Method and composition for treating obesity and / or skin disorders

Abstract

To provide a method for treating obesity.SOLUTION: A method for treating obesity or obesity-related disorders includes a step of locally administering a pharmaceutically effective amount of vitamin D3 analog to a subject. In another aspect, the method in which a TSLP level systemically increases in the subject. The obesity-related disorder is at least one disorder selected from non-alcoholic steatohepatitis (NASH), metabolic disease, type I diabetes mellitus, type II diabetes mellitus, hypertension, dyslipidemia, coronary artery disease, cerebral stroke, gallbladder disease, kidney disease, osteoarthritis, sleep apnea and respiratory disorder, and cancer.SELECTED DRAWING: Figure 32-2

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 849,656, filed May 17, 2019, and U.S. Provisional Patent Application No. 62 / 972,462, filed February 10, 2020, each of which is incorporated by reference in its entirety.

[0002] Statement regarding federally funded research This invention was made with Government support under HL111501 awarded by the National Institutes of Health. The Government has certain rights in the invention. [Background technology]

[0003] background Obesity and obesity-related disorders, such as type 2 diabetes (T2D) and nonalcoholic steatohepatitis (NASH), are major health threats worldwide, especially in developed countries. Apart from lifestyle modifications and significant surgical procedures, there are not many treatment options available for these conditions.

[0004] Thus, there is a need in the art for new compositions and / or methods that allow for controlled weight loss. The present invention addresses this need. Summary of the Invention

[0005] BRIEF SUMMARY OF THE INVETION The present invention provides a method for treating obesity and / or obesity-related disorders. The present invention further provides a method for treating skin disorders and / or improving scalp health. The present invention further provides a method for treating eye disorders. The present invention further provides a method for treating, improving, and / or preventing skin disorders. The present invention further provides a method for treating, improving, and / or preventing alopecia in a subject.

[0006] In certain embodiments, the method comprises administering a pharma- tically effective amount of a vitamin D3 analog to a subject in need thereof. In certain embodiments, the method comprises administering a pharma- tically effective amount of a TSLP inhibitor to a subject in need thereof. Further non-limiting embodiments that define the present invention are listed elsewhere herein. [Brief description of the drawings]

[0007] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects of the present application.

