Programming adipocytes with RNA

Abstract

The disclosure provides methods and compositions for inducing beige and brown fat cell differentiation through modulation of one or more biomarkers, including Zfp423, Ebf2, and Prdm16. Also provided are methods for preventing or treating obesity or an obesity-related disorder in a subject through modulation of a combination of these biomarkers.

Description

[0001] Attorney Docket No.: 44807-0493WO1 / P18367-01

[0002] PROGRAMMING ADIPOCYTES WITH RNA CLAIM OF PRIORITY

[0003] This application claims the benefit of U. S. Provisional Application Serial No.

[0004] 63 / 723,655, filed on November 22, 2024. The entire contents of the foregoing are incorporated herein by reference.

[0005] SEQUENCE LISTING

[0006] This application contains a Sequence Listing that has been submitted electronically as an XML file named “44807-0493WO1_SL_ST26.xml.” The XML file, created on November 20, 2025, is 4,532 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0007] STATEMENT REGARDING FEDERAL FUNDING

[0008] This invention was made with government support under Grant No. R01 DE031488, awarded by National Institutes of Health. The government has certain rights in the invention.

[0009] TECHNICAL FIELD

[0010] The present disclosure relates to the area of treatment of obesity and metabolic disease.

[0011] BACKGROUND

[0012] Obesity represents the most prevalent of body weight disorders, affecting an estimated 30 to 50% of the middle-aged population in the western world. Obesity, defined as a body mass index (BMI) of 30 kg / m2or more, contributes to diseases such as coronary artery disease, hypertension, stroke, diabetes, hyperlipidemia and some cancers. Obesity is the end product of metabolic disease that can manifest as metabolic syndrome or diabetes mellitus (DM II).

[0013] Adipose tissue consists primarily of adipocytes. Vertebrates possess two distinct types of adipose tissue: white adipose tissue and beige / brown adipose tissue. White adipose tissue stores and releases energy according to the nutritional needs of the animal. This stored energy is used by the body for heat insulation (e.g., subcutaneous fat), mechanical cushion (e.g., surrounding internal organs), and as a source of energy. Beige or brown adipose tissue, on the other hand, burns fat, releasing the energy as heat through thermogenesis. Beige or brown adipose tissue thermogenesis is used both (1) to maintain homeothermy by increasing thermogenesis in response to lower temperatures and (2) to maintain energy balance by increasing energy Attorney Docket No.: 44807-0493WO1 / P18367-01

[0014] expenditure in response to increases in caloric intake (Sears, T. B. et al. (1996) Mol. Cell. Biol.

[0015] 16(7):3410-3419). Beige or brown adipose tissue is also the major site of thermogenesis in rodents and plays an important role in thermogenesis in human infants. In humans, and to a lesser extent rodents, beige or brown fat diminishes with age, but can be re-activated under certain conditions, such as prolonged exposure to cold, maintenance on a high fat diet and in the presence of noradrenaline producing tumors. Inducing brown or beige fat in adults could be therapeutically beneficial by increasing expenditure of excess energy, improving metabolic parameters, regulating glucose homeostasis, reducing insulin resistance, and treating metabolic syndrome, diabetes mellitus, and obesity.

[0016] Thermogenic beige and brown adipocytes are characterized by the expression of the gene UCP1. Several transcriptional regulators have been shown to regulate beige and brown fat identity and thereby regulate the expression of UCP1. As one example, Zfp423 is a master regulator transcription factor that drives the differentiation of white adipocytes and plays a key role in their maintenance as white adipocytes. In genetically modified mice, adipose tissuespecific knock-out of Zfp423 showed desired upregulation of UCP1 in mice. There remains an urgent need for novel therapeutics to treat obesity and metabolic disease through the induction of brown and beige fat.

[0017] REFERENCES

[0018] 1. Thyagarajan, B, and Foster, M. T. (2017). Beiging of white adipose tissue as a therapeutic strategy for weight loss in humans. Preprint at Walter de Gruyter GmbH, doi.org / 10.1515 / hmbci-2017-0016 doi.org / 10.1515 / hmbci-2017-0016.

[0019] 2. Wu, R., Park, J., Qian, Y., Shi, Z., Hu, R., Yuan, Y., Xiong, S., Wang, Z., Yan, G., Ong, S. G., et al. (2023). Genetically prolonged beige fat in male mice confers long-lasting metabolic health. NatCommun 14. doi.org / 10.1038 / s41467-023-38471-z.

[0020] 3. Rosenwald, M., Perdikari, A., Rtilicke, T., and Wolfrum, C. (2013). Bi-directional interconversion of brite and white adipocytes. Nat Cell Biol 15, 659-667.

[0021] doi.org / 10.1038 / ncb2740.

[0022] 4. Gupta, R. K., Arany, Z., Seale, P., Mepani, R. J., Ye, L., Conroe, H. M., Roby, Y. A., Kulaga, H., Reed, R. R., and Spiegelman, B. M. (2010). Transcriptional control of preadipocyte determination by Zfp423. Nature 464, 619-623. doi.org / 10.1038 / nature08816. Attorney Docket No.: 44807-0493WO1 / P18367-01

[0023] 5. Shao, M., Ishibashi, J., Kusminski, C M., Wang, Q. A., Hepler, C., Vishvanath, L, MacPherson, K. A., Spurgin, S. B., Sun, K., Holland, W. L., et al. (2016). Zfp423 Maintains White Adipocyte Identity through Suppression of the Beige Cell Thermogenic Gene Program. Cell Metab 23, 1167-1184. doi.org / 10.1016 / j.cmet.2016.04.023.

[0024] 6. Shao, M., Zhang, Q., Truong, A., Shan, B, Vishvanath, L., Li, L., Seale, P, and Gupta, R. K. (2021). ZFP423 controls EBF2 coactivator recruitment and PPARy occupancy to determine the thermogenic plasticity of adipocytes. Genes Dev 35, 1461-1474.

[0025] doi.org / 10.1101 / GAD.348780.121.

[0026] 7. Harder, L., Puller, A. C., and Horstmann, M. A. (2014). ZNF423: Transcriptional modulation in development and cancer. Mol Cell Oncol 7.

[0027] doi.org / 10.4161 / 23723548.2014.969655.

[0028] 8. Rakhra, G., and Rakhra, G. (2021). Zinc finger proteins: insights into the transcriptional and post transcriptional regulation of immune response. Preprint at Springer Science and Business Media B. V., doi.org / 10.1007 / s11033-021-06556-x doi.org / 10.1007 / s11033-021-06556-x.

[0029] 9. Wang, C.-H., Lundh, M., Fu, A., Kriszt, R., Huang, T. L., Lynes, M. D., Leiria, L. O., Shamsi, F., Darcy, J., Greenwood, B. P., et al. (2020). CRISPR-engineered human brown-like adipocytes prevent diet-induced obesity and ameliorate metabolic syndrome in mice.

[0030] 10. Basta, J., and Rauchman, M. (2015). The nucleosome remodeling and deacetylase complex in development and disease. Preprint at Mosby Inc., doi.org / 10.1016 / j.trsl.2014.05.003 doi.org / 10.1016 / j.trsl.2014.05.003.

[0031] 11. Alfert, A., Moreno, N., and Kerl, K. (2019). The BAF complex in development and disease. Preprint at BioMed Central Ltd., doi.org / 10.1186 / s13072-019-0264-y

[0032] doi.org / 10.1186 / s13072-019-0264-y.

[0033] 12. Stine, R. R., Shapira, S. N., Lim, H. W., Ishibashi, J., Harms, M„ Won, K. J., and Seale, P. (2016). EBF2 promotes the recruitment of beige adipocytes in white adipose tissue. Mol Metab 5, 57-65. doi.org / 10.1016 / j.molmet.2015.11.001.

[0034] 13. Wang, W., Ishibashi, J., Trefely, S., Shao, M., Cowan, A. J., Sakers, A., Lim, H. W., O’Connor, S., Doan, M. T., Cohen, P., et al. (2019). A PRDM16-Driven Metabolic Signal from Adipocytes Regulates Precursor Cell Fate. Cell Metab 30, 174-189. e5.

[0035] doi.org / 10.1016 / j.cmet.2019.05.005. Attorney Docket No.: 44807-0493WO1 / P18367-01

[0036] SUMMARY OF THE DISCLOSURE

[0037] Disclosed herein are methods of treating obesity and metabolic diseases. Induction of energy expending beige and brown adipocytes in adults could greatly ameliorate obesity and metabolic disease. Yet currently existing methods to achieve this - such as prolonged cold exposure or beta-adrenergic agonism - are impractical or unsafe. Leveraging recent developments in RNA biology, we have developed safe and effective methods to produce metabolically-beneficial beige and brown fat directly.

[0038] Obesity and metabolic disease are major problems worldwide. Incretin drugs have had a major impact on obesity treatment but need to be administered weekly for life. Discontinuation of these medicines leads to rebound weight gain. Furthermore, long term use of these drugs is associated with loss of lean muscle mass. We have disclosed an approach that can complement or supplant these drugs through the use of RNA delivery and cell programming.

[0039] Delivery of small RNAs (siRNA and miRNA) as well as mRNAs, both singularly and in combination can affect cell fate transitions in other contexts (Rossi ref). Here, we demonstrate the utility of siRNA and siRNA / mRNA combinations in inducing beige fat gene expression and phenotypic change in white adipocytes. In exemplary manifestations inhibition of the white adipocyte regulator Zfp423 alone or in combination with enhancement of the brown / beige regulatory Ebf2 confers beiging of white adipocytes with distinct advantages over prior chemical or physical approaches. First, since Zfp423 is a master regulator, even a transient knock-down can have long-lasting cascade effects on the effector genes. Second, the switch from NuRD to BAF is the key here. Once the trithorax group of genes instills an activation mark, the locus maintains a state of activation for as long as a strong repressor reverses the activation marks. Finally, Ebf2 is an efficient transcriptional activator and therefore boosting with Ebf2, can potentially exclude Zfp423 in re-repressing the complex.

[0040] In some instances, the methods disclosed herein include a method for increasing energy expenditure in a subject, the method comprising generating differentiated beige or brown fat cells by the steps of: inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation, wherein energy expenditure in the subject is increased relative to a subject whose (1) one or more biomarkers sufficient to maintain white fat Attorney Docket No.: 44807-0493WO1 / P18367-01

[0041] cell expression is not inhibited and (2) one or more biomarkers sufficient to activate beige or brown fat cell differentiation, thereby increasing energy expenditure in the subject.

[0042] In some instances, the methods disclosed herein include a method for increasing energy expenditure in a subject, the method comprising generating differentiated beige or brown fat cells, the method comprising inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; wherein energy expenditure in the subject is increased relative to a subject whose (1) one or more biomarkers sufficient to maintain white fat cell expression is not inhibited, thereby increasing energy expenditure in the subject.

[0043] In some instances, the methods disclosed herein also include a method for increasing energy expenditure in a subject, the method comprising generating differentiated beige or brown fat cells, the method comprising inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation, wherein energy expenditure in the subject is increased relative to a subject whose one or more biomarkers sufficient to activate beige or brown fat cell differentiation, thereby increasing energy expenditure in the subject.

[0044] In some instances, increasing energy expenditure comprises increasing respiration in the subject. In some instances, the respiration is total respiration. In some instances, the respiration is uncoupled respiration. In some instances, the respiration is measured by oxygen consumption or positron emission tomography.

[0045] Also disclosed is a method for preventing or treating obesity or an obesity-related disorder in a subject, the method comprising generating differentiated beige or brown fat cells, the method comprising: inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and / or inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation, thereby preventing or treating obesity or the obesity-related disorder in the subject.

[0046] In some instances, disclosed is a method for preventing or treating obesity or an obesity-related disorder in a subject, the method comprising generating differentiated beige or brown fat cells by inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression, thereby preventing or treating obesity or the obesity-related disorder in the subject.

[0047] Also disclosed is a method for preventing or treating obesity or an obesity-related disorder in a subject, the method comprising generating differentiated beige or brown fat cells by inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell Attorney Docket No.: 44807-0493WO1 / P18367-01

[0048] differentiation, thereby preventing or treating obesity or the obesity-related disorder in the subject.

