWAT, iBAT with WAT, and iBAT from 16-wk-old male mice.

(Figures 6C, 6D, 6E, 6F, 6G and 6H) H&E staining of sections from different adipose tissues of 16-wk-old male mice. Scale bar: 100 ^m.

(Figure 6I) Representative photographs of liver from 16-wk-old male mice.

(Figures 6J, 6K, 6L and 6M) Liver sections of 16-wk-old male mice stained with H&E (Figures 6J and 6K) and Oil Red O (Figures 6L and 6M). Scale bar: 100 ^m.

(Figure 6N) Growth curve of male Ovol2^boh/- compound heterozygous mice (n=4) and WT littermates (n=4) from 6 wk to 13 wk of age.

(Figures 6O and 6P) Food intake of male Ovol2^boh/- compound heterozygous mice (n=4) mouse per day (g/day) (Figure 6O) or average food intake per body weight per day (g food/g body weight/day) (Figure 6P). Figures 7A-7J. Metabolic cage testing of boh mice. Body weight (Figure 7A), fat to lean weight ratio (Figure 7B), fat weight (Figure 7C) and lean weight (Figure 7D) of 9-wk-old homozygous boh mice (n=6) and WT littermates (n=6). These mice were tested in metabolic cages for food intake (Figure 7E), oxygen consumption (Figure 7F), carbon dioxide production (Figure 7G), heat production (Figure 7H), ambulatory motion (Figure 7I), and rearing/jumping motion (Figure 7J) for a 72h period. Figures 8A, 8B, 8C and 8D. OVOL2 interaction inhibits the binding of C/EBPa to its target DNA regions.

(Figure 8A) Mass spectrometry identification of OVOL2 interacting proteins. Silver staining (Top) and immunoblot analysis (Middle and Bottom) of immunoprecipitates of 3T3- L1 adipocytes expressing 3xFLAG-tagged OVOL2.

(Figure 8B) Mass spectrometry identified hits that were only found in 3xFLAG- OVOL2 immunoprecipitates.

(Figures 8C and 8D) C/EBPa ChIP assay of fully differentiated Ovol2 KO 3T3-L1 adipocytes expressing WT or boh mutant OVOL2. Two classic C/EBPa transcriptional targets (Fabp4 and Glut4) were detected by real-time PCR. Figure 9A-9M. OVOL2 deficiency causes reduced thermogenic gene expression and cold intolerance in brown/beige fat.

(Figures 9A, 9B, 9C and 9D) Liver sections of 16-wk-old male mice stained with UCP1. Scale bar: 100 ^m.

(Figures 9E and 9F) Relative mRNA level of thermogenic genes in iBAT (Figure 9E) and iWAT (Figure 9F) of 16-wk-old female mice (n=2, mRNA level was normalized to Polr2a and shown as fold change to WT controls).

(Figure 9G) Internal temperature of 16-wk-old female mice housed at cold environment in the absence of food (n=4 WT, 3 Ovol2^boh/-).

(Figures 9H and 9I) Relative Ucp1 mRNA level in iBAT (Figure 9H) and iWAT (Figure 9I) of 5-wk-old female mice treated with different conditions for 10 days. (n=3, mRNA level was normalized to Polr2a). with different conditions for 10 days.

(Figures 9K, 9L and 9M) Body weight (Figure 9K), fat weight (Figure 9L), and lean weight (Figure 9M) change of 5-wk-old female mice housed at thermoneutral environment.

Data points represent individual mice (Figures 9K, 9L and 9M). Data are representative of two independent experiments (Figures 9A-9G) or one experiment (Figures 9H-9M). All data are presented as means ± SEM. * P £ 0.05; ** P £ 0.01; *** P £ 0.001; **** P £ 0.0001; ns, not significant with P > 0.05. Figures 10A-10I. The expression of thermogenic genes, acute cold tolerance, and body composition of 5-wk-old Ovol2^boh/boh mice after chronic cold challenge.

(Figures 10A and 10B) Relative mRNA level of thermogenic genes in iBAT (Figure 10A) and iWAT (Figure 10B) of 5-wk-old female mice. (n=3, mRNA level was normalized to Polr2a and shown as fold change to WT controls).

(Figure 10C) Internal temperature change of 5-wk-old female mice housed at cold environment in the absence of food (n=4).

(Figures 10D, 10E, 10F, 10G, 10H and 10I) Body weight, fat weight, and lean weight of 5-wk-old female mice treated with different conditions for 10 days.

Data points represent individual mice (Figures 10D, 10E, 10F, 10G, 10H and 10I). Data are representative of two independent experiments (Figures 10A, 10B and 10C) or one experiment (Figures 10D, 10E, 10F, 10G, 10H and 10I). All data are presented as means ± SEM. * P £ 0.05; ** P £ 0.01; *** P £ 0.001; **** P £ 0.0001; ns, not significant with P > 0.05. Figures 11A-11S. OVOL2 overexpression in adipocyte reduces high-fat diet-induced obesity in mice.

(Figure 11A) Generation of adipocyte-specific OVOL2 expression mouse model induced by doxycycline.

(Figure 11B) Experimental design of TRE-Ovol2; Apn-rtTA mice feeding with HFD and doxycycline.

(Figure 11C) Immunoblots of lysates of two TRE-Ovol2; Apn-rtTA mice and two Apn- rtTA littermates under HFD-Dox feeding for 12 weeks.

(Figure 11D) Photograph of a male TRE-Ovol2; Apn-rtTA mice and Apn-rtTA littermate under HFD-Dox feeding for 12 weeks. (Figure 11F), fat weight (Figure 11G), and lean weight (Figure 11H) of mice under HFD-Dox feeding for 12 weeks.

(Figure 11I) Representative photographs of eWAT, iWAT, and iBAT from mice under HFD-Dox feeding for 12 weeks.

(Figures 11J, 11K, 11L, 11M and 11N) Serum glucose (Figure 11J), insulin (Figure 11K), leptin (Figure 11L), cholesterol (Figure 11M), and triglyceride (Figure 11N) in 12 weeks HFD-Dox feeding mice after a 6-h fast.

(Figure 11O) Representative photographs of liver from 12 weeks HFD-Dox feeding mice.

(Figures 11P, 11Q, 11R and 11S) Liver sections of 16-wk-old male mice stained with H&E (Figures 11P and 11Q) and Oil Red O (Figures 11R and 11S). Scale bar: 100 ^m.

Data points represent individual mice (Figures 11E-11H, 11J-11N). Data are representative of two independent experiments (Figures 11A-11O) or one experiment (Figures 11P-11S). All data are presented as means ± SEM. * P £ 0.05; ** P £ 0.01; *** P £ 0.001; **** P £ 0.0001; ns, not significant with P > 0.05. Figures 12A and 12B. Verification of the inducible adipocyte-specific OVOL2 expression mouse model.

(Figure 12A) Ovol2 mRNA levels normalized to Polr2a mRNA in different tissues of male mice after 4 weeks high-fat diet doxycycline feeding.

(Figure 12B) Immunoblots of lysates of different tissues of male mice after 4 weeks high-fat diet doxycycline feeding.

Data are representative of two independent experiments. All data are presented as means ± SEM. DETAILED DESCRIPTION

Using random germline mutagenesis, a fundamentally new form of obesity has been identified, involving Ovo like zinc finger 2 (OVOL2). It has also been surprisingly discovered that OVOL2 binds to CCAAT (SEQ ID NO.: 1)/enhancer-binding protein a (C/EBPa), one of two key effectors of adipocyte differentiation, and prevents it from engaging its transcriptional targets. The existence of a protein that restrains adipocyte differentiation points to the existence of a previously unknown regulatory checkpoint, limiting adiposity both as it occurs in the basal state and as it is driven by excessive caloric consumption. brown adipocytes and for thermogenesis under cold stress. OVOL2 deficiency can result in both an abundance of white adipocytes and a deficiency of brown adipocytes. Thus, delivering OVOL2 in human adipocytes can be used to treat obesity, e.g., by overexpressing the gene (via gene therapy, or drugs targeting the OVOL2 promoter, or intervention by inhibiting a repressor of OVOL2), or by administration of the protein in a form that can be taken up by adipocytes or precursors.

Consequently, provided herein are methods and compositions for reducing or inhibiting adipogenesis of a cell, such as:

correcting a mutation in Ovo like zinc finger 2 (Ovol2) gene present in the cell;

increasing an amount of OVOL2 protein or a functional fragment thereof (e.g., that binds to C/EBPa, thereby increasing OVOL2 binding to C/EBPa);

enhancing an activity of OVOL2 protein (e.g., thereby increasing OVOL2 binding to C/EBPa); and

administering a C/EBPa inhibitor that mimics OVOL2 binding to C/EBPa. Definitions

Certain terms are defined herein below. Additional definitions are provided throughout the application.

As used herein, the articles“a” and“an” refer to one or more than one, e.g., to at least one, of the grammatical object of the article. The use of the words "a" or "an" when used in conjunction with the term "comprising" herein may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

As used herein,“about” and“approximately” generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given range of values. The term“substantially” means more than 50%, preferably more than 80%, and most preferably more than 90% or 95%.

As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are present in a given embodiment, yet open to the inclusion of unspecified elements.

As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

As used herein, "adipocyte" refers to any cell in the adipocyte lineage, including precursor cells that differentiate into mature adipocytes. In certain embodiments, the adipocyte includes, e.g., a mesenchymal stem cell, a preadipocyte, a mature white adipocyte, a mature beige adipocyte, or a mature brown adipocyte. In some embodiments, adipocytes also include precursor cells in early stages of development, including stem cell adipocyte precursors (e.g., embryonic stem cells or induced pluripotent stem cells). In a particular embodiment, the adipocyte is a human adipocyte.