[0008] [Figure 1A] Figures 1A-1D show prevention of weight gain and improvement of metabolic parameters in HFD-fed mice as a result of administering MC903. WT C57BL / 6 mice were fed normal chow (NC) or 40% high fat diet (HFD) for 12 weeks. Mice were treated topically in both ears with either vehicle (EtOH) or MC903 (2 nmol / ear) on Mondays, Wednesdays, and Fridays of odd-numbered weeks. (Figure 1A) Mice were weighed weekly and % change from baseline is plotted as mean ± SEM. (Figure 1B) Mice were euthanized at week 12 and epididymal fat pads were weighed. (Figure 1C, Figure 1D) Glucose tolerance test was performed at week 12 by injection of glucose and subsequent measurement of blood glucose levels. HOMA-IR analysis was performed after overnight fasting at week 12. *, **, and *** indicate statistical significance of p<0.05, p<0.01, and p<0.001 by Student's t-test or ANOVA, respectively. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Diagram 2]Figure 2 shows prevention of HFD-induced fatty liver by MC903. WT C57BL / 6 mice were fed normal chow (NC) or 40% high fat diet (HFD) for 12 weeks. Mice were treated topically in both ears with either vehicle (EtOH) or MC903 (2 nmol / ear) on Mondays, Wednesdays, and Fridays of odd-numbered weeks. Representative histology of livers at 12 weeks is shown. [Diagram 3] Figure 3 shows that MC903 does not prevent weight gain or improve metabolic parameters in HFD-fed TSLP-R KO mice. TSLP-R KO mice were fed normal chow (NC) or 40% high fat diet (HFD) for 12 weeks. Mice were treated topically in both ears with either vehicle (EtOH) or MC903 (2 nmol / ear) on Mondays, Wednesdays, and Fridays of odd-numbered weeks. Mice were weighed at the indicated time points and plotted as mean ± SEM. Glucose tolerance tests were performed at week 12 by injection of glucose and subsequent measurement of blood glucose levels. AUC analysis is performed on GTT curves. * indicates statistical significance of p<0.05 by Student's t-test. [Figure 4] Figure 4 shows that AAV8-TSLP induces weight loss in HFD-fed mice. WT C57BL / 6 mice were fed a 40% high fat diet (HFD) for 4 weeks. On day 0, mice were injected with either AAV8 control or AAV8-TSLP. Mice were weighed weekly and % change from baseline is plotted as mean ± SEM. ** and *** indicate statistical significance of p<0.01 and p<0.001, respectively. [Diagram 5]FIG. 5 shows that AAV8-TSLP induces weight loss in previously obese HFD-fed mice. WT C57BL / 6 mice were fed a 40% high fat diet (HFD) for 10 weeks. Mice were then injected with either AAV8 control or AAV8-TSLP at week 10. Mice were maintained on a HFD and weighed weekly, and % change from baseline is plotted as mean±SEM. *, **, and *** indicate statistical significance of p<0.05, p<0.01, and p<0.001 by Student's t-test or ANOVA, respectively. [Figure 6] Figure 6 shows that AAV8-TSLP induces loss of subcutaneous fat. WT C57BL / 6 mice were fed a 40% high-fat diet (HFD) for 10 weeks. Mice were then injected with either AAV8 control or AAV8-TSLP at week 10. Mice were maintained on a HFD for 6 weeks and euthanized for histological analysis. Representative histological images of mouse skin are shown. [Figure 7] Figure 7 shows that AAV8-TSLP induces weight loss in Ob / Ob mice. Ob / Ob mice were injected with either AAV8 control or AAV8-TSLP and fed regular chow for 6 weeks. Mice were weighed weekly and the % change from baseline is plotted as mean ± SEM. * and *** indicate statistical significance of p<0.05 and p<0.001, respectively, by Student's t-test. [Figure 8A] Figures 8A-8E show that AAV8-TSLP induces selective adipose tissue loss in mice fed a normal chow diet. WT C57BL / 6 mice were injected with either AAV8 control or AAV8-TSLP and fed a normal chow diet for 14 days. (Figure 8A) Mice were weighed on the indicated days and the % change from baseline is plotted as the mean ± SEM. Mice were euthanized on the indicated days and (Figure 8B) eWAT, (Figure 8C) iWAT, (Figure 8D) BAT, and (Figure 8E) quadriceps muscle weights were measured. ** and *** indicate statistical significance of p<0.01 and p<0.001, respectively, by Student's t-test. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 8E] See legend to Figure 8A. [Figure 9A] Figures 9A-9C show that AAV8-TSLP does not alter food intake in intestinal absorption. WT C57BL / 6 mice were injected with either AAV8 control or AAV8-TSLP and fed regular chow or HFD for 12 weeks. (Figure 9A) Average food intake per week is plotted as mean ± SEM. Ten days after AAV8 injection, mice were gavaged with a fixed amount of (Figure 9B) glucose or (Figure 9C) olive oil. Subsequent glucose and triglyceride levels were measured and plotted over time. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 10] Figures 10A-10B show that mice treated with AAV8-TSLP excrete less fecal energy compared to mice treated with AAV8 control. WT C57BL / 6 mice were injected with either AAV8 control or AAV8-TSLP and fed regular chow for 9 days. Feces were collected and food intake was measured in individually caged mice from days 9 to 11. Collected feces were subjected to bomb calorimetry to measure fecal calories. (Figure 10A) Fecal energy content per day for AAV8 control or AAV8-TSLP treated mice. (Figure 10B) Net energy intake per day, calculated by food calorie intake minus fecal calories for AAV8 control or AAV8-TSLP treated mice. Statistical analysis was performed by Student's t-test. [Figure 11-1]Figure 11 shows that AAV8-TSLP increases food intake between days 7 and 10 post-injection. WT C57BL / 6 mice were injected with either AAV8 control or AAV8-TSLP and placed in metabolic chambers on days 7-10. Hourly and cumulative food intake is plotted over the final 48 hours. [Figure 11-2] See description of Figure 11-1. [Figure 12-1] Figure 12 shows that AAV8-TSLP does not increase oxygen consumption between days 7 and 10 post-injection. WT C57BL / 6 mice were injected with either AAV8 control or AAV8-TSLP and placed in metabolic chambers on days 7 to 10. Hourly (left plots) and daily / half-day (right plots) oxygen consumption are plotted over the final 48 and 72 hours, respectively. [Figure 12-2] See description of Figure 12-1. [Figure 13-1] Figure 13 shows that AAV8-TSLP does not increase carbon dioxide output between days 7 and 10 post-injection. WT C57BL / 6 mice were injected with either AAV8 control or AAV8-TSLP and placed in metabolic chambers on days 7 to 10. Hourly (left plots) and daily / half-daily (right plots) carbon dioxide output are plotted over the final 48 and 72 h, respectively. [Figure 13-2] See description of Figure 13-1. [Figure 14-1] Figure 14 shows that AAV8-TSLP does not increase locomotor activity between days 7 and 10 post-injection. WT C57BL / 6 mice were injected with either AAV8 control or AAV8-TSLP and placed in metabolic chambers on days 7 to 10. Hourly (left plots) and daily / half-day (right plots) locomotor activity are plotted over the final 48 and 72 hours, respectively. [Figure 14-2] See description of Figure 14-1. [Figure 15]Figure 15 shows that AAV8-TSLP produces oily coat in previously obese HFD-fed mice. WT C57BL / 6 mice were fed a 40% high fat diet (HFD) for 10 weeks. Mice were then injected with AAV8-TSLP at week 10. Mice were kept on HFD for 6 weeks. Representative pictures of mice at week 6 are shown. [Figure 16] Figure 16 shows that AAV8-TSLP does not induce adipose tissue loss in SCD1 KO mice fed normal chow. WT C57BL / 6 or SCD1 KO mice were injected with either AAV8 control or AAV8-TSLP and fed normal chow for 14 days. Mice were euthanized and eWAT, body weight were measured. Statistical analysis was performed by Student's t-test. [Figure 17] Figures 17A-17B show that AAV8-TSLP induces weight loss in a T cell-dependent manner. (Figure 17A) RAG KO mice and (Figure 17B) TCR-β KO mice were injected with either AAV8 control or AAV8-TSLP. Epididymal white adipose tissue (eWAT) weight was measured 14 days after injection. Data are plotted as mean ± SEM. [Figure 18] Without being bound by theory, Figure 18 shows a non-limiting proposed model of TSLP-induced adipose tissue loss. Systemic TSLP elevation can be achieved by topical treatment with MC903, injection of recombinant TSLP, or gene therapy using viral vectors. Elevated systemic TSLP levels directly or indirectly lead to cutaneous lipid secretion, which increases the organism's fat energy demand. This causes the release of fat stores (lipolysis) in adipose and other fat-containing tissues. This ultimately leads to weight loss and reversal of obesity. [Figure 19] FIG. 19 shows the finding that subcutaneous fat is significantly reduced in mice expressing high levels of TSLP. [Figure 20] FIG. 20 shows the finding that visceral fat was significantly reduced in mice expressing high levels of TSLP in a dose-dependent manner. [Figure 21]Figure 21 shows that mice injected with AAV8-TSLP exhibit increased sebum components in their fur. WT C57BL / 6 mice were injected with either AAV8 control or AAV8-TSLP and fed normal chow for 10 days. Fixed areas of dorsal fur were scraped from mice and lipids were extracted. Extracted lipids were subjected to thin layer chromatography to separate lipid components into wax monoesters (WME), wax diesters (WDE), free fatty acids (FFA), and free cholesterol (FC). Amounts were quantified by Image J software and plotted. ** indicates statistical significance of p<0.01 by Student's t-test. [Figure 22] Figure 22 shows that mice injected with AAV8-TSLP show an increase in Ki67+ basal layer sebocytes. WT C57BL / 6 mice were injected with either AAV8 control or AAV8-TSLP and fed normal chow for 10 days. Skin was removed and fixed. Paraffin-embedded skin tissue sections were stained with anti-Ki67 antibody. The number of Ki67+ basal layer sebocytes was counted and the percentage of Ki67+ / total sebocytes was calculated. ** indicates statistical significance of p<0.01 by Student's t-test. [Diagram 23] Figure 23 shows that TSLP-R KO mice show a reduction in sebum lipid composition in their fur compared to WT mice. Fixed areas of dorsal fur were scraped from WT C57BL / 6 or TSLP-R KO mice, and lipids were extracted. The extracted lipids were subjected to thin layer chromatography to separate the lipid components into wax monoesters (WME), wax diesters (WDE), free fatty acids (FFA), and free cholesterol (FC). The amounts were quantified and plotted by Image J software. ** indicates statistical significance of p<0.01 by Student's t-test. [Figure 24]Figure 24 shows increased expression of antimicrobial peptides in the skin of mice injected with AAV8-TSLP. WT C57BL / 6 mice were injected with either AAV8 control or AAV8-TSLP and fed regular chow for 10 days. Skin was removed and RNA was extracted. Transcript levels of two sebum-associated antimicrobial peptides, cathelicidin antimicrobial peptide (CAMP) and defensin B4 (DEF4B), were measured by qPCR. * indicates statistical significance of p<0.05 by Student's t-test. [Diagram 25] Figure 25 shows that topical application of MC903 remotely increases sebum secretion in a human individual. MC903 was applied topically to the right arm of an individual daily. Sebum measurements were taken by cleaning the forehead with an ethanol wipe, resting for 15 minutes, and placing Sebtape on the forehead for 2 minutes. Black dots on the blue and white background of the tape indicate the presence of sebum on each day after treatment. [Figure 26] Figure 26 shows that topical application of MC903 remotely heals the dry skin of the hand of a human individual.MC903 is topically applied to the right arm of an individual every day.Photographs of the right hand are shown before treatment and after 3 or 5 days of treatment.The individual is treated for a total of 14 days, and does not require hand cream for at least 9 weeks after treatment is stopped.In certain embodiments, sebum secretion continues after treatment is stopped. [Figure 27] Figure 27 shows that topical application of MC903 remotely promotes hair growth on the scalp of a human individual. MC903 is topically applied to the left arm of an individual every day for 14 days (first treatment). Treatment is stopped for 4 weeks, and resumed daily on the right arm for another 2 weeks (second treatment). Photographs of the scalp are shown before treatment, 2 weeks after the first treatment, 11 days after the second treatment, and 8 weeks after the second treatment, as well as 3 days or 5 days after treatment. [Figure 28]Figure 28 shows that topical application of MC903 remotely improves dry eye symptoms in two human individuals. MC903 was topically applied to the left arm of one individual and to the right arm of the other individual for 14 days daily (first treatment). The presence of dry eye symptoms was subjectively assessed in two individuals before and after treatment. Responses are plotted in a bar graph. [Figure 29] Figures 29A-29F show that one TSLP induces selective white fat loss and reverses obesity and associated metabolic complications. Figure 29A: Body weight of HFD-fed mice after AAV (control AAV or TSLP-AAV). (n=5 mice / group). (Figure 29B) Body weight of obese mice, (Figure 29C) Glucose tolerance test (GTT), and (Figure 29D) Liver TG (HFD 10 weeks before AAV, n=5 NC, 10 HFD control, and 8 HFD-TSLP mice). Figure 29E: Serum ALT of MCDD-fed mice 325 injected with AAV (n=7 mice / group). Figure 29F: Tissue mass of NC-fed mice 2 weeks after AAV (n=6 mice / group). Data are mean ± sem from two independent experiments. ANOVA (Figures 29A-29C) or Student's t-test (Figures 29D-29F). Statistics: (Figure 29A) F=18.96, dfn=1, dfd=8. (Figure 29B) F=32.29, dfn=1, dfd=16. (Figure 29C) NC vs. HFD control: F=14.33, dfn=1, dfd=13, NC vs. HFD-TSLP: F=1.753, dfn=1, dfd=11, HFD control vs. HFD-TSLP: F=27.71, dfn=1, dfd=16. (Figure 29D) t=9.835, df=23. (Figure 29E) t=2.560, df=12. (Figure 29F) eWAT: t = 8.128, iWAT: t = 7.226, BAT: t = 1.150, and muscle: t = 0.2566, df = 10 for all tissues. [Diagram 30]Figures 30A-30F show that TSLPR signaling in T cells is required for fat loss by TSLP. (Figure 30A) E-β KO mice (n=5 mice / group), (Figure 30B) WT mice injected with aCD4±aCD8 (n=4 control and 6 TSLP mice in all groups), (Figures 30C-30D) RAG KO mice transferred with CD4+ or CD8+ T cells (n=5 mice / group in all groups except TSLP mice transferred with CD8+ T from n=6 mice) and WT or TSLPR KO T cells (n=8 WT T and 10 TSLPR KO T transferred mice), and (Figure 30E) eWAT volume in TSLPR KO mice transferred with WT or TSLPR KO T cells 2 weeks after AAV (n=8 mice / group). (Figure 30F) eWAT volume of WT mice injected with AAV before antibody injection (2 weeks after AAV, n=4 mice / group), 2 weeks after isotype injection (4 weeks after AAV, n=5 mice / group) or 2 weeks after aCD4+aCD8 antibody injection (4 weeks after AAV, n=8 control and 7 TSLP mice). Data are mean ± sem from two (Figures 30A-C, 30E-30F) or three (Figure 30D) independent experiments. Student's t-test. Statistics: (Figure 30A) t=0.0466, df=8. (Figure 30B) aCD4: t=6.231, df=8, aCD8: t=5.032, df=8, and aCD4±aCD8: t=0.1433, df=8. (Figure 30C) CD4+: t=8.787, df=8, CD8+: t=3.598, df=9. (Figure 30D) WT: t=7.103, df=12, TSLPR KO: t=0.2950, ​​df=18. (Figure 30E) WT: t=6.255, df=14, TSLPR KO: t=0.8481, df=14. (FIG. 30F) Before Ab: t=3.994, df=6, isotype Ab: t=34.95, df=8, aCD4+aCD8: t=4.641, df=13, before Ab TSLP vs aCD4+aCD8 TSLP: t=3.151, df=9, isotype Ab TSLP vs aCD4+aCD8 TSLP: t=11.17, df=10. [Figure 31-1]Figures 31A-31J show that TSLP induces fat loss by promoting T cell-mediated sebum secretion. (Figure 31A) 3-day food intake (n=7 control and 8 TSLP mice), (Figure 31B) fecal calories (n=5 mice / group), and (Figure 31C) energy expenditure (n=7 control and 8 TSLP mice) of NC-fed mice for 3 days 9-11 days after AAV. (Figure 31D) Gross appearance and (Figure 31E) TLC quantification (CE=cholesterol esters, WE=wax esters, TG=triglycerides, FFA=free fatty acids, FC=free cholesterol, n=9 mice / group) of HFD-fed mice 4 weeks after AAV. (Figures 31F-31G) Ki67 staining and quantification of sebaceous glands of NC-fed mice 10 days after AAV. (n=78 control and 50 TSLP sebaceous glands from 3 mice / group). (FIG. 31H) TLC quantification of hair lipids from WT and RAG KO mice 2 weeks after AAV (n=5 mice / group). (FIG. 31I) CD3 staining of skin 10 days after AAV. (FIG. 31J) eWAT volume of WT vs. SCD1 KO mice 2 weeks after AAV (n=5 WT mice / group and 6 SCD1 KO mice / group). Data are mean±sem from two independent experiments. Student's t-test (FIGS. 31A-31B, 31E, 31G-31H, 31J) or ANOVA (FIG. 31C). Statistics. (FIG. 31A) t=2.624, df=13. (FIG. 31B) t=3.046, df=9. (Figure 31C) F=0.9142, dfn=1, dfd=13. (Figure 31E) CE: t=4.079, WE: t=2.931, TG: t=3.721, FFA: t=3.346, FC; t=4.273, df=16 for all comparisons. (Figure 31G) t=3.763, df=12. (Figure 31H) WT CE: t=2.561, RAG KO CE: t=0.4456, WT WE: t=4.380, RAG KO WE: t=1.163, WT FFA: t=5.333, RAG KO FFA: t=1.990, WT FC: t=3.388, KO FC: t=1.632, df=8 for all comparisons. (FIG. 31J) For WT control vs. TSLP: t=4.858, df=8. For SCD1 KO control vs. TSLP: t=1.440, df=10.For WT-TSLP vs. SCD1 KO-TSLP: t = 4.650, df = 9. [Figure 31-2] See description of Figure 31-1. [Figure 32-1] Figures 32A-32F show that TSLP and T cells regulate sebum secretion in homeostasis. (Figure 32A) TLC quantification of WT vs. TSLPR KO mice (n=11 mice / group). (Figures 32B-32C) Ki67 staining and quantification of WT vs. TSLPR KO sebaceous glands (n=132 WT and 109 TSLPR KO sebaceous glands from n=3 mice / group). (Figure 32D) TLC quantification of WT vs. E-β KO mice (n=4 WT and 3 E-β KO mice). (Figure 32E) Linear regression analysis of TSLP expression vs. sebaceous gland (SG) gene expression in publicly available human skin microarray data. (n=36 healthy individuals). (Figure 32F) An exemplary non-limiting model for the pharmacological and homeostatic role of TSLP-driven sebum secretion. Data are means ± sem from two independent experiments (Figures 32A-32D). Student's t-test (Figures 32A, 32C-32D) or Pearson's r-test and linear regression slope test (Figure 32E). Statistics: (Figure 32A) CE: t = 1.530, WE: t = 3.924, FFA: t = 1.748, FC: t = 1.052, df = 20 for all comparisons. (Figure 32C) t = 3.139, df = 239. (Figure 32D) CE: t = 4.219, WE: t = 4.234, FFA: t = 1.555, FC: t = 1.636, df = 5 for all comparisons. (Figure 32E) F = 9.393, dfn = 1, dfd = 34. [Figure 32-2] See description of Figure 32-1. [Diagram 33]Figures 33A-33J show that TSLP stimulates weight loss and improves metabolic parameters. (Figure 33A) % weight change in HFD-fed mice after AAV (n=5 mice / group). (Figure 33B) Weight and (Figure 33C) % weight change in NC-fed ob / ob mice after AAV (n=4 mice / group). (Figure 33D) % body weight change (n=9 mice / group), (Figure 33E) eWAT volume (n=9 control and 8 TSLP mice), (Figure 33F) fasting glucose (n=8 mice / group), (Figure 33G) fasting insulin (n=8 mice / group), (Figure 33H) HOMA-IR (n=8 mice / group), (Figure 33I) GTT quantification (n=5 NC, 10 HFD control, and 8 HFD-TSLP mice), and (Figure 33J) H&E histology of liver of obese mice (previously fed HFD for 10 weeks) 4 weeks after AAV. Data are shown as mean ± sem from two independent experiments. ANOVA with Sidak's post-hoc test (Figures 33A-33D) or Student's t-test (Figures 33E-33I). Statistics. (Figure 33A) F=19.27, dfn=1, dfd=8. (Figure 33B) F=1.911, dfn=1, dfd=6. 1st week: t=0.4474, df=4.910, 2nd week: t=0.1274, df=4. 365, 3rd week: t=1.543, df=3.482, 4th week: t=2.524, df=3.217, 5th week: t=3.282, df=3.365. (Figure 33C) F=5.139, dfn=1, dfd=6. Week 1: t=1.857, df=4.992, week 2: t=0.1761, df=4, week 3: t=2.445, df=5.674, week 4: t=2.342, df=5.967, week 5: t=2.833, df=5.914. (Figure 33D) F=10.05, dfn=1, dfd=16. (Figure 33E) t=10.81, df=15. (Figure 33F) NC vs. HFD control: t=6.692, NC vs. HFD-TSLP: t=0.2312, HFD control vs. HFD-TSLP: t=6.129, df=14 for all comparisons. (Figure 33G) NC vs. HFD control: t = 2.757, fNC vs. HFD-TSLP: t = 0.5797, HFD control vs. HFD-TSLP: t = 3.144, df = 14 for all comparisons.(Figure 33H) NC vs HFD control: t = 4.381, NC vs HFD-TSLP: t = 1.899, HFD control vs HFD-TSLP: t = 3.783, df = 14 for all comparisons. (Figure 33I) NC vs HFD control: t = 3.688, df = 13, NC vs HFD-TSLP: t = 1.296, df = 11, HFD control vs HFD647 TSLP: t = 5.141, df = 16. [Diagram 34]Figures 34A-34L show that TSLP ameliorates MCDD-induced liver injury and induces selective white fat loss in NC-fed mice. (Figure 34A) Liver TG (n=5 NC, 10 MCDD control, and 10 MCDD TSLP mice) of MCDD-fed mice after AAV, (Figure 34B-34D) picrosirius red liver staining and quantification (n=15 mice / group). (Figure 34E) eWAT, (Figure 34F) iWAT, (Figure 34G) BAT, and (Figure 34H) femoral appendicular muscle mass (n=6 mice / group for all tissues), (Figure 34I) NMR analysis before (day 0) and after (day 14) AAV (n=5 mice / group), (Figure 34J) BCS (n=10 mice / group), and (Figure 34K) % body weight change in NC-fed mice 2 weeks after AAV (n=6 mice / group). (Figure 34L) eWAT volume in TSLPR KO mice 2 weeks after AAV (n=7 mice / group). Data are shown as mean ± sem from two (Figures 34A, 34E-34H, 34J-34L) or three (Figures 34C-34D) independent experiments. Student's t-test (Figures 34A, 34C-34I, 34L) or ANOVA with Sidak's post-hoc test (Figures 34J-34K). Statistics. (Figure 34A) NC vs MCDD control: t = 9.703, df = 13, NC vs MCDD TSLP: t = 13.17, df = 13, MCDD control vs MCDD TSLP: t = 4.768, df = 18. (Figures 34B-34D) Area %: t = 5.399, df = 28, Integrated Density: t = 5.640, df = 28. (Figure 34E) Day 3: t = 0.9539, Day 7: t = 0.6498, Day 10: t = 2.183, Day 14: t = 8.128. (Figure 34F) Day 3: t = 0.3344, day 7: t = 0.6961, day 10: t = 5.972, day 14: t = 7.226. (Figure 34G) Day 3: t = 1.355, day 7: t = 0.1674, day 10: t = 2.829, day 14: t = 1.150. (Figure 34H) Day 3: t = 0.4126, day 7: t = 0.0925, day 10: t = 0.5618, day 14: t = 0.2566, df = 10 for all comparisons. (Figure 34I) Before vs. After Control: t = 0.3241, Before vs. After TSLP: t = 3.736, After Control vs. After TSLP: t = 3.111, df = 8 for all comparisons.(Figure 34J) F=0.000, dfn=1, dfd=18. (Figure 34K) F=2.635, dfn=1, dfd=10. (Figure 34L) t=0.5779, df=12. [Diagram 35] Figures 35A-35E show that TSLPR signaling in hematopoietic cells is required for TSLP-induced white fat loss and weight loss. WT or TSLPR KO mice were irradiated and reconstituted with WT or TSLPR KO bone marrow: (Figure 35A) schematic and (Figure 35B) eWAT volume of NC-fed mice 2 weeks after AAV (n=7 WT→WT control, 9 WT→WT TSLP, 9 KO→WT control, 9 KO→WT TSLP, 7 WT→KO control, and 5 WT→KO TSLP mice). (Figures 35C-35E) Body weight of HFD-fed chimeras after AAV (n=6 WT→WT control, 10 WT→WT TSLP, 10 KO→WT control, 11 KO→WT TSLP, 11 WT→KO control, and 14 WT→KO TSLP mice). Data are means ± sem from three independent experiments. Student's t-test (Figure 35B) or ANOVA with Sidak's post-hoc test (Figures 35C-35E). Statistics. (Figure 35B) WT → WT: t = 3.959, df = 14, KO → WT: t = 0.0661, df = 16, WT → KO: t = 4.807, df = 10. (Figure 35C) F = 8.120, dfn = 1, dfd = 14. (Figure 35D) F = 2.727, dfn = 1, dfd = 19. (Figure 35E) F = 22.97, dfn = 1, dfd = 23. [Diagram 36]Figures 36A-36E show that adaptive immune cells, but not DCs, eosinophils, or Treg cells, are required for fat loss by TSLP. (Fig. 36A) ROSA-Stop-flox-DTA mice (n=5 control and 4 TSLP mice) and CD11cCre-ROSA-Stop-flox-DTA mice (n=5 control and 6 TSLP mice) 2 weeks after AAV, (Fig. 36) ROSA-Stop-flox-DTA mice (n=5 control and 4 TSLP mice) and EoCre-ROSA-Stop-flox-DTA mice (n=6 control and 6 TSLP mice), (Fig. 36C) FOXP3-GFP and FOXP3-DTR mice treated with PBS or diphtheria toxin (DT) (n=6 mice / group except for FOXP3-DTR PBS mice where n=5 mice), (Fig. 36D) RAG / IL2rg DKO mice injected with PBS or WT T cells (n=6 PBS control, 8 PBS eWAT volume in 5 control, 5 T cell TSLP mice, and 5 T cell TSLP mice, as well as (Figure 36E) RAG KO mice (n=10 mice / group). Data are shown as mean ± sem from two (Figures 36A-36D) or three (Figure 36E) independent experiments. Student's t-test. Statistics. (Figure 36A) DTA: t=5.135, df=7, CD11cCre-DTA: t=7.114, df=9. (Figure 36B) DTA: t=5.135, df=7, EoCre-DTA: t=3.928, df=10. (Figure 36C) FOXP3-GFP PBS: t=5.375, df=10, FOXP3-GFP DT: t=3.954, df=10, FOXP3-DTR PBS: t=6.100, df=9, FOXP3-DTR DT: t=4.593, df=10. (Figure 36D) PBS: t=0.6571, df=10, T cells: t=7.710, df=6. (Figure 36E) t=0.4072, df=18. [Figure 37]Figures 37A-37L show that TSLP does not affect energy intake, metabolic rate, locomotor activity, or urinary metabolite excretion. NC-fed mice were placed in individual metabolic chambers 9-11 days post-AAV and monitored for: (Figure 37A) food intake (n=7 mice / group), (Figure 37D-37E) locomotor activity. (Figure 37F) O2 consumption, (Figure 37G) CO2 production, and (Figure 37H) respiratory exchange rate (n=7 control and 8 TSLP mice). (Figure 37B-37C) Plasma glucose and TG levels after oral GTT and OFTT (n=5 mice / group). (Figure 37I) eWAT volume in WT and UCP1 KO mice 2 weeks post-AAV (n=5 mice / group, except UCP1 KO TSLP mice where n=7 mice). (Figure 37J) Urinary glucose, (Figure 37K) Urinary ketones, and (Figure 37L) Urinary protein 10 days after AAV (n=10 mice / group). Data are mean ± sem from two independent experiments. ANOVA (Figures 37A-37H) or Student's t-test (Figure 37I). Statistics. (Figure 37A) F=0.8248, dfn=1, dfd=12. (Figure 37B) F=0.3628, dfn=1, dfd=8. (Figure 37C) F=3.811, dfn=1, dfd=8. (Figure 37D) F = 0.5290, (Figure 37E) F = 0.0148, (Figure 37F) F = 0.0851, (Figure 37G) F = 0.0374, and (Figure 37H) F = 0.8375, dfn = 1 and dfd = 12 for all comparisons in Figures 37D-37H. (Figure 37I) WT: t = 9.401, df = 8, UCP1 KO: t = 10.26, df = 10. [Figure 38]Figures 38A-38K show that TSLP increases sebum secretion. (Figure 38A) Lipid mass and (Figure 38B) TLC plots from HFD-fed mice 4 weeks after AAV (n=9 mice / group). (Figure 38C) Lipid mass and (Figure 38D-38E) TLC plots and quantification from NC-fed mice 10 days after AAV (n=9 mice / group). (Figure 38F) Dorsal skin H&E histology, sebaceous gland size (red dashed line) 10 days after AAV. (Figure 38G) Quantification of gland size (n=101 control and 100 TSLP sebaceous glands from 3 mice / group). (Figure 38H) Oil Red O (ORO) staining of dorsal skin 10 days after AAV. (Figure 38I) ORO integrated density (n=95 control and 131 TSLP sebaceous glands from 3 mice / group). (Figures 38J-38K) Number of Ki67+ and Ki67- basal cells in sebaceous glands 10 days after AAV (n=78 control and 50 TSLP sebaceous glands from 3 mice / group). Data are shown as mean ± sem from two independent experiments. Student's t-test. Statistics. (Figure 38A) t=6.897, df=16. (Figure 38C) t=2.252, df=16. (Figure 38E) CE: t=0.6720, WE: t=3.829, FFA: t=2.238, FC: t=1.426, df=18 for all comparisons. (Figure 38G) t=6.749, df=199. (Figure 38I) t=0.0985, df=232. (Figure 38J) t = 2.863, df = 126. (Figure 38K) t = 0.7165, df = 126. [Figure 39]Figures 39A-39C show that TSLPR signaling in T cells is required for TLSP-driven sebum secretion. (Figure 39A) TLC plots from WT vs. RAG KO mice 2 weeks after AAV. (Figures 39B-39C) TLC plots and quantification from RAG KO mice adoptively transferred with WT or TSLPR KO T cells 2 weeks after AAV (n=6 WT T-transferred control, 9 WT T-transferred TSLP, 9 TSLPR KO T-transferred control, and 11 TSLPR KO T-transferred TSLP mice). Data are shown as mean ± sem from two independent experiments. Student's t-test. Statistics. (Figure 39C) WT CE:t=1.196, df=13, TSLPR KO CE:t=0.6370, df=18, WT WE:t=\2.695, df=13, TLSPR KO WE:t=1.122, df=18, WT FFA:t=2.057, df=13, TSLPR KOFFA:t=0.6613, df=18, WT FC:t=3.111, df=13, TSLPR KO FC:t=0.6130, df=18. [Figure 40-1] Figures 40A-40I show that TSLP increases T cells in the skin. (Figure 40A) Representative cytometry flow plots of CD4+ and CD8+ T cells in the skin 10 days after AAV. (Figures 40B-40C) % and number of skin T cells. (Figures 40D-40E) % and number of skin CD4+ T cells. (Figures 40F-40G) % and number of skin CD8+ T cells. (n=11 control and 9 TSLP mice). (Figures 40H-40I) CD4 and CD8 staining in the skin 10 days after AAV. Data are shown as mean ± sem from two experiments. Student's t-test. Statistics. (Figure 40B) t = 3.331, (Figure 40C) t = 3.249, (Figure 40D) t = 4.355, (Figure 40E) t = 3.325, (Figure 40F) t = 2.886, (Figure 40G) t = 3.401, df = 18 for all comparisons. [Figure 40-2] See description of Figure 40-1. [Diagram 41]Figures 41A-41H show that TSLPR KO, RAG KO, and E-β KO mice have reduced sebum secretion. (Figure 41A) Representative TLC plots of hair lipids from WT vs. TSLPR KO mice. (Figure 41B) H&E histology of dorsal skin from WT vs. TSLPR KO mice. (Figure 41C) Sebaceous gland size quantification (n=106 WT and 95 TSLPR KO sebaceous glands from 3 mice / group). (Figure 41D) ORO staining and (Figure 41E) ORO quantification of dorsal skin from WT vs. TSLPR KO mice (n=149 WT and 129 TSLPR KO sebaceous glands from 3 mice / group). (Figures 41F-41G) Representative TLC plots and quantification of hair lipids from WT vs. RAG KO (n=5 mice / group). (Figure 41H) Representative TLC plots of WT vs. E-β KO coat lipids. Data are shown as mean ± sem from two experiments. Student's t-test. Statistics. (Figure 41C) t = 0.9520, df = 199. (Figure 41E) t = 3.3460, df = 276. (Figure 41G) CE: t = 1.520, WE: t = 3.924, FFA: t = 1.748, FC: t = 1.052, df = 20 for all comparisons. [Figure 42-1]Figures 42A-42R show that TSLP expression is positively correlated with the expression of multiple sebaceous gland genes in healthy human skin. Linear regression analysis of TSLP expression versus expression of a panel of SG-associated genes: (Figure 42A) SCD, (Figure 42B) FADS2, (Figure 42C) PPARg, (Figure 42D) FA2H, (Figure 42E) DGAT1, (Figure 42F) DGAT2, (Figure 42G) FABP4, (Figure 42H) FABP5, (Figure 42I) ACACA, (Figure 42J) FASN, (Figure 42K) AWAT1, (Figure 42L) ELOVL1, (Figure 42M) ELOVL3, (Figure 42N) ELOVL4, (Figure 42O) ELOVL5, (Figure 42P) MOGAT1, (Figure 42Q) MOGAT2, (Figure 42R) MOGAT3 (n=36 healthy individuals). Pearson's r test and linear regression slope test. Statistics. (Figure 42A) F = 5.691, (Figure 42B) F = 5.09, (Figure 42C) F = 1.202, (Figure 42D) F = 6.386, (Figure 42E) F = 0.9058, (Figure 42F) F = 8.930, (Figure 42G) F = 0.0543, (Figure 42H) F = 6.016, (Figure 42I) F = 4.342, (Figure 42J) F = 4.1 38, (Figure 42K) F = 5.363, (Figure 42L) F = 6.801, (Figure 42M) F = 9.739, (Figure 42N) F = 3.467, (Figure 42O) F = 3.903, (Figure 42P) F = 3.156, (Figure 42Q) F = 3.872, (Figure 42R) F = 0.0108, dfn = 1 and dfd = 34 for all comparisons. [Figure 42-2] See description of Figure 42-1. [Figure 42-3] See description of Figure 42-1. [Figure 42-4] See description of Figure 42-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Detailed Description of the Invention Reference will now be made in detail to certain aspects of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the recited claims, it will be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0010] Throughout this document, values ​​expressed in range format should be interpreted flexibly to include not only the numerical values ​​expressly recited as the limits of the range, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were expressly recited. For example, the range "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also the individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement "from about X to Y" has the same meaning as "from about X to about Y" unless otherwise specified. Similarly, the statement "from about X, Y, or about Z" has the same meaning as "from about X, about Y, or about Z" unless otherwise specified.