[0049] In some instances, the obesity-related disorder is selected from the group consisting of: obesity, type II diabetes, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Lawrence-Moon syndrome, Prader-Labhart-Willi syndrome, anorexia, and cachexia.

[0050] Also provided herein is a method for inducing beige or brown fat cell differentiation in a subject, the method comprising: (a) providing white fat cells, cells to be differentiated into beige or brown fat cells or adipocyte cells; (b) inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation; (c) injecting the white fat cells or differentiated beige or brown fat cells from (b) into the subject; and (d) measuring differentiation of beige or brown fat cells in the subject.

[0051] In some instances, disclosed is a method for inducing beige or brown fat cell differentiation in a subject, the method comprising: (a) providing white fat cells, cells to be differentiated into beige or brown fat cells or adipocyte cells; (b) inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; (c) injecting the white fat cells from (b) into the subject; and (d) measuring differentiation of beige or brown fat cells in the subject. In some instances, disclosed is a method for inducing beige or brown fat cell differentiation in a subject, the method comprising: (a) providing white fat cells, cells to be differentiated into beige or brown fat cells or adipocyte cells; (b) inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation; (c) injecting the white fat cells or differentiated beige or brown fat cells or adipocyte cells from (b) into the subject; and (d) measuring differentiation of beige or brown fat cells in the subject.

[0052] In some instances, the cells to be differentiated into beige or brown fat cells are selected from the group consisting of fibroblasts and myoblasts. In some instances, the fibroblasts are selected from the group consisting of skin fibroblasts, dermal fibroblasts, primary embryonic fibroblasts, immortalized embryonic fibroblasts, and human foreskin fibroblasts. In some instances, the cells to be differentiated into beige or brown fat cells are autologous, allogeneic, syngeneic, xenogeneic, or HLA compatible with the subject. In some instances, the injecting is by a subcutaneous injection. In some instances, the injecting is by an intravenous injection. Attorney Docket No.: 44807-0493WO1 / P18367-01

[0053] Also disclosed herein is a method for inducing beige or brown fat cell differentiation in a subject, the method comprising: (a) obtaining cells from the subject; (b) inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation; (c) injecting the differentiated cells from (b) into the subject; and (d) measuring differentiation of beige or brown fat cells in the subject.

[0054] In some instances, the disclosure includes a method for inducing beige or brown fat cell differentiation in a subject, the method comprising: (a) obtaining cells from the subject; (b) inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; (c) injecting the differentiated cells from (b) into the subject; and (d) measuring differentiation of beige or brown fat cells in the subject. In some instances, disclosed is a method for

[0055] inducing beige or brown fat cell differentiation in a subject, the method comprising: (a) obtaining cells from the subject; (b) inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation; (c) injecting the differentiated cells from (b) into the subject; and (d) measuring differentiation of beige or brown fat cells in the subject. In some instances, the injecting is by a subcutaneous injection. In some instances, the injecting is by an intravenous injection.

[0056] In some instances, disclosed is a method for identifying a cocktail capable of inducing differentiation white fat cells into beige or brown fat cells in a subject, the method comprising: (a) providing white adipocyte cells; (b) inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation; and (c) measuring differentiation of beige or brown fat cells in the subject. Also provided is a method for identifying a cocktail capable of inducing differentiation white fat cells into beige or brown fat cells in a subject, the method comprising: (a) providing white adipocyte cells; (b) inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and (c) measuring differentiation of beige or brown fat cells in the subject.

[0057] In some instances, provided herein is a method for identifying a cocktail capable of inducing differentiation white fat cells into beige or brown fat cells in a subject, the method comprising: (a) providing white adipocyte cells; (b) inducing expression of one or more Attorney Docket No.: 44807-0493WO1 / P18367-01

[0058] biomarkers sufficient to activate beige or brown fat cell differentiation; and (c) measuring differentiation of beige or brown fat cells in the subject.

[0059] In some instances, the beige or brown fat cell differentiation is monitored by measuring the expression of a marker selected from the group consisting of: Ppar alpha, perilipin, Zic1, Lhx8, Eva1, Epsti1, PD-L1, cidea, adiponectin, adipsin, otopetrin, type II deiodinase, cig30, ppar gamma 2, pgc1a, ucp1, elovB, cAMP, Prdm1β, cytochrome C, cox4il, coxIII, cox5b, cox7al, cox8b, glut4, atpase b2, cox II, atp5o, ndufb5, and any combination thereof.

[0060] In some instances, the beige or brown fat cell differentiation is monitored by measuring degree of cellular locality. In some instances, the differentiation of beige or brown fat cells in monitored by measuring respiration within the beige or brown fat cells. In some instances, the respiration is total respiration. In some instances, the respiration is uncoupled respiration. In some instances, the respiration is measured by oxygen consumption or positron emission tomography.

[0061] In some instances, inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression comprises administering to the subject one or more siRNA and / or shRNA sequences that inhibit the one or more biomarkers sufficient to maintain white fat cell expression. In some instances, inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression comprises administering to the subject a first vector comprising a nucleic acid that encodes for an inhibitor of one or more biomarkers sufficient to maintain white fat cell expression.

[0062] In some instances, the first vector comprises one or more promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, or a combination thereof. In some instances, inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression comprising administering to a subject a second vector comprising a nucleic acid that encodes for one or more biomarkers sufficient to activate beige or brown fat cell differentiation. In some instances, the second vector comprises one or more promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, or a combination thereof. In some instances, the first vector and / or the second vector is an adeno-associated viral (AAV) vector. In some instances, the AAV vector is any one of AAV1, AAV2, Attorney Docket No.: 44807-0493WO1 / P18367-01

[0063] AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9. In some instances, the AAV vector is AAV8. In some instances, the AAV vector is AAV2. In some instances, the AAV vector is AAV9. In some instances, the first vector and / or the second vector is a recombinant adeno-associated virus (rAAV).

[0064] In some instances, the one or more biomarkers sufficient to maintain white fat cell expression comprises a transcription factor that drives differentiation of white adipocytes. In some instances, the one or more biomarkers sufficient to maintain white fat cell expression comprises a transcription factor that maintains white adipocytes. In some instances, expression of the one or more biomarkers sufficient to maintain white fat cell expression upregulates UCP1, optionally wherein the one or more biomarkers sufficient to maintain white fat cell expression comprises a transcription factor that recruits NuRD repressor complex to the Ebf2-bound thermogenic gene enhancers. In some instances, inhibition of the one or more biomarkers sufficient to maintain white fat cell expression causes expression of Brahma Associated Factor (BAF). In some instances, the one or more biomarkers sufficient to maintain white fat cell expression comprises a transcription factor that comprises at least 20, at least 25, or at least 30 C2H2-type zinc fingers. In some instances, the one or more biomarkers sufficient to maintain white fat cell expression comprises Zfp423. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Ebf2. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Prdml6. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Tle3. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Plinl. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Fabp4. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Klf9. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises cidea. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Ucpl. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Cox8b. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Cptlb. Attorney Docket No.: 44807-0493WO1 / P18367-01

[0065] In some instances, the methods include measuring protein or mRNA expression level of one or more of Zfp423, Ebf2, or Prdm16 in the subject. In some instances, the measuring protein or mRNA expression level of one or more of Zfp423, Ebf2, or Prdm16 in the subject occurs prior to the inhibiting and inducing steps. In some instances, the measuring protein or mRNA expression level of one or more of Zfp423, Ebf2, or Prdm16 in the subject occurs after the inhibiting and inducing steps. In some instances, the methods include measuring protein or mRNA expression level of one or more of Tle3, Plin1, Fabp4, Klf9, cidea, Ucp1, Cox8b, or Cpt1b in the subject. In some instances, the measuring protein or mRNA expression level of one or more of Tle3, Plin1, Fabp4, Klf9, cidea, Ucp1, Cox8b, or Cpt1b in the subject occurs prior to the inhibiting and inducing steps. In some instances, the measuring protein or mRNA expression level of one or more of Tle3, Plin1, Fabp4, Klf9, cidea, Ucp1, Cox8b, or Cpt1b in the subject occurs after the inhibiting and inducing steps.

[0066] In some instances, the subject is a mammal. In some instances, the subject is a mouse. In some instances, the subject is a human.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the disclosure, suitable methods and materials are described below.

[0068] Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

[0069] The terms “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.

[0070] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Attorney Docket No.: 44807-0493WO1 / P18367-01

[0071] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.

[0072] BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows a graph of percentage change of Zfp423 mRNA expression in NIH-3T3-treated cells. scRNA-Sgl: scrambled siRNA; siRNA-Sgl: Zfp423 siRNA; Prdm: Prdml6; p-values: * - 0.0455 (<0.05), ** - 0.0033 (<0.005), **** - P<0.0001.

[0073] FIG. 2 shows a graph of percentage change of UCP1 mRNA expression in NIH-3T3-treated cells. scRNA-Sgl: scrambled siRNA; siRNA-Sgl: Zfp423 siRNA; Prdm: Prdml6; ns: not significant; p-values: ** - 0.0018 (<0.005), *** - 0.0003 (0.0005).

[0074] FIG. 3 shows graphs depicting the fold change of Zfp423 (left panel) and Tle3 (right panel) mRNA expression in mature, differentiated 3T3-L1 cells. X-axis: scrRNA = scrambled siRNA; Zfp423 = Zfp423 siRNA (siRNA-1); Tle3 = Tle3 siRNA (siRNA-2); Zfp423, Tle3 = a combination of siRNA-1 and siRNA-2 (1:1). p-values: ** p0.005; *** p0.0005; **** pO. OOOl.

[0075] FIG. 4 shows graphs depicting the fold change of Klfl5 (left panel), Plinl (middle panel), and Fabp4 (right panel) mRNA expression in mature, differentiated 3T3-L1 cells. X-axis: scrRNA = scrambled siRNA; Zfp423 = Zfp423 siRNA (siRNA-1); Tle3 = Tle3 siRNA (siRNA-2); Zfp423, Tle3 = a combination of siRNA-1 and siRNA-2 (1:1). p-values: * pO.05; ** p0.005; *** p0.0005.

[0076] FIG. 5 shows graphs depicting the fold change of Klf9 (left panel), Cidea (middle panel), and UCP1 (right panel) mRNA expression in mature, differentiated 3T3-L1 cells. X-axis: scrRNA = scrambled siRNA; Zfp423 = Zfp423 siRNA (siRNA- 1); Tle3 = Tle3 siRNA (siRNA-2); Zfp423, Tle3 = a combination of siRNA-1 and siRNA-2 (1:1). p-values: * pO.05; ** p0.005; *** p0.0005; **** pO. OOOl.

[0077] FIG. 6 shows graphs depicting the fold change of Cox8b (left panel) and Cptlb (right panel) mRNA expression in mature, differentiated 3T3-L1 cells. X-axis: scrRNA = scrambled siRNA; Zfp423 = Zfp423 siRNA (siRNA- 1); Tle3 = Tle3 siRNA (siRNA-2); Zfp423, Tle3 = a combination of siRNA-1 and siRNA-2 (1:1). p-values: ** p<0.005; **** p<0.0001. Attorney Docket No.: 44807-0493WO1 / P18367-01

[0078] FTG. 7 shows a schematic of a Seahorse XF Cell Mito Stress Test profile, depicting the key parameters of mitochondrial function.

[0079] FIG. 8 shows a graph measuring the oxygen consumption rate in mature, differentiated 3T3-L1 cells that were treated with: scrRNA (control, scrambled siRNA); siZfp423 (Zfp423 siRNA (siRNA- 1)); siTle3 (Tle3 siRNA (siRNA-2)); or siZfp423, siTle3 (a combination of siRNA-1 and siRNA-2 (1:1)). p-values: **** p<0.0001.

[0080] FIG. 9 shows a graph measuring the oxygen consumption rate in mature, differentiated 3T3-L1 cells that were treated with: scrRNA (control, scrambled siRNA); siZfp423, siTle3 (a combination of siRNA-1 and siRNA-2 (1:1)); or siAlk7 (Alk7 siRNA), p-values: **** p<0.0001.