By "adipogenesis" is meant the process in which a preadipocyte differentiates into an adipocyte.

As used herein, "protein" and "polypeptide" are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). The term "protein" encompasses a naturally-occurring full-length protein as well as a functional fragment of the protein.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. In certain embodiments, the fragment retains the activity of the polypeptide or nucleic acid molecule of which it is a fragment.

The term "functional fragment" refers to a portion of a protein that retains some or all of the activity or function (e.g., biological activity or function) of the full-length protein, such as, e.g., the ability to bind and/or interact with or modulate another protein or nucleic acid. The functional fragment can be any size, provided that the fragment retains, e.g., the ability to bind and interact with another protein or nucleic acid.

As used herein, "recombinant" includes reference to a polypeptide produced using cells that express a heterologous polynucleotide encoding the polypeptide. The cells produce the recombinant polypeptide because they have been genetically altered by the introduction of the appropriate isolated nucleic acid sequence. The term also includes reference to a cell, or nucleic acid, or vector, that has been modified by the introduction of a heterologous the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non- recombinant) form of the cell, express mutants of genes that are found within the native form, or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all.

By "promoter" is meant a promoter, e.g., a viral promoter, that is capable of initiating expression in a cell. Such cells include cells selected from the group consisting of a preadipocyte, an adipocyte, a hepatocyte (e.g., an HepG2 cell) and precursors thereof. In various embodiments, cell specific promoters are capable of initiating expression of that cell. In certain embodiments, such cells are mammalian cells (e.g., human cells).

The terms "operably linked" is intended to mean that molecules are functionally coupled to each other in that the change of activity or state of one molecule is affected by the activity or state of the other molecule.

As used herein, the term "nucleic acid" refers to a polymer comprising multiple nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). "Nucleic acid" includes, for example, genomic DNA, cDNA, RNA, and DNA-RNA hybrid molecules. Nucleic acid molecules can be naturally occurring, recombinant, or synthetic. In addition, nucleic acid molecules can be single-stranded, double-stranded or triple-stranded. In some embodiments, nucleic acid molecules can be modified. Nucleic acid modifications include, for example, methylation, substitution of one or more of the naturally occurring nucleotides with a nucleotide analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like), charged linkages (e.g., phosphorothioates, phosphorodithioates, and the like), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, and the like), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, and the like). "Nucleic acid" does not refer to any particular length of polymer and therefore, can be of substantially any length, typically from about six (6) nucleotides to about 10⁹ nucleotides or larger. In the case of a double-stranded polymer, "nucleic acid" can refer to either or both strands of the molecule.

The term "nucleotide sequence," or "sequence" in reference to a nucleic acid, refers to a contiguous series of nucleotides that are joined by covalent linkages, such as phosphorus linkages (e.g., phosphodiester, alkyl and aryl-phosphonate, phosphorothioate, phosphotriester bonds), and/or non-phosphorus linkages (e.g., peptide and/or sulfamate bonds).

The terms "nucleotide" and "nucleotide monomer" refer to naturally occurring derivatives and analogs thereof. Accordingly, nucleotides can include, for example, nucleotides comprising naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or

deoxycytidine) and nucleotides comprising modified bases (e.g., 2-aminoadenosine, 2- thiothymidine, pyrrolo-pyrimidine, 3-methyl adenosine, C5- propynylcytidine, C5- propynyluridine, C5-bromouridine, C5-fluorouridine, C5- iodouridine, C5-methylcytidine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2- thiocytidine).

The term "identity" or "sequence identity" means that two nucleotide or amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least, e.g., 70% sequence identity, or at least 80% sequence identity, or at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity or more. For sequence comparison, typically one sequence acts as a reference sequence (e.g., parent sequence), to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math.2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., Current Protocols in Molecular Biology). One example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol.215:403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (publicly accessible through the National Institutes of Health NCBI internet server).

Typically, default program parameters can be used to perform the sequence comparison, although customized parameters can also be used. For amino acid sequences, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

As used herein, "contacting" refers to any one or more known methods of introducing an agent to, e.g., a cell (e.g., adipocyte or its precursor). For example, various transfection methods for introducing nucleic acid agents (e.g., siRNA or components of the CRISPR/Cas9 system) are known in the art. In some embodiments, the term "contacting" can be used synonymously with, e.g., "introducing" or "transfecting." For example, the one or more agents can be introduced into a cell by viral delivery including retrovirus, adenovirus, lentivirus, herpes simplex virus, vaccinia, and adeno-associated virus. In further examples, the one or more agents can be introduced by one or more of injection of naked DNA, electropermeabilization (e.g., electroporation), a biolistic particle delivery system (e.g., gene gun), cellular sonication (sonoporation), magnetic field-based transfection (magnetifection), and may further include use of oligonucleotides, lipoplexes, dendrimers, and inorganic nanoparticles.

As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the terms "subject" and "subjects" refer to an animal, preferably a mammal including a non-primate (e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey or a human), and more preferably a human.

As used herein, "treat," " treating," "treatment," and the like refer to reducing, ameliorating, or delaying a disorder mediated by OVOL2. As will be appreciated by those of skill in the relevant art, treating a disorder mediated by OVOL2 does not require that the disorder be completely eliminated. Thus, a treatment is not necessarily curative, and may reduce the effect of a disorder mediated by the thermogenesis pathway by a certain percentage over an untreated disorder mediated by OVOL2. In particular embodiments, the percentage reduction or diminution can be from 10% up to 20, 30, 40, 50, 60, 70, 80, 90, 95, 99 or 100%.

As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

In certain embodiments, the disorder includes, e.g., obesity, cardiovascular disease, type 2 diabetes, high blood pressure, stroke, abnormal blood fats, osteoarthritis, sleep apnea, obesity hypoventilation syndrome, or other metabolic syndrome. In some embodiments, the thrombocytosis, heart arrhythmia, endocrine disorders including hypothyroidism, or opportunistic infections.

The term "obesity" as used herein, refers to a condition characterized by the accumulation of excess body fat. Obesity can have a negative effect on health, leading to reduced life expectancy and/or increased health problems. Obesity may be evaluated by assessing a subject's body mass index (BMI), which is obtained by dividing a subject's weight by the square of the subject's height and/or by assessing fat distribution via the waist-hip ratio and total cardiovascular risk factor. A BMI between 18.50-24.99 kg/m² classifies an individual as having normal weight, between 25.00- 29.99 kg/m² as being overweight, and exceeding 30 kg/m² as being obese.

Various aspects of the disclosure are described in further detail below. Additional definitions are set out throughout the specification. OVOL2

Using random germline mutagenesis, a fundamentally new form of obesity has been identified, involving Ovo like zinc finger 2 (OVOL2). OVOL2 is a member of the Ovo family of zinc finger transcription factors and is highly conserved in invertebrates and vertebrates, including mammals (Kumar et al., 2012). Ovol2 was first cloned as a murine homolog of Drosophila ovo, which is highly expressed in the testis (Masu et al., 1998). Later studies revealed that Ovol2 is involved in neural development (Mackay et al., 2006), vascular angiogenesis (Unezaki et al., 2007) and inhibition of epithelial-mesenchymal transition (EMT) in normal tissue (Lee et al., 2014; Watanabe et al., 2014) and tumors (Bai et al., 2018; Song and Faber, 2018; Wang et al., 2017; Wu et al., 2017). M OVOL2 f ll l th (SEQ ID NO 2)

In some embodiments, provided herein is an inhibitory function of OVOL2 in adipogenesis, exercised through direct binding and inhibition of C/EBPa. In various embodiments, the functional fragment of OVOL2 can include the amino acid sequence from H118 to K274 of the OVOL2 protein, or a fragment thereof. This functional fragment may interact with C/EBPa. C/EBPa

It has been surprisingly discovered that OVOL2 binds to CCAAT/enhancer-binding protein a (C/EBPa), one of two key effectors of adipocyte differentiation, and prevents it from engaging its transcriptional targets. C/EBPa is a leucine zipper protein that is conserved across humans and rats. This nuclear transcription factor is enriched in hepatocytes, myelomonocytes, adipocytes, as well as other types of mammary epithelial cells [Lekstrom- Himes et al, J. Bio. Chem, vol.273, 28545-28548 (1998)]. It is composed of two

transactivation domains in the N-terminal part, and a leucine zipper region mediating dimerization with other C/EBP family members and a DNA-binding domain in the C- terminal part. The binding sites for the family of C/EBP transcription factors are present in the promoter regions of numerous genes that are involved in the maintenance of normal hepatocyte function and response to injury. C/EBPa has a pleiotropic effect on the transcription of several liver-specific genes implicated in the immune and inflammatory responses, development, cell proliferation, anti-apoptosis, and several metabolic pathways [Darlington et al., Current Opinion of Genetic Development, vol.5(5), 565-570 (1995)]. It is essential for maintaining the differentiated state of hepatocytes. It activates albumin transcription and coordinates the expression of genes encoding multiple ornithine cycle enzymes involved in urea production, therefore playing an important role in normal liver function.