[0011] In this document, the terms "a" or "an" or "the" are used to include one or more unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise specified. The statements "at least one of A and B" or "at least one of A or B" have the same meaning as "A, B, or A and B". Furthermore, expressions or terms used herein and not otherwise defined should be understood to be for descriptive purposes only and not limiting. The use of section headings is intended to aid in the reading of the document and should not be construed as limiting; information associated with a section heading may occur within or outside that particular section. All publications, patents, and patent documents referred to in this document are incorporated herein by reference in their entirety, as if individually incorporated by reference.

[0012] In the methods described herein, acts may be performed in any order unless a temporal or operational order is explicitly recited. Moreover, certain acts may be performed simultaneously unless express claim language dictates that they be performed separately. For example, a claimed act of performing X and a claimed act of performing Y may be performed simultaneously in a single operation, and the resulting process is included within the literal scope of the claimed process.

[0013] definition The term "about" as used herein allows for some variability in values ​​or ranges, for example, variability within 10%, within 5%, or within 1% of the stated value or limits of the stated range, and includes the precisely stated value or range.

[0014] As used herein, the term "substantially" refers to a majority or majority, such as at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, at least about 99.99%, or at least about 99.999% or more, or 100%. As used herein, the term "substantially free" means that the composition contains from about 0% to about 5% by weight, or from about 0% to about 1% by weight, or about 5% or less than about 5% by weight, or less than about 4.5% by weight, less than about 4% by weight, less than about 3.5% by weight, less than about 3% by weight, less than about 2.5% by weight, less than about 2% by weight, less than about 1.5% by weight, less than about 1% by weight, less than about 0.9% by weight, less than about 0.8% by weight, less than about 0.7% by weight, less than about 0.6% by weight, less than about 0.5% by weight, less than about 0.4% by weight, less than about 0.3% by weight, less than about 0.2% by weight, less than about 0.1% by weight, less than about 0.01% by weight, or about 4.5% by weight, about 4% by weight, about 3.5% by weight, about 3% by weight, about 2.5% by weight, about 2% by weight, about 1.5% by weight, about 1% by weight, about 0.9% by weight, about 0.8% by weight, about 0.7% by weight, about 0.6% by weight, about 0.5% by weight, about 0.4% by weight, about 0.3% by weight, about 0.2% by weight, about 0.1% by weight, about 0.01% by weight, or greater than about 4.5% by weight, greater than about 4% by weight, greater than about 3.5% by weight, greater than about 3% by weight, greater than about 2.5% by weight, greater than about 2% by weight, greater than about 1% by weight It can mean having no or only a negligible amount, such as greater than about 0.5 weight percent, greater than about 1 weight percent, greater than about 0.9 weight percent, greater than about 0.8 weight percent, greater than about 0.7 weight percent, greater than about 0.6 weight percent, greater than about 0.5 weight percent, greater than about 0.4 weight percent, greater than about 0.3 weight percent, greater than about 0.2 weight percent, greater than about 0.1 weight percent, greater than about 0.01 weight percent, or about 0.001 weight percent or less.The term "substantially free" means that the composition contains between about 0% and about 5% by weight of a substance, or between about 0% and about 1% by weight, or about or less than about 5% by weight, or less than about 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.01% by weight, or about 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5% by weight %, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.01% by weight, or greater than about 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.01% by weight, or greater than about 0.001% or less by weight.

[0015] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin from natural or recombinant sources, or an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. The antibody of the present invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY;Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York;Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883;Bird et al., 1988, Science 242: 423-426).

[0016] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, single domain antibodies such as sdAb (either VL or VH), e.g., camelid antibodies (Riechmann, 1999, J. Immunol. Meth. 231:25-38), camelid VHH domains composed of either VL or VH domains that show sufficient affinity to a target, and multispecific antibodies formed from antibody fragments such as bivalent fragments comprising two Fab fragments linked by disulfide bridges at the hinge region, and isolated complementarity determining regions (CDRs) or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFvs (see, e.g., Hollinger & Hudson, 2005, Nature Biotech. 23: 1126-1136). Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds such as fibronectin type III (Fn3) (U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies). Antibody fragments also include human or humanized antibodies or portions of human or humanized antibodies.

[0017] The term "antigen" or "Ag" as used herein is defined as a molecule that elicits an immune response. This immune response may include antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Thus, those skilled in the art will understand that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will encode an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded only by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0018] The term "coding sequence" as used herein refers to a sequence of a nucleic acid or its complement, or a portion thereof, that can be transcribed and / or translated to produce an mRNA and / or a polypeptide or a fragment thereof. Coding sequences include exons of genomic DNA or premature primary RNA transcripts, which are joined by the biochemical machinery of the cell to provide a mature mRNA. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced. In contrast, the term "non-coding sequence" as used herein refers to a sequence of a nucleic acid or its complement, or a portion thereof, that is not translated into amino acids in vivo or does not interact or attempt to position amino acids with tRNA. Non-coding sequences include both intron sequences of genomic DNA or premature primary RNA transcripts, and gene-associated sequences such as promoters, enhancers, silencers, etc.

[0019] As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., sequences of nucleotides) related by base pairing rules. For example, the sequence "AGT" is complementary to the sequence "TCA." Complementarity may be "partial," in which only a portion of the bases of the nucleic acid match according to the base pairing rules. Alternatively, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and detection methods that depend on binding between nucleic acids.

[0020] As used herein, the term "conservative mutation" or "conservative substitution" refers to the replacement of an amino acid residue with another biologically similar residue. Conservative mutation or substitution is unlikely to change the shape of peptide chain. Examples of conservative mutation or substitution include the replacement of one hydrophobic residue with another residue, such as isoleucine, valine, leucine or methionine, or the replacement of one polar residue with another residue, such as the replacement of lysine with arginine, the replacement of aspartic acid with glutamic acid, or the replacement of asparagine with glutamine.

[0021] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties resulting therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually listed in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to code for the protein or other product of that gene or cDNA.

[0022] As used herein, the term "fragment" as applied to a nucleic acid refers to a subsequence of a larger nucleic acid. A "fragment" of a nucleic acid can be at least about 15 nucleotides in length; e.g., at least about 50 nucleotides to about 100 nucleotides; at least about 100 to about 500 nucleotides, at least about 500 to about 1000 nucleotides; at least about 1000 nucleotides to about 1500 nucleotides; about 1500 nucleotides to about 2500 nucleotides; or about 2500 nucleotides (and any integer value in between). As used herein, the term "fragment" as applied to a protein or peptide refers to a subsequence of a larger protein or peptide. A "fragment" of a protein or peptide can be at least about 20 amino acids in length; e.g., at least about 50 amino acids in length; at least about 100 amino acids in length; at least about 200 amino acids in length; at least about 300 amino acids in length; or at least about 400 amino acids in length (and any integer value in between).

[0023] As used herein, the term "heavy chain antibody" or "multiple heavy chain antibodies" includes immunoglobulin molecules derived from Camelidae species, either by immunization with an antigen and subsequent isolation of serum, or by cloning and expression of nucleic acid sequences encoding such antibodies. The term "heavy chain antibody" or "multiple heavy chain antibodies" refers to antibodies isolated from or derived from animals with heavy chain disease. H It further encompasses immunoglobulin molecules prepared by cloning and expression of (variable heavy chain immunoglobulin) genes.

[0024] The term "immunoglobulin" or "Ig" as used herein is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response of most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin with no known antibody function, but may function as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

[0025] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes production of the gene product in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0026] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or polypeptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or polypeptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0027] The term "specifically binds" as used herein with respect to an antibody means an antibody that recognizes a particular antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind that antigen from one or more species. However, such species cross-reactivity does not change the classification of the antibody as specific in itself. In another example, an antibody that specifically binds to an antigen may also bind to a different allelic form of the antigen. However, such cross-reactivity does not change the classification of the antibody as specific in itself. In some cases, the terms "specific binding" or "specifically binds" may be used with respect to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) of the chemical species; for example, an antibody recognizes and binds to a particular protein structure, rather than to proteins broadly. If an antibody is specific for epitope "A", then in a reaction involving labeled "A" and the antibody, the presence of a molecule containing epitope A (or free unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0028] The term "synthetic antibody" as used herein means an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein, or an amino acid sequence that specifies the antibody, where the DNA or amino acid sequence is available and has been obtained using synthetic DNA or amino acid sequence techniques well known in the art.

[0029] As used herein, the term "wild type" refers to a gene or gene product isolated from a naturally occurring source. A wild type gene is the gene most frequently observed in a population and is therefore arbitrarily designated as the "normal" or "wild type" form of the gene. In contrast, the term "modified" or "mutant" refers to a gene or gene product that exhibits altered sequence and / or functional properties (i.e., altered properties) when compared to the wild type gene or gene product. Note that naturally occurring mutants can be isolated; they are identified by the fact that they have altered properties (including altered nucleic acid sequences) when compared to the wild type gene or gene product.

[0030] As used herein, the term "independently selected" refers to a referenced group that is the same, different, or a mixture thereof, unless the context clearly dictates otherwise. Thus, under this definition, "X 1 , X 2 , and X 3 is independently selected from the noble gases" is intended to include, for example, 1 , X 2 , and X 3 are all the same, X 1 , X 2 , and X 3 are all different, X 1 and X 2 is the same, but X 3 , and other similar permutation scenarios.

[0031] As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one compound described herein and a pharma- ceutical acceptable carrier. Pharmaceutical compositions facilitate the administration of compounds to patients or subjects. There are multiple techniques of administering compounds in the art, including but not limited to intravenous, oral, aerosol, parenteral, ocular, pulmonary and topical administration.

[0032] A "disease" is a condition in the health of an animal where the animal is unable to maintain homeostasis and where, if the disease is not ameliorated, the animal's health continues to deteriorate.

[0033] In contrast, a "disorder" in an animal is one in which the animal is able to maintain homeostasis, but the animal's health status is less favorable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily cause a further deterioration in the animal's health status.

[0034] As used herein, the terms "effective amount," "pharmacologically effective amount," and "therapeutically effective amount" refer to a non-toxic but sufficient amount of an agent to provide a desired biological result. The result may be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or other desired alteration of a biological system. The appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0035] As used herein, the term "efficacy" refers to the maximum effect (Emax) achieved within an assay.

[0036] As used herein, the term "HFD" refers to a high fat diet.

[0037] As used herein, the term "NC" refers to normal chow, the usual diet.

[0038] As used herein, the term "pharmaceutical acceptable" refers to a substance, such as a carrier or diluent, that does not abolish the biological activity or properties of the compound and is relatively non-toxic, i.e., that may be administered to an individual without causing undesirable biological effects or interacting in a deleterious way with any of the components of the composition in which it is contained.

[0039] As used herein, the term "pharmaceutically acceptable salts" refers to salts of the compound being administered prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids or bases, organic acids or bases, solvates, hydrates, or clathrates thereof.

[0040] Suitable pharma- ceutically acceptable acid addition salts may be prepared from inorganic or organic acids. Examples of inorganic acids include hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric (including sulfate and hydrogen sulfate), and phosphoric acid (including hydrogen phosphate and dihydrogen phosphate). Suitable organic acids may be selected from the aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, malonic acid, saccharin, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, trifluoromethanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, galactaric acid and galacturonic acid.

[0041] Suitable pharmaceutically acceptable base addition salts of the compounds described herein include, for example, ammonium salts, alkali metal salts, alkaline earth metal salts, and metal salts including transition metal salts such as calcium salts, magnesium salts, potassium salts, sodium salts, and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts can be prepared from the corresponding compound, for example, by reacting the compound with an appropriate acid or base.

[0042] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent or encapsulating material, that is involved in carrying or transporting a compound described herein into or to a patient so that it can perform its intended function. Typically, such constructs are carried or transported from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation containing the compound(s) described herein and not harmful to the patient. Some examples of materials which may serve as pharma- ceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; and other non-toxic compatible substances used in pharmaceutical preparations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial agents, antifungal agents, absorption delaying agents, and the like, that are compatible with the activity of the compound(s) described herein and are physiologically acceptable to the patient. Supplementary active compounds can also be incorporated into the composition. "Pharmaceutically acceptable carrier" can further include pharmaceutically acceptable salts of the compound(s) described herein.Other additional ingredients that may be included in the pharmaceutical compositions used in the methods or compounds described herein are known in the art and described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.), which is incorporated herein by reference.

[0043] The terms "patient," "subject," or "individual" are used interchangeably herein and refer to any animal or cells thereof suitable for the methods described herein, whether in vitro or in situ. In non-limiting aspects, the patient, subject, or individual is a human.

[0044] As used herein, the term "efficacy" refers to the half-maximal response (ED 50 ) is the amount of water needed to produce

[0045] A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of a pathology with the intent of reducing or eliminating those signs.

[0046] As used herein, the term "treatment" or "treating" is defined as the application or administration of a therapeutic agent, i.e., one or more compounds described herein (alone or in combination with another agent), to a patient, or to a tissue or cell line isolated from a patient having a condition contemplated herein or a symptom of a condition contemplated herein (e.g., for diagnostic or ex vivo applications) for the purpose of curing, curing, mitigating, alleviating, altering, correcting, ameliorating, improving, or affecting the condition contemplated herein or a symptom of the condition contemplated herein. Such treatments may be specifically tailored or modified based on knowledge gained from the field of pharmacogenomics.

[0047] As used herein, the term "TSLP" refers to thymic stromal lymphopoietin.

[0048] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0049] Preparation of compounds The compounds described herein can be prepared according to the general schemes described herein using synthetic methods known to those skilled in the art. The following examples illustrate non-limiting embodiments of the compounds described herein and their preparation.

[0050] The compounds described herein may have one or more stereocenters, and each stereocenter may exist independently in either the (R) or (S) configuration. In certain embodiments, the compounds described herein exist in optically active or racemic forms. It is understood that the compounds described herein encompass racemic, optically active, regioisomeric and stereoisomeric forms, or combinations thereof, that have the therapeutically useful properties described herein. Preparation of optically active forms is accomplished in any suitable manner, including, by way of non-limiting examples, resolution of racemic forms by recrystallization techniques, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using chiral stationary phases. In certain embodiments, a mixture of one or more isomers is utilized as the therapeutic compounds described herein. In other embodiments, the compounds described herein include one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis, and / or separation of mixtures of enantiomers and / or diastereomers. Resolution of the compounds and their isomers can be accomplished by any means, including, but not limited to, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.

[0051] The methods and formulations described herein include the use of N-oxides (where appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and / or pharma- ceutically acceptable salts of compounds having the structure of any compound(s) described herein, as well as metabolites and active metabolites of these compounds having the same type of activity. Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates, and the like. In certain embodiments, the compounds described herein exist in solvated forms with pharma- ceutically acceptable solvents such as water and ethanol. In other embodiments, the compounds described herein exist in unsolvated forms.

[0052] In certain embodiments, the compound(s) described herein can exist as tautomers. All tautomers are included within the scope of the compounds presented herein.

[0053] In certain embodiments, the compounds described herein are prepared as prodrugs. "Prodrug" refers to an agent that is converted to a parent drug in vivo. In certain embodiments, upon in vivo administration, the prodrug is chemically converted to a biologically, pharmacologic, or therapeutically active form of the compound. In other embodiments, the prodrug is enzymatically metabolized by one or more steps or processes to a biologically, pharmacologic, or therapeutically active form of the compound.

[0054] In certain embodiments, for example, sites on the aromatic ring portion of the compound(s) described herein are susceptible to various metabolic reactions. The incorporation of suitable substituents into the aromatic ring structure can reduce, minimize or eliminate this metabolic pathway. In certain embodiments, suitable substituents for reducing or eliminating the susceptibility of the aromatic ring to metabolic reactions are, by way of example only, deuterium, halogen, or alkyl groups.

[0055] The compounds described herein also include isotopically labeled compounds in which one or more atoms are replaced with an atom having the same atomic number but an atomic mass or mass number different from that normally found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include: 2 H, 3 H, 11 C. 13 C. 14 C. 36 Cl, 18 F, 123 I, 125 I, 13 N, 15 N, 15 O. 17 O. 18 O. 32 P, and 35Isotopically labeled compounds include, but are not limited to, S. In certain embodiments, isotopically labeled compounds are useful in drug and / or substrate tissue distribution studies. In other embodiments, substitution with heavier isotopes such as deuterium provides greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements). In yet other embodiments, 11 C. 18 F, 15 O and 13 Substitution with positron emitting isotopes, such as N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically labeled compounds are prepared by any suitable method or process in which a suitable isotopically labeled reagent is substituted for a non-labeled reagent used in other methods.

[0056] In certain aspects, the compounds described herein are labeled by other means, including but not limited to the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

[0057] The compounds described herein are synthesized using any suitable procedure starting from compounds available from commercial sources or prepared using the procedures described herein.

[0058] composition In various embodiments, the compositions useful in the methods described herein comprise at least one vitamin D3 analog. In certain embodiments, the vitamin D3 analog is at least one analog selected from the group consisting of: 26,27-cyclo-22-ene-1α,24S-dihydroxyvitamin D3 (also known as MC903, calcipotriene, or calcipotriol); 1α,18,25-(OH)3D3; 23-(m-(dimethylhydroxymethyl)-22-yne-24,25,26,27(tetranor)-1α-OH)2D3; 1α,25-dihydroxy-trans-isotachysterol (also known as 1,25-trans-iso-T); (1S,3R,6S)-7,19-retro-1,25-(OH)2D3; (1S,3R,6R)-7,19-retro-1,25-(OH)2D3; 22-(p-(hydroxyphenyl)-23,24,25,26,27-pentanol-D3; 22-(m-(hydroxyphenyl)-23,24,25,26,27-pentanol-D3; 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3'(E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (also known as EB1089); 1α,25-(OH)-20-epi-22-oxa-24,26,27-trisoximovitamin D (also known as KH1060); 22-Oxa-1α,25(OH)2D3 (also known as OCT or 22-OXA); 1R,25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D3; and any combination thereof.