[0081] DETAILED DESCRIPTION

[0082] Overview

[0083] Disclosed herein are methods and compositions for treating obesity and metabolic disease. The present disclosure is based on the discovery that manipulation of white and brown fat transcriptional regulators using therapeutically actionable RNA molecules can induce metabolically beneficial gene expression. These results indicate a regulatory role of these biomarkers in brown fat differentiation and thermogenesis.

[0084] One of the biomarkers disclosed in this application is zinc finger protein 423 (Zfp423; human gene: Human NCBI Gene ID: 23090, Ensembl: ENSG00000102935, MIM:604557, AllianceGenome: HGNC: 16762; Mouse NCBI Gene ID: 94187, Ensembl: ENSMUSG00000045333, AllianceGenome: MGI: 1891217). Zfp423 recruits the Nucleosome Remodeling and Deacetylase (NuRD) repressor complex to the Ebf2-bound thermogenic gene enhancers. Ebf2 (EBF transcription factor 2; human NCBI Gene ID: 64641; Ensembl: ENSG00000221818, MIM:609934, AllianceGenome: HGNC: 19090; early B cell factor 2, mouse NCBI Gene ID: 13592, Ensembl: ENSMUSG00000022053, AllianceGenome: MGI:894332) is a major browning factor that binds to other transcription factors, such as Prdml6 (PR / SET domain 16, human NCBI Gene ID: 63976, EnsembkENSGOOOOOl 42611, M1M:6O5557, AllianceGenome: HGNC: 14000; mouse NCBI Gene ID: 70673, Ensembl: ENSMUSG00000039410 AllianceGenome: MGI: 1917923), to drive Attorney Docket No.: 44807-0493WO1 / P18367-01

[0085] the expression of thermogenic genes, including peroxisome proliferator activated receptor gamma (Pparg; NCBI Gene ID: 5468) and uncoupling protein 1 (UCP1; NCBI Gene ID: 7350).

[0086] In the absence of Zfp423, NuRD is replaced by an activating chromatin remodeling complex called BAF. While NuRD is a chromatin remodeller associated with repression, BAF, or Brahma Associated Factor is largely associated with gene activation. The NuRD to BAF switch in Zfp423 knock-out mice leads to Ebf2-mediated activation of thermogenic genes.

[0087] Additionally, Zfp423 has 30 C2H2-type zinc fingers, as opposed to a single C5-type zinc finger in Ebf2, implying a higher affinity towards DNA at common sites. The competitive exclusion of Ebf2 by Zfp423 can contribute to the maintenance of white adipocyte. In the absence of Zfp423 in the knock-out model, Ebf2 can bind to its target regions freely, thereby upregulating browning pathways.

[0088] Embodiments and aspects of the disclosure are further provided.

[0089] Methods and Uses

[0090] The present disclosure provides methods for preventing or treating obesity, an obesity related disorder, and / or metabolic disease in a subject (e.g., a human) comprising modulating expression of one or more biomarkers sufficient to activate brown fat cell gene expression, wherein the resultant cells increase energy expenditure to thereby prevent or treat obesity or an obesity related disorder in the subject. The obesity related disorder may be insulin resistant obesity, type II diabetes, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Lawrence-Moon syndrome, Prader-Labhart-Willi syndrome, anorexia, and cachexia.

[0091] In some instances, the method can treat one or more metabolic disorders or one or more obesity related disorders. The terms “metabolic disorder” and “obesity related disorders” are used interchangeably herein and include a disorder, disease or condition which is caused or characterized by an abnormal metabolism (i.e., the chemical changes in living cells by which energy is provided for vital processes and activities) in a subject. Metabolic disorders include diseases, disorders, or conditions associated with aberrant thermogenesis or aberrant adipose cell (e.g., brown or white adipose cell) content or function. Metabolic disorders can detrimentally affect cellular functions such as cellular proliferation, growth, differentiation, or migration, cellular regulation of homeostasis, inter- or intra-cellular communication; tissue function, such as Attorney Docket No.: 44807-0493WO1 / P18367-01

[0092] liver function, muscle function, or adipocyte function; systemic responses in an organism, such as hormonal responses (e.g., insulin response). Examples of metabolic disorders include obesity, including insulin resistant obesity, diabetes, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Lawrence-Moon syndrome, Prader-Labhart-Willi syndrome, anorexia, and cachexia.

[0093] As used herein, “obesity” refers to a body mass index (BMI) of 30 kg / m2or more (National Institute of Health, Clinical Guidelines on the Identification, Eva1uation, and Treatment of Overweight and Obesity in Adults (1998)). However, the present disclosure is also intended to include a disease, disorder, or condition that is characterized by a body mass index (BMI) of 25 kg / m2or more, 26 kg / m2or more, 27 kg / m2or more, 28 kg / m2or more, kg / m2or more, 29.5 kg / m2or more, or 29.9 kg / m2or more, all of which are typically referred to as overweight (National Institute of Health, Clinical Guidelines on the Identification, Eva1uation, and Treatment of Overweight and Obesity in Adults (1998)). The obesity described herein may be due to any cause, whether genetic or environmental. Examples of disorders that may result in obesity or be the cause of obesity include overeating and bulimia, polycystic ovarian disease, craniopharyngioma, the Prader-Willi Syndrome, Frohlich's syndrome, Type II diabetics, GH-deficient subjects, normal variant short stature, Turner's syndrome, and other pathological conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total fat-free mass, e.g., children with acute lymphoblastic leukemia.

[0094] “Treatment” refers to reducing the BMI of the mammal. In some instances, it can include maintaining that weight for a period of time, e.g., for at least about 6 months. The treatment suitably results in an increase in metabolic activity.

[0095] “Prevention” refers to preventing obesity or an obesity related disorder from occurring if the treatment is administered prior to the onset of the obese condition. Moreover, if treatment is commenced in subjects already suffering from or having symptoms of obesity or an obesity related disorder, such treatment is expected to prevent, or to prevent the progression of obesity or the obesity related disorder, and the medical sequelae of obesity, such as, e g., arteriosclerosis, Type II diabetes, polycystic ovarian disease, cardiovascular diseases, osteoarthritis, dermatological disorders, hypertension, insulin resistance, hypercholesterolemia, hypertriglyceridemia, and cholelithiasis. Attorney Docket No.: 44807-0493WO1 / P18367-01

[0096] In some instances, disclosed are methods for preventing or treating obesity or an obesity-related disorder in a subject. In some instances, the methods include inducing brown and beige fat effector gene expression and / or generating differentiated beige or brown fat cells. In some instances, the methods are achieved by the steps of: inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and / or inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation, thereby preventing or treating obesity or an obesity-related disorder in the subject. In some instances, the one or more biomarkers sufficient to maintain white fat cell expression includes Zfp423. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation includes Ebf2. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation includes Prdml6.

[0097] In some instances, disclosed are methods for increasing energy expenditure in cells in a subject. In some instances, the cells include mature white fat cells, mature adipocytes, and / or pre-adipocytes (e.g., fibroblasts that can differentiate into adipocytes.

[0098] In some instances, the methods include generating differentiated beige or brown fat cells by the steps of: inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation, wherein energy expenditure in the subject is increased relative to a subject whose (1) one or more biomarkers sufficient to maintain white fat cell expression is not inhibited and / or (2) one or more biomarkers sufficient to activate beige or brown fat cell differentiation, thereby increasing energy expenditure in the subject. In some instances, the one or more biomarkers sufficient to maintain white fat cell expression includes Zfp423. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation includes Ebf2. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation includes Prdml6.

[0099] In some instances, disclosed are methods for inducing beige or brown fat cell differentiation in a subject. In some instances, the methods include (a) providing white fat cells or cells to be differentiated into beige or brown fat cells. The cells provided include mature white fat cells, mature adipocytes, and / or pre-adipocytes (e.g., fibroblasts that can differentiate into adipocytes. In some instances, the methods further include (b) inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and / or inducing expression of Attorney Docket No.: 44807-0493WO1 / P18367-01

[0100] one or more biomarkers sufficient to activate beige or brown fat cell differentiation; (c) injecting the newly differentiated beige adipocytes or therapeutically intervened white adipocytes from (b) into the subject; and (d) measuring differentiation of beige or brown fat cells in the subject. In some instances, the one or more biomarkers sufficient to maintain white fat cell expression includes Zfp423. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation includes Ebf2. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation includes Prdml6.

[0101] In some instances, disclosed are methods for inducing beige or brown fat cell differentiation in a subject. In some instances, the methods include (a) obtaining cells from the subject; (b) inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and / or inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation; (c) injecting the newly differentiated beige adipocytes or therapeutically intervened white adipocytes from (b) into the subject; and (d) measuring differentiation of beige or brown fat cells in the subject. In some instances, the one or more biomarkers sufficient to maintain white fat cell expression includes Zfp423. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation includes Ebf2. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation includes Prdml6.

[0102] In some instances, disclosed are methods for identifying a cocktail capable of inducing differentiation white fat cells into beige or brown fat cells. In some instances, the methods include (a) providing cells; (b) inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and / or inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation; and (c) measuring differentiation of beige or brown fat cells in the subject. In some instances, the one or more biomarkers sufficient to maintain white fat cell expression includes Zfp423. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation includes Ebf2. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation includes Prdml6.

[0103] Another aspect of the disclosure relates to a method for inducing brown fat cell differentiation in a mammal comprising inhibiting Zfp423 expression and expressing Ebf2 or Prdml6 in white fat cells or cells to be differentiated into beige or brown fat cells; injecting the Attorney Docket No.: 44807-0493WO1 / P18367-01

[0104] white fat cells or cells to be differentiated into beige or brown fat cells expressing Prdml6 into the mammal; and monitoring the differentiation of brown fat cells. Injection of newly differentiated beige adipocytes or therapeutically intervened white adipocytes may be by a subcutaneous or intravenous injection. Increased brown adipose tissue in the mammal will warm up the body and blood of the mammal resulting in an increased energy expenditure from the cells. The increased energy expenditure will increase the metabolic rate of the subject and may be used for the treatment and / or prevention of obesity and obesity related disorders. In some instances, the one or more biomarkers sufficient to maintain white fat cell expression includes Zfp423. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation includes Ebf2. In some instances, the one or more biomarkers sufficient to activate beige or brown fat cell differentiation includes Prdml6.

[0105] The term “admini stering” is intended to include routes of administration which allow the agent to perform its intended function of modulating the one or more biomarkers to change expression and / or activity. Examples of routes of administration which can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, etc ), oral, inhalation, and transdermal. The injection can be bolus injections or can be continuous infusion. Depending on the route of administration, the agent can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. The agent may be administered alone, or in conjunction with a pharmaceutically acceptable carrier Further the agent may be coadministered with a pharmaceutically acceptable carrier. The agent also may be administered as a prodrug, which is converted to its active form in vivo.

[0106] It will be appreciated that individual dosages may be varied depending upon the requirements of the subject in the judgment of the attending clinician, the severity of the condition being treated and the particular compound being employed. In determining the therapeutically effective amount or dose, a number of additional factors may be considered by the attending clinician, including, but not limited to: the pharmacodynamic characteristics of the particular respiration uncoupling agent and its mode and route of administration; the desired time course of treatment, the species of mammal; its size, age, and general health, the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual subject; the particular compound administered; the mode of administration; the Attorney Docket No.: 44807-0493WO1 / P18367-01

[0107] bioavail ability characteristics of the preparation administered; the dose regimen selected; the kind of concurrent treatment; and other relevant circumstances.