In the adult liver, C/EBPa is defined as functioning in terminally differentiated hepatocytes whilst rapidly proliferating hepatoma cells express only a fraction of C/EBPa [Umek et al, Science, vol.251, 288-292 (1991)]. C/EBPa is known to up-regulate p21, a strong inhibitor of cell proliferation through the up-regulation of retinoblastoma and inhibition of Cdk2 and Cdk4 [Timchenko et al, Genes & Development, vol.10, 804-815 (1996); Wang et al, Molecular Cell, vol.8, 817-828 (2001)]. In hepatocellular carcinoma (HCC), C/EBPa functions as a tumor suppressor with anti-pro liferative properties [Iakova et Different approaches are carried out to study C/EBPa mRNA or protein modulation. It is known that C/EBPaa protein is regulated by post-translational phosphorylation and sumoylation. For example, FLT3 tyrosine kinase inhibitors and extra-cellular signal-regulated kinases 1 and/or 2 (ERK1/2) block serine-21 phosphorylation of C/EBPa, which increases the granulocytic differentiation potential of the C/EBPa protein [Radomska et al., Journal of Experimental Medicine, vol.203(2), 371-381 (2006) and Ross et al, Molecular and Cellular Biology, vol.24(2), 675-686 (2004)]. In addition, C/EBPa translation can be efficiently induced by 2-cyano-3,12-dioxoolean-l ,9-dien-28-oic acid (CDDO), which alters the ratio of the C/EBPa protein iso forms in favor of the full-length p42 form over p30 form thereby inducing granulocytic differentiation [Koschmieder et al, Blood, vol.110(10), 3695-3705 (2007)]. Therapeutic Methods and Compositions

In various embodiments, provided herein are methods, and related compositions, for reducing or inhibiting adipogenesis of a cell. In various embodiments, the cell is a preadipocyte such as a pluripotent stem cell, a mesenchymal stem cell, or an adipocyte progenitor cell.

In some embodiments, the methods and compositions can be used for correcting a mutation in the Ovol2 gene present in the cell. For example, the mutation can be a substitution of the cysteine at position 120 in OVOL2 isoform A. In certain embodiments, said correcting is via gene therapy such as gene editing. Exemplary promoters suitable for such gene therapy include the PdgfRa or PdgfRb promoter.

In some embodiments, the methods and compositions can be used for increasing an amount of OVOL2 protein or a functional fragment thereof that binds to C/EBPa, thereby increasing OVOL2 binding to C/EBPa. For example, functional OVOL2 can be expressed in the cell, from an engineered nucleic acid encoding the OVOL2 protein or functional fragment thereof.

In some embodiments, a recombinant OVOL2 protein or functional fragment thereof can be contacted and introduced into the cell. In one example, the recombinant OVOL2 protein or functional fragment thereof can be engineered to be operably linked to a cell- penetrating peptide (CPP) such as penetratin, Tat peptide and R7. Other CPPs are disclosed in, e.g., Guidotti et al., Trends in Parmacological Sciences, 38 (4), 406-424, 2017

incorporated herein by reference in its entirety. activity of OVOL2 protein, thereby increasing OVOL2 binding to C/EBPa. For example, a phosphotransferase such as a kinase that phosphorylates OVOL2 can be blocked or inhibited.

In some embodiments, a C/EBPa inhibitor that mimics OVOL2 binding to C/EBPa can be administered into a patient in need thereof. In some embodiments, said C/EBPa inhibitor is a small molecule drug.

In some embodiments, one or more genome editing systems can be used to correct Ovol2 mutations and/or express functional OVOL2. Various methods for genome editing are known and available in the art (Gaj et al., Trends Biotech.31 (7):397-405, 2013), and include, e.g., transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease (e.g., Cas9)

("CRISPR/Cas9"). In a particular embodiment, the CRISPR/Cas9 system can be used to express OVOL2. As those of skill in the art would appreciate, other forms of the

CRISPR/Cas system can also be used.

CRISPR together with cas (CRISPR-associated) genes was first identified as an adaptive immune system that provides acquired resistance against invading foreign nucleic acids in bacteria and archaea (Barrangou et al. Science 315: 1709-12 (2007)). CRISPR consists of arrays of short conserved repeat sequences interspaced by unique variable DNA sequences of similar size called spacers, which often originate from phage or plasmid DNA (Barrangou et al. Science 315: 1709-12 (2007); Bolotin et al. Microbiology 151 :2551-61 (2005); Mojica et al. J Mol Evol 60: 174-82 (2005)). In its native environment, the

CRISPR/Cas system functions by acquiring short pieces of foreign DNA (spacers) which are inserted into the CRISPR region and provide immunity against subsequent exposures to phages and plasmids that carry matching sequences (Barrangou et al. Science 315: 1709-12 (2007)). The CRISPR/Cas9 system from Streptococcus pyogenes was first characterized as involving only a single gene encoding the Cas9 protein and two RNAs - a mature CRISPR RNA (crRNA) and a partially complementary trans-acting RNA (tracrRNA) - which were identified as necessary and sufficient for RNA-guided silencing of foreign DNAs.

Since its discovery, the CRISPR/Cas9 system has been developed to modify or silence various genes of interest in many organisms (see, e.g., WO 2014/018423; WO 2014/011237; WO 2013/176772; and WO 2013/169398). In its most widely used form, Cas9 nuclease is directed by a synthetic guide RNA (sgRNA or guide or guide RNA) to perform site-specific double-strand DNA breaks at a target nucleotide sequence within a gene of interest. Specificity is conferred by homology (or identity) of the sgRNA to the target et al. Science 343, 84-87 (2014); Wang, T. et al. Science 343, 80-84 (2014)). The break at the target nucleotide sequence is repaired with a repair template, which can be used to insert a desired mutation or sequence into the target site, including tethering a polypeptide to a target site within the genome (the gene). Methods of using the CRISPR/Cas9 system, which comprises the cas enzyme, sgRNA, and repair template, are known in the art. In addition, methods of designing suitable sgRNA sequences are also known.

In one specific aspect, provided herein is a composition for expressing OVOL2 in a cell, comprising an engineered nucleic acid encoding an amino acid sequence having at least 80%, at least 85%, at least 95% or at least 98% sequence identity to SEQ ID NO.: 2 or 3, or a functional fragment thereof. In some embodiments, the engineered nucleic acid encodes the amino acid sequence of SEQ ID NO.: 2 or 3. In some embodiments, expression of the engineered nucleic acid can be under the control of an adipocyte-specific promoter or is activated by a trans factor in adipocytes. In some embodiments, the engineered nucleic acid can include a PdgfRa or PdgfRb promoter. In some embodiments, the cell is a preadipocyte such as a pluripotent stem cell, a mesenchymal stem cell, a white adipocyte progenitor cell, or a brown adipocyte progenitor cell. In some embodiments, the cell is a white adipocyte or a brown adipocyte.

In some embodiments, recombinant AAV vectors can be used, such as those disclosed in Jimenez et al., EMBO Mol Med.2018 Aug; 10(8): e8791, incorporated herein by reference in its entirety.

In various embodiments, the compositions disclosed herein can be administered to a cell, a tissue, or a patient as a pharmaceutical composition, as described herein.

As used herein, "a patient in need thereof” refers to any human subject receiving or who may receive medical treatment, in need of treatment, or desires treatment - e.g., voluntary weight loss. Therefore, in one embodiment, a patient in need thereof includes a patient desiring weight loss.

In various embodiments, the methods and compositions disclosed herein can be used for the treatment of a metabolic disorder, such as obesity, type II diabetes, insulin resistance, hyperinsulinemia, hypertension, hyperlipidemia, hepatosteatosis, fatty liver, non-alcoholic fatty liver disease, hyperuricemia, polycystic ovarian syndrome, acanthosis nigricans, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Laurence-Moon syndrome, and Prader-Willi syndrome.

As used herein, a "therapeutically effective amount" refers to the amount of an agent symptom of a disorder, in a clinically relevant manner. Any improvement in the patient is considered sufficient to achieve treatment. As those of skill in the art will appreciate, a sufficient amount of an active agent used to practice the present disclosure for the treatment of a disorder mediated by OVOL2 will vary depending upon, e.g., the manner of

administration, the age, body weight, genotype, and general health of the patient. Moreover, an effective amount will also depend upon whether an agent is administered as the sole therapeutic agent, or in combination with another agent of the present disclosure (as described herein), or in combination with other therapeutics known to have a beneficial effect on the disorder. Ultimately, the prescribers or researchers will decide the appropriate amount and dosage regimen. Such determinations are routine to those of skill in the art.

Various therapeutics (additional therapeutic agents) beneficial for the disorders disclosed herein are known, and can be used in combination with any one or more of the agents described herein. From a therapeutic perspective, antihypertensives (such as diuretic medicines, beta-blocking agents, calcium-channel blockers, renin-angiotensin system agents), lipid-modifying medicines, nitrates, and antiarrhythmic medicines are considered strong candidates for a disorder mediated by OVLO2. Further aspects of the disclosure relate to the administration of antihypertensives (such as diuretic medicines, beta-blocking agents, calcium-channel blockers, renin-angiotensin system agents), lipid-modifying medicines, nitrates, and antiarrhythmic medicines separately to individuals in need thereof that may also possess different gene variants associated with a favorable response to each type of administration.

In other embodiments, treatment of the disorder may also include administration of, e.g., aspirin, statins and/or epigenetic modifiers. The epigenetic modifiers may be nonspecific DNA synthesis inhibitors, such as DNA methyltransferase inhibitors (such as, but not limited to 5-aza-2'-deoxycytidine or 5-azacytidine) or histone deacetylase inhibitors (such as varinostat, romidepsin, panobinostat, belinostat and entinostat).