[0059] In certain embodiments, the vitamin D3 analog is MC903. In certain embodiments, the vitamin D3 analog is the only biologically active agent administered to the subject. In other embodiments, MC903 is the only biologically active agent administered to the subject. In still other embodiments, the analog is the only biologically active agent administered to the subject in an amount sufficient to increase systemic TSLP levels in the subject. In still other embodiments, MC903 is the only biologically active agent administered to the subject in an amount sufficient to increase systemic TSLP levels in the subject.

[0060] TSLP levels can be elevated in subjects by inducing its production by local administration of MC903. Compared to vehicle (EtOH), local administration of MC903 for 12 weeks results in a significant reduction in weight gain in mice fed a high-fat diet (HFD) (Figure 1A). The reduction in weight gain is associated with a significant reduction in epididymal white adipose tissue (eWAT) weight and improved metabolic parameters, as judged by glucose tolerance test (GTT) and homeostasis model assessment of insulin resistance (HOMA-IR) (Figures 1B-1D). Hepatic fat deposition is also markedly reduced in mice treated with MC903 (Figure 2). The loss of eWAT, improved GTT and HOMA-IR values, and reduced hepatic deposition resulting from local administration of MC903 are dependent on the presence of TSLP in the body, as MC903 treatment does not prevent weight gain or improve metabolic parameters in TSLP-R-deficient mice (Figure 3).

[0061] Composition of TSLP In various embodiments, compositions useful in the methods described herein include viral vectors that contain a TSLP polypeptide isoform or a TSLP expression sequence.

[0062] TSLP isoforms In various embodiments, the subject is a human and the TSLP isoform is At least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to at least one polypeptide of TIFF2025032239000002.tif34158.

[0063] In other embodiments, the subject is a human and the TSLP isoform is at least one selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2.

[0064] TSLP isoforms can be stabilized for in vitro and / or in vivo administration of the peptide to provide a stabilized form suitable for a particular route of administration, such as intravenous, subcutaneous, or other intraperitoneal routes of administration. Stabilized TSLP isoforms of SEQ ID NO:1, SEQ ID NO:2, and peptides having at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:1 or SEQ ID NO:2, include peptides having substitutions of one or more residues in the primary peptide sequence. Substitutions can include changing one or more amino acids to another homologous naturally occurring amino acid, a D-form of an amino acid, a synthetic derivative of an amino acid, or a peptidomimetic moiety that mimics the physicochemical properties of an amino acid. Stabilized TSLP isoforms can also include the addition of a cysteine ​​residue or the substitution of a non-cysteine ​​residue with a cysteine ​​so that disulfide bonds can form in solution, both in vitro and in vivo. Stabilized TSLP isoforms can also include the addition of chemical moieties at the N-terminal or C-terminal residues, or both, such as by addition of one or more polyethylene glycol (PEG) moieties. Suitable PEG moieties can include about to about 100 ethylene glycol units. PEG moieties can include amide bonds, C1-C 10 Covalent attachment to the peptide can be by any suitable chemical means, including via alkyl linkers, carbamates, carbonates, etc. In various embodiments, the stabilized TSLP isoforms of SEQ ID NO:1 or SEQ ID NO:2, and peptides having at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:1 or SEQ ID NO:2, retain at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.5% of the activity of the unmodified / unstabilized peptide.

[0065] TSLP-containing viral vectors In various embodiments, TSLP expression can be enhanced in vitro or in vivo using a viral vector incorporating a TSLP expression sequence and at least one promoter. The viral vector can be any suitable adeno-associated virus (AAV), such as the AAV1-AAV8 family of adeno-associated viruses. In some embodiments, the viral vector is a viral vector capable of infecting humans. The TSLP expression sequence can be inserted between the inverted terminal repeats (ITRs) of AAV. The TSLP expression sequence can be any suitable mammalian TSLP sequence. Non-limiting examples of suitable mammalian TSLP expression sequences include mouse, human, porcine, canine, bovine, equine, feline, or equine TSLP expression sequences. In certain embodiments, the TSLP expression sequence is a human sequence. In various embodiments, the viral vector is AAV8. The promoter can be a thyroxine-binding globulin (TBG) promoter. In various embodiments, the promoter is a human promoter sequence that allows TSLP expression in the liver. The AAV can be a recombinant AAV, with the capsid being from one AAV serotype and the ITR being from another AAV serotype.In various embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8 capsid.In various embodiments, the ITR in the AAV is at least one ITR selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8 ITR.Suitable AAV expressing human or mouse TSLP using TBG promoter can be purchased from Vector Biolabs (Malvern, PA).In various embodiments, TSLP expression can be enhanced in vitro or in vivo using AAV8-TSLP vector. As used herein, the term "AAV8-TSLP" refers to an AAV8 viral vector (recombinant or non-recombinant) that contains a TSLP expression sequence and at least one promoter sequence that increases systemic expression of TSLP when administered to a subject.In some embodiments, the viral vector is a recombinant or non-recombinant AAV2 or AAV5 that contains any of the TSLP expression sequences described herein.

[0066] The TSLP expression sequence, in some embodiments, can be a long form mammalian TSLP expression sequence or a short form mammalian TSLP expression sequence, hi some embodiments, the TSLP expression sequence is the long form human TSLP expression sequence of SEQ ID NO:3: TIFF2025032239000003.tif51142.

[0067] In various embodiments, the TSLP expression sequence is the long form human TSLP expression sequence of SEQ ID NO:4 as follows: TIFF2025032239000004.tif16159.

[0068] In various embodiments, the TSLP expression sequence is the short form human TSLP expression sequence of SEQ ID NO:5 as follows: TIFF2025032239000005.tif21128.

[0069] In various embodiments, the TSLP expression sequence is the short form human TSLP expression sequence of SEQ ID NO:6: TIFF2025032239000006.tif3147.

[0070] In various embodiments, the TSLP expression sequence is the long form mouse TSLP expression sequence of SEQ ID NO:7 as follows: TIFF2025032239000007.tif45146.

[0071] In various embodiments, the TSLP expression sequence is the long form mouse TSLP expression sequence of SEQ ID NO:8 as follows: TIFF2025032239000008.tif16159.

[0072] To test whether TSLP alone is sufficient to prevent weight gain in HFD-fed mice, mice were injected with an adeno-associated virus expressing TSLP (AAV8-TSLP). Injection of AAV8-TSLP drives expression of TSLP in the liver for 6–8 weeks using the thyroxine-binding globulin (TBG) promoter. Compared to AAV8 controls, mice administered AAV8-TSLP lost weight despite consuming a HFD (Figure 4). Furthermore, AAV8-TSLP injection caused significant weight loss in already overweight mice (previously fed a HFD for 10 weeks) despite continuing the HFD (Figure 5). At the end of the 5 weeks, skin histology revealed a complete loss of subcutaneous fat from these mice (Figure 6). The effect of TSLP was also tested in Ob / Ob mice. Ob / Ob mice are leptin deficient, which leads to hyperphagia (overeating) and consequent weight gain despite being fed a normal chow diet. Similar to HFD-fed mice, weight loss was also observed when Ob / Ob mice were treated with AAV8-TSLP (Figure 7). AAV8-TSLP also induced adipose tissue loss in normal chow-fed mice. Although only slight weight loss was observed in normal chow-fed mice treated with AAV8-TSLP (Figure 8A), a near complete loss of eWAT and inguinal white adipose tissue (iWAT) weight was observed between days 7 and 14 after injection of AAV8-TSLP compared to AAV8 controls (Figure 8B, 8C). This effect was specific to white adipose tissue, as no differences in brown adipose tissue (BAT) or muscle weight were observed in mice injected with AAV8-TSLP compared to mice injected with AAV8 controls (Figure 8D, 8E).

[0073] Compositions comprising the compound(s), peptides, and / or viral vectors described herein include pharmaceutical compositions comprising at least one compound, peptide, and / or viral vector described herein and at least one pharma- ceutically acceptable carrier. In certain embodiments, the compositions are formulated for routes of administration such as oral or parenteral, e.g., transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, ​​vaginal (e.g., vaginal and perivaginal), nasal (intra) and (trans)rectal, intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration.

[0074] Methods for Treating, Ameliorating, and / or Preventing Obesity and / or Skin Disorders by Increasing TSLP Levels obesity: MC903 In various aspects, a method for treating obesity or obesity-related disorders is provided. The method comprises administering at least one vitamin D3 analogue to a subject suffering from obesity or obesity-related disorders, as described herein. The type of obesity-related disorder that can be treated by these methods is not particularly limited, and any disorder resulting from a subject having excess white adipose tissue in the body can be treated by the compositions and methods described herein. Non-limiting examples of obesity-related disorders include non-alcoholic steatohepatitis (NASH), metabolic disease, diabetes (type I and type II), hypertension, dyslipidemia (high LDL cholesterol, low HDL cholesterol, or high levels of triglycerides), coronary heart disease, stroke, gallbladder disease, kidney disease, osteoarthritis, sleep apnea and respiratory disorders, and cancer, including endometrial cancer, breast cancer, colon cancer, kidney cancer, gallbladder cancer, and liver cancer. In various aspects, the obesity-related disorder is NASH.

[0075] Obesity can be determined by a variety of art-recognized methods, including measuring a subject's body mass index (BMI), waist circumference, waist-to-hip ratio, skinfold thickness, dual energy x-ray absorptiometry (DEXA), blood triglyceride levels, blood cholesterol levels, and the like. Criteria for determining whether a human subject is obese include those promulgated by the Centers for Disease Control and Prevention (CDC). In various embodiments, an obese human subject has a BMI of at least about 25.0, at least about 30.0, at least about 35.0, or at least about 40.0. Generally, a human subject with a BMI of less than about 25.0 is not considered obese, although the compositions described herein may also be used by non-obese individuals to reduce the amount of white adipose tissue in the body for aesthetic, cosmetic, athletic, or other purposes.

[0076] In various embodiments, the method of treating obesity or obesity-related disorder causes weight loss in the subject.The weight loss resulting from applying the method described herein can cause weight loss of at least about 5%, at least about 7.5%, at least about 10%, at least about 12.5%, at least about 15%, at least about 17.5%, or at least about 20%, more than about 5%, more than about 7.5%, more than about 10%, more than about 12.5%, more than about 15%, more than about 17.5%, or more than about 20%, or less than about 5%, less than about 7.5%, less than about 10%, less than about 12.5%, less than about 15%, less than about 17.5%, or less than about 20%, for a period of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks. As shown in FIG. 1A, mice on a high fat diet (HFD) administered MC903 experienced significantly less weight gain over 12 weeks compared to control mice not administered MC903. FIG. 2 shows that HFD control mice (not administered MC903) developed significant amounts of white adipose tissue (bottom left image), whereas HFD mice administered MC903 had a distribution of white adipose tissue that appeared very similar to that of mice fed a normal chow diet. Administration of MC903, in various embodiments, does not result in a substantial loss of muscle mass in the subject. In various embodiments, administration causes the secretion of lipids from the skin of the subject. In certain embodiments, the loss of white adipose tissue from the subject's body results in the secretion of lipid components of white adipose tissue from the skin of the subject.

[0077] TSLP peptides and TSLP-containing viral vectors In various embodiments, a method for treating obesity or an obesity-related disorder is provided, comprising administering to a subject suffering from obesity or an obesity-related disorder at least one viral vector comprising at least one TSLP polypeptide isoform or a TSLP expression sequence as described herein.

[0078] The types of obesity-related disorders that can be treated by these methods are not particularly limited, and any disorder caused by subjects with excess white adipose tissue in the body can be treated by the compositions and methods described herein.Non-limiting examples of obesity-related disorders include non-alcoholic steatohepatitis (NASH), metabolic disease, diabetes (type I and type II), hypertension, dyslipidemia (high LDL cholesterol, low HDL cholesterol, or high levels of triglycerides), coronary heart disease, stroke, gallbladder disease, kidney disease, osteoarthritis, sleep apnea and respiratory disorders, and cancer, including endometrial cancer, breast cancer, colon cancer, kidney cancer, gallbladder cancer, and liver cancer.In various embodiments, the obesity-related disorder is NASH.

[0079] In various embodiments, the method of treating obesity or obesity-related disorders results in weight loss in a subject.The weight loss resulting from administering at least one viral vector comprising at least one TSLP polypeptide isoform or TSLP expression sequence using the methods described herein can result in a weight loss of at least about 5%, at least about 7.5%, at least about 10%, at least about 12.5%, at least about 15%, at least about 17.5%, or at least about 20%, more than about 5%, more than about 7.5%, more than about 10%, more than about 12.5%, more than about 15%, more than about 17.5%, or more than about 20%, or less than about 5%, less than about 7.5%, less than about 10%, less than about 12.5%, less than about 15%, less than about 17.5%, or less than about 20%, over a period of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks.

[0080] For a subject to lose weight, a net negative energy imbalance must be present, i.e., decreased energy intake (food intake / absorption), increased energy expenditure (heat generation, increased locomotor activity), and / or increased energy output (excretion or secretion outside the body). Energy intake with AAV8 control and AAV8-TSLP was similar based on food intake (Figure 9A), glucose absorption (Figure 9B), and lipid absorption (Figure 9C). Additionally, bomb calorimetry of 1-day fecal collections from AAV8 control and AAV8-TSLP-treated mice revealed significantly less energy in the feces, indicating a trend toward a net increase in energy intake (Figure 10). To more precisely measure energy intake and expenditure, AAV control and AAV8-TSLP-treated mice were placed in metabolic chambers from days 7 to 10 after AAV8 injection. According to these precise measurements, indeed, food intake increased by approximately 20% in the AAV8-TSLP-treated mice over the final 48 hours in the metabolic chamber (Figure 11). Despite losing 30% of fat mass during the 72 hours the mice were placed in the metabolic chamber, O2 consumption and CO2 production (both measures of energy expenditure), and locomotor activity were all similar between AAV8 control and AAV8-TSLP-treated mice (Figures 12-14). Taken together, these data suggest, without being bound by theory, that TSLP does not induce white fat loss by decreasing energy intake or increasing energy expenditure. Weight loss in mice is further illustrated in Figure 19 and Figure 20.

[0081] Although not evident until approximately week 4 when mice were fed a normal chow diet, obese mice treated with AAV8-TSLP develop a very oily coat as they lose weight and fat mass (Figure S15). This sebum secretion is responsible for adipose tissue loss, as stearoyl-CoA desaturase 1 (SCD1) knockout mice, which have defective sebaceous glands, do not lose adipose tissue upon AAV8-TSLP treatment (Figure S31J).

[0082] Thus, without wishing to be bound by theory, TSLP induces lipid secretion through the skin (e.g., through sebum), thereby causing fat loss. In various embodiments, administration causes lipid secretion from the subject's skin. In certain embodiments, loss of white adipose tissue from the subject's body results in the secretion of lipid components of white adipose tissue from the subject's skin.

[0083] To test whether TSLP responsiveness is required in the hematopoietic or non-hematopoietic compartment, a bone marrow (BM) transplantation study was performed. BM transplantation allows the replacement of the hematopoietic compartment with the BM of another mouse. Thus, B6.SJL mice transplanted with TSLP-R KO BM (TSLP-R KO → B6.SJL) respond to TSLP in the non-hematopoietic compartment but not in the hematopoietic compartment. Conversely, TSLP-R KO mice transplanted with B6.SJL BM (B6.SJL → TSLP-R KO) respond to TSLP in the hematopoietic compartment but not in the non-hematopoietic compartment. When treated with AAV8-TSLP, B6.SJL → B6 and B6.SJL → TSLP-R KO mice lost weight, whereas TSLP-R KO → B6.SJL mice failed to lose weight, suggesting that TSLP signaling must occur in the hematopoietic compartment (Figure 16). To more precisely identify the cell types involved, RAG KO mice (mice with no T or B cells) and TCR-β KO mice (with no T cells) were treated with AAV8-TSLP. Both RAG KO and TCR-β KO mice failed to lose weight when treated with AAV8-TSLP, suggesting that T cells are required for TSLP-induced weight loss (Figure 17). Without being bound by theory, it is possible that T cells stimulated with TSLP can produce factors that induce lipid secretion from the skin, which drives the weight loss in these mice, thereby causing TSLP to cause selective loss of white fat from mice.

[0084] The methods described herein increase TSLP levels in a subject. In various embodiments, TSLP levels in a subject are increased by at least about 5%, more than about 5%, less than about 5%, between about 5% and about 40%, between about 5% and about 30%, between about 5% and about 20%, or between about 5% and about 10% compared to a control. In various embodiments, TSLP levels in a subject are increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40%, more than about 5%, more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, or more than about 40%, or less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, or less than about 40% compared to a control. Administration of at least one TSLP polypeptide isoform or at least one viral vector comprising a TSLP expression sequence, in various embodiments, does not result in a substantial loss of muscle mass in a subject.

[0085] TSLP can be elevated in mice via topical MC903 treatment, recombinant TSLP polypeptide isoforms, or viral vectors (gene therapy). Elevated TSLP triggers dermal secretion of lipids, which consumes energy and reduces circulating lipid levels. Lipolysis occurs from fat stores to replenish consumed lipids, ultimately leading to selective depletion of white adipose tissue and weight loss (Figure 18).

[0086] The method described herein comprises administering to the subject a therapeutically effective amount of at least one compound described herein, which is optionally formulated into a pharmaceutical composition.In various embodiments, the therapeutically effective amount of at least one compound described herein present in the pharmaceutical composition is the only therapeutically active compound in the pharmaceutical composition.In certain embodiments, the method further comprises administering to the subject a further therapeutic agent for treating or preventing obesity or obesity-related disorders.

[0087] In certain embodiments, administering a compound(s) described herein to a subject allows for a lower dose of the additional therapeutic agent to be administered compared to the dose of the additional therapeutic agent alone required to achieve the same result in treating or preventing obesity or obesity-related disorders in the subject.For example, in certain embodiments, a compound(s) described herein enhances the activity of the additional therapeutic compound, thereby allowing a lower dose of the additional therapeutic compound to provide the same effect.

[0088] In certain embodiments, the compound(s) described herein and the therapeutic agent are co-administered to the subject. In other embodiments, the compound(s) described herein and the therapeutic agent are co-formulated and co-administered to the subject.

[0089] In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.