[0108] Any means for the introduction of a polynucleotide into mammals, human or non-human. or cells thereof may be adapted to the practice of this disclosure for the delivery of the various constructs of the disclosure into the intended recipient. In one embodiment of the disclosure, the DNA constructs are delivered to cells by transfection, i.e., by delivery of “naked” DNA or in a complex with a colloidal dispersion system. A colloidal system includes macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this disclosure is a lipid-complexed or liposome-formulated DNA. In the former approach, prior to formulation of DNA, e.g., with lipid, a plasmid containing a transgene bearing the desired DNA constructs may first be experimentally optimized for expression (e g., inclusion of an intron in the 5' untranslated region and elimination of unnecessary sequences (Feigner, et al., Ann NY Acad Sci 126-139, 1995). Formulation of DNA, e.g. with various lipid or liposome materials, may then be effected using known methods and materials and delivered to the recipient mammal. See, e g,, Canonico et al, Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et al, Am J Physiol 268; Alton et al., Nat. Genet. 5:135-142, 1993 and U. S. Pat. No. 5,679,647 by Carson et al.

[0109] The targeting of liposomes can be classified based on anatomical and mechanistic factors Anatomical classification is based on the level of selectivity, for example, organ-specific, cell¬ specific, and organelle-specific Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs, which contain sinusoidal capillaries.

[0110] Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

[0111] Nucleic acids can be delivered in any desired vector. These include viral or non-viral vectors, including adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and plasmid vectors. Exemplary types of viruses include HSV (herpes simplex virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia vims). Nucleic acids can be administered Attorney Docket No.: 44807-0493WO1 / P18367-01

[0112] in any desired format that provides sufficiently efficient delivery levels, including in vims particles, in liposomes, in nanoparticles, and complexed to polymers.

[0113] In some instances, the vector is an adeno-associated viral (AAV) vector. In some instances, the AAV vector is any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9. In some instances, the AAV vector is AAV8. In some instances, the vector is a recombinant adeno-associated virus (rAAV).

[0114] In one embodiment of the disclosure, the gene delivery vehicle comprises a promoter and a demethylase coding sequence. Preferred promoters are tissue-specific promoters and promoters which are activated by cellular proliferation, such as the thymidine kinase and thymidylate synthase promoters. Other preferred promoters include promoters which are activatable by infection with a virus, such as the a- and p-interferon promoters, and promoters which are activatable by a hormone, such as estrogen. Other promoters which can be used include the Moloney virus LTR, the CMV promoter, and the mouse albumin promoter. A promoter may be constitutive or inducible.

[0115] A gene delivery vehicle can optionally comprise viral sequences such as a viral origin of replication or packaging signal. These viral sequences can be selected from viruses such as astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picomavirus, poxvirus, retrovirus, togavirus or adenovirus. In a preferred embodiment, the growth factor gene delivery vehicle is a recombinant retroviral vector. Other viral vector systems that can be used to deliver a polynucleotide of the disclosure have been derived from herpes virus, e.g., Herpes Simplex Virus and vaccinia virus. In some instances, the viruses include an alphavirus, a poxivirus, an arena virus, a vaccinia virus, a polio virus, and the like.

[0116] In another aspect, the present disclosure provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of an agent that induces Prdml 6 expression and / or activity formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. As described in detail below, the pharmaceutical compositions of the present disclosure may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as Attorney Docket No.: 44807-0493WO1 / P18367-01

[0117] a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.

[0118] The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) al inic acid; (16) pyrogen- free water; (17) isotonic saline; (18) Ringer's solution, (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0119] The term “pharmaceutically-acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of the agents that induce Prdml6 expression and / or activity encompassed by the disclosure These salts can be prepared in situ during the final isolation and purification of the respiration uncoupling agents, or by separately reacting a purified respiration uncoupling agent in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and lauiyl sulphonate salts and the like (See, for example, Berge et al. (1977) “Pharmaceutical Salts'”, J. Pharm. Sci. 66:1-19) Attorney Docket No.: 44807-0493WO1 / P18367-01

[0120] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0121] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha¬ tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like

[0122] Methods of preparing these formulations or compositions include the step of bringing into association an agent that modul ted expression and / or activity of one or more of the biomarkers described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a respiration uncoupling agent with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product

[0123] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a respiration uncoupling agent as an active ingredient. A compound may also be administered as a bolus, electuary or paste.

[0124] Detection of Biomarkers

[0125] In order to determine whether a cell or sample has transitions from a white fat cell(s) to a beige or brown fat cell(s), a variety of different assays can be performed.

[0126] Expression of any one of the biomarkers described herein may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed molecule or protein. Non-limiting examples of such methods include immunological methods for detection of proteins, protein purification methods, protein function or activity assays, nucleic acid Attorney Docket No.: 44807-0493WO1 / P18367-01

[0127] hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.

[0128] In some instances, mRNA levels are ascertained by measuring gene transcript (e.g. mRNA), by a measure of the quantity of translated protein, or by a measure of gene product activity. Expression levels can be monitored in a variety of ways, including by detecting mRNA levels, protein levels, or protein activity, any of which can be measured using standard techniques Detection can involve quantification of the level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can be a qualitative assessment of the level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear from the context.

[0129] In some instances, mRNA expression level can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art. The term “biological sample” is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from cells (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987- 1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U. S. Pat. No. 4,843,155).

[0130] The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding one of the biomarkers. Other suitable probes for use in the diagnostic assays of the disclosure are described herein. Hybridization of an mRNA with the probe indicates that, one of the biomarkers is expressed. Attorney Docket No.: 44807-0493WO1 / P18367-01

[0131] In some instances, the methods for determining Prdml6 mRNA expression level in a sample involves the process of nucleic acid amplification, e.g., by PCR, ligase chain reaction, rolling circle replication, or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[0132] In some instances, biomarker expression (nucleic acid expression (e.g., mRNA expression) or protein expression is determined. In some instances, the biomarker includes one or more of Ppar alpha, perilipin, Zic1, Lhx8, Eva1, Epsti1, PD-L1, cidea, adiponectin, adipsin, otopetrin, type II deiodinase, cig30, ppar gamma 2, pgc1α, ucpl, elovB, cAMP, Prdml0, cytochrome C, cox4il, coxIII, cox5b, cox7al, cox8b, glut4, atpase b2, cox II, atp5o, or ndufb5.

[0133] In some instances, biomarker expression (nucleic acid expression (e.g, mRNA expression) or protein expression is determined. In some instances, the biomarker includes one or more of Tle3, Plinl, Fabp4, Klf9, cidea, Ucpl, Cox8b, or Cptlb.

[0134] Table 1 below provides a list of biomarkers that can be expressed to induce beiging. Table 1.

[0135] Number Gene Function Gene Reference

[0136] ID

[0137] (NCBI)

[0138] 1 Ebf2 (EBF Transcriptional doi.org / 10.1073 / pnas.1412685111 transcription factor 2) regulator of 64641

[0139] beiging

[0140] 2 Prdml6 (PR / SET Transcriptional doi.org / 10.1038 / nature07182 domain 16) regulator of 63976

[0141] beiging

[0142] 3 Slit2-C Positive doi.org / 10.1016 / j.cmet.2016.01.008 regulator of 9353

[0143] beiging

[0144] 4 PM20D1 UCP1 doi.org / 10.10I6 / j.cell.20I6.05.071 independent 148811

[0145]

[0146] Attorney Docket No.: 44807-0493WO1 / P18367-01

[0147] (peptidase M20 mitochondrial

[0148] domain containing 1) uncoupler

[0149] Adiponectin Induced upon doi.org / 10.1016 / j.cmet.2015.06.004 cold exposure

[0150] in WAT and 9370

[0151] promotes

[0152] beiging

[0153] CIDEA (cell death Inhibits the

[0154] inducing DFFA like inhibitor of

[0155] effector a) UCP1, thereby 1149

[0156] positively

[0157] regulating

[0158] UCP1

[0159] UCP1 (uncoupling Hallmark of doi.org / 10.3389 / fendo.2020.00498

[0160] 7350

[0161] protein 1) biogenesis

[0162] CKB (creatine Plays a role in doi.org / 10.1016 / j.cmet.2024.01.001 kinase B) cold-induced 1152

[0163] thermogenesis

[0164] PPARA (peroxisome Upregulates doi.org / 10.1 194 / jlr. M01 1320 proliferator activated metabolic

[0165] receptor alpha) pathways 5465

[0166] associated with

[0167] thermogenesis

[0168] CPT1A (carnitine Implantation of doi.org / 10.1016 / j.ymben.2023.04.010 palmitoyltransferase CPT1AM- 1A) expressing

[0169] adipocytes

[0170] 1374

[0171] reduces obesity?

[0172] and glucose

[0173] intolerance in

[0174] mice

[0175] TLE3 (TLE family Transcriptional

[0176] member 3, co-rcprcssor

[0177] transcriptional protein 7090

[0178] corepressor)

[0179] PLIN1 (perilipin 1) Coats lipid

[0180] storage droplets

[0181] 5346

[0182] FABP4 (fatty acid Fatty acid

[0183] binding protein 4) binding protein

[0184] found in 2167

[0185] adipocytes

[0186]

[0187] Attorney Docket No.: 44807-0493WO1 / P18367-01

[0188] 14 KLF9 (KLF Transcription dot.org / 10.1016 / j.mce.2020.111028, transcription factor 9) factor that doi.org / 10.1038 / cdd.2010.100 binds to GC 687

[0189] box elements

[0190] located in a

[0191] promoter

[0192] 15 Cox8b (cytochrome c Involved in

[0193] oxidase subunit 8B) mitochondrial

[0194] electron 12869

[0195] transport

[0196] 16 CPT1B (carnitine Rate- palmitoyltransferase controlling

[0197] IB) enzyme of the

[0198] long-chain fatty

[0199] acid beta1375

[0200] oxidation

[0201] pathway in

[0202] muscle

[0203]

[0204] mitochondria

[0205] The level or activity of any one of the biomarkers can also be detected and / or quantified by detecting or quantifying the expressed polypeptide The polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. These may include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, and the like.

[0206] Design of Inhibitors

[0207] In some instances, disclosed herein are methods of designing one or more inhibitors of any one of the biomarkers, particularly if they promote maintenance of white fat cells. In order to design an inhibitor, one can use the sequence (e.g., the mRNA sequence of one or more inhibitors). In some instances, the inhibitor sequence and the mRNA target sequence have 100% sequence identity or homology. “Sequence identity or homology”, as used herein, refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino Attorney Docket No.: 44807-0493WO1 / P18367-01

[0208] acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identical at that position. The percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by the two sequences divided by the number of positions compared^ 100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences are 60% homologous or have 60% sequence identity By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology Unless otherwise specified “loop out. regions”, e.g., those arising from, from deletions or insertions in one of the sequences are counted as mismatches.

[0209] Table 2 below provides a list, of biomarkers that can be targeted in white fat cells to induce beiging.

[0210] Table 2.

[0211] Gene ID

[0212] Number Gene Function

[0213] (NCBI) Reference

[0214] Maintains white www cell.com / cell- 1 ZFP423 fat depot by 23090 metabolism / fulltext / S 1550- repressing Ebf2 4131(16)30175-9

[0215] Subcutaneous fat

[0216] 2 KLF15 requires Klfl5 for 28999 doi.org / 10.1172 / JCI172360

[0217] its maintenance

[0218] Target of MiR- 142-3p; regulates

[0219] white fat doi.org / 10.1016 / j.mce.2020.111028, 3 KLF9 687

[0220] development by doi.org / 10.1038 / cdd.2010.100 transactivation of

[0221] PparG

[0222] Inhibits PPARy's

[0223] role in driving the

[0224] thermogenic

[0225] program.

[0226] 4 TLE3 Repression of 7090 doi.org / 10.1016 / j.cmct.2011.02.014 TLE3 can

[0227] promote the

[0228] beiging of white

[0229] adipocvtes.

[0230] Target of MiR- 455; repression of

[0231] 5 Necdin 4692 doi.org / I0. I5252 / embr.201540837

[0232] Necdin promotes

[0233]

[0234] beiging Attorney Docket No.: 44807-0493WO1 / P18367-01

[0235] Target of MiR- 455; repression of

[0236] 6 Runxltl 862 doi.org / 10.15252 / embr.201540837

[0237] Runxltl promotes

[0238] beiging

[0239] Repression of

[0240] FOXO1 enhances

[0241] PGCla and UCP1

[0242] 7 FoxOl 2308 doi.org / 10.2337 / db08-1001

[0243] expression,

[0244] facilitating

[0245] thermogenesis.