In related embodiments, the additional therapeutic agent can be administered before, simultaneously with, or after the administration of a composition comprising one or more agent of the present disclosure. Accordingly, a composition of the present disclosure and an additional therapeutic agent can be administered together in a single formulation (e.g., a tablet, capsule, powder, injectable liquid, etc.), or can be administered in separate

formulations, e.g., either simultaneously or sequentially, or both. The duration of time between the administration of a composition of the present disclosure and one or more addition, a composition of the present disclosure and the additional therapeutic agent(s) may or may not be administered on similar dosing schedules. For example, one or more agents of a composition of the present disclosure and the additional therapeutic agent may have different half-lives and/or act on different time-scales such that the composition of the present disclosure is administered with greater frequency than the additional therapeutic agent, or vice-versa. The number of days in between administration of therapeutic agents can be appropriately determined by persons of ordinary skill in the art according to the safety and pharmacodynamics of each drug.

As described herein, therapy or treatment according to the disclosure may be performed alone or in conjunction with another therapy (e.g., adjuvant therapy), and may be provided at home, the doctor's office, a clinic, a hospital's outpatient department, or a hospital. Treatment generally begins at a hospital so that the doctor can observe the therapy's effects closely and make any adjustments that are needed. The duration of the therapy depends on the age and condition of the patient, the stage of the disorder, and how the patient responds to the treatment. Additionally, a patient having a greater risk of developing the disorder can receive prophylactic treatment to inhibit or delay symptoms of the disorder.

In related aspects, the present disclosure also provides compositions, e.g., pharmaceutical compositions, comprising an effective amount of one or more agents that modulate the function of OVOL2 and/or C/EBPa.

In related embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, vehicles diluents, stabilizers, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. For example, such pharmaceutical compositions can include diluents of various buffer content (e.g., Tris-HCl, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Polysorbate 20, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). The

medicaments of the disclosure are prepared in a manner known to those skilled in the art, for example, by means of conventional dissolving, lyophilizing, mixing, granulating or confectioning processes. Methods well known in the art for making formulations are found, for example, in Remington: The Science and Practice of Pharmacy, 20th ed., ed. A. R.

Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of

Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York. See also, e.g., Remington's Pharmaceutical Sciences, 18th Edition (1990, Mack Depending on the intended mode of administration, the pharmaceutical formulations can be in a solid, semi-solid, or liquid dosage form, such as, for example, tablets, pills, capsules, microspheres, powders, liquids, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols or the like, possibly contained within an artificial membrane, preferably in unit dosage form suitable for single administration of a precise dosage.

Administration of pharmaceutical compositions according to the methods of the present disclosure may be by any suitable means that results in a compound concentration that is effective for treating or inhibiting (e.g., by delaying) the development of the disorder. The agent is admixed with a suitable carrier substance, e.g., a pharmaceutically acceptable excipient that preserves the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable excipient is physiological saline. The suitable carrier substance is generally present in an amount of 1-95% by weight of the total weight of the medicament. The composition may be provided in a dosage form that is suitable for parenteral or non-parenteral administration, e.g., oral, rectal, intravenous, intramuscular, subcutaneous, inhalation, nasal, topical, transdermal, or ophthalmic administration. EXAMPLES

Example 1: OVOL2 limits adipogenesis through negative regulation of C/EBPa

Summary

Using random germline mutagenesis in mice, we identified a fundamentally new form of obesity in which excessive adiposity initially developed despite normal caloric intake and energy expenditure. Although normophagic, normally active, and normal in total body weight for the first nine weeks of postpartum life, homozygotes for the boh mutation showed a 2.3- fold increase in fat:lean body composition. Subsequently, despite only a modest increase in food consumption, adiposity progressively increased, reaching levels seven-fold higher than normal by 12 weeks. Ultimately the mice became obese, hepatosteatotic, insulin resistant, and diabetic. The causative mutation was a missense error in Ovol2, a gene with no previously known function in energy metabolism. We determined that OVOL2 binds to C/EBPa, a key effector of adipocyte differentiation, and prevents it from engaging its transcriptional targets. Our data point to the existence of a genetic mechanism that may explain in part the variation individuals. Results

The boh phenotype

A phenotype observed among third-generation (G3) C57BL/6J mice heterozygous or homozygous for mutations induced by N-ethyl-N-nitrosourea (ENU), termed boh, was characterized by increased body weight (approximately 20% increased at 12 wk of age) (Figures 1A and 1B) first detected at 10 wk of age (Figure 5A). Heterozygous and homozygous boh mice were born at expected Mendelian frequencies and survived into adulthood. Magnetic resonance imaging (MRI) showed that boh/boh mice had an extremely high ratio of fat to lean weight compared with wild type (WT) littermates by 12 wk of age (Figure 1C); indeed, higher than the comparatively small increase in total body weight at this age (Figure 1B) would suggest. Approximately 442% increase in fat weight (Figure 1D) was accompanied by a 37% decline in lean weight (Figure 1E). Necropsy revealed that adipose tissue beds of boh/boh mice were increased in size relative to those in WT littermates, including epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) (Figure 1F). An overgrowth of both interscapular brown adipose tissue (iBAT) and the outside covering white adipose tissue (WAT) was associated with a“hump” and a hairless region on the backs of boh/boh mice (Figures 1A and 1F). Hematoxylin and eosin (H&E) staining revealed enlarged adipocytes, indicative of hypertrophy (Figures 1G-1L). In addition to the increased adipocyte size, we also counted greater numbers of adipocytes in boh/boh mice, indicative of hyperplasia (Figures 5D and 5E). At 12 wk of age, fasting blood glucose levels were comparable between boh/boh mice and WT littermates, while fasting insulin levels were dramatically increased in boh/boh mice (Figures 1M and 1N). Glucose intolerance (Figure 1O) and insulin resistance (Figure 1P) were both observed in 12-wk-old boh/boh mice. Presumably as a result of increased adiposity, boh/boh mice had increased leptin in the serum (Figure 1Q). Interestingly, while 12-wk-old boh mice had increased fasting cholesterol levels (Figure 1R), no significant differences in the fasting triglyceride levels were observed (Figure 1S). In addition, 16-wk-old boh/boh mice had large, pallid livers (Figure 1T), and Oil Red O (ORO) staining showed an abundance of stored lipid (Figures 1U-1X). Transmission electron microscope (TEM) images of liver sections showed“adipocyte-like” abnormal hepatocytes, each containing a single large lipid droplet (Figures 5F and 5G). The boh phenotype was mapped as a quantitative trait. A total of 92 G3 mice were weighed, and weights were scaled with respect to age and sex (Figure 2A). Automated mapping (Wang et al., 2015b) implicated a missense allele of Ovo like zinc finger 2 (Ovol2) as the causative mutation, displaying strong linkage in a recessive model of inheritance (P=1.17x10- ¹²) (Figures 2A and 2B). The boh mutation caused substitution of a tyrosine for a highly conserved cysteine at position 120 (C120Y) in OVOL2 isoform A (OVOL2-A) and position 87 (C87Y) in the shorter OVOL2 isoform B (OVOL2-B), within the first zinc finger domain of both isoforms (Figures 2C and 6A). Overexpression of the WT and boh mutant forms of OVOL2 in 293T cells revealed a similar expression level by immunoblot, suggesting that the boh mutation does not affect the stability of OVOL2 protein (Figure 2D). By CRISRP/Cas9 gene targeting, a null allele of Ovol2 was made. No phenotype was apparent in heterozygotes for the null allele (Ovol2^+/-). The Ovol2^-/- genotype caused fully penetrant embryonic lethality as previously reported (Mackay et al., 2006; Unezaki et al., 2007). However, compound heterozygotes with the boh allele and a null allele of Ovol2 (Ovol2^boh/-) were viable and displayed all aspects of the boh phenotype (Figures 2E-2O and 6B-6M). Ovol2^boh/- mice had increased body weight beginning at 9 wk of age (Figure 6N), when body weights of homozygous boh mice were still normal (Figure 5A). At 12 wk of age, Ovol2^boh/- mice had overall stronger phenotypes than boh/boh mice. These data are consistent with the interpretation that the boh allele confers a viable hypomorphic effect.

Food intake of Ovol2^boh/boh and Ovol2^boh/- mice was monitored beginning at 6 wk of age (Figures 5B, 5C, 6O and 6P). No significant differences in food intake were found between boh/boh mice and WT littermates before 9 wk of age (Figure 5B), when body weight was also similar (Figure 5A). At 9 wk of age, although there were no significant differences of body weight between boh/boh mice and WT littermates (Figure 7A), boh/boh mice already had a 2.3-fold increased fat to lean weight ratio (Figure 7B) resulting from an increased fat weight (Figure 7C) and decreased lean weight (Figure 7D). Measurement of energy intake and energy expenditure using metabolic cages revealed no significant differences in food intake, oxygen consumption, carbon dioxide production, heat production, and motion activities between 9-wk- old boh/boh mice and WT littermates (Figures 7E-7J). Beginning at 10 wk of age, increased body weight of boh/boh mice was accompanied by slightly increased food intake per mouse per day (Figure 5B); however, the elevation of food intake was not significant when normalized for body weight (Figure 5C). A similar trend of food intake change was observed in Ovol2^boh/- mice (Figures 6O and 6P). These data indicate that despite normal energy intake and lean weight, in which fat is increased and lean weight is reduced. Subsequently, these mice increase food intake in proportion to their increase in body weight. By 12 wk of age, the fat to lean weight ratio is even more skewed towards fat in boh/boh mice (Figure 1C), with 40% and 45% of body weight contributed by fat and lean weight, respectively, compared to 10% and 75% in WT littermates (Figures 1D and 1E). OVOL2 directly interacts with C/EBPa