[0090] skin disorders In various embodiments, a method for treating skin disorders or improving scalp health is provided. The method includes topically administering at least one vitamin D3 analogue or directly administering TSLP isoform to a subject suffering from skin disorders as described herein. The type of skin disorder that can be treated by these methods is not particularly limited, and is any disorder that benefits from increasing sebum secretion and / or increasing skin barrier function. In certain embodiments, skin disorders include eczema / atopic dermatitis, dry skin-related dermatitis, dry skin (xeroderma), ichthyosis (all forms), recurrent skin infections, wrinkles (skin aging), and hair loss and / or hair growth failure (including but not limited to alopecia, such as but not limited to androgenetic alopecia).

[0091] Without being bound by theory, the mechanism by which this loss of adipose tissue occurred may be related to increased sebum production from TSLP-overexpressing mice. Although not clear until approximately week 4, mice treated with AAV8-TSLP develop an oily coat. To test this more directly, lipids were extracted from the shaved coat of mice injected with AAV8-TSLP compared to mice injected with AAV8 control. Consistent with the oily coat, the mice showed an increase in lipids (wax monoesters and wax diesters) found specifically in the sebum of the coat, suggesting that the mice produced more sebum when TSLP was overexpressed (Figure 21). Furthermore, immunohistochemistry of sebaceous glands revealed an increased proportion of KI67+ basal layer sebocytes (Figure 22), suggesting that the glands were more active. Next, to test whether TSLP has a physiological role in controlling sebum release, we shaved the coat of unmanipulated WT or TSLP-R KO mice and quantified the amount of sebum lipid components. TSLP-R KO mice showed reduced amounts of sebum-specific lipid components (wax monoesters and wax diesters) compared to WT mice (Figure 23). These data suggest that sebum release can be pharmacologically increased by TSLP elevating agents (or TSLP itself) and inhibited by blocking TSLP. TSLP also promoted barrier function by increasing the expression of antimicrobial peptides from the skin (Figure 24).

[0092] Topical MC903 exerts desirable effects in humans. One individual showed increased amount of sebum on the forehead after topical application of MC903 on the arm (Figure 25). Eczematous dry skin of the hand showed marked improvement after topical application of MC903 on the arm (Figure 26). Considering that a healthy scalp provides a better environment for hair growth and that eczema / atopic dermatitis results in incomplete hair growth (onlinelibrary dot wiley dot com / doi / full / 10 dot 1002 / jemt dot 21101), hair growth would be remotely promoted by topical MC903 treatment as well. Indeed, substantial restoration of hair growth was observed after two cycles of topical MC903 application on the arm for two weeks (Figure 27). Taken together, these data suggest that direct administration of at least one vitamin D3 analog or TSLP isoform as described herein remotely improves eczema / atopic dermatitis, dry skin-related dermatitis, dry skin (xeroderma), wrinkles (skin aging), and ichthyosis (all forms) by promoting sebum secretion. In certain embodiments, direct administration of at least one vitamin D3 analog or TSLP isoform as described herein remotely treats, improves, and / or prevents alopecia (such as, but not limited to, male pattern baldness). Such treatment (direct administration of vitamin D3 analog and / or TSLP isoform) treats or prevents recurrent skin infections. In certain embodiments, such treatment promotes antimicrobial peptides contained within sebum. In certain embodiments, such treatment promotes hair growth or prevents or minimizes hair loss by creating a healthier scalp environment.

[0093] Unexpectedly, the methods described herein can treat skin disorders when at least one vitamin D3 analog is administered to a healthy area of ​​the skin. That is, the vitamin D3 analog is not administered to a skin lesion or other symptom of a disorder on the skin, but rather to a site on the skin of a healthy subject. In one embodiment, the vitamin D3 analog is calcipotriol. The vitamin D3 analog can be administered in a topical composition, such as a cream, gel, lotion, or patch. In some embodiments, the vitamin D3 analog is the only active agent in the topical composition. The vitamin D3 analog can be present in an amount of about 0.0001% to about 10% (w / w) relative to the amount of inactive ingredients in the topical composition. In various embodiments, the vitamin D3 analog can be present in an amount of about 0.0001% to about 10%, about 0.001% to about 10%, about 0.01% to about 10%, about 0.1% to about 10%, about 0.0001% to about 5%, about 0.0001% to about 2%, about 0.0001% to about 1%, about 0.001% to about 1%, or about 0.01% to about 1% (w / w). Topical compositions can include pharma- ceutically or cosmetically acceptable excipients, such as, but not limited to, fragrances, colorants, emulsifiers, skin permeation enhancers, viscosity modifiers, and the like.

[0094] Eye disorders In various embodiments, a method of treating an eye disorder is provided. The method includes administering at least one vitamin D3 analog locally or directly administering a TSLP isoform to a subject suffering from an eye disorder, as described herein. In the case of local administration, the vitamin D3 analog can be administered distal to the eye, so that there is no direct contact between the vitamin D3 analog and the eye. The type of eye disorder that can be treated by these methods is not particularly limited, and is any disorder that benefits from increased meibum secretion and / or increased eye barrier function. In one embodiment, the eye disorder includes dry eye syndrome, keratoconjunctivitis sicca, keratitis sicca, dysfunctional tear syndrome, age-related dry eye syndrome, drug-related dry eye syndrome, menopausal dry eye syndrome, contact lens-related dry eye, environmentally induced dry eye, dysfunctional eyelid-induced dry eye, autoimmune-related dry eye (Sjogren's syndrome, rheumatoid arthritis, systemic lupus erythematosus), and infection-related conjunctivitis.

[0095] Given that meibomian gland function, which increases ocular moisturization, is regulated in a manner similar to that of sebaceous glands, it was possible that dry eye symptoms might also be remotely improved by topical treatment with MC903. Indeed, there was subjective improvement in dry eye symptoms in two individuals who reported dry eye symptoms prior to the start of treatment (Figure 28).

[0096] Methods for Treating, Ameliorating, and / or Preventing Skin Disorders by Reducing TSLP Levels In various embodiments, the method of treating, improving, and / or preventing skin disorders comprises administering a TSLP inhibitor to a subject. Without being bound by theory, the TSLP inhibitor can inhibit the secretion of sebum by the skin. Sebum is an oily substance secreted from hair follicles. Sebum is important for maintaining moisture in the skin and providing a barrier function against pathogens. However, increased secretion of sebum can lead to undesirable diseases such as acne vulgaris, hidradenitis suppurativa, and seborrheic dermatitis. This specification identifies a novel factor that controls the production of sebum.

[0097] The first indication of the role of TSLP in controlling sebum production came from studies showing that forced expression of TSLP in the liver of mice by adeno-associated virus 8 expressing TSLP (AAV8-TSLP) caused selective adipose tissue loss. Although only slight weight loss was observed in normal chow-fed mice treated with AAV8-TSLP (Figure 8A), a nearly complete loss of epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) was observed in a dose-dependent manner between days 7 and 14 after injection of AAV8-TSLP compared to AAV8 controls (Figure 8B-8C). This effect was specific to white adipose tissue, as no differences in brown adipose tissue (BAT) or muscle weight were observed in mice injected with AAV8-TSLP compared to mice injected with AAV8 controls (Figure 8D-8E). The loss of white adipose tissue caused by AAV8-TSLP occurred in a dose-dependent manner (Figure 20).

[0098] Taken together, these data suggest that TSLP positively controls sebum release. Therefore, blocking TSLP (e.g., by neutralizing antibody) reduces sebum release and may be beneficial for treating disorders caused by increased sebum release. Skin disorders that can be treated with TSLP inhibitors include acne vulgaris, seborrheic dermatitis, and hidradenitis suppurativa. Non-limiting examples of TSLP inhibitors include antibodies, small molecules, siRNA, shRNA, and miRNA. In humans, TSLP exists in two forms: short form (sfTSLP) and long form (lfTSLP). In one embodiment, TSLP inhibitors can bind and inhibit sfTSLP, lfTSLP, or both.

[0099] Antibody inhibitors The present disclosure also provides inhibitors of sfTSLP or lfTSLP, including antibodies or antibody fragments specific to sfTSLP or lfTSLP. That is, the antibodies can inhibit sfTSLP or lfTSLP to provide beneficial effects, such as reduced sebum secretion. Suitable antibodies include, for example, tezepelumab (also known as MEDI9929 and AMG 157; CAS number 1572943-04-4).

[0100] Antibodies include intact monoclonal or polyclonal antibodies, as well as immunologically active fragments (e.g., Fab or (Fab)2 fragments), antibody heavy chains, antibody light chains, humanized antibodies, engineered single-chain Fs, and the like. V The antibody may be a murine antibody (Ladner et al., U.S. Pat. No. 4,946,778), or a chimeric antibody, e.g., an antibody that contains the binding specificity of a mouse antibody, but the remainder of which is of human origin. Antibodies, including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art.

[0101] Antibodies can be prepared using intact polypeptides or fragments that contain the immunizing antigen of interest. The polypeptides or oligopeptides used to immunize animals can be obtained from the translation of RNA or can be chemically synthesized, and can be bound to carrier proteins as desired. Suitable carriers that can be chemically bound to peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin. The bound polypeptides can then be used to immunize animals (e.g., mice, rats, or rabbits).

[0102] As a non-limiting example, an antibody useful within the present invention can bind to circulating sfTSLP or lfTSLP. As will be appreciated by one of skill in the art, any antibody capable of recognizing and specifically binding to circulating sfTSLP or lfTSLP is useful in the present invention. The present invention should not be construed as being limited to any one type of antibody, known or heretofore unknown, provided that the antibody specifically binds to circulating sfTSLP or lfTSLP and can prevent or minimize the biological activity of sfTSLP or lfTSLP.

[0103] Methods for making and using such antibodies are well known in the art. For example, the production of polyclonal antibodies can be accomplished by inoculating the desired animal with an antigen and isolating therefrom antibodies that specifically bind to the antigen. Monoclonal antibodies against full-length or peptide fragments of a protein or peptide can be prepared using any well-known monoclonal antibody preparation procedure, such as those described, for example, in Harlow et al. (1989, Antibodies, A Laboratory Manual, Cold Spring Harbor, New York) and Tuszynski et al. (1988, Blood 72:109-115). Quantities of the desired peptide can also be synthesized using chemical synthesis techniques. Alternatively, DNA encoding the desired peptide can be cloned and expressed from an appropriate promoter sequence in a cell suitable for making large amounts of the peptide. Monoclonal antibodies against the peptide are made from mice immunized with the peptide using standard procedures referenced herein. However, the present invention should not be construed as being limited solely to methods and compositions comprising these antibodies, but should be construed to include other antibodies, as that term is defined elsewhere herein.

[0104] In some cases, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as rodents (e.g., mice), primates (e.g., humans), etc. Descriptions of techniques for preparing such monoclonal antibodies are well known and can be found, for example, in Harlow et al., ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988); Harlow et al., Using Antibodies: A Laboratory Manual, (Cold Spring Harbor Press, New York, 1998); Breitling et al., Recombinant Antibodies (Wiley-Spektrum, 1999); and Kohler et al., 1997 Nature 256: 495-497; and U.S. Patent Nos. 5,693,762; 5,693,761; 5,585,089; and 6,180,370.

[0105] Nucleic acids encoding the antibodies obtained using the procedures described herein are available in the art and may be cloned and sequenced, for example, using the techniques described in Wright et al. (Critical Rev. Immunol. 1992,12:125-168) and the references cited therein. Furthermore, antibodies useful within the invention may be "humanized" using the techniques described in Wright et al. (supra) and the references cited therein, and Gu et al. (Thrombosis and Hematocyst 1997,77:755-759).

[0106] Alternatively, antibodies can be produced using phage display technology. To produce a phage antibody library, first obtain a cDNA library from the mRNA isolated from cells, such as hybridomas, that express the desired protein, such as the desired antibody, expressed on the surface of phage. A cDNA copy of the mRNA is generated using reverse transcriptase. The cDNA that specifies immunoglobulin fragments is obtained by PCR, and the resulting DNA is cloned into a suitable bacteriophage vector to produce a bacteriophage DNA library that contains DNA that specifies immunoglobulin genes. Procedures for producing a bacteriophage library that contains heterologous DNA are well known in the art and are described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York).

[0107] Bacteriophage encoding a desired antibody can be engineered to display the protein on its surface in such a way that it is available for binding to its corresponding binding protein, e.g., the antigen against which the antibody is directed. Thus, when bacteriophage expressing a particular antibody are incubated in the presence of a cell expressing the corresponding antigen, the bacteriophage will bind to the cell. Bacteriophage that do not express the antibody will not bind to the cell. Such panning techniques are well known in the art and are described, for example, in Wright et al. (Critical Rev. Immunol. 1992, 12:125-168).

[0108] The process as described herein was developed to produce human antibodies using M13 bacteriophage display (Burton et al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library is made from mRNA obtained from a population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin genes, and thus the cDNA encodes the same. The amplified cDNA is cloned into an M13 expression vector, creating a library of phages that express human Fab fragments on their surface. Phages that display the antibody of interest are selected by antigen binding and propagate in bacteria to produce soluble human Fab immunoglobulins. Thus, in contrast to conventional monoclonal antibody synthesis, this procedure immortalizes the DNA encoding the human immunoglobulins, rather than the cells that express the human immunoglobulins.

[0109] The procedure just presented describes the generation of phages encoding the Fab portion of an antibody molecule. However, the present invention should not be construed as being limited to the generation of phages encoding Fab antibodies only. Rather, phages encoding single chain antibodies (scFv / phage antibody libraries) are also included in the present invention. Fab molecules contain the entire Ig light chain, i.e., they contain both the variable and constant regions of the light chain, but only the variable region and the first constant region domain (CH1) of the heavy chain. Single chain antibody molecules contain a single chain of protein that contains an Ig Fv fragment. An Ig Fv fragment contains only the variable regions of the heavy and light chains of an antibody, but not the constant regions. Phage libraries containing scFv DNA can be generated according to the procedure described in Marks et al. (1991, J Mol Biol 222:581-597). Panning of the phages so generated for isolation of the desired antibodies is performed in a similar manner as described for the phage libraries containing Fab DNA.

[0110] The invention should also be construed to include synthetic phage display libraries in which heavy and light chain variable regions can be synthesized to include almost every possible specificity (Barbas, 1995, Nature Medicine 1:837-839; de Kruif et al., 1995, J Mol Biol 248:97-105).

[0111] The present invention encompasses polyclonal, monoclonal, synthetic antibodies, etc. One of skill in the art will understand, based on the disclosure provided herein, that an important feature of an antibody useful within the present invention is that the antibody specifically binds to a circulating protein.

[0112] Small molecule inhibitors In certain embodiments, the TSLP inhibitor comprises a small molecule. When the inhibitor is a small molecule, the small molecule can be obtained using standard methods known to those skilled in the art. Such methods include chemical organic synthesis or biological means. Biological means include purification from biological sources, recombinant synthesis and in vitro translation systems using methods well known in the art. In certain embodiments, the small molecule inhibitor of the present invention comprises organic molecules, inorganic molecules, biomolecules, synthetic molecules, etc.

[0113] Combinatorial libraries of molecularly diverse compounds that are potentially useful for treating various diseases and conditions are well known in the art, as are the methods for creating libraries.These methods may use a variety of techniques well known to those skilled in the art, including solid-phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and the generation of unbiased molecular landscapes for lead discovery and biased structures for lead development.

[0114] A common method for small library synthesis involves condensing an activated core molecule with several building blocks to generate a combinatorial library of covalently linked core-building block assemblies. The shape and rigidity of the core determine the orientation of the building blocks in shape space. Libraries can be biased by modifying the core, linkages, or building blocks to target characterized biological structures ("focused libraries"), or can be synthesized with less structural bias by using flexible cores.

[0115] Nucleic Acid Inhibitors In certain embodiments, the TSLP inhibitor comprises an isolated nucleic acid. In other embodiments, the inhibitor is an siRNA or antisense molecule that inhibits sfTSLP or lfTSLP. In yet other embodiments, the nucleic acid preferably comprises a promoter / regulatory sequence so as to direct expression of the nucleic acid. Thus, the present invention provides expression vectors and methods for the introduction of exogenous DNA into cells with co-expression of the exogenous DNA in the cells, as described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley&Sons, New York), and elsewhere herein.

[0116] In certain embodiments, sfTSLP or lfTSLP can be inhibited by inactivating and / or sequestering sfTSLP or lfTSLP. Thus, inhibiting the activity of sfTSLP or lfTSLP can be achieved by using a transdominant negative mutant.

[0117] In certain embodiments, siRNA is used to reduce the levels of sfTSLP or lfTSLP proteins. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into various organisms and cell types causes the degradation of complementary mRNA. In cells, long dsRNA is cleaved into short 21-25 nucleotide small interfering RNAs or siRNAs by a ribonuclease known as Dicer. The siRNAs then assemble into RNA-induced silencing complexes (RISCs) along with protein components and unwind in the process. The activated RISC then binds to the complementary transcripts through base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved, and sequence-specific degradation of the mRNA results in gene silencing. See, for example, U.S. Patent No. 6,506,559;Fire et al., 1998, Nature 391(19): 306-311;Timmons et al., 1998, Nature 395: 854;Montgomery et al., 1998, TIG 14(7): 255-258;Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003);and Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003).Soutschek et al. (2004, Nature 432: 173-178) describe chemical modifications to siRNA that aid in intravenous systemic delivery. Optimization of siRNA includes consideration of overall G / C content, terminal C / T content, Tm, and nucleotide content of 3' overhangs. See, e.g., Schwartz et al., 2003, Cell, 115: 199-208 and Khvorova et al., 2003, Cell 115: 209-216.Thus, the present invention also includes methods of reducing levels of sfTSLP or lfTSLP using RNAi technology.

[0118] In certain embodiments, the present invention provides a vector comprising siRNA or antisense polynucleotide.In other embodiments, the siRNA or antisense polynucleotide inhibits the expression of sfTSLP, lfTSLP, or both.The incorporation of desired polynucleotides into vectors and the selection of vectors are well known in the art.

[0119] In certain embodiments, the expression vector described herein encodes a short hairpin RNA (shRNA) inhibitor. shRNA inhibitors are well known in the art and are directed to target mRNA, thereby reducing the expression of the target. In certain embodiments, the encoded shRNA is expressed by cells and then processed into siRNA. For example, in certain instances, cells have a natural enzyme (e.g., dicer) that cuts shRNA to form siRNA.

[0120] The siRNA, shRNA, or antisense polynucleotides can be cloned into several types of vectors, as described elsewhere herein. For expression of the siRNA or antisense polynucleotide, at least one module of each promoter functions to position the start site for RNA synthesis.