[0246] Inhibitor of the

[0247] Wnt signaling

[0248] pathway,

[0249] influencing

[0250] 8 SFRP5 adipose 6425 doi.org / 10.1016 / j.lfs.2020.117338

[0251] differentiation.

[0252] Downregulation is

[0253] associated with

[0254] enhanced beiging.

[0255] Direct repressor of

[0256] UCP1.

[0257] Downregulation

[0258] 9 LxRa 10062 doi.org / 10.1128 / MCB.01479-07

[0259] of LxRa can

[0260] potentially cause

[0261] beiging

[0262] Responsible for

[0263] CUL2- 8453 and www.nature.com / articles / s41586-022- 10 repression of

[0264] APPBP2 10513 05067-4

[0265]

[0266] Prdml6.

[0267] In some instances, the inhibitors include any one or more of SEQ ID NOs: 1-4, as shown below:

[0268] siRNA-1: mZfp423

[0269] Sense - GGAUGUUGCGUC ACCUACGtt (SEQ ID NO: 1 )

[0270] Antisense - CGUAGGUGACGCAACAUCCtt (SEQ ID NO:2)

[0271] siRNA-2: mTle3

[0272] Sense - GCAUACUCCUUCCAUGUGAtt (SEQ ID NO:3)

[0273] Antisense - UCACAUGGAAGGAGUAUGCcg (SEQ ID NO:4)

[0274] In some instances, the inhibitor includes SEQ ID NO: 1, or a complement thereof. In some instances, the inhibitor includes SEQ ID NO:2, or a complement thereof. In some instances, the inhibitor includes SEQ ID NO:3, or a complement thereof. In some instances, the inhibitor includes SEQ ID NO: 4, or a complement thereof. Attorney Docket No.: 44807-0493WO1 / P18367-01

[0275] In some instances, the inhibitor includes a sequence that is 80%, 85%, 90%, or 95% identical to SEQ ID NO.1, or a complement thereof. In some instances, the inhibitor includes a sequence that is 80%, 85%, 90%, or 95% identical to SEQ ID NO:2, or a complement thereof. In some instances, the inhibitor includes a sequence that is 80%, 85%, 90%, or 95% identical to SEQ ID NO:3, or a complement thereof. In some instances, the inhibitor includes a sequence that is 80%, 85%, 90%, or 95% identical to SEQ ID NO:4, or a complement thereof.

[0276] In some embodiments, inhibitors are designed to target and hybridize to a plurality of nucleic acids (e.g., to one target or a plurality of similar targets). Disclosed herein are methods to design and test candidate inhibitor sequences. Candidate inhibitors are designed so that each inhibitor sequence theoretically hybridizes to a unique target of interest. To identify an inhibitor, at unique nucleotide sections (e.g., 20, 25, 30, 35, 40, etc. nucleotides) of the human transcriptome are identified and aligned to the genome using an aligner designed for aligning RNA-seq data. In some embodiments inhibitors are designed to hybridize to a particular exon. In some embodiments, inhibitors are designed to span an exon-exon junction. In some embodiments, inhibitors can hybridize to a target, allowing for identification of splicing and alternative splicing transcripts in the transcriptome. Using the alignments, sequences that align to the genome one or more times are identified and cataloged. Each inhibitor designed can be tested against (i.e., compared to) a sequence identified in the genome. If the sequences in the inhibitors and in the genome do not match, then the bait can be tested in one or more panels as disclosed herein.

[0277] The present disclosure provides novel antisense RNA molecules, including siRNAs and shRNAs, and methods for their use in modulating gene expression. These molecules are designed to target specific nucleotide sequences of mRNA, resulting in reduced expression of the corresponding protein. The disclosure also includes compositions and delivery systems for enhancing the stability, specificity, and cellular uptake of these molecules.

[0278] The disclosure provides siRNA molecules comprising a double-stranded RNA structure with 19-25 base pairs, including a guide strand complementary to the target mRNA and a passenger strand. Chemical modifications such as 2'-O-methyl and phosphorothioate linkages are incorporated to enhance stability and reduce immunogenicity. The disclosure also provides shRNA molecules comprising a stem-loop structure. The stem includes a sequence Attorney Docket No.: 44807-0493WO1 / P18367-01

[0279] complementary to the target mRNA, and the loop facilitates Dicer processing. shRNAs can be encoded in expression vectors for sustained gene silencing.

[0280] In some instances, the antisense RNA molecules are designed to target specific sequences in mRNA transcripts, particularly regions such as the 3'-untranslated region (3'-UTR) or coding sequence (CDS). Bioinformatics tools are employed to minimize off-target interactions.

[0281] Chemical modifications are incorporated into the siRNAs and shRNAs to enhance nuclease resistance and binding affinity. Examples include: 2'-O-methyl and 2'-fluoro modifications on the ribose sugar, locked nucleic acids (LNAs) to enhance thermal stability, and phosphorothioate backbone modifications to improve nuclease resistance.

[0282] In some embodiments, the modification is a pseudouracil, Ml -pseudouracil, 5-m ethoxy uridine (5moU), or N4-acetylcytidine modification. In some embodiments, the mRNA comprises a nucleotide modification. In some embodiments, the nucleotide modification is a Nl-m ethyladenosine (ml A), N6-methyladenosine (m6A), or adenosine to inosine (A-to-I) modification. In some embodiments, the nucleotide modification is a pseudouracil, Ml-pseudotiracil, 5-methoxyuridine (5moU), or N4-acetylcytidine. In some embodiments, the nucleotide modification is a l,2’-O-dimethyladenosine (ml Am), l,2’-O-dimethylguanosine (m 1 Gm), 1,2’-O-dimethylinosine (m 11m), 1 -methyl-3 -(3-amino-3-carboxypropyl)pseudouridine (mlacp3Y), 1 -methyladenosine (ml A), 1 -methylguanosine (mlG), I -methylinosine (mil), 1-methylpseudouridine (mlY), 2,8-dimethyladenosine (m2,8A), 2-geranylthiouridine (ges2U), 2-lysidine (k2C), 2-methyladenosine (m2A), 2-methylthiomethylenethio-N6-isopentenyl-adenosine (msms2i6A), 2-methylthio-cyclic-N6-threonylcarbamoyladenosine (ms2ct6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)-adenosine (ms2io6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine (ms2hn6A), 2-methylthio-N6-isopentenyladenosine (ms2i6A), 2-methylthio-N6-methyladenosine (ms2m6A), 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A), 2-selenouridine (se2U), 2-thio-2’-O-methyluridine (s2Um), 2 -thiocytidine (s2C), 2 -thiouridine (s2U), 2’-O-methyladenosine (Am), 2’-O-methylcytidine (Cm), 2’-O-methylguanosine (Gm), 2’-O-methylinosine (Im), 2’-O-methylpseudouridine (Ym), 2’-O-methyluridine (Um), 2’-O-methyluridine 5-oxyacetic acid methyl ester (mcmo5Um), 2’-O-ribosyladenosine (phosphate) (Ar(p)), 2’-O-ribosylguanosine (phosphate) (Gr(p)), 3,2’-O-dimethyluridine (m3Um), 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine (acp3D), 3-(3-amino-3-carboxypropyl)pseudouridine (acp3Y), 3-(3-amino-3- Attorney Docket No.: 44807-0493WO1 / P18367-01

[0283] carboxypropyl (uridine (acp3U), 3 -methyl cytidine (m3C). 3-methylpseudouridine (m3Y), 3-methyluridine (m3U), 4-demethylwyosine (imG-14), 4-thtouridine (s4U), 5,2’-O-dimethylcytidine (m5Cm), 5,2’-O-dimethyluridine (m5Um), 5-(carboxyhydroxymethyl)-2’-O-methyluridine methyl ester (mchm5Um), 5-(carboxyhydroxymethyl)uridine methyl ester (mchm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U), 5-(isopentenylaminomethyl)-2’-O-methyluridine (inm5Um), 5-(isopentenylaminomethyl)uridine (inm5U). 5 -aminomethyl -2-geranylthiouridine (nm5ges2U), 5-aminomethyl-2-selenouridine (nm5se2U), 5-aminomethyl-2-thiouridine (nm5s2U), 5-aminomethyluridine (nm5U), 5-carbamoylhydroxymethyluridine (nchm5U), 5-carbamoylmethyl-2-thiouridine (ncm5s2U), 5-carbamoylmethyl-2’-O-methyluridine (ncm5Um), 5 -carbamoylmethyluridine (ncm5U), 5-carboxyhydroxymethyluridine (chm5U), 5-carboxytnethyl-2-thiouridine (cm5s2U), 5-carboxymethylaminomethyl-2-geranylthiouridine (cmnm5ges2U), 5-carboxymethylaminomethyl-2-selenouridine (cmnm5se2U), 5-carboxymethylaminomethyl-2 -thiouridine (cmnm5s2U), 5-carboxymethylaminomethyl-2’-O-methyluridine (cmnm5lJm), 5-carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethyluridine (cm5U), 5- cyanomethyluridine (cnm5U), 5-formyl-2’-O-methylcytidine (f5Cm), 5 -formyl cytidine (f5C), 5-hydroxycytidine (ho5C), 5-hydroxymethylcytidine (hm5C), 5-hydroxyuridine (ho5U), 5-methoxy carbonyl ethyl-2-thiouridine (mcm5s2U), 5-methoxy carbonylmethyl -2’ -O-methyluridine (mcm5Um), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxyuridine (mo5U), 5-methyl-2-thiouridine (ni5s2U), 5-methylaminomethyl-2 -geranyl thiouridine (mnm5ges2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-methylaminomethyl-2- thiouridine (mnm5s2U), 5-methylaminomethyluridine (mnm5U), 5-methylcytidine (m5C), 5-methyldihydrouridine (m5D), 5-methyluridine (m5U), 5-taurinomethyl-2-thiouridine (tm5s2U), 5-taurinomethyluridine (tm5U), 7-aminocarboxypropyl-demethylwyosine (yW-86), 7-aminocarboxypropylwyosine (yW-72), 7-aminocarboxypropylwyosine methyl ester (yW-58), 7-aminomethyl-7-deazaguanosine (preQltRNA), 7-cyano-7-deazaguanosine (preQOtRNA), 7-methylguanosine (m7G), 8-methyl adenosine (m8A), N2,2’-O-dimethylguanosine (m2Gm), N2,7,2’-O-trimethylguanosine (m2,7Gm), N2,7-dimethylguanosine (m2,7G), N2?N2,2’-O-trimethylguanosine (m2,2Gm), N2, N2,7-trimethylguanosine (m2,2,7G), N2, N2- dimethylguanosine (m2,2G), N2-methylguanosine (m2G), N4,2'-O-dimethylcytidine (m4Cm), N4, N4,2’-O-trimethyl cytidine (m4,4Cm), N4, N4-dimethylcytidine (m4,4C), N4-acetyl-2’-O- Attorney Docket No.: 44807-0493WO1 / P18367-01

[0284] methylcytidine (ac4Cm), N4-acetyl cytidine (ac4C), N4-methylcytidine (m4C), N6,2’-O-dimethyladenosine (m6Am), N6, N6,2’-O-trimethyladenosine (m6,6Am), N6, N6-dimethyladenosine (m6,6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), N6-acetyladenosine (ac6A), N6-formyladenosine (f6A), N6-glycinylcarbamoyladenosine (g6A), N6- hydroxymethyladenosine (hm6A), N6-hydroxynorvalylcarbamoyladenosine (hn6A), N6-isopentenyladenosine (i6A), N6-methyl-N6-threonylcarbamoyladenosine (m6t6A), N6-methyladenosine (m6A), N6-threonylcarbamoyladenosine (t6A), Agmatidine (C+), Archaeosine (G+), cyclic N6-threonylcarbamoyladenosine (ct6A), dihydrouridine (D), epoxyqueuosine (oQtRNA), galactosyl-queuosine (galQtRNA), glutamyl-queuosine (gluQtRNA), hydroxy-N6-threonylcarbamoyladenosine (ht6A), hydroxywybutosine (OHyW), inosine (I), isowyosine (imG2), mannosyl-queuosine (manQtRNA), methylated undermodified hydroxywybutosine (OHyWy), methylwyosine (mimG), peroxywybutosine (o2yW), pseudouridine (Y), queuosine (QtRNA), undermodified hydroxywybutosine (OHyWx), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), wybutosine (yW), or wyosine (imG) modification.