The elevated fat weight in boh/boh or Ovol2^boh/- mice was due to both adipocyte hypertrophy and hyperplasia (Figures 1F-1L and 5D-5E), and we sought to understand the mechanism of OVOL2 in blocking adipogenesis. We identified OVOL2 interacting protein(s) using mass spectrometry analysis of immunoprecipitates of 3xFlag tagged OVOL2-A (3xFlag- OVOL2-A) from differentiated 3T3-L1 adipocytes (Figure 8A), a well-established cultured adipocyte system. Several OVOL2 specific interacting proteins were identified, among which C/EBPa was the top candidate (Figure 8B). OVOL2 interacted with C/EBPa when expressed in 293T cells (Figure 3A). In vitro GST pull-down experiments supported a direct interaction between OVOL2 and C/EBPa (Figure 3B). Further mapping experiments revealed the C- terminal zinc finger domains of OVOL2, and C-terminal DNA-binding (DBD) and leucine zipper (ZIP) domains of C/EBPa were required for this interaction (Figures 3C-3E). Unlike the WT OVOL2 protein, the mutant OVOL2^boh protein (C120Y) had a much lower affinity for C/EBPa (Figure 3F). Collectively, these data demonstrate that C/EBPa is an interaction partner of OVOL2, and that this interaction is disrupted by the boh mutation. OVOL2 regulates adipogenesis through inhibition of the transcriptional activity of C/EBPa The C/EBPa binding ability of OVOL2 suggested a potential regulatory role of OVOL2 during adipocyte differentiation. To test this, we generated Ovol2 KO 3T3-L1 cells by CRISPR/Cas9 targeting and then re-introduced CRISPR resistant (CR) transgenes encoding WT OVOL2 or OVOL2^boh of both isoforms. Although both WT and boh mutant OVOL2 proteins were expressed at similar levels (Figure 4C), WT OVOL2-A dramatically inhibited adipogenesis as revealed by decreased Oil Red O staining and expression of major mature adipocytes markers, while the boh mutant OVOL2-A had no such effect (Figures 4A-4C). Compared with OVOL2-A, OVOL2-B had a weak inhibitory effect (Figures 4A-4C). Since OVOL2 interacted with the C-terminal portion of C/EBPa containing the DBD and ZIP domains (Figures 3C-3E), we hypothesized that OVOL2 binding occupies the DNA binding Electrophoretic mobility shift assay (EMSA) showed that purified His-OVOL2 protein inhibited the binding of His-C/EBPa protein to a biotin-labeled DNA probe containing a classic C/EBPa binding motif (Figure 4D). Moreover, chromatin immunoprecipitation (ChIP) assay revealed that WT OVOL2 but not boh mutant OVOL2 inhibited the binding of C/EBPa to the promoter region of C/EBPa target genes Fabp4 and Glut4 in 3T3-L1 cells (Figures 8C and 8D). Taken together, these findings indicate that OVOL2 regulates adipogenesis through direct inhibition of the transcriptional effects of C/EBPa. Discussion

Here we have shown that OVOL2 is a negative regulator of adipogenesis. Adipogenesis is controlled by several key transcription factors, including PPARg and the C/EBP family proteins (Farmer, 2006). Once adipocyte differentiation has been launched, cross-regulation between PPARg and C/EBPa amplifies the differentiation signal through a unified pathway to activate expression of hundreds of adipogenic genes (Rosen et al., 2002; Wu et al., 1999). By directly interacting with the DBD and ZIP domains of C/EBPa, OVOL2 limits the DNA binding ability of C/EBPa. In boh mice, a point mutation in Ovol2 results in substitution of tyrosine for cysteine 120 (C120Y in isoform A, and C87Y in isoform B), a residue conserved in vertebrate and invertebrate species we examined (Fig. 8). OVOL2^boh fails to interact with C/EBPa and leads to a super-active status of C/EBPa, causing increased adipogenesis at the expense of lean tissue and subsequent obesity in boh mice. Although several KO mouse models suggest that C/EBPa regulates white but not brown adipose tissue differentiation in vivo (Linhart et al., 2001; Wang et al., 1995; Yang et al., 2005), we observed that both white and brown adipose tissues were increased in quantity in boh/boh mice, suggesting a more complex regulatory role for OVOL2 in vivo.

In mice homozygous for the boh allele, a progressive increase in adiposity and decrease in lean tissue occur, leading to fat to lean weight ratios about seven-fold higher than normal. Beginning at 8-9 wk of age, body weight increase exceeds that observed in normal growth. Boh/boh mice exhibited increased body weights only as adults (beginning at 10 wk of age), consistent with a recent study suggesting the importance of C/EBPa in white adipogenesis in the adult mouse (Wang et al., 2015a). Insulin resistance, diabetes, and hepatic steatosis are observed in boh/boh mice by 12 wk of age. Obesity typically occurs when energy intake exceeds energy expenditure, as observed in many well-studied mouse obesity models, including Lep^ob/ob, Lepr^db/db, Mc4r^-/-, Pomc^-/- mice (Lutz and Woods, 2012), in which the deficient mice, the result is hyperphagia prior to weaning age and increased body fat content together with visible obesity by 3-4 wk of age (Chlouverakis et al., 1970; Coleman, 1978). An early sign of accommodation of increased food intake may be the adipocyte hypertrophy observed by two wk of age in these mice (Joosten and van der Kroon, 1974). In contrast, elevated adiposity and fat to lean weight ratios observed in Ovol2^boh/boh mice precede excessive food intake or diminished energy expenditure. We hypothesize that this initial adiposity stimulates an increase in food intake, leading to adipocyte hypertrophy and subsequent body weight gain. Asprosin is secreted by adipocytes and stimulates feeding (Duerrschmid et al., 2017), and this orexigenic hormone or others like it may link the skewed fat to lean tissue ratio of boh/boh mice to increased food consumption. Importantly, food intake normalized to body weight remained normal in OVOL2-deficient mice, even as their body weight became significantly elevated compared to WT mice. Our findings suggest that irrespective of a primary drive to increase caloric consumption and/or diminish energy expenditure, there can be marked variability in the avidity of fat storage between individuals. This variability is subject to genetic control by OVOL2. OVOL2 activity prevents excessive fat storage, minimally through regulation of C/EBPa function, and ultimately, by limiting the number of adipocytes available for fat storage. OVOL2 may also limit adipocyte differentiation as it occurs in response to chronic overfeeding. The regulation of OVOL2 activity therefore emerges as a topic for further inquiry. Experimental Procedures

Mice

C57BL/6J mice were purchased from The Jackson Laboratory. The boh strain (C57BL/6J-Ovol2^boh) was generated by ENU mutagenesis and is described at http://mutagenetix.utsouthwestern.edu. Heterozygous Ovol2 knockout (Ovol2^+/-) mice were generated in our laboratory using the CRISPR/Cas9 system as described previously (Ran et al., 2013) with the Ovol2 (5¢- AGAGTTGTCGCATGTGCCGG-3¢) (SEQ ID NO.: 4) small base- pairing guide RNA. Compound heterozygous mice for the boh allele and the null allele (Ovol2^boh/-) were generated by breeding. Mice were maintained at the University of Texas Southwestern Medical Center and studies were performed in accordance with institutionally approved protocols. All experiments in this study were approved by the University of Texas Southwestern Medical Center Institutional Animal Care and Use Committee. All mice were fed standard chow diet (2016 Teklad Global 16% Protein Rodent Diet). Metabolic analysis

Mice were fasted for fasted for 6 h (7:00 AM - 1:00 PM) for glucose tolerance tests (GTT) and insulin tolerance tests (ITT). Blood glucose was tested with the AlphaTRAK glucometer and test strips. After the measurement of the first blood glucose, the GTT was initiated by i.p. injection with 10% glucose solution (1 g/kg; Sigma-Aldrich) and blood glucose was measured at set time points over the next 2 h. The ITT was initiated by i.p. injection with human insulin (0.75 U/kg; Sigma-Aldrich) and blood glucose was measured at set time points over the next 2 h. MRI of live mice was measured by EchoMRI Body Composition Analyzers with default settings. Metabolic cage studies were conducted using a Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments) at UT Southwestern Metabolic Phenotyping Core. Mice were acclimated in the metabolic chambers for 5 days before the start of the experiments. Food intake, movement, and CO₂ and O₂ levels were measured every 74 min for each mouse over a period of 4 days. Blood/Serum chemistries and ELISA

Mice were fasted for 6 h (7:00 AM - 1:00 PM) for all blood sample collections. ELISA kits were used to measure insulin (Crystal Chem) and leptin (Crystal Chem) in the serum according to the manufacturer’s instructions. Triglyceride was measured with Infinity Triglycerides Liquid Stable Reagent (Thermo Fisher Scientific). Matrix Plus Chemistry Reference Kit (Verichem Laboratories) was used as the standard for triglyceride measurement. Cholesterol was measured with Infinity Cholesterol Liquid Stable Reagent (Thermo Fisher Scientific). Matrix Plus Cholesterol Reference Kit (Verichem Laboratories) was used as the standard for cholesterol measurement. Immunohistochemistry