[0121] To evaluate the expression of siRNA, shRNA, or antisense polynucleotide, the expression vector introduced into the cell can also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the population of cells to be transfected or infected using the viral vector. In certain embodiments, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences that allow expression in the host cell. Useful selectable markers are known in the art and include, for example, antibiotic resistance genes, such as neomycin resistance.

[0122] Thus, in another aspect, the present invention relates to a vector comprising the nucleotide sequence of the present invention or the construct of the present invention. The choice of vector depends on the host cell into which it will subsequently be introduced. In a particular embodiment, the vector of the present invention is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In a particular embodiment, the expression vector is selected from the group consisting of viral vectors, bacterial vectors and mammalian cell vectors. Systems based on prokaryotic and / or eukaryotic vectors are available for use with the present invention to produce polynucleotides or their cognate polypeptides. Many such systems are commercially available and widely available.

[0123] Furthermore, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers. (See, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193.)

[0124] By way of example, the vector into which the nucleic acid sequence is introduced may be a plasmid that may or may not be integrated into the genome of the host cell when it is introduced into the cell. Illustrative, non-limiting examples of vectors into which the nucleotide sequence of the invention or the genetic construct of the invention may be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0125] The vector can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In a particular embodiment, the vector is a vector useful for transforming animal cells.

[0126] In certain aspects, the recombinant expression vector may also include a nucleic acid molecule encoding a peptide or peptidomimetic inhibitor of the invention, as described elsewhere herein.

[0127] A promoter may be one that is naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequence located upstream of the coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a polynucleotide sequence located either downstream or upstream of that sequence. Alternatively, certain advantages may be obtained by placing the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring", i.e., that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to synthetically producing promoter and enhancer nucleic acid sequences, sequences may be produced using nucleic acid amplification techniques, including recombinant cloning and / or PCR™, in conjunction with the compositions disclosed herein (U.S. Patent No. 4,683,202; U.S. Patent No. 5,928,906).Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, etc., may be used as well.

[0128] It is important to use a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism selected for expression.Those skilled in the art of molecular biology generally know how to use a combination of promoter, enhancer, and cell type for protein expression.The promoter used can be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, which is advantageous in large-scale production of recombinant protein and / or peptide.The promoter can be heterologous or endogenous.

[0129] Recombinant expression vectors can also contain selectable marker genes that facilitate the selection of transformed or transfected host cells.Suitable selectable marker genes are genes that code for proteins such as G418 and hygromycin that confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or immunoglobulins or Fc portions of immunoglobulins, preferably IgG, etc.Selectable markers can be introduced into a vector separate from the nucleic acid of interest.

[0130] Following generation of a siRNA polynucleotide, one of skill in the art will appreciate that the siRNA polynucleotide has certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Thus, siRNA polynucleotides can be further designed to resist degradation by modification to include phosphorothioates, or other linkages, methylphosphonates, sulfones, sulfates, ketyl, phosphorodithioates, phosphoramidates, phosphate esters, and the like (see, e.g., Agrwal et al., 1987, Tetrahedron Lett. 28: 3539-3542; Stec et al., 1985 Tetrahedron Lett. 26: 2191-2194; Moody et al., 1989 Nucleic Acids Res. 12:4769-4782; Eckstein, 1989 Trends Biol. Sci. 14: 97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp. 97-117 (1989).

[0131] Any polynucleotide can be further modified to increase its stability in vivo.Possible modifications include, but are not limited to, adding flanking sequences at 5' and / or 3' ends; using phosphorothioate or 2'O-methyl instead of phosphodiester linkage in backbone; and / or containing non-traditional bases such as inosine, queosine and vybdosine, and acetyl, methyl, thio and other modified forms of adenine, cytidine, guanine, thymine and uridine.

[0132] In certain embodiments, antisense nucleic acid sequences expressed by plasmid vectors are used to inhibit expression of sfTSLP or lfTSLP proteins. Antisense expression vectors are used to transfect mammalian cells or the mammal itself, thereby causing a reduction in endogenous expression of sfTSLP or lfTSLP.

[0133] Antisense molecules and their use to inhibit gene expression are well known in the art (see, for example, Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press).Antisense nucleic acid, as that term is defined elsewhere herein, is a DNA or RNA molecule that is complementary to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262: 40).Intracellularly, antisense nucleic acid hybridizes to corresponding mRNA, forming a double-stranded molecule, thereby inhibiting the translation of genes.

[0134] The use of antisense methods to inhibit the translation of genes is known in the art and is described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172: 289). Such antisense molecules can be provided to cells via gene expression using DNA encoding the antisense molecule, as taught by Inoue, 1993, U.S. Patent No. 5,190,931.

[0135] Alternatively, the antisense molecules of the present invention can be synthetically produced and then provided to cells. Antisense oligomers of about 10 to about 30, more preferably about 15, nucleotides are preferred because they are easily synthesized and introduced into target cells. Synthetic antisense molecules contemplated by the present invention include oligonucleotide derivatives known in the art that have improved biological activity compared to unmodified oligonucleotides (see U.S. Pat. No. 5,023,243).

[0136] In certain embodiments, ribozymes are used to inhibit the expression of sfTSLP or lfTSLP proteins. Ribozymes useful for inhibiting the expression of target molecules can be designed, for example, by incorporating target sequences complementary to the mRNA sequence encoding sfTSLP or lfTSLP into a basic ribozyme structure. Ribozymes targeting sfTSLP or lfTSLP can be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or can be genetically expressed from the DNA that encodes them.

[0137] In various embodiments, a method of treating a skin disorder comprises administering a TSLP stimulator to a subject.

[0138] Combination therapy The compound useful in the method described herein can be used in combination with one or more additional therapeutic agents useful for treating obesity or obesity-related disorders.These additional therapeutic agents can include compounds that are commercially available or synthetically available to those skilled in the art.These additional therapeutic agents are known to treat, prevent or alleviate the symptoms of obesity or obesity-related disorders.

[0139] In various embodiments, when the compounds described herein are administered with one or more additional therapeutic agents or compounds, synergistic effects are observed. Synergistic effects can be observed, for example, in combination with sigmoid-E maxThe above-mentioned equations can be applied to experimental data to generate corresponding graphs to help evaluate the effect of drug combinations. The corresponding graphs related to the above-mentioned equations are concentration-effect curves, isobologram curves and combination index curves, respectively.

[0140] Administration / Dosage / Formulation Dosage regimen can affect what constitutes an effective amount.Therapeutic preparation can be administered to subject before or after the onset of obesity or obesity-related disorder.Furthermore, several divided doses and staggered doses can be administered daily or continuously, or the dose can be continuously infused or bolus injection.Furthermore, the dosage of therapeutic preparation can be increased or decreased proportionally as indicated by the exigencies of therapeutic or preventive situation.

[0141] Administration of the compositions described herein to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures at dosages and for periods of time effective to treat the patient's obesity or obesity-related disorder. The effective amount of the therapeutic compound required to achieve a therapeutic effect may vary depending on factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat the patient's obesity or obesity-related disorder. Dosage regimens may be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as dictated by the exigencies of the treatment situation. Non-limiting examples of effective dose ranges of the therapeutic compounds described herein are from about 1 to about 5,000 mg / kg body weight / day. One of skill in the art would be able to consider the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0142] Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied to provide an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration without toxicity to the patient.

[0143] In particular, the selected dosage level will depend on a variety of factors, including the activity of the particular compound used, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors known in the medical arts.

[0144] A medical practitioner, such as a physician or veterinarian, having ordinary skill in the art can easily determine and prescribe the effective amount of the pharmaceutical composition required.For example, the physician or veterinarian can start the dose of the compound described herein used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0145] In certain embodiments, it is particularly advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to a physically separate unit suitable as a unitary dosage for the patient to be treated; each unit contains a predetermined amount of therapeutic compound calculated to produce a desired therapeutic effect together with the necessary pharmaceutical vehicle.The dosage unit form of the compound(s) described herein is determined by and directly depends on (a) the inherent characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations of the technology of compounding / formulating such therapeutic compound.

[0146] In certain embodiments, the compositions described herein are formulated using one or more pharma- ceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions described herein comprise a therapeutically effective amount of the compounds described herein and a pharma- ceutically acceptable carrier.

[0147] The carrier may be a solvent or dispersion medium, for example, containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. Prevention of microbial action may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar, sodium chloride, or a polyalcohol such as mannitol and sorbitol, in the composition. Prolonged absorption of an injectable composition may be achieved by including an agent that delays absorption, for example, aluminum monostearate or gelatin, in the composition.

[0148] In certain embodiments, the compositions described herein are administered to patients in dosages ranging from once a day to five or more times a day. In other embodiments, the compositions described herein are administered to patients in dosages ranging from, but not limited to, once a day, once every two days, once every three days to once a week, and once every two weeks. It is readily apparent to those skilled in the art that the frequency of administration of the various combination compositions described herein varies from individual to individual depending on many factors, including but not limited to, age, disease or disorder being treated, sex, general health, and other factors. Thus, administration of the compounds and compositions described herein should not be construed as being limited to a particular dosing regimen, and the exact dosage and composition administered to a patient is determined by the attending physician, taking into account all other factors about the patient.

[0149] The compound(s) described herein for administration may be in the form of a compound or compounds in the form of a mixture of compounds in the form of a medicament, such as from about 1 μg to about 10,000 mg, from about 20 μg to about 9,500 mg, from about 40 μg to about 9,000 mg, from about 75 μg to about 8,500 mg, from about 150 μg to about 7,500 mg, from about 200 μg to about 7,000 mg, from about 350 μg to about 6,000 mg, from about 500 μg to about 5,000 mg, from about 750 μg to about 4,000 mg, from about 1 mg about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.

[0150] In some embodiments, the dose of the compounds described herein is about 1 mg to about 2,500 mg. In some embodiments, the dose of the compounds described herein used in the compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, the dose of the second compound described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0151] In certain embodiments, the compositions described herein are packaged pharmaceutical compositions comprising a container holding a therapeutically effective amount of a compound described herein, alone or in combination with a second agent, and instructions for using the compound to treat, prevent, or ameliorate one or more symptoms of a disease or disorder in a patient.

[0152] The formulations may be used in admixture with conventional excipients, i.e., pharma- ceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable administration method known in the art. Pharmaceutical preparations may be sterilized and may be mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, colorants, flavorings and / or aromatic substances, if desired. They may also be combined with other active agents, such as other analgesics, if desired.

[0153] The route of administration of any of the compositions described herein includes oral, nasal, rectal, vaginal, parenteral, buccal, sublingual or topical.The compounds for use in the compositions described herein can be formulated for administration by any suitable route, such as oral or parenteral, for example, transdermal, transmucosal (e.g. sublingual, lingual, (trans)buccal, (trans)urethral, ​​vaginal (e.g. vaginal and perivaginal), nasal (intra) and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration.

[0154] In certain aspects, the compositions described herein are applied to the skin of a subject using, for example, a patch, an adhesive membrane, a cohesive membrane, a lotion, a paste, etc. In some aspects, the compositions described herein are administered by injection, such as subcutaneous injection, intramuscular injection, or intravenous injection.

[0155] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gelcaps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosol formulations for inhalation, compositions and formulations for intravesical administration, etc. It should be understood that the formulations and compositions described herein are not limited to the specific formulations and compositions described herein.

[0156] Oral route For oral application, tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps are particularly suitable. Compositions intended for oral use can be prepared according to any method known in the art, and such compositions may contain one or more agents selected from the group consisting of inert and non-toxic pharmaceutical excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricants such as magnesium stearate. Tablets may be uncoated or may be coated by known techniques for elegance or to delay the release of the active ingredient. Preparations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert diluent.

[0157] For oral administration, the compound(s) described herein may be in the form of a tablet or capsule prepared by conventional means with pharma- ceutically acceptable excipients, such as binders (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., corn starch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). If desired, tablets may be coated using suitable methods and coating materials, such as the OPADRY™ film coating system available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY type, OYC type, organic enteric OY-P type, aqueous enteric OY-A type, OY-PM type, and OPADRY™ White, 32K18400). Liquid preparations for oral administration may be in the form of a solution, syrup, or suspension. Liquid preparations may be prepared by conventional means with pharma- ceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

[0158] Parenteral Administration For parenteral administration, the compounds described herein may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and / or continuous infusion. Suspensions, solutions or emulsions in oily or aqueous vehicles, optionally containing other formulating agents such as suspending agents, stabilizing agents and / or dispersing agents, may be used.

[0159] Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as, for example, solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharma- ceutical acceptable oils, such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as Swiss Pharmacopoeia or similar alcohols.

[0160] Further dosage forms Additional dosage forms suitable for use with the compound(s) and compositions described herein include those described in U.S. Patent Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms suitable for use with the compound(s) and compositions described herein also include those described in U.S. Patent Application Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms suitable for use with the compound(s) and compositions described herein include those described in PCT Application Publication Nos. WO 03 / 35041; WO 03 / 35040; WO 03 / 35029; WO 03 / 35177; WO 03 / 35039; WO 02 / 96404; WO 02 / 32416; WO 01 / 97783; WO 01 / 56544; WO 01 / 32217; WO 98 / 55107; WO 98 / 11879; WO 97 / 47285; WO 93 / 18755; and WO 90 / 11757.

[0161] Sustained Release Formulations and Drug Delivery Systems In certain aspects, the formulations described herein can be, but are not limited to, short-term formulations, fast-eliminating formulations, and sustained release formulations, including sustained release, delayed release, and pulsatile release formulations.

[0162] The term sustained release is used in its conventional sense to refer to a drug formulation that provides gradual release of drug over an extended period of time, resulting in substantially constant blood levels of drug over an extended period of time, which may be, but is not necessarily, a month or longer, and should be a longer release than the same amount of agent administered in bolus form.

[0163] In the case of sustained release, the compound can be formulated with suitable polymer or hydrophobic material that provides the compound with sustained release properties.Therefore, the compound for use in the method(s) described herein can be administered in the form of microparticles, for example, by injection, or in the form of wafer or disk by implantation.

[0164] In some cases, the dosage form used can be provided as a sustained or controlled release of one or more active ingredients therein, for example, by using hydropropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multi-layer coatings, microparticles, liposomes, or microspheres, or combinations thereof that provide desired release profile in various ratios.Suitable sustained release formulations known to those skilled in the art, including those described herein, can be easily selected for use with the pharmaceutical compositions described herein.Therefore, the single unit dosage forms suitable for oral administration, such as tablets, capsules, gel caps, and caplets, that are adapted for sustained release, are included in the compositions and dosage forms described herein.

[0165] Most sustained release pharmaceutical products have a common goal of improving drug therapy over that achieved by non-controlled pharmaceutical products. Ideally, the use of optimally designed sustained release formulations in medicine is characterized by the use of a minimum of drug substance to cure or control a condition in a minimum amount of time. Advantages of sustained release formulations include extended activity of the drug, reduced dosing frequency, and improved patient compliance. In addition, sustained release formulations can be used to affect other characteristics such as the time of onset of action or blood levels of the drug, and therefore the occurrence of side effects.

[0166] Most sustained release formulations are designed to initially release an amount of drug that rapidly produces the desired therapeutic effect, and then slowly and continuously release other amounts of drug to maintain this level of therapeutic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body.

[0167] The sustained release of active ingredients can be stimulated by various inducers, such as pH, temperature, enzymes, water, or other physiological conditions or compounds. The term "sustained release component" is defined herein as one or more compounds, including but not limited to polymers, polymer matrices, gels, permeable membranes, liposomes, or microspheres, or combinations thereof, that facilitate sustained release of active ingredients. In one embodiment, the compound(s) described herein are administered to a patient using sustained release formulations, alone or in combination with another agent. In one embodiment, the compound(s) described herein are administered to a patient using sustained release formulations, alone or in combination with another agent.

[0168] The term delayed release is used herein in its conventional sense to refer to drug formulations that provide an initial release of drug after some delay following drug administration, including, but not necessarily, a delay of from about 10 minutes to about 12 hours.

[0169] The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of drug in a manner that results in a pulsatile plasma profile of the drug following drug administration.

[0170] The term immediate release is used in its conventional sense to refer to a drug formulation that provides release of the drug immediately following drug administration.

[0171] As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration, and any or all full or partial increments thereof.

[0172] As used herein, rapid clearance refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration, and any and all whole or partial increments thereof.

[0173] dosage The therapeutically effective amount or dose of the compounds described herein will depend on the age, sex and weight of the patient, the patient's current medical condition, and the progression of obesity or obesity-related disorders in the patient being treated. Those skilled in the art will be able to determine appropriate dosages depending on these and other factors.

[0174] Suitable doses of the compounds described herein may range from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, such as from about 1 mg to about 500 mg, such as from about 5 mg to about 250 mg per day. The dose may be administered in a single dose or multiple doses, such as from one to four or more times per day. When multiple doses are used, the amount of each dose may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses with a dosing interval of about 12 hours.

[0175] In various embodiments, a therapeutic amount of a vitamin D3 analog is administered per cm of treated skin. 2 The dose of vitamin D3 analog is about 0.05 nmol / cm to about 20 nmol / cm. 2 ~about 10nmol / cm 2 , 0.1 nmol / cm 2 ~about 5nmol / cm 2 , or 0.1 nmol / cm 2 ~about 2.5nmol / cm 2 The dose of the vitamin D3 analog may be about 0.01 nmol / cm, where the area corresponds to the surface area of ​​the skin to be treated. 2 , about 0.05nmol / cm 2 , about 0.1nmol / cm 2 , about 0.2nmol / cm 2 , about 0.4nmol / cm2 , about 0.6nmol / cm 2 , about 0.8nmol / cm 2 , about 1nmol / cm 2 , about 1.5nmol / cm 2 , about 2nmol / cm 2 , about 3nmol / cm 2 , about 4nmol / cm 2 , about 5nmol / cm 2 , about 6nmol / cm 2 , about 7nmol / cm 2 , about 8nmol / cm 2 , about 9nmol / cm 2 , about 10nmol / cm 2 , about 12nmol / cm 2 , about 14nmol / cm 2 , about 16nmol / cm 2 , about 18nmol / cm 2 , or about 20 nmol / cm 2 , where the area corresponds to the surface area of ​​the skin to be treated.