[0285] In some instances, the inhibitor is delivered using a delivery system. Delivery systems are critical for efficient cellular uptake and endosomal escape of antisense RNAs. In some instances, the delivery system includes lipid nanoparticles (LNPs) encapsulating antisense RNA, polymer conjugates, such as polyethylene glycol (PEG)-linked RNAs, and viral vectors for in vivo expression of shRNAs.

[0286] Combination treatment

[0287] In addition to modulating one or more biomarkers, the methods described herein can include treatment with one or more additional therapeutic molecule to treat obesity or an obesity-related disease.

[0288] In some instance, the additional therapeutic molecule includes a molecule approved for nutrition and weight loss. In some instances, the additional therapeutic molecule includes one or more of Bydureon, Bydureon BCise (exenatide extended-release for injectable suspension), Contrave (naltrexone HC1 and bupropion HC1), Jardiance (empagliflozin), Nascobal (Cyanocobalamin) Nasal Spray, Nesina (alogliptin), Qsymia (phentermine + topiramate extended-release), Redux (dexfenfluramine hydrochloride), Saxenda (liraglutide [rDNA origin] Attorney Docket No.: 44807-0493WO1 / P18367-01

[0289] injection), Tanzeum (albiglutide), Semaglutide (Ozempic®, Rybelsus®, Wegovy®) and Trulicity (dulaglutide).

[0290] In some instance, the additional therapeutic molecule includes a molecule approved for cardiology or vascular diseases. In some instances, the additional therapeutic molecule includes one or more of Adcirca (tadalafil), Adempas (riociguat), Agrylin (anagrelide), Altocor (lovastatin) Extended-Release Tablets, Androderm (Testosterone Transdermal System), Arcalyst (rilonacept), Atacand (candesartan cilexetil), Atryn (antithrombin recombinant lyophilized powder for reconstitution), Azor (amlodipine besylate; olmesartan medoxomil), Benicar (olmesartan medoxomil), Benicar HCT (olmesartan medoxomil and hydrochlorothiazide), Betapace, Betapace AF (sotalol hydrochloride), BiDil (isosorbide dinitrate / hydralazine hydrochloride), Brilinta (ticagrelor), Caduet (amlodipine besylate and atorvastatin calcium tablet), Camzyos (mavacamten) capsules, CellCept (mycophenolate mofetil), Cleviprex

[0291] (cl evi dipine), Corlanor (ivabradine), Corlopam (fenoldopam mesylate) injection, Convert Injection (ibutilide fumarate injection), Crestor (rosuvastatin calcium), Diovan (valsartan), Diovan HCT (valsartan and hydrochlorothiazide USP), Edarbi (azilsartan medoxomil), Edarbyclor (azilsartan medoxomil / chlorthalidone), edex (alprostadil for injection), Effient (prasugrel), Eliquis (apixaban), Entresto (sacubitril and valsartan), Fragmin (dalteparin sodium), Injectafer (ferric carboxymaltose injection), Inpefa (sotagliflozin), Inspra (eplerenone) tablets, Invokana (canagliflozin), Juxtapid (lomitapide), Kanuma (sebelipase alfa), Kengreal (cangrelor), Kerendia (finerenone), Kynamro (mipomersen sodium), Leqvio (inclisiran), Lescol XL (fluvastatin sodium) tablet, extended release, Letairis (ambrisentan), Levitra (vardenafil), Lipitor (atorvastatin calcium), Liptruzet (ezetimibe and atorvastatin), Livalo (pitavastatin), Micardis (telmisartan), MicardisPlus, Micardis HCT (telmisartan and hydrochlorothiazide), Multaq (dronedarone), Muse (alprostadil), Nitrostat (nitroglycerin) Tablets, Nymalize (nimodipine), Opsumit (macitentan), Opsynvi (macitentan and tadalafil), Plavix (clopidogrel bisulfate), Pradaxa (dabigatran etexilate mesylate), Praluent (alirocumab), Prestalia (perindopril arginine and amlodipine besylate), Prograf (tacrolimus), Ranexa (ranolazine), Remodulin (treprostinil), Repatha (evolocumab), Retavase (reteplase) for injection, Savaysa (edoxaban), Soliris (eculizumab), Tekturna (aliskiren), Tiazac (diltiazem hydrochloride) XR, ToprolXL (metoprolol succinate), Tribenzor (olmesartan medoxomil + amlodipine + hydrochlorothiazide), Tricor (fenofibrate) tablets, Trilipix (fenofibric acid), Trulicity (dulaglutide), Tryvio (aprocitentan), Attorney Docket No.: 44807-0493WO1 / P18367-01

[0292] Tyvaso (treprostinil) inhalation solution, Uptravi (selexipag), Varithena (polidocanol injectable foam), Vascepa (icosapent ethyl), Viagra (sildenafil citrate), Vyndaqel (tafamidis meglumine) and Vyndamax (tafamidis), Welchol (colesevelam hydrochloride), Xarelto (rivaroxaban), Zocor (simvastatin) tablets, and Zontivity (vorapaxar).

[0293] In some instance, the additional therapeutic molecule includes a molecule approved for treatment of high blood pressure. In some instances, the additional therapeutic molecule includes one or more of Atacand (candesartan cilexetil), Azor (amlodipine besylate; olmesartan medoxomil), Benicar (olmesartan medoxomil), Benicar HCT (olmesartan medoxomil and hydrochlorothiazide), Caduet (amlodipine besylate and atorvastatin calcium tablet), Cleviprex (clevidipine), Corlopam (fenoldopam mesylate) injection, Diovan (valsartan), Diovan HCT (valsartan and hydrochlorothiazide USP), Edarbi (azilsartan medoxomil), Edarbyclor (azilsartan medoxomil / chlorthalidone), Inspra (eplerenone) tablets, Micardis (telmisartan), MicardisPlus, Micardis HCT (telmisartan and hydrochlorothiazide), Prestalia (perindopril arginine and amlodipine besylate), Tekturna (aliskiren), Tiazac (diltiazem hydrochloride) XR, Toprol-XL (metoprolol succinate), Tribenzor (olmesartan medoxomil + amlodipine + hydrochlorothiazide), and Tryvio (aprocitentan).

[0294] In some instance, the additional therapeutic molecule includes a molecule approved for treatment of diseases that affect the endocrine system. In some instances, the additional therapeutic molecule includes one or more of Activella (Estradiol / Norethindrone Acetate) Tablets, Actonel, Actos (pioglitazone); ACTOplus met (pioglitazone and metformin HC1), Addyi (flibanserin), Adlyxin (lixisenatide), Afrezza (insulin human) Inhalation Powder, Aggrenox (aspirin / extended-release dipyridamole capsules), Aldurazyme (laronidase), Amaryl (glimepiride), Androderm (Testosterone Transdermal System), AndroGel testosterone gel, Arava, Aredia (pamidronate disodium for injection), Arimidex (anastrozole), Aromasin Tablets, Atacand (candesartan cilexetil), Avandia (rosiglitazone maleate), Aveed (testosterone undecanoate) injection, Benicar (olmesartan medoxomil), Benicar HCT (olmesartan medoxomil and hydrochlorothiazide), Brenzavvy (bexagliflozin), Brisdelle (low-dose paroxetine mesylate), Bydureon, Bydureon BCise (exenatide extended-release for injectable suspension), Byetta (exenatide), Cetrotide (cetrorelix acetate for injection), Climara Pro (estradiol / levonorgestrel transdermal system), Cometriq (cabozantinib), Corlopam (fenoldopam mesylate) injection, Crinone (progesterone gel) 4% and 8%, Cycloset (bromocriptine mesylate), Cymbalta Attorney Docket No.: 44807-0493WO1 / P18367-01

[0295] (duloxetine delayed-release capsules), Duavee (conjugated estrogens / bazedoxifene), Elelyso (taliglucerase alfa), Elestrin (estradiol gel), Ellence (epirubicin hydrochloride injection), Estrostep Fe (Norethindrone Acetate and Ethinyl Estradiol, USP and Ferrous Fumarate), Evamist (estradiol transdermal spray), Eylea (aflibercept), Fabrazyme (agalsidase beta), Farxiga (dapagliflozin), Femara (letrozole), FemPatch, Forteo (teriparatide), Fosamax (alendronate sodium), Genotropin (somatropin) injection, Glucagon, Glyset (miglitol), Gonal-F (follitropin alfa for injection), Hectorol (doxercalciferol), Humalog (insulin lispro), Humatrope (somatropin [rDNA origin] for injection), Increlex (mecasermin), Invokana (canagliflozin), Januvia (sitagliptin phosphate), Jardiance (empagliflozin), Jatenzo (testosterone undecanoate) capsules, Jentadueto (linagliptin plus metformin hydrochloride), Korlym (mifepristone), Lantidra (donislecel-jujn), Lantus (insulin glargine) injection, Lenvima (lenvatinib), Lescol XL (fluvastatin sodium) tablet, extended release, Levoxyl, Lucentis (ranibizumab), Lupron Depot (leuprolide acetate for depot suspension), Macrilen (macimorelin), Miacalcin (calcitonin-salmon), Mirena (levonorgestrel-releasing intrauterine system), Mounjaro (tirzepatide), Naprelan (naproxen sodium), Nascobal (Cyanocobalamin) Nasal Spray, Natesto, (testosterone) nasal gel, Natpara (parathyroid hormone), Nesina (alogliptin), Ngenla (somatrogon-ghla), Norditropin (somatropin) injection, Novolog MIX 70 / 30 (insulin aspart protamine and insulin aspart), Nutropin (somatropin-rDNA origin), Onglyza (saxagliptin), Oriahnn (elagolix, estradiol, and norethindrone acetate capsules; elagolix capsules), Osphena (ospemifene), Ovidrel (choriogonadotropin alfa injection), OxyContin (oxycodone hydrochloride) extended-release tablets, Ozempic (semaglutide) subcutaneous injection, Parsabiv (etelcalcetide), Prempro & Premphase (conjugated estrogens / medroxyprogesterone acetate tablets), Prolia (denosumab), Prometrium (progesterone), Qsymia (phentermine + topiramate extended-release), Qtem (dapagliflozin and saxagliptin), Recorlev (levoketoconazole), Redux (dexfenfluramine hydrochloride), REGRANEX (becaplermin) Gel, Retevmo (selpercatinib), Rybelsus (semaglutide) oral tablets, Saizen (somatropin) injection, Signifor (pasireotide diaspartate), Signifor LAR (pasireotide), Simulect (basiliximab), Skytrofa (lonapegsomatropin-tcgd), Soliqua 100 / 33 (insulin glargine and lixisenatide injection), Somatuline Depot (lanreotide acetate), Somavert (pegvisomant), Steglatro (ertugliflozin), Supprelin LA (histrelin acetate), Symlin (pramlintide), Synjardy (empagliflozin and metformin hydrochloride), Synthroid (levothyroxine sodium), Tanzeum (albiglutide), Taxotere (Docetaxel), Testim, Tradjenta (linagliptin), Trelstar Attorney Docket No.: 44807-0493WO1 / P18367-01

[0296] (triptorelin pamoate), Tresiba (insulin degludec injection), Trulicity (dulaglutide), Tzield (teplizumab-mzwv), Victoza (liraglutide), Viokace (pancrelipase) tablets, Vogelxo (testosterone) gel, Voxzogo (vosoritide) for Injection, Welchol (colesevelam hydrochloride), Xeloda (capecitabine), Xenical (orlistat) Capsules, Xigduo XR (dapagliflozin + metformin hydrochloride), Xultophy 100 / 3.6 (insulin degludec and liraglutide injection), Yasmin (drospirenone / ethinyl estradiol), Zemplar (paricalcitol) capsules, and Zoladex (goserelin acetate implant), and Zometa (zoledronic acid).