Samples for routine histology and special stains were harvested from anesthetized mice and fixed according to standard procedures (Sheehan and Hrapchak, 1980; Woods and Ellis, 1996) with modifications for tissue size and stains. Samples for routine Hematoxylin and Eosin (H&E) staining were fixed for 48 h in 10% (vol/vol) neutral-buffered formalin and stored briefly in 50% (vol/vol) ethanol, and samples for Oil Red O (ORO) staining were fixed in methanol-free 4% (vol/vol) paraformaldehyde for 48 h before equilibration in 18% (wt/vol) sucrose. Subsequent paraffin processing and embedding (H&E) and cryoembedding (ORO) were carried out, and sections were cut on a rotary microtome and cryostat, respectively. The a Sakura Finetek DRS-601 robotic staining system using Leica SelecTech reagents (hematoxylin 560 and alcoholic eosin Y 515). ORO staining was performed manually according to established protocols. Cell culture, transfection, infection, adipocyte differentiation, Oil Red O staining, and primary adipocytes isolation

The 293T cells were purchased from American Type Culture Collection (ATCC) and grown at 37 °C in DMEM (Life Technologies)/10% (vol/vol) FBS (ATCC)/antibiotics (Life Technologies) in 5% CO2. Transfection of plasmids was carried out using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. Cells were harvested between 36 and 48 h posttransfection. The 3T3-L1 cells were purchased from ATCC and grown at 37 °C in DMEM (Life Technologies)/10% (vol/vol) calf serum (ATCC)/antibiotics (Life Technologies) in 5% CO2. Infections of 3T3-L1 cells were carried out using a 3^rd generation lentiviral system packaged in 293T cells. The Ovol2 KO 3T3-L1 cells were generated with sgRNA 5’-CACGACGCCCAAGGCACCGA-3’ (SEQ ID NO.: 5) and 5’- GAAGACTGCCGCAGCGACGG-3’ (SEQ ID NO.: 6) constructed in pLentiCRISPR-v2 vector. For adipocyte differentiation, 2-d post-confluent cells (day 0) were treated with 293T grown medium containing 10 mg/mL insulin, 1 mM dexamethasone, and 0.5 mM methylisobutylxanthine (all from Sigma-Aldrich). On day 2, cells were changed to medium containing 10 mg/mL insulin (Sigma-Aldrich). From day 4, cells were maintained in growth medium, which was changed every other day. 3T3-L1 cells were fully differentiated and harvested at day 10. Oil Red O staining was performed with a lipid staining kit (BioVision) and quantified by measuring optical density at 492 nm (OD492). Isolation and separation of murine primary adipocytes from different adipose tissues (iBAT, eWAT, iWAT) was performed as described previously (Viswanadha and Londos, 2008). Sample preparation, immunoprecipitation, mass spectrometric analysis, GST pull-down, and western blot analysis

For regular western blot analysis, cells were harvested in 1× NuPAGE LDS sample buffer (Life Technologies) with 2.5% (vol/vol) 2-mercaptoethanol (Sigma-Aldrich). For regular immunoprecipitation, cells were lysed in Nonidet P-40 lysis buffer [50 mM Tris-Cl, pH 8.0, 0.1 M NaCl, 10 mM sodium fluoride, 1 mM sodium vanadate, 1% (vol/vol) Nonidet P-40, 10% (vol/vol) glycerol, 1.5 mM EDTA, and Protease Inhibitor Mixture] for 30 min at 4 °C. 4 °C. Beads were washed three times with 1 mL of Nonidet P-40 lysis buffer and then eluted with 3xFlag peptides for 30 min at 4 °C. For immunoprecipitation for mass spectrometric analysis, the procedure was almost the same except for the increased cell number and lysis time. Mass spectrometric analysis was performed as previously described (Zhang et al., 2016). GST fusion proteins were produced in Escherichia coli BL21 and purified with glutathione agarose beads (GE Healthcare). His fusion proteins were produced in Escherichia coli BL21 and purified with Ni-NTA agarose beads (QIAGEN). GST fusion protein-loaded beads were incubated with eluted His fusion proteins in GST pull-down buffer [20 mM Tris-Cl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% (vol/vol) Nonidet P-40, and PMSF] at 4 °C for 1 h. The beads were washed three times with GST pull-down buffer, followed by western blot analysis. In a typical Western blot, samples were resolved by NuPAGE 4–12% (wt/vol) Bis-Tris gels (Thermo Fisher Scientific), transferred to NC membranes (Bio-Rad), blotted with the primary antibody at 4 °C overnight and the secondary antibody for 1 h at room temperature, and then visualized by chemiluminescent substrate (Thermo Fisher Scientific). The following primary antibodies were used in this study: mouse anti-HA, anti-FLAG, anti-His (Sigma-Aldrich), rabbit anti-Adiponectin, anti-Gapdh, anti-b-actin, anti-PPARg, anti-CEBP/a, anti-FAS, anti- ACC (Cell Signaling Technology), and anti-Ovol2 (Invitrogen). Electrophoretic mobility shift assay (EMSA) and Chromatin Immunoprecipitation (ChIP) assay

EMSA was carried out using LightShift Chemiluminescent EMSA Kit (Thermo Scientific) according to the manufacturer’s instructions. The sequence of C/EBP ^ target DNA is 5’-AAGCTGCAGATTGCGCAATCTGCAGCTT-3’ (SEQ ID NO.: 7). Target DNA was synthesized as single-stranded DNA with biotin labeling on the 5’ end, and then boiled and slowly cooled down to form double-stranded DNA for use in EMSA. In brief, the following components were mixed in 20 mL binding reactions: ultrapure water, binding buffer (1x), poly (dI-dC) (50 ng/ ^L), unlabeled target DNA (4 pmol), purified proteins, and biotin-labeled target DNA (20 fmol). After incubation at room temperature for 20 min, 5 ^L 5x loading buffer was added to each 20 ^L binding reaction. Samples were loaded into a 6% DNA retardation gel (Invitrogen) for electrophoresis and then transferred to a nylon membrane for UV crosslinking and biotin-labeled DNA detection by chemiluminescence. ChIP was carried out using SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology) according to the manufacturer’s instructions. In brief, fully differentiated 3T3-L1 adipocytes were fixed with anti-Flag IgG were used for IP reactions. After washes, chromatin was eluted from the beads and crosslinks were reversed. DNA was purified with spin columns and analyzed by real-time PCR. The primer pairs used were as follows: Fabp4, 5’-CCAGGGAGAACCAAAGTTGAG- 3’ (forward) (SEQ ID NO.: 8), 5’- CTGACCATGTGACTGTAGGAG-3’ (reverse) (SEQ ID NO.: 9); Glut4, 5’-GGCTCTATGTCATCCTGCTG-3’ (forward) (SEQ ID NO.: 10), 5’- CTTGGACTGCGAAATTTCTG-3’(reverse) (SEQ ID NO.: 11). Example 2: Cell-Penetrating Peptide (CPP) - OVOL2

To introduce OVOL2 protein into cells, CPP can be used as a tag fused with OVOL2 protein. Three different CPPs can be used to make CPP-OVOL2 fusion proteins:

1. Penetratin, derived from Antennapedia protein (43-58aa), the amino acid sequence is RQIKIWFQNRRMKWKK (SEQ ID NO.: 12).

2. Tat peptide, derived from Tat protein (48-60aa), the amino acid sequence is

GRKKRRQRRRPPQ (SEQ ID NO.: 13).

3. R7, an artificial peptide, the amino acid sequence is RRRRRRR (SEQ ID NO.: 14).

CPP-OVOL2 protein can be expressed in E. coli or 293T cells. MBP tag or other affinity tags can be fused with CPP-OVOL2 protein to facilitate the purification of fusion proteins. The purified CPP-OVOL2 fusion proteins can be added into the medium of undifferentiated Ovol2 KO 3T3-L1 cells at different dosages.3T3-L1 differentiation can be induced for a total of 10 days. The medium can be changed every two days with new CPP- OVOL2 fusion proteins. Adipogenesis can be tested by measuring the triglyceride level in these differentiated mature adipocytes. With the addition of CPP-OVOL2 into the medium, it is expected there is an inhibition of adipogenesis in a dose-dependent manner.

Following in vitro experiments, the effect of CPP-OVOL2 in mice can be tested. CPP-OVOL2 fusion protein can be delivered in mice through different ways, including intravenous (i.v.), intraperitoneal (i.p.), intramuscular (i.m.), and subcutaneous (s.c.) injections. The fusion protein can also be injected directly into the brown and white fat pads. The injection can be repeated daily to maintain the level of CPP-OVOL2 protein in mice. The experimental mice can be put on a normal chow diet or a high-fat diet to study the effect of CPP-OVOL2 injection. The body weight, body composition as well as blood glucose, insulin, leptin can be monitored to check the development of obesity. It is expected that CPP-OVOL2 administration in mouse decreases the fat weight of mice on a normal chow diet and inhibits the development of obesity of mice on a high-fat diet. Example 3: Treatment of Obesity by Overexpression of OVOL2 in Adipocytes

Previously we found that OVOL2 limited the development of adipocytes by blocking C/EBPa engagement of its transcriptional targets. The following data suggest that OVOL2 was also necessary for the development of brown adipocytes and for thermogenesis under cold stress. These two functions resulted in an abundance of white adipocytes and a deficiency of brown adipocytes in OVOL2 deficient mice. To test the potential application of OVOL2 in treating obesity in vivo, we generated a mouse model that could specifically overexpress OVOL2 in adipocytes (fat cells) under the induction of doxycycline. These mice were put on a high fat diet (HFD) to mimic human obesity caused by overeating. We found that overexpression of OVOL2 in the adipocytes of HFD feeding mice reduced total body and liver fat and improved insulin sensitivity. We could use the similar concept to deliver OVOL2 in human adipocytes to treat obesity either by overexpressing the gene (via gene therapy, or drugs targeting the OVOL2 promoter, or intervention by inhibiting a repressor of OVOL2), or by administration of the protein in a form that could be taken up by adipocytes or precursors. Results