[0176] The vitamin D3 analog can be applied to any particular skin surface area depending on the needs of the subject. In various embodiments, the vitamin D3 analog is applied to an area of ​​5 cm 2 ~About 2500cm 2 , about 10cm 2 ~about 2000cm 2 , about 20cm 2 ~Approx. 1500cm 2 , about 30cm 2 ~About 1000cm 2 , 40cm 2 ~Approx. 750cm 2 , 50cm 2 ~about 700cm 2 , 60cm 2 ~Approx. 650cm 2 , about 70cm 2 ~about 600cm 2 , 80cm 2 ~Approx. 550cm 2 , 90cm 2 ~about 500cm 2 , or 100cm 2 ~about 450cm 2It can be applied to the skin area within the range. Vitamin D3 analogs, in various aspects, are about 5 cm 2 , about 10 cm 2 , about 15 cm 2 , about 20 cm 2 , about 25 cm 2 , about 30 cm 2 , about 35 cm 2 , about 40 cm 2 , about 45 cm 2 , about 50 cm 2 , about 55 cm 2 , about 60 cm 2 , about 65 cm 2 , about 70 cm 2 , about 75 cm 2 , about 80 cm 2 , about 85 cm 2 , about 90 cm 2 , about 95 cm 2 , about 100 cm 2 , about 110 cm 2 , about 120 cm 2 , about 130 cm 2 , about 140 cm 2 , about 150 cm 2 , about 160 cm 2 , about 170 cm 2 , about 180 cm 2 , about 190 cm 2 , about 200 cm 2 , about 225 cm 2 , about 250 cm 2 , about 275 cm 2 , about 300 cm 2 , about 350 cm 2 , about 400 cm 2 , about 450 cm 2 , 500 cm 2 , about 550 cm 2 , about 600 cm 2 , about 650 cm 2 , about 700 cm 2 , about 750 cm 2 , about 800 cm 2 , about 850 cm 2 , about 900 cm 2 , about 950 cm 2 , about 1000 cm 2 , about 1250 cm 2, about 1500cm 2 , about 1750cm 2 , about 2000cm 2 , about 2250cm 2 , or about 2500 cm 2 can be applied to any skin area.

[0177] The amount of vitamin D3 analog applied to a given skin area can be about 0.005% to about 10% by weight, about 0.01% to about 5% by weight, about 0.05% to about 5% by weight, about 0.1% to about 5% by weight, or about 0.5% to about 5% by weight for any pharmaceutical composition of D3 analog described herein. Thus, for example, a topical pharmaceutical composition of a vitamin D3 analog can include 0.005% to about 10% by weight of vitamin D3 analog, with the remainder being pharma- ceutical acceptable carriers and / or excipients. In various embodiments, the amount of vitamin D3 analog applied to a given skin area can be about 0.005%, about 0.01%, about 0.05%, about 0.1%, about 0.%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.2%, about 1.4%, about 1.6%, about 1.8%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight for any pharmaceutical composition of D3 analog described herein.

[0178] The amount of a TSLP polypeptide isoform or viral vector containing a TSLP expression sequence and effective to achieve the weight loss described herein is about 0.1 mg / kg to about 500 mg / kg, about 0.5 mg / kg to about 400 mg / kg, about 1 mg / kg to about 300 mg / kg, about 5 mg / kg to about 200 mg / kg, or about 10 mg / kg to about 100 mg / kg. Effective amounts of TSLP polypeptide isoforms or viral vectors comprising a TSLP expression sequence for achieving weight loss as described herein, in various embodiments, include at least about 0.1 mg / kg, at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 3 mg / kg, at least about 4 mg / kg, at least about 5 mg / kg, at least about 0.1 mg / kg, at least about 6 mg / kg, at least about 7 mg / kg, at least about 8 mg / kg, at least about 9 mg / kg, at least about 10 mg / kg, at least about 15 mg / kg, at least about 20 mg / kg, at least about 40 mg / kg, at least about 60 mg / kg, at least about 80 mg / kg, at least about 100 mg / kg, at least about 150 mg / kg, at least about 200 mg / kg, at least about 250 mg / kg, at least about 300 mg / kg, at least about 350 mg / kg, g, at least about 400 mg / kg, at least about 450 mg / kg, or at least about 500 mg / kg, greater than about 0.1 mg / kg, greater than about 0.5 mg / kg, greater than about 1 mg / kg, greater than about 2 mg / kg, greater than about 3 mg / kg, greater than about 4 mg / kg, greater than about 5 mg / kg, greater than about 0.1 mg / kg, greater than about 6 mg / kg, greater than about 7 mg / kg, greater than about 8 mg / kg, greater than about 9 mg / kg, greater than about 10 mg / kg, greater than about 15 mg / kg, greater than about 20 mg / kg, or greater than about 40 mg / kg , greater than about 60 mg / kg, greater than about 80 mg / kg, greater than about 100 mg / kg, greater than about 150 mg / kg, greater than about 200 mg / kg, greater than about 250 mg / kg, greater than about 300 mg / kg, greater than about 350 mg / kg, greater than about 400 mg / kg, greater than about 450 mg / kg, or greater than about 500 mg / kg, or less than about 0.1 mg / kg, less than about 0.5 mg / kg, less than about 1 mg / kg, less than about 2 mg / kg, less than about 3 mg / kg, less than about 4 mg / kg, less than about 5 mg / kg, less than about 0.Less than 1 mg / kg, less than about 6 mg / kg, less than about 7 mg / kg, less than about 8 mg / kg, less than about 9 mg / kg, less than about 10 mg / kg, less than about 15 mg / kg, less than about 20 mg / kg, less than about 40 mg / kg, less than about 60 mg / kg, less than about 80 mg / kg, less than about 100 mg / kg, less than about 150 mg / kg, less than about 200 mg / kg, less than about 250 mg / kg, less than about 300 mg / kg, less than about 350 mg / kg, less than about 400 mg / kg, less than about 450 mg / kg, or less than about 500 mg / kg. In various embodiments, the amount of TSLP polypeptide isoform or viral vector comprising a TSLP expression sequence to achieve weight loss as described herein is an amount that results in a circulating TSLP level in a subject of about 5 to about 40 ng / mL.

[0179] It is understood that the amount of compound administered per day can be, in non-limiting examples, administered every day, every other day, every 2nd day, every 3rd day, every 4th day, or every 5th day. For example, every other day administration can be starting with a 5mg / day dose on Monday, administering a 5mg / day dose on Wednesday as the first subsequent dose, administering a 5mg / day dose on Friday as the second subsequent dose, etc.

[0180] If the patient's condition improves, at the discretion of the physician, administration of the compound(s) described herein is optionally continued; alternatively, the dose of the administered drug is temporarily reduced or temporarily suspended for a certain period of time (i.e., a "drug holiday"). The length of the drug holiday optionally varies between 2 days to 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Dose reductions during drug holidays include 10% to 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0181] In various embodiments, the vitamin D3 analog is administered using a dosing schedule that has a treatment week followed by a non-treatment week. During the non-treatment week, the vitamin D3 analog is not administered. During the treatment week, the vitamin D3 analog can be administered every day (7 days of treatment) or every other day (3 or 4 days of treatment).

[0182] Once the patient's condition has improved, a maintenance dose is administered as needed.Then, the dosage or frequency of administration, or both, is reduced to a level at which the improved disease persists.In certain embodiments, patients require intermittent treatment on a long-term basis upon recurrence of symptoms and / or infection.

[0183] The compounds described herein can be formulated in unit dosage form. The term "unit dosage form" refers to a physically discrete unit suitable as a unitary dosage for a patient to be treated, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect, optionally together with a suitable pharmaceutical carrier. The unit dosage form can be for a single daily dose or for one of multiple daily doses (e.g., about 1-4 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose.

[0184] The toxicity and therapeutic efficacy of such treatment regimens may optionally be evaluated using LD 50 (a dose lethal to 50% of the population) and ED 50 These methods include, but are not limited to, determining the dose that is therapeutically effective in 50% of a population in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 and ED 50 The data obtained from cell culture assays and animal studies are optionally used to formulate a range of dosages for use in humans. The dosage of such compounds is preferably administered at a dose that is consistent with the ED 50 The dosage amount optionally varies within this range depending upon the dosage form employed and the route of administration utilized. EXAMPLES

[0185] Various aspects of the present application can be better understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the scope of the present application to the examples provided herein.

[0186] Example 1: MC903 prevents weight gain and improves metabolic parameters in HFD-fed mice. Wild-type C57BL / 6 mice were fed normal chow (NC) or 40% high fat diet (HFD) for 12 weeks. Mice were treated topically in both ears with either vehicle (EtOH) or MC903 (2 nmol / ear) on Mondays, Wednesdays, and Fridays of odd-numbered weeks. (A) Mice were weighed weekly and % change from baseline is plotted as mean ± SEM. (B) Mice were euthanized at week 12 and epididymal fat pads were weighed. (C, D) Glucose tolerance test (GTT) was performed at week 12 by injection of glucose and subsequent measurement of blood glucose levels. After overnight fasting at week 12, homeostasis model assessment-insulin resistance (HOMA-IR) analysis was performed. * , ** , and *** indicates statistical significance of p<0.05, p<0.01, and p<0.001 by Student's t test or ANOVA, respectively.

[0187] Example 2. MC903 prevents HFD-induced hepatic steatosis. C57BL / 6 mice were fed normal chow (NC) or 40% high fat diet (HFD) for 12 weeks. Mice were treated topically in both ears with either vehicle (EtOH) or MC903 (2 nmol / ear) on Mondays, Wednesdays, and Fridays of odd-numbered weeks. Representative histology of liver at 12 weeks is shown.

[0188] Example 3. MC903 does not prevent weight gain or improve metabolic parameters in HFD-fed TSLP-R KO mice. TSLP-R knockout (KO) mice were fed normal chow (NC) or 40% high fat diet (HFD) for 12 weeks. Mice were treated topically in both ears with either vehicle (EtOH) or MC903 (2 nmol / ear) on Mondays, Wednesdays, and Fridays of odd-numbered weeks. Mice were weighed at the indicated time points and plotted as mean ± SEM. Glucose tolerance test (GTT) was performed at week 12 by injection of glucose followed by measurement of blood glucose levels. AUC analysis is performed on the GTT curves. * indicates statistical significance of p<0.05 by Student's t test.

[0189] Example 4. AAV8-TSLP induces weight loss in HFD-fed mice. C57BL / 6 mice were fed a 40% high fat diet (HFD) for 4 weeks. Mice were injected intravenously with either AAV8 control (control AAV) or AAV8-TSLP (mTSLP-AAV) on day 0. Mice were weighed weekly and the % change from baseline is plotted as the mean ± SEM. ** and *** indicates statistical significance of p<0.01 and p<0.001, respectively.

[0190] Example 5. AAV8-TSLP induces weight loss in previously obese HFD-fed mice. C57BL / 6 mice were fed a 40% high fat diet (HFD) for 10 weeks. Mice were then injected intravenously with either AAV8 control or AAV8-TSLP at week 10. Mice were maintained on a HFD, weighed weekly, and the % change from baseline is plotted as the mean ± SEM. * , ** , and *** indicates statistical significance of p<0.05, p<0.01, and p<0.001 by Student's t test or ANOVA, respectively.

[0191] Example 6. AAV8-TSLP induces subcutaneous fat loss. C57BL / 6 mice were fed a 40% high fat diet (HFD) for 10 weeks. Mice were then intravenously injected with either AAV8 control or AAV8-TSLP at week 10. Mice were maintained on a HFD for 6 weeks and euthanized for histological analysis. Representative histological images of mouse skin are shown.

[0192] Example 7. AAV8-TSLP induces weight loss in Ob / Ob mice. Ob / Ob mice were injected with either AAV8 control or AAV8-TSLP and fed regular chow for 6 weeks. Mice were weighed weekly and the % change from baseline is plotted as the mean ± SEM. * and *** indicates statistical significance of p<0.05 and p<0.001, respectively, by Student's t test.

[0193] Example 8. AAV8-TSLP induces selective adipose tissue loss in mice fed a normal chow diet. C57BL / 6 mice were injected intravenously with either AAV8 control or AAV8-TSLP and fed regular chow for 14 days. (A) Mice were weighed on the indicated days and the % change from baseline is plotted as the mean ± SEM. Mice were euthanized on the indicated days and (B) epididymal white adipose tissue (eWAT), (C) inguinal white adipose tissue (iWAT), (D) brown adipose tissue (BAT), and (E) quadriceps muscle weights were measured. ** and *** indicates statistical significance of p<0.01 and p<0.001, respectively, by Student's t test.

[0194] Example 9. AAV8-TSLP does not alter intestinal feeding. C57BL / 6 mice were injected intravenously with either AAV8 control or AAV8-TSLP and fed regular chow or HFD for 12 weeks. (A) Average weekly food intake is plotted as mean ± SEM. Ten days after AAV8 injection, mice were gavaged with a fixed amount of (B) glucose or (C) olive oil. Subsequent glucose and triglyceride levels were measured and plotted over time.

[0195] Example 10. Mice treated with AAV8-TSLP excrete less fecal energy compared to mice treated with the AAV8 control. C57BL / 6 mice were intravenously injected with either AAV8 control or AAV8-TSLP and fed normal chow for 9 days. Feces were collected and food intake was measured in individually caged mice from days 9 to 11. Collected feces were subjected to bomb calorimetry to measure fecal calories. (A) Daily fecal energy content of AAV8 control or AAV8-TSLP treated mice. (B) Daily net energy intake, calculated by food calorie intake minus fecal calories, of AAV8 control or AAV8-TSLP treated mice is shown. Statistical analysis was performed by Student's t-test.

[0196] Example 11. AAV8-TSLP increases food intake 7-10 days after injection. C57BL / 6 mice were injected intravenously with either AAV8 control or AAV8-TSLP and placed into metabolic chambers on days 7–10. Hourly and cumulative food intake is plotted over the final 48 h.

[0197] Example 12. AAV8-TSLP does not increase oxygen consumption 7-10 days after injection. C57BL / 6 mice were injected intravenously with either AAV8 control or AAV8-TSLP and placed in metabolic chambers on days 7–10. Hourly (left plots) and daily / half-day (right plots) oxygen consumption are plotted over the final 48 and 72 h, respectively.

[0198] Example 13. AAV8-TSLP does not increase carbon dioxide output 7-10 days after injection. WT C57BL / 6 mice were injected intravenously with either AAV8 control or AAV8-TSLP and placed in metabolic chambers on days 7–10. Hourly (left plots) and daily / half-daily (right plots) carbon dioxide excretion are plotted over the final 48 and 72 h, respectively.

[0199] Example 14. AAV8-TSLP does not increase locomotor activity 7-10 days after injection. WT C57BL / 6 mice were injected intravenously with either AAV8 control or AAV8-TSLP and placed in metabolic chambers on days 7–10. Hourly (left plots) and daily / half-day (right plots) locomotor activity are plotted over the final 48 and 72 h, respectively.

[0200] Example 15. AAV8-TSLP produces oily coat in previously obese HFD-fed mice. C57BL / 6 mice were fed a 40% high fat diet (HFD) for 10 weeks. Then, at week 10, mice were intravenously injected with AAV8-TSLP. Mice were kept on HFD for an additional 6 weeks. Representative photographs of mice 6 weeks after AAV8 injection are shown.

[0201] Example 16. AAV8-TSLP induces weight loss in HFD-fed mice with a TSLP-responsive hematopoietic system. C57BL6 or TSLP-R KO mice were irradiated with 1000 cGy and injected intravenously with bone marrow cells from C57BL / 6 or TSLP-R knockout (KO) mice. Four weeks after bone marrow injection, mice were injected intravenously with either AAV8 control or AAV8-TSLP and fed a 40% high fat diet (HFD) for 3 weeks. Mice were weighed weekly and the % change from baseline is plotted as the mean ± SEM.

[0202] Example 17. AAV8-TSLP induces weight loss in a T cell-dependent manner. (A) Recombinase activating gene 1 (RAG1) KO mice and (B) T cell receptor (TCR)-β KO mice were injected with either AAV8 control or AAV8-TSLP. RAG1 KO mice lack T and B cells. TCR-β KO mice lack only T cells. Epididymal white adipose tissue (eWAT) weight was measured 14 days after injection. Data are plotted as mean ± SEM.

[0203] Example 18. Non-limiting model of TSLP-induced adipose tissue loss. Systemic TSLP elevation can be achieved by topical treatment with MC903, injection of recombinant TSLP, or gene therapy using viral vectors. Elevated systemic TSLP levels lead to lipid secretion in the skin, which increases the organism's fat energy demand. This triggers the release of fat stores (lipolysis) in adipose and other fat-containing tissues. This ultimately leads to weight loss and reversal of obesity. This strategy is therefore beneficial for the treatment of obesity and obesity-related disorders such as nonalcoholic steatohepatitis (NASH) and type 2 diabetes (T2D).

[0204] Example 19: Thymic stromal lymphopoietin-1 prevents obesity via sebum hypersecretion. The current results show that thymic stromal lymphopoietin (TSLP), a type II cytokine that activates ILC2s and eosinophils, induces selective white fat loss and prevents obesity and associated complications, including type II diabetes and nonalcoholic steatohepatitis (NASH). However, surprisingly, the induction of fat loss was dependent on T cells, not ILC2s or eosinophils. Furthermore, fat loss was not secondary to increased thermogenesis and energy expenditure, but rather related to sebum hypersecretion. Inhibition of sebum secretion or depletion of T cells prevented TSLP-induced fat loss. In addition to the pharmacological effects of TSLP, the current data revealed a homeostatic role for TSLP and T cells in regulating sebum secretion. Taken together, this study demonstrates that fat loss can be achieved by sebum hypersecretion and reveals a previously unknown role for TSLP and T cells in controlling sebum release.

[0205] method Mice. C57BL / 6 and B6-LY5.1 / Cr mice were purchased from Charles River Laboratories (stock numbers 556 and 564). ob / ob, SCD1 KO, RAG / IL2Ri DKO, ROSA-DTA (ROSA-Stop-flox-DTA), and CD11cCre mice were purchased from Jackson Laboratories (stock numbers 000632, 005956, 014593, 009669, and 008068). EoCre mice were generated as previously described (Doyle, et al., 2013, Leukoc Biol 94: 17-24). CD11cCre mice were crossed with ROSA-DTA mice to generate DC-deficient mice, and EoCre mice were crossed with ROSA-DTA mice to generate eosinophil-deficient mice. TSLPR KO mice were a gift from Warren Leonard (NIH) and were bred and maintained elsewhere, as previously described (Al-Shami, et al., 2004, J Exp Med 200: 159-168). E-β KO, RAG2 KO (RAG KO), FOXP3-GFP, and FOXP3-DTR mice were also bred and maintained in the same facility. CD4 + and CD8 +For T cell depletion, antibodies against CD4 (BioXCell, BE0003-1 clone GK1.5) and CD8 (BioXCell, BE006, clone 2.43) were administered by intraperitoneal injection at a dose of 200 μg / mouse on days 0, 2, 4, 8, and 12, or 14, 16, 18, 22, and 26 after AAV injection. FOXP3-GFP and FOXP3-DTR mice were administered diphtheria toxin (Santa Cruz Biotechnology, sc-391135) intraperitoneally at a dose of 10 μg / kg body weight on days -2, 0, 2, 4, 6, 8, 10, and 12 after AAV injection. In all NC-fed mice experiments, mice were harvested 2 weeks after AAV injection to measure the volume of eWAT (bilateral), iWAT (bilateral), BAT (bilateral), and quadriceps (left quadriceps). ob / ob mice received AAV injections for 5 weeks starting at the age of 12 weeks. Unless otherwise stated, all mice were male, 8–10 weeks old at the time of use, and were housed under pathogen-free conditions.