[0297] EXAMPLES

[0298] Example 1. Co-treatment of siRNA targeting Zfp423 and Ebf2 or Prdml 6 mRNAs leads to induction ofUCPl

[0299] This experiment demonstrates that modulation of expression of a combination of white fat cells biomarkers and beige or brown fat cell biomarkers. 3T3-L1 (ATCC# CL-173) fibroblast cells were seeded in a 12-well plate at 70-80% confluency. Growth media for differentiation containing DMEM / F12 with IX antibiotic-antimycotic (anti-anti) and fetal bovine serum (FBS, final concentration 10% v / v) was added to the cells. After 24 hrs, the growth media was replaced by pre-differentiation media containing growth media with 0.5 mM IBMX, 1 mM dexamethasone and 20 mg / ml insulin. The pre-differentiation media was replaced by differentiation media containing 20 mg / ml insulin and 2 mM D-biotin after 3 days. The differentiation media was replaced every 3 days for 3 times.

[0300] The cells next were transfected with nucleic acids to inhibit Zfp423, express Ebf2, or express Prdml 6. Specifically, four formulations of lipid nanoparticles (LNPs) were produced to deliver nucleic acids of the following groups:

[0301] • 1.5 mg scrambled siRNA (“scRNA-Sgl”);

[0302] • 1.5 mg siRNA directed to Zfp423 (“siRNA- Sgl”);

[0303] • 1.5 mg siRNA-Sgl and 1.5 mg with 500ng Ebf2 mRNA;

[0304] • 1.5 mg siRNA- Sgl and 1.5 mg with 500ng Prdml 6 mRNA (“Prdm mRNA” or “Prdml 6 mRNA”);

[0305] • 500ng Ebf2 mRNA; and

[0306] • 500ng Prdm 16 mRNA. Attorney Docket No.: 44807-0493WO1 / P18367-01

[0307] Each of the six conditions was performed in triplicate. Procedurally, the LNPs were gently added on top of the cells drop by drop and the plate was swirled for even mixing. The cells were harvested for RNA isolation after 6 days of transfection.

[0308] As shown in FIG. 1, a single dose of siRNA targeting Zfp423 — either alone or in combination with Ebf2 mRNA or Prdml6 mRNA — significantly downregulated Zfp423.

[0309] Additionally, introduction of Ebf2 and Prdml6 mRNAs also caused partial downregulation of Zfp423. These data demonstrate that manipulation of these biomarkers leads to down-regulation of a white fat cell biomarker (Zfp423).

[0310] Next, we considered whether this biomarker manipulation would affect beige or brown fat cell gene expression. To test this, mRNA expression of a marker of beige or brown fat, UCP1, was examined. As shown in FIG. 2, Zfp423 siRNA-treated samples show upregulation of UCP1 mRNA. Additionally, treatment of cells only with Ebf2 or PRdml6 mRNAs could not upregulate UCP1. On the other hand, while Zfp423 siRNA alone could elicit a non-significant 3 -fold (300%) higher expression of UCP1, siRNA in combination with Ebf2 could elicit a significant 5.74-fold (574%) induction of UCP1 mRNA. Further, siRNA with Prdml6 mRNA showed 4.8 fold (481%) upregulation of UCP1.

[0311] Taken together, these data demonstrate that inhibiting Zfp423 and overexpressing either Ebf2 or Prdml6 show that white cells and transition to beige or brown cells. Therefore, modulation of these biomarkers can be used therapeutically to increase beige or brown fat cells in a subject.

[0312] Example 2. Co-treatment of siRNA targeting Zfp423 and Tle3 synergistically repressed white fat genes and synergistically upregulated beige fat genes

[0313] 3T3-L1 fibroblasts were differentiated into mature adipocytes using a standard 10-day differentiation protocol similar to that described in Strnadova M et al., STAR Protoc. 2024 Jun 21;5(2): 103075. The resulting mature, differentiated 3T3-L1 cells were then treated with different combinations of siRNA and tested for white fat, beige fat, and mitochondrial markers. In particular, the cells were treated with siRNA packaged in LNPs that targeted the Zfp423 and or Tle3 genes. Zfp423 is a master regulator of adipocyte development and maintenance of white adipocytes (Shao M et al., Cell Metab. 2016 Jun 14;23(6): 1167-1184) and Tle3 is required for maturation of pre-adipocytes and maintenance of white fat depot (Pearson et al., Genes Dev. Attorney Docket No.: 44807-0493WO1 / P18367-01

[0314] 2019 Jul 1;33(13-14):747-762). To date, these factors have been studied in silos and in different systems. Here, we combined them (siRNA 1:1) and achieved inter-dependent activation of beiging factors. Sequences of the siRNA used in this experiment were as follows:

[0315] siRNA-1: mZfp423

[0316] Sense - GGAUGUUGCGUCACCUACGtt (SEQ ID NO: 1)

[0317] Antisense - CGUAGGUGACGCAACAUCCtt (SEQ ID NO:2)

[0318] siRNA-2: mTle3

[0319] Sense - GCAUACUCCUUCCAUGUGAtt (SEQ ID NO:3)

[0320] Antisense - UCACAUGGAAGGAGUAUGCcg (SEQ ID NO:4)

[0321] siRNA-1, siRNA-2, or a combination of siRNA-1 and siRNA-2 (1: 1) were packaged in LNPs according to methods known in the art (see, e.g., Yonezawa et al., Adv Drug Deliv Rev.

[0322] 2020;154-155:64-78; Jurgens et al., OpenNano 2023;12, Article 100161; Haque et al., Int. J. PharmX. 2024; 8, 100283).

[0323] As demonstrated in FIG.3, the target genes Zfp423 (left panel) and Tle3 (right panel) were successfully downregulated using either siRNA- 1, siRNA-2, or a 1:1 combination of siRNA- 1 and siRNA-2.

[0324] Obesity is linked with sustained expression of genes that are responsible for white adipocyte growth, development and functioning. Here, we examined the effect of siRNA treatment on three genes that are known canonical markers of white adipocytes - Klfl5, Plinl, and Fabp4 - in mature, differentiated 3T3-L1 cells (i.e., mature adipocytes). Klf 15 is a transcription factor required for white fat maintenance and Plinl and Fabp4 are required for triglyceride synthesis and sequestering. As demonstrated in FIG. 4, the white adipocyte genes Klfl5 (left panel), Plinl (middle panel), and Fabp4 (right panel) were successfully downregulated upon downregulation of Zfp423 and / or Tle3 using either siRNA-1, siRNA-2, or a 1:1 combination of siRNA-1 and siRNA-2. Co-treatment with Zfp423 and Tle3 siRNA (i.e., the 1:1 combination of siRNA- 1 and siRNA-2) was synergistic in the downregulation of Klfl5, demonstrating a higher downregulation of Klfl5 in co-treatment (FIG. 4, left panel) implying better control over white fat homeostasis.

[0325] We also examined the effect of siRNA treatment on three genes that are known canonical markers of beige adipocytes - Klfl9, Cidea, and UCP1 - in mature, differentiated 3T3-L1 cells (i.e., mature adipocytes). Klf9, a transcription factor regulator of beiging, was downregulated Attorney Docket No.: 44807-0493WO1 / P18367-01

[0326] following independent treatment with either siRNA-1 (Zfp423) or siRNA-2 (Tle3) (FIG. 5, left panel). Co-treatment with Zfp423 and Tle3 siRNA (i.e., the 1:1 combination of siRNA-1 and siRNA-2) acted in a synergistic manner to result in the desired effect of upregulating the beige fat maintenance gene (FIG. 5, left panel). This result is demonstrative of a classic repressor of a repressor mechanism. Cidea, a canonical marker of beige adipocytes, was also upregulated in the mature adipocytes following co-treatment with Zfp423 and Tle3 siRNA (FIG. 5, middle panel). UCP1 is the hallmark of beiging. Downregulation of Zfp423 and Tle3 were complementary and together synergistically upregulated UCP1 (FIG. 5, right panel). Indeed, the UCP1 gene showed a very high fold change of the transcript in co-treatment condition. Note that Tle3 (siRNA-2) alone upregulated UCP1 but did not have as good an effect on Cidea (FIG. 5).

[0327] Additionally, we examined the effect of siRNA treatment on Cox8b and Cptlb, two genes that are known markers of mitochondrial biogenesis, in mature, differentiated 3T3-L1 cells (i.e., mature adipocytes). Cox8b is a mitochondrial electron transport chain protein which is specific to beiging cells. While Cox8b expression did not increase following individual Zfp423 siRNA or Tle3 siRNA treatment, co-treatment with Zfp423 and Tle3 siRNA resulted in a synergistic increase in Cox8b mRNA (FIG. 6, left panel). The observed Cox8b-increase only in the co-treatment condition demonstrated that functional beiging was achieved in the treated differentiated 3T3-L1 cells. Cptlb is the rate limiting step of functional beiging and essentially defines the rate of thermogenesis by channelling lipids for UCP1 activation and sustenance. Note that while siZfp423 siRNA treatment alone repressed Cptlb, Tle3 siRNA treatment alone increased Cptlb expression and co-treatment with Zfp423 and Tle3 siRNA synergistically increased in Cptlb mRNA in the treated cells (FIG. 6, right panel).

[0328] Together, the above data demonstrated successful downregulation of target genes Zfp423 and Tle3 using siRNA packaged in LNPs. Co-treatment of mature differentiated 3T3-L1 cells (i.e., mature adipocytes) with Zfp423 and Tle3 siRNA synergistically repressed white fat genes and synergistically upregulated beige fat genes. The data also demonstrated that mitochondrial biogenesis and functional changes were required to achieve meaningful beiging, which was shown in co-treatment conditions. Attorney Docket No.: 44807-0493WO1 / P18367-01

[0329] Example 3. Functional Analysis of Mitochondrial upregulation and Beiging

[0330] To assess the effects of siRNA treatment of mature differentiated 3T3-L1 cells (i.e., mature adipocytes) on functional beiging, treated cells were subjected to a Seahorse assay. A Seahorse assay functionally assays respiration capacity, uncoupling and spare oxygen. Cells that are functionally uncoupled (undergoing thermogenesis), should have lower ATP-linked respiration and higher maximal respiration. Lower ATP-linked respiration shows higher uncoupling.

[0331] In brief, the Seahorse XF Cell Mito Stress Test used herein measured key parameters of mitochondrial function by directly measuring the oxygen consumption rate (OCR) of cells on Seahorse XFe and XF Pro Extracellular Flux Analyzers. The assay used the built-in injection ports on XF sensor cartridges to add modulators of respiration into the cell well during the assay to reveal the key parameters of mitochondrial function by shutting down each of the complexes in the electron transport chain in reverse order. The modulators included in this assay were Oligomycin, Carbonyl cyanide-4 (trifluorom ethoxy) phenylhydrazone (FCCP), Rotenone, and Antimycin. FIG. 7 shows the injection sequence of these modulators and the parameters obtained with this Seahorse assay.

[0332] Mature differentiated 3T3-L1 cells (i.e., mature adipocytes) were first treated with LNPs comprised of either scrambled siRNA (control), Zfp423 (siRNA-1), Tle3 (siRNA-2), or a 1:1 combination of siRNA-1 and siRNA-2. Cells were then subjected to a Seahorse assay following treatment. Co-treatment with Zfp423 and Tle3 siRNA (i.e., the 1:1 combination of siRNA-1 and siRNA-2) showed higher uncoupling compared to scrambled siRNA (FIG. 8). Co-treatment with Zfp423 and Tle3 siRNA also demonstrated the highest spare oxygen capacity compared to any treatment group (FIG. 8). The combined effect of lower ATP-respiration and higher spare oxygen demonstrated functional beiging.