Defective thermogenesis in OVOL2 deficient mice

Compared to the iBAT of WT littermates, the color of iBAT from 16-wk-old Ovol2^boh/boh and Ovol2^boh/- mice appeared abnormally white and H&E staining revealed that adipocytes within Ovol2^boh/boh and Ovol2^boh/- iBAT were heterogeneous in size, with many containing a large unilocular lipid droplet, suggesting the function of brown fat might be compromised. Immunohistochemical staining of UCP1 in iBAT of Ovol2^boh/- mice showed that UCP1 appeared to be absent from the larger adipocytes containing unilocular lipid droplets, while the smaller adipocytes with multilocular lipid droplets were positive for UCP1 expression (Figures 9A and 9B). Beige adipocytes were visible as a cluster of UCP1 positive cells in the iWAT of WT mice (Figure 9C), but no UCP1 positive beige adipocytes were found in the iWAT of Ovol2^boh/- mice (Figure 9D). Consistent with the histological analysis, quantification of Ucp1, Prdm16, Cidea, and Ppargc1a by real-time PCR showed a reduction of these brown/beige cell markers in iBAT of 16-wk-old Ovol2^boh/- mice (Figure 9E), as well as in iWAT, where absolute levels of these mRNAs were lower than in iBAT (Figure 9F). Moreover, 16-wk-old Ovol2^boh/- mice had normal basal core body temperature at room temperature but failed to maintain their core body temperature during an acute cold stress in thermogenesis.

Because obesity of Ovol2^boh/boh and Ovol2^boh/- mice develops after 9 wk of age and progresses with age, we examined thermogenic gene expression and resistance to cold stress in 5-wk-old Ovol2^boh/boh mice, which had similar body weight and fat weight as WT littermates (Figures 10D and 10E). At 5 wk of age, Ovol2^boh/boh iBAT and iWAT showed slightly reduced expression levels of thermogenic genes compared to iBAT and iWAT from WT littermates, but this was not statistically significant (Figures 10A and 10B); tolerance to acute cold stress was similar in 5-wk-old Ovol2^boh/boh and WT mice (Figure 10C). After chronic (10 days) cold stress or treatment with CL316,243, an agonist that directly stimulates b3-adrenergic receptors on adipocytes to induce UCP1 and promote adipose tissue browning, Ovol2^boh/boh and WT mice had similar body weights and lean weight percentages, and showed similar reductions in fat weight percentages (Figures 10D-10I). UCP1 mRNA and protein were upregulated similarly in Ovol2^boh/boh and WT iBAT in response to both treatments (Figure 9H and 9J), but this response was reduced in iWAT of Ovol2^boh/boh mice compared to WT mice (Figure 9I and 9J). These data suggest that while perception of cold is preserved in 5-wk-old Ovol2^boh/boh mice, their ability to induce browning of WAT is impaired in response to chronic stimuli. The ambient temperature of our vivarium is below the thermoneutral zone of mice, providing a chronic cold stress that results in BAT activation and consequent elevated metabolism and food intake. Eliminating this stress by housing mice at thermoneutral temperature for 6 wk beginning at 5 wk of age not only failed to prevent, but rather increased the development of obesity in Ovol2^boh/boh mice (Figures 9K-9M; compare Figure 1B and 6 wk timepoint in Figure 9K; and Figure 1D and 6 wk timepoint in Figure 9L). Together, these data suggest that reduced formation and functionality of brown and beige adipocytes resulted in a defect of thermogenesis in OVOL2 deficient mice that was observed prior to and plausibly linked with the development of massive obesity. Adipocyte-specific transgenic expression of OVOL2 reduces high-fat diet-induced obesity The elevated fat weight of OVOL2 deficient mice (Ovol2^boh/boh or Ovol2^boh/-) was due to both adipocyte hypertrophy and hyperplasia, and we sought to determine the effect of Ovol2 overexpression using a tetracycline responsive element (TRE)-driven Ovol2 transgene (TRE- Ovol2) (Figure 11A). An adiponectin promoter-driven reverse tetracycline-dependent transcriptional activator (Apn-rtTA) (Sun et al., 2012) was also used (Figure 11A). In the presence of doxycycline (Dox), rtTA binds TRE and activates Ovol2 expression (Figure 11A). and Dox (HFD-Dox) feeding, and selected TG line #38015 because it showed the highest inducible TRE-Ovol2-derived mRNA (Figure 12A) and protein levels (Figure 12B) in iBAT, eWAT, and iWAT, but not in liver. 6-wk-old mice expressing TRE-Ovol2 and Apn-rtTA transgenes were fed HFD-Dox for 12 weeks (Figure 11B) to induce adipocyte specific Ovol2 expression (Figure 11C). Before HFD-Dox feeding, the body weights of TRE-Ovol2; Apn-rtTA mice and Apn-rtTA littermates were similar. However, after 12 weeks HFD-Dox feeding the body weights of TRE-Ovol2; Apn-rtTA mice averaged 22% lower than those of Apn-rtTA littermates (Figures 11D and 11E). MRI showed that TRE-Ovol2; Apn-rtTA mice had reduced ratios of fat to lean weight (Figure 11F) compared with Apn-rtTA littermates, which resulted from a 40% average reduction in absolute fat weight (Figure 11G) but no change in lean weight (Figure 11H). Necropsy revealed that adipose tissue beds (eWAT, iWAT, iBAT) of TRE-Ovol2; Apn-rtTA mice were decreased in size relative to those in Apn-rtTA littermates (Figure 11I). In addition to the decreased obesity, adipocyte-specific OVOL2 expression reduced both fasting glucose (Figure 11J) and insulin levels (Figure 11K). Presumably due to decreased adiposity, TRE-Ovol2; Apn-rtTA mice had less leptin in the serum (Figure 11L). Adipocyte-specific OVOL2 overexpression also reduced fasting cholesterol levels (Figure 11M), while no significant differences in the fasting triglyceride levels were observed (Figure 11N). HFD-Dox induced large, pallid fatty livers in Apn-rtTA control mice whereas TRE-Ovol2; Apn-rtTA mice had smaller, reddish livers (Figure 11O), suggesting less storage of fat in the livers of these mice. In addition, Oil Red O (ORO) staining showed little stored lipid in TRE-Ovol2; Apn-rtTA mice (Figures 11P-11S). These findings indicate that OVOL2 overexpression in adipocytes limits high fat diet-induced obesity and adiposity in mice. Experimental Procedures

Mice

C57BL/6J mice (stock# 000664) and the ob strain (B6.Cg-Lep^ob/J, stock# 000632) were purchased from The Jackson Laboratory. The boh strain (C57BL/6J-Ovol2^boh) was generated by ENU mutagenesis and is described at http://mutagenetix.utsouthwestern.edu. Heterozygous Ovol2 knockout (Ovol2^+/-) mice were generated in our laboratory using the CRISPR/Cas9 system as described previously (Ran et al., 2013) with the Ovol2 (5¢- AGAGTTGTCGCATGTGCCGG-3¢) (SEQ ID NO.: 4)  small base-pairing guide RNA. Compound heterozygous mice for the boh allele and the null allele (Ovol2^boh/-) were generated by breeding. The TRE-Ovol2 transgenic mice were generated in our laboratory using standard (University of Texas Southwestern Medical Center). TRE-Ovol2; Apn-rtTA mice were generated by breeding hemizygous TRE-Ovol2 mice with homozygous Apn-rtTA mice. All mice were fed standard chow diet (2016 Teklad Global 16% Protein Rodent Diet) except mice with diet-induced obesity, which were fed with high-fat diet (60 kcal% fat) with 625mg Doxycycline/kg (D11051103, Research Diets) from 6 wk of age. All mice were housed at room temperature (22°C) unless indicated. Mice were maintained at the University of Texas Southwestern Medical Center and studies were performed in accordance with institutionally approved protocols. All experiments in this study were approved by the University of Texas Southwestern Medical Center Institutional Animal Care and Use Committee. Metabolic Analysis

Mice were fasted for 6 h (7:00 AM - 1:00 PM) for glucose tolerance tests (GTT) and insulin tolerance tests (ITT). Blood glucose was tested with the AlphaTRAK glucometer and test strips. After the measurement of the first blood glucose, the GTT was initiated by i.p. injection with 10% glucose solution (1 g/kg; Sigma-Aldrich) and blood glucose was measured at set time points over the next 2 h. The ITT was initiated by i.p. injection with human insulin (0.75 U/kg; Sigma-Aldrich) and blood glucose was measured at set time points over the next 2 h. MRI of live mice was measured by EchoMRI Body Composition Analyzers with default settings. Internal temperature of mouse was obtained through implanting a temperature transponder (IPTT-300) under the skin, and measured with a portable reader (DAS-8007-IUS, BioMedic Data Systems). For acute cold exposure, mice were single housed in 6°C cold chambers in the absence of food, and body temperature was measured at the indicated time points. For chronic cold exposure, mice were single housed in 6°C cold chambers with free access to food for a total of 10 days. CL 316,243 was i.p. injected at the dose of 1mg/kg body weight daily for a total of 10 days. For thermoneutral experiments, mice were group housed in 29°C warm chambers. Blood/Serum Chemistries and ELISA

Mice were fasted for 6 h (7:00 AM - 1:00 PM) for all blood sample collections. ELISA kits were used to measure insulin (Crystal Chem) and leptin (Crystal Chem) in the serum according to the manufacturer’s instructions. Triglyceride was measured with Infinity Triglycerides Liquid Stable Reagent (Thermo Fisher Scientific). Matrix Plus Chemistry Reference Kit (Verichem Laboratories) was used as the standard for triglyceride measurement. Scientific). Matrix Plus Cholesterol Reference Kit (Verichem Laboratories) was used as the standard for cholesterol measurement. Immunohistochemistry and Immunostaining