[0333] Next, we compared the beiging effect of co-treatment with Zfp423 and Tle3 siRNA (siZfp423-siTle3) with treatment of the cells with an Alk7 siRNA (siAlk7). Alk7 is a membrane receptor that plays a role in lipid synthesis and has been previously shown to achieve meaningful beiging. Mature differentiated 3T3-L1 cells treated with siZfp423-siTle3 demonstrated superior uncoupling and very high spare oxygen capacity when compared to siAlk7-treated cells in a Seahorse assay (FIG. 9).

Claims

Attorney Docket No.: 44807-0493WO1 / P18367-01What is Claimed is:

1. A method for increasing energy expenditure in a subject, the method comprising generating differentiated beige or brown fat cells by the steps of:inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; andinducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation,wherein energy expenditure in the subject is increased relative to a subject whose (1) one or more biomarkers sufficient to maintain white fat cell expression is not inhibited and (2) one or more biomarkers sufficient to activate beige or brown fat cell differentiation,thereby increasing energy expenditure in the subject.

2. A method for increasing energy expenditure in a subject, the method comprising generating differentiated beige or brown fat cells, the method comprising inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression,wherein energy expenditure in the subject is increased relative to a subject whose (1) one or more biomarkers sufficient to maintain white fat cell expression is not inhibited,thereby increasing energy expenditure in the subject.

3. A method for increasing energy expenditure in a subject, the method comprising generating differentiated beige or brown fat cells, the method comprising inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation, wherein energy expenditure in the subject is increased relative to a subject whose one or more biomarkers sufficient to activate beige or brown fat cell differentiation,thereby increasing energy expenditure in the subject.

4. The method of any one of claims 1-3, wherein increasing energy expenditure comprises increasing respiration in the subject.

5. The method of claim 4, wherein the respiration is total respiration.Attorney Docket No.: 44807-0493WO1 / P18367-016. The method of claim 4, wherein the respiration is uncoupled respiration.

7. The method of any one of claims 4-6, wherein the respiration is measured by oxygen consumption or positron emission tomography.

8. A method for preventing or treating obesity or an obesity-related disorder in a subject, the method comprising generating differentiated beige or brown fat cells, the method comprising: inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and / orinducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation,thereby preventing or treating obesity or the obesity -related disorder in the subject.

9. A method for preventing or treating obesity or an obesity -related disorder in a subject, the method comprising generating differentiated beige or brown fat cells by inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression, thereby preventing or treating obesity or the obesity-related disorder in the subject.

10. A method for preventing or treating obesity or an obesity -related disorder in a subject, the method comprising generating differentiated beige or brown fat cells by inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation, thereby preventing or treating obesity or the obesity -related disorder in the subject.

11. The method of any one of claims 8-10, wherein the obesity-related disorder is selected from the group consisting of: obesity, type II diabetes, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Lawrence-Moon syndrome, Prader-Labhart-Willi syndrome, anorexia, and cachexia.

12. A method for inducing beige or brown fat cell differentiation in a subject, the method comprising:Attorney Docket No.: 44807-0493WO1 / P18367-01(a) providing white fat cells, cells to be differentiated into beige or brown fat cells or adipocyte cells;(b) inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation;(c) injecting the white fat cells or differentiated beige or brown fat cells from (b) into the subject; and(d) measuring differentiation of beige or brown fat cells in the subject.

13. A method for inducing beige or brown fat cell differentiation in a subject, the method comprising:(a) providing white fat cells, cells to be differentiated into beige or brown fat cells or adipocyte cells;(b) inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression;(c) injecting the white fat cells from (b) into the subject; and(d) measuring differentiation of beige or brown fat cells in the subject.

14. A method for inducing beige or brown fat cell differentiation in a subject, the method comprising:(a) providing white fat cells, cells to be differentiated into beige or brown fat cells or adipocyte cells;(b) inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation;(c) injecting the white fat cells or differentiated beige or brown fat cells or adipocyte cells from (b) into the subject; and(d) measuring differentiation of beige or brown fat cells in the subject.

15. The method of claim 12 or 14, wherein the cells to be differentiated into beige or brown fat cells are selected from the group consisting of fibroblasts and myoblasts.Attorney Docket No.: 44807-0493WO1 / P18367-0116. The method of claim 15, wherein the fibroblasts are selected from the group consisting of skin fibroblasts, dermal fibroblasts, primary embryonic fibroblasts, immortalized embryonic fibroblasts, and human foreskin fibroblasts.

17. The method of any one of claims 12, 14, or 15, wherein the cells to be differentiated into beige or brown fat cells are autologous, allogeneic, syngeneic, xenogeneic, or HLA compatible with the subject.

18. The method of any one of claims 12-17, wherein the injecting is by a subcutaneous injection.

19. The method of any one of claims 12-17, wherein the injecting is by an intravenous injection.

20. A method for inducing beige or brown fat cell differentiation in a subject, the method comprising:(a) obtaining cells from the subject;(b) inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation;(c) injecting the differentiated cells from (b) into the subject; and(d) measuring differentiation of beige or brown fat cells in the subject.

21. A method for inducing beige or brown fat cell differentiation in a subject, the method comprising:(a) obtaining cells from the subject;(b) inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression;(c) injecting the differentiated cells from (b) into the subject; and(d) measuring differentiation of beige or brown fat cells in the subject.Attorney Docket No.: 44807-0493WO1 / P18367-0122. A method for inducing beige or brown fat cell differentiation in a subject, the method comprising:(a) obtaining cells from the subject;(b) inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation;(c) injecting the differentiated cells from (b) into the subject; and(d) measuring differentiation of beige or brown fat cells in the subject.23 The method of any one of claims 20-22, wherein the injecting is by a subcutaneous injection.

24. The method of any one of claims 20-22, wherein the injecting is by an intravenous injection.

25. A method for identifying a cocktail capable of inducing differentiation white fat cells into beige or brown fat cells in a subject, the method comprising:(a) providing white adipocyte cells;(b) inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation; and(c) measuring differentiation of beige or brown fat cells in the subject.

26. A method for identifying a cocktail capable of inducing differentiation white fat cells into beige or brown fat cells in a subject, the method comprising:(a) providing white adipocyte cells;(b) inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression; and(c) measuring differentiation of beige or brown fat cells in the subject.

27. A method for identifying a cocktail capable of inducing differentiation white fat cells into beige or brown fat cells in a subject, the method comprising:Attorney Docket No.: 44807-0493WO1 / P18367-01(a) providing white adipocyte cells;(b) inducing expression of one or more biomarkers sufficient to activate beige or brown fat cell differentiation; and(c) measuring differentiation of beige or brown fat cells in the subject.

28. The method of any one of the preceding claims, wherein the beige or brown fat cell differentiation is monitored by measuring the expression of a marker selected from the group consisting of: Ppar alpha, perilipin, Zic1, Lhx8, Eva1, Epsti1, PD-L1, cidea, adiponectin, adipsin, otopetrin, type II deiodinase, cig30, ppar gamma 2, pgc1a, ucp1, elovB, cAMP, Prdm1β, cytochrome C, cox4il, coxIII, cox5b, cox7al, cox8b, glut4, atpase b2, cox II, atp5o, ndufb5, and any combination thereof.

29. The method of any one of the preceding claims, wherein the beige or brown fat cell differentiation is monitored by measuring the expression of a marker selected from the group consisting of: Tle3, Plinl, Fabp4, Klf9, cidea, Ucpl, Cox8b, and Cptlb.

30. The method of any one of the preceding claims, wherein the beige or brown fat cell differentiation is monitored by measuring degree of cellular locality or by measuring respiration within the beige or brown fat cells.

31. The method of claim 30, wherein the respiration is total respiration.

32. The method of claim 30, wherein the respiration is uncoupled respiration.

33. The method of any one of claims 30-32, wherein the respiration is measured by oxygen consumption or positron emission tomography.

34. The method of any one of the preceding claims, wherein inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression comprises administering to the subject one or more siRNA and / or shRNA sequences that inhibit the one or more biomarkers sufficient to maintain white fat cell expression.Attorney Docket No.: 44807-0493WO1 / P18367-0135. The method of any one of the preceding claims, wherein inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression comprises administering to the subject a first vector comprising a nucleic acid that encodes for an inhibitor of one or more biomarkers sufficient to maintain white fat cell expression, optionally wherein the inhibitor comprises one or more of SEQ ID NOs: 1-4.

36. The method of claim 35, wherein the first vector comprises one or more promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRES), enhancers, or a combination thereof.

37. The method of any one of the preceding claims, wherein inhibiting expression of one or more biomarkers sufficient to maintain white fat cell expression comprising administering to a subject a second vector comprising a nucleic acid that encodes for one or more biomarkers sufficient to activate beige or brown fat cell differentiation.

38. The method of claim 37, wherein the second vector comprises one or more promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRES), enhancers, or a combination thereof.

39. The method of any one of claims 35-38, wherein the first vector and / or the second vector is an adeno-associated viral (AAV) vector.

40. The method of claim 39, wherein the AAV vector is any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.

41. The method of claim 39 or 40, wherein the AAV vector is AAV8.Attorney Docket No.: 44807-0493WO1 / P18367-0142. The method of any one of claims 35-41, wherein the first vector and / or the second vector is a recombinant adeno-associated virus (rAAV).

43. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to maintain white fat cell expression comprises a transcription factor that drives differentiation of white adipocytes.

44. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to maintain white fat cell expression comprises a transcription factor that maintains white adipocytes.

45. The method of any one of the preceding claims, wherein expression of the one or more biomarkers sufficient to maintain white fat cell expression upregulates UCP1, optionally wherein the one or more biomarkers sufficient to maintain white fat cell expression comprises a transcription factor that recruits NuRD repressor complex to the Ebf2 -bound thermogenic gene enhancers.

46. The method of any one of the preceding claims, wherein inhibition of the one or more biomarkers sufficient to maintain white fat cell expression causes expression of Brahma Associated Factor (BAF).

47. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to maintain white fat cell expression comprises a transcription factor that comprises at least 20, at least 25, or at least 30 C2H2-type zinc fingers.

48. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to maintain white fat cell expression comprises Zfp423.

49. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Ebf2.Attorney Docket No.: 44807-0493WO1 / P18367-0150. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Prdml6.

51. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Tle3.

52. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Klfl5, Plinl, and / or Fabp4.

53. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Klfl5.

54. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Plinl.

55. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Fabp4.

56. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Klf9, Cidea, and / or Ucpl.

57. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Klf9.

58. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Cidea.

59. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Ucpl.Attorney Docket No.: 44807-0493WO1 / P18367-0160. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Cox8b and / or Cptlb.

61. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Cox8b.

62. The method of any one of the preceding claims, wherein the one or more biomarkers sufficient to activate beige or brown fat cell differentiation comprises Cptlb.

63. The method of any one of the preceding claims, further comprising measuring protein or mRNA expression level of one or more of Zfp423, Ebf2, or Prdml6 in the subject.

64. The method of claim 63, wherein the measuring protein or mRNA expression level of one or more of Zfp423, Ebf2, or Prdml6 in the subject occurs prior to the inhibiting and inducing steps.

65. The method of claim 63 or 64, wherein the measuring protein or mRNA expression level of one or more of Zfp423, Ebf2, or Prdml6 in the subject occurs after the inhibiting and inducing steps.

66. The method of any one of the preceding claims, further comprising measuring protein or mRNA expression level of one or more of Tle3, Plinl, Fabp4, Klf9, cidea, Ucpl, Cox8b, or Cptlb in the subject.

67. The method of claim 66, wherein the measuring protein or mRNA expression level of one or more of Tle3, Plinl, Fabp4, Klf9, cidea, Ucpl, Cox8b, or Cptlb in the subject occurs prior to the inhibiting and inducing steps.

68. The method of claim 66 or 67, wherein the measuring protein or mRNA expression level of one or more of Tle3, Plinl, Fabp4, Klf9, cidea, Ucpl, Cox8b, or Cptlb in the subject occurs after the inhibiting and inducing steps.Attorney Docket No.: 44807-0493WO1 / P18367-0169. The method of any one of the preceding claims, wherein the subject is a mammal.

70. The method of any one of the preceding claims, wherein the subject is a mouse.

71. The method of any one of the preceding claims, wherein the subject is a human.

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