Samples for routine histology and special stains were harvested from anesthetized mice and fixed according to standard procedures (Sheehan and Hrapchak, 1980; Woods and Ellis, 1996) with modifications for tissue size and stains. Samples for routine Hematoxylin and Eosin (H&E) staining and UCP1 staining were fixed for 48 h in 10% (vol/vol) neutral-buffered formalin and stored briefly in 50% (vol/vol) ethanol, and samples for Oil Red O (ORO) staining were fixed in methanol-free 4% (vol/vol) paraformaldehyde for 48 h before equilibration in 18% (wt/vol) sucrose. Subsequent paraffin processing and embedding (H&E, UCP1) and cryoembedding (ORO) were carried out, and sections were cut on a rotary microtome and cryostat, respectively. The resulting sections were stained for routine histopathological evaluation by regressive H&E on a Sakura Finetek DRS-601 robotic staining system using Leica SelecTech reagents (hematoxylin 560 and alcoholic eosin Y 515). The resulting sections were stained for UCP1 with a rabbit polyclonal antibody (Abcam, ab10983) at 1:600 dilution. ORO staining was performed manually according to established protocols. For immunostaining, 3T3-L1 cells cultured in chambers were washed with PBS and fixed in freshly made 4% (vol/vol) formaldehyde in PBS buffer at room temperature for 10 min, then washed again with PBS, treated with PBST [PBS and 0.25% (vol/vol) Triton X-100] for permeabilization, and blocked with PBSA [PBS and 1% (wt/vol) BSA] for 15 min. Cells were incubated with primary antibody diluted in PBSA overnight at 4 °C, then washed with PBS and incubated with secondary antibody diluted in PBSA for 30 min at room temperature, and finally mounted in mounting medium(Life Technologies). Sample Preparation, Immunoprecipitation, Mass Spectrometric Analysis, GST Pull-down, and Western Blot Analysis

For regular western blot analysis, cells were harvested in 1× NuPAGE LDS sample buffer (Life Technologies) with 2.5% (vol/vol) 2-mercaptoethanol (Sigma-Aldrich). For regular immunoprecipitation, cells were lysed in Nonidet P-40 lysis buffer [50 mM Tris-Cl, pH 8.0, 0.1 M NaCl, 10 mM sodium fluoride, 1 mM sodium vanadate, 1% (vol/vol) Nonidet P-40, 10% (vol/vol) glycerol, 1.5 mM EDTA, and Protease Inhibitor Mixture] for 30 min at 4 °C. After centrifugation, lysates were incubated with Flag antibody-conjugated beads for 2 h at with 3xFlag peptides for 30 min at 4 °C. For immunoprecipitation for mass spectrometric analysis, the procedure was almost the same except for the increased cell number and lysis time. Mass spectrometric analysis was performed as previously described (Zhang et al., 2016). GST fusion proteins were produced in Escherichia coli BL21 and purified with glutathione agarose beads (GE Healthcare). His fusion proteins were produced in Escherichia coli BL21 and purified with Ni-NTA agarose beads (QIAGEN). GST fusion protein-loaded beads were incubated with eluted His fusion proteins in GST pull-down buffer [20 mM Tris-Cl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% (vol/vol) Nonidet P-40, and PMSF] at 4 °C for 1 h. The beads were washed three times with GST pull-down buffer, followed by western blot analysis. In a typical Western blot, samples were resolved by NuPAGE 4–12% (wt/vol) Bis-Tris gels (Thermo Fisher Scientific), transferred to NC membranes (Bio-Rad), blotted with the primary antibody at 4 °C overnight and the secondary antibody for 1 h at room temperature, and then visualized by chemiluminescent substrate (Thermo Fisher Scientific). The following primary antibodies were used in this study: mouse anti-HA, anti-FLAG, anti-His (Sigma-Aldrich), rabbit anti-Adiponectin, anti-Gapdh, anti-b-actin, anti-PPARg, anti-CEBP/a, anti-FAS, anti- ACC, anti-UCP1 (Cell Signaling Technology), and anti-Ovol2 (Invitrogen). RNA Isolation, Reverse Transcription, and RT-qPCR

Tissue samples or cells were lysed in TRIzol (Invitrogen) for RNA isolation following a standard protocol, and 1 mg of RNA was used for reverse transcription by SuperScript III First-Strand Synthesis SuperMix (Life Technologies). RT-qPCR was performed with ABI StepOnePlus with Powerup SYBR Green Master Mix (Life Technologies). The 2-DDCt method was used for relative quantification. The following primer pairs were used: Ovol2, 5’- AATCAAGTTTACCACCGGCA-3’ (forward) (SEQ ID NO.: 15), 5’- CTCTTCAGGTCGAAGGTGTC-3’ (reverse) (SEQ ID NO.: 16); Polr2a, 5’- CAAGATGCAAGAGGAGGAAGAG-3’ (forward) (SEQ ID NO.: 17), 5’- TGTTGTCTGTCTGAGGTAAGTG-3’ (reverse) (SEQ ID NO.: 18); Ucp1, 5’- AAATACTGGCAGATGACGTC-3’ (forward) (SEQ ID NO.: 19), 5’- GTACAATCCACTGTCTGTCTG-3’ (reverse) (SEQ ID NO.: 20); Prdm16, 5’- CCATTCATATGCGAGGTCTG-3’ (forward) (SEQ ID NO.: 21), 5’- GTGTAATGGTTCTTGCCCTC-3’ (reverse) (SEQ ID NO.: 22); Cidea, 5’- TGCTCTTCTGTATCGCCCAGT-3’ (forward) (SEQ ID NO.: 23), 5’- GCCGTGTTAAGGAATCTGCTG-3’ (reverse) (SEQ ID NO.: 24); Ppargc1a, 5’- GCTGCATGGTTCTGAGTGCTAAG-3 (reverse) (SEQ ID NO.: 26). References

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All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A method for reducing or inhibiting adipogenesis of a cell, comprising one or more of:

(a) correcting a mutation in Ovo like zinc finger 2 (Ovol2) gene present in the cell; (b) increasing an amount of OVOL2 protein or a functional fragment thereof in the cell;

(c) enhancing an activity of OVOL2 protein in the cell; and

(d) administering to the cell a CCAAT enhancer-binding protein a (C/EBPa) inhibitor that mimics OVOL2 binding to C/EBPa.

2. The method of claim 1, wherein the cell is a preadipocyte such as a pluripotent stem cell, a mesenchymal stem cell, a white adipocyte progenitor cell, a brown adipocyte progenitor cell, or a white adipocyte or a brown adipocyte.

3. The method of claim 1, wherein the mutation is a substitution of the cysteine at position 120 in OVOL2 isoform A.

4. The method of claim 1, wherein said correcting is via gene therapy such as gene editing.

5. The method of claim 4, wherein said gene therapy comprises a construct under the control of PdgfRa or PdgfRb promoter.

6. The method of claim 1, wherein said functional fragment binds to C/EBPa, thereby increasing OVOL2 binding to C/EBPa.

7. The method of claim 1, wherein said increasing comprises expressing OVOL2 from an engineered nucleic acid encoding the OVOL2 protein or functional fragment thereof.

8. The method of claim 7, wherein said engineered nucleic acid comprises a PdgfRa or PdgfRb promoter.

9. The method of claim 7, wherein said engineered nucleic acid is provided by

10. The method of claim 1, wherein said increasing comprises introducing into the cell a recombinantly produced OVOL2 protein or functional fragment thereof.

11. The method of claim 10, wherein said recombinantly produced OVOL2 protein or functional fragment thereof is engineered to be operably linked to a cell- penetrating peptide such as penetratin, Tat peptide and R7.

12. The method of claim 1, wherein the functional fragment comprises amino acids H118 to K274 of the OVOL2 protein.

13. The method of claim 1, wherein said enhancing comprises blocking a phosphotransferase such as a kinase that phosphorylates OVOL2.

14. The method of claim 1, wherein said C/EBPa inhibitor is a small molecule drug.

15. A composition for use in the method of any one of claims 1-14.

16. A composition for expressing OVOL2 in a cell, comprising an engineered nucleic acid encoding an amino acid sequence having at least 80%, at least 85%, at least 95% or at least 98% sequence identity to SEQ ID NO.: 2 or 3, or a functional fragment thereof, wherein preferably expression of the engineered nucleic acid is under the control of an adipocyte-specific promoter or is activated by a trans factor in adipocytes.

17. The composition of claim 16, wherein the engineered nucleic acid encodes the amino acid sequence of SEQ ID NO.: 2 or 3.

18. The composition of claim 16, wherein the cell is a preadipocyte such as a pluripotent stem cell, a mesenchymal stem cell, a white adipocyte progenitor cell, or a brown adipocyte progenitor cell.

19. The composition of claim 16, wherein the cell is a white adipocyte or a brown adipocyte. medicament for the treatment of a metabolic disorder. 21. The composition of claim 16, wherein the metabolic disorder is one or more of obesity, type II diabetes, insulin resistance, hyperinsulinemia, hypertension, hyperlipidemia, hepatosteatosis, fatty liver, non-alcoholic fatty liver disease, hyperuricemia, polycystic ovarian syndrome, acanthosis nigricans, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Laurence-Moon syndrome, and Prader-Willi syndrome.