[0206] AAV injections. Control AAV (AAV8.TBG.PI.Null.bGH) and TSLP-AAV (AAV8.TBG.PI.mTSLP.IRES.eGFP.WPRE.bGH) were generated by the Penn Vector Core. Mice were injected with 3 × 10 of AAV, corresponding to a serum TSLP level of 40 ng / mL. 10 genome copies were injected intravenously.

[0207] BM chimeras. Femurs and hip bones were isolated from donor mice and crushed with a mortar and pestle to obtain bone marrow. Red blood cells were lysed with ACK lysis buffer (ThermoFisher, A1049201), and bone marrow was filtered through a 70 μm filter (Sigma, CLS431751). Host mice were irradiated with 1,000 cGy and 2 × 10 6 donor bone marrow cells were injected intravenously. Four weeks later, BM chimeras were injected with AAV.

[0208] Adoptive transfer. WT or TSLPR KO splenic T cells were isolated using a T cell negative selection kit (STEMCELL Technologies, 19851) and then purified using a FACSAria cell sorter (BD Biosciences) to screen for CD19 - / B220 - / NK1.1 - / CD11c - / CD11b - and CD90.2 + / CD4 + or CD8 + Cells were sorted. 2 × 10 6 Sorted cells were transferred intravenously into RAG KO or TSLPR KO mice. Four weeks later, the adoptively transferred mice were injected with AAV.

[0209] HFD and MCDD models. Mice were fed either a HFD consisting of 45 kcal% fat (Research Diets, D12451) or a choline-free MCDD consisting of 60 kcal% fat and 0.1% methionine (Research Diets, A06071302). For the HFD model, mice were injected with AAV on day 0 or injected with AAV after 10 weeks of HFD feeding and continued on HFD for an additional 4 weeks (HFD for 14 weeks, AAV injected at week 10). Mice were weighed weekly. For the MCDD model, mice were injected with AAV after 4 weeks of diet and continued on MCDD for an additional 4 weeks (MCDD for 8 weeks, AAV injected at week 4).

[0210] In vivo metabolic analysis. Mice were individually housed and monitored for 9–11 days after AAV injection using an OxyMax Comprehensive Laboratory Animal Monitoring System (CLAMS) by the University of Pennsylvania's Rodent Metabolic Phenotyping Core. Mice were fed NC and maintained on a standard 12:12 light:dark cycle at 24 °C with food and water available ad libitum. Accumulated feces were collected from individually housed mice on days 9–11 after AAV injection, and fecal bomb calorimetry was performed by the University of Michigan Mouse Phenotyping Core. Fat and lean mass were measured on days 0 and 14 after AAV injection. 1Blood glucose levels were measured by H-NMR spectroscopy. For GTT, mice were fasted overnight for 14–16 h and then injected intraperitoneally with 10 μl / g body weight of 20% w / v dextrose solution. Blood glucose levels were measured from blood taken from the tail vein at 0, 15, 30, 60, 90, and 120 min after dextrose challenge. The 0 min reading was used to determine fasting blood glucose and insulin levels. Blood glucose measurements were performed using a handheld blood glucose meter (Contour, 7151G), and plasma insulin levels were determined by ELISA (Crystal Chem, 90080). HOMA-IR index values ​​were calculated as previously described (Matthews, et al., 1985, Diabetologia 28:412-419). OGTT was performed in a similar manner to GTT, except that dextrose solution was administered by oral gavage. OFTT was performed as previously described (Wada, et al., 2013, Gastroenterology 144:369-380), and blood triglyceride levels were measured by the Infinity Triglyceride kit (ThermoFisher, TR22421). Serum ALT assay was performed by the Translational Core Laboratory at the Children's Hospital of Philadelphia (CHOP) Research Institute. For urinalysis, urine was collected from mice 10 days after AAV injection, and 100 μl of urine was loaded onto urine test strips (Ketostix, 2881; Chemstrip 2GP, 11895397) for colorimetric analysis.

[0211] Body condition scoring. BCS scoring (Cooke, et al., 1996, Blood 88: For the 3230-3239), mice were scored on days 0, 3, 7, 10, and 14 after AAV injection with a grade of 0–2 in five categories as follows: 1) weight loss: 2 points if >25%, 1 point if 10–25%, 0 point if <10%, 2) hunching: 2 points if severe hunching impairing movement, 1 point if hunching at rest, 0 point if normal posture, 3) visible lethargy: 2 points if still unless stimulated, 1 point if mild to moderately reduced movement, 0 point if normal, 4) lethargy to touch: 2 points if still when stimulated, 1 point if mild to moderately reduced movement when stimulated, 1 point if normal, 5) disheveled coat: 2 points if severely disheveled and poorly groomed, 1 point if mild to moderately disheveled, 0 point if normal. Scores from each category were summed to calculate a total score.

[0212] Liver TG. 20–100 mg of liver tissue was homogenized in 10 μl / mg liver of 5% NP-40 (Igepal CA630, Sigma-Aldrich, I8896). The resulting homogenate was boiled at 80 °C for 10 min, cooled to room temperature, boiled again at 80 °C for 10 min, and centrifuged. The supernatant was then diluted 1:20 in dH2O. Triglyceride levels were quantified using Infinity Triglyceride Reagent.

[0213] Lipid isolation and TLC of hair. A 2.5 cm × 5 cm area of ​​hair was shaved from the back skin and immersed in 4 mL of chloroform / methanol (2:1 v / v) followed by 4 mL of acetone. The lipid extracts were pooled, syringe filtered, dried overnight under a stream of N2 gas, and resuspended in an equal volume of chloroform (Sigma, 288306) / methanol (Sigma, 322415) (4:1, v / v) for loading onto a TLC plate (Sigma, 100390). The TLC plate was developed three times using: 1) hexane (Sigma, 296090):isopropyl diether (Sigma, 673803):acetic acid (80:20:1) to 50% of the plate height, 2) hexane:benzene (Sigma, 401765) (1:1) to 80% of the plate height, and 3) hexane to 90% of the plate height. The plate was allowed to dry between each development solution. Lipid species were visualized by spraying the plates evenly with 10% copper sulfate (Sigma, 451657) / 8% phosphoric acid (Sigma, P6560) solution and then baking at 120 °C for 20 min. Band intensity and area were quantified using ImageJ (NIH). TLC non-polar lipid mixture A (Matreya, 1129) was used as a standard to identify lipid classes.

[0214] Flow cytometry analysis. Ear skin skin sheets were separated, minced, and incubated in Hanks Balanced Salt Solution (ThermoFisher, 24020117) containing 0.25 mg / ml Liberase TL (Roche, 5401020001), 0.1 mg / ml DNase I (Roche, 10104159001), and 0.7 mg / ml Collagenase D (Roche, 11088882001) for 2 hours with shaking at 37°C. The contents were then strained through a 70 μm filter into a new tube containing 10 ml PBS, centrifuged, and resuspended in PBS. Cells were stained with live / dead and cell surface stains for 15 minutes in PBS at 4°C. Flow cytometry was performed using an LSR II or LSR Fortessa (BD Biosciences). Data were analyzed using FlowJo software (TreeStar). Staining antibodies used included CD4 (BioLegend, 100406 and 100510), CD8α (BioLegend, 100753), CD90.2 (BioLegend, 140322), TCRb (BioLegend, 109241), CD19 (BioLegend, 115520), B220 (BioLegend, 103222), NK1.1 (BioLegend, 108714), CD3 (BioLegend, 100218), CD45.1 (BioLegend, 110739), CD45.2 (eBioscience, 56-0454-82), CD11b (BioLegend, 101215), and CD11c (BioLegend, 117318). Dead cells were excluded using live / dead near-infrared staining (ThermoFisher, L10119).

[0215] Histology. Tissues were fixed in 10% formalin overnight at 4°C and embedded in paraffin prior to H&E or IHC staining. Liver sections were processed and stained by the University of Pennsylvania Cardiovascular Institute's Histology and Gene Expression Core. Picrosirius red staining was performed as previously described (Jeong, et al., 2018, J Clin Invest 128:1010-1025). Skin samples were processed and stained (IHC for H&E, Ki67, ORO, CD3, CD4, and CD8) by the University of Pennsylvania's Skin Histology and Characterization Core. For ORO staining of skin samples, frozen skin samples were embedded in cryosection medium prior to sectioning and staining. For quantification of sebocyte size, ORO lipid content, and fibrosis for picrosirius red, 8–10 sections were captured at 20x magnification per animal. For H&E skin sections, ImageJ was used to draw circumscribing ellipses around sebaceous cells and quantitate the area and intensity of staining. For ORO skin and picrosirius red liver sections, ImageJ was used to split RGB channels, threshold positive staining, and measure % area and intensity. Integrated density was calculated as area × intensity. For quantification of Ki67 sebaceous basal cell staining, 8–10 sections per animal were imaged at 40x magnification and Ki67 staining was determined for each sebaceous gland. + and Ki67 - Basal cells were counted.

[0216] Human gene expression analysis. Publicly available human skin gene expression data was obtained from www dot ncbi dot nlm dot nih dot gov / geo / query / acc dot cgi?acc=GSE98774. Gene expression values ​​for sebum production-related genes (SCD, FADS2, PPARg, FA2H, DGAT1, DGAT2, FABP4, FABP5, ACACA, FASN, AWAT1, ELOVL1, ELOVL3, ELOVL4, ELOVL5, MOGAT1, MOGAT2, MOGAT3) were plotted against the corresponding TSLP expression values ​​for each sample. For normalized SG gene expression, the expression of each of the sebum-related gene datasets was normalized to a mean of 0 and a standard deviation of 1, and then all SG genes from each sample were averaged together and plotted against the corresponding TSLP expression. Pearson's r and Pearson linear regression analyses were performed using Prism (GraphPad Sofware, Inc.).

[0217] Statistical analysis. Data are reported as mean ± sem. All measurements were performed from separate biological samples. For normally distributed data, statistical significance was determined by Student's t-test (two-tailed) or two-way analysis of variance with Sidak's post-hoc test with correction for multiple comparisons. Correlation analysis was performed using Pearson linear regression. Statistical analysis was performed using Prism 8.

[0218] result Selected results are presented herein.

[0219] TSLP protects against obesity and obesity-related complications The potential effect of TSLP on obesity was tested by injecting TSLP-expressing adeno-associated virus serotype 8 (TSLP-AAV, expression targeted to the liver) to induce systemic expression of TSLP in mice fed a high-fat diet (HFD). Strikingly, HFD-fed mice injected with TSLP-AAV lost weight over a 4-week period compared to control AAV-injected mice, which gained weight (Figures 29A, 33A). TSLP-AAV injection also caused weight loss in hyperphagic ob / ob mice fed a normal chow diet (NC) (Figures 33B-33C). TSLP not only prevented but also reversed obesity. Obese mice injected with TSLP-AAV (initially fed a HFD for 10 weeks and then injected with TSLP-AAV while continuing the HFD) showed weight loss, reduced visceral fat mass, and marked improvements in metabolic parameters including fasting glucose and insulin levels, homeostasis model assessment of insulin resistance (HOMA-IR), glucose tolerance, hepatic steatosis, and hepatic triglyceride (TG) levels (Figures 29B-29D, 33D-33J).

[0220] Considering the reduction of hepatic steatosis, the effect of TSLP was also tested in mouse models of nonalcoholic fatty liver disease (NAFLD) and NASH. To this end, mice were fed a methionine-choline deficient diet (MCDD) for 4 weeks and then injected with either control AAV or TSLP-AAV for an additional 4 weeks. MCDD-fed mice injected with TSLP-AAV had lower hepatic TG levels, lower serum alanine aminotransferase levels (ALT), and reduced liver fibrosis compared to controls (Figures 29E, 34A-33D).

[0221] The effect of TSLP on fat loss was also observed in NC-fed mice. Two weeks after TSLP-AAV injection, NC-fed mice exhibited significantly reduced epididymal white adipose tissue mass (eWAT, visceral fat) and inguinal white adipose tissue mass (iWAT, subcutaneous fat) without a corresponding reduction in brown adipose tissue (BAT) or muscle mass (Figures 29F, 34E-34H). Body composition analysis also showed that TSLP-AAV reduced the total body fat mass percentage (Figure 34I). Thus, TSLP-AAV-injected mice are not cachectic per se because white fat was selectively reduced. Furthermore, body conditioning scoring (BCS) of TSLP-AAV-injected mice remained normal, and the mice lost only 5% of their total body weight, despite losing a significant amount of white fat, indicating that TSLP-AAV-injected mice are not clinically diseased (Figures 34J-34K). Finally, the effect of TSLP on white fat loss depends on signaling through the TSLP receptor (TSLPR), as TSLPR KO mice did not lose fat upon TSLP-AAV injection (Figure 34L).

[0222] T cells mediate TSLP-induced fat loss To determine which cell type(s) are involved in mediating white fat loss by TSLP, we first tested whether TSLP causes fat loss via TSLPR signaling in hematopoietic (radiosensitive) or non-hematopoietic (radioresistant) cells. Three types of bone marrow (BM) irradiated chimeric mice were generated (Figure 35A) and injected with control AAV or TSLP-AAV after BM reconstitution. TSLP-induced fat loss was observed in TSLPR KO recipient mice transplanted with WT donor BM, but not in WT recipient mice transplanted with TSLPR KO donor BM (Figure 35B), indicating that the effect is mediated by TSLPR signaling in hematopoietic cells. Similar results were seen in BM chimeras fed a HFD (Figures 35C-35E).

[0223] To identify hematopoietic cells required for TSLP-induced fat loss, we injected control or TSLP AAV into various mouse strains lacking different immune cell types. Injection of TSLP-AAV caused significant fat loss in mice lacking dendritic cells (DCs), eosinophils, or Tregs (Figures 36A-36C). However, RAG / common γ chain DKO (lacking B, T, and innate lymphocytes), RAG KO (lacking B and T cells), and E-β KO (lacking αβ T cells) mice did not lose fat upon TSLP-AAV injection (Figures 36D-36E and Figure 30A), suggesting that αβ T cells are required for fat loss induced by TSLP. CD4 + or CD8 + To test whether T cells are required for this effect, we used antibodies to CD4 + and / or CD8 + Strikingly, CD4 + and CD8 + We found that depletion of both T cells was necessary to prevent TSLP-induced white fat loss (Figure 30B), suggesting that either T cell subset is sufficient to induce fat loss in response to TSLP. + or CD8 + Reconstitution of RAG KO mice with either 115 T cells restored their ability to lose fat in response to TSLP (Figure 30C). Importantly, RAG / common γ chain DKO mice with selective reconstitution of T cells became susceptible to TSLP-induced fat loss (Figure 36D), suggesting that T cells, but not B cells, NK cells, or ILCs, were required for this effect.

[0224] To test whether TSLP acts directly on T cells to induce fat loss, we adoptively transferred WT or TSLPR KO T cells into RAG KO mice. TSLP-AAV-injected RAG KO mice reconstituted with WT but not TSLPR KO T cells lost white fat mass (Figure 30D). Furthermore, TSLPR KO mice adoptively transferred with WT but not TSLPR KO T cells also lost white fat mass upon TSLP-AAV injection (Figure 30E). Taken together, these results indicate that TSLP functions directly through the TSLPR on T cells and that TSLPR signaling in T cells is sufficient to mediate TSLP-induced white fat loss. Interestingly, depletion of T cells 2 weeks after TSLP-AAV injection allowed partial but significant recovery of white fat mass, suggesting that T cells are continually required to maintain fat loss in TSLP-AAV-injected mice (Figure 30F).

[0225] TSLP increases sebum secretion The reductions in body weight and white adipose tissue suggest that TSLP causes a net negative energy balance in mice, resulting in caloric output being greater than caloric intake. However, reduced caloric intake was not responsible for the TSLP-induced fat loss, as mice injected with TSLP AAV consumed more feed (Figures 31A, 37A), absorbed and eliminated orally administered glucose and olive oil equally (Figures 37B-37C), and excreted fewer calories / day in feces as measured by bomb calorimetry (Figure 31B) compared with mice injected with control AAV. Because TSLP is a known activator of type II responses that can cause adipose browning, it was hypothesized that TSLP would cause a net negative energy balance by increasing caloric output via increased thermogenic energy expenditure. Surprisingly, however, there was no evidence of increased energy metabolism in mice injected with TSLP-AAV. Despite losing approximately 30% of total fat during the 3-day metabolic cage measurement period, locomotor activity and oxygen consumption rates, carbon dioxide production, energy expenditure, and respiratory exchange rates were similar between control AAV and TSLP-AAV injected mice (Figures 31C, 37D-37H). Consistent with these results, uncoupling protein 1 (UCP1) KO mice, which have reduced fat browning capacity, were not resistant to fat loss induced by TSLP (Figure 37I). Overall, these data indicated that TSLP does not increase thermogenic energy expenditure.

[0226] We next considered that TSLP-AAV-injected mice might be losing energy through secretion or excretion of metabolites, including calories. The fecal caloric content as well as urinary protein, glucose, and ketones were similar between control-AAV and TSLP-AAV-injected mice (Figure 31B, 37J-37L). Interestingly, however, TSLP-AAV-injected mice began to develop a prominent macroscopically greasy coat phenotype approximately 4-5 weeks after injection (Figure 31D). This occurred in both HFD- and NC-fed mice, but was more pronounced in HFD-fed mice. To determine the identity of the greasy material in the coat of TSLP-AAV-injected mice, lipids from their coat were extracted and analyzed by thin-layer chromatography (TLC). Compared to mice injected with control AAV, HFD-fed mice injected with TSLP-AAV showed a significant increase in the amount of hair lipids (Figure 38A), which consist of a mixture of wax esters, cholesterol esters, triglycerides, free fatty acids, and free cholesterol (Figure 31E, 38B). The increase in wax esters, a sebum-specific lipid, implied the presence of enhanced sebum secretion in mice injected with TSLP-AAV. Similar to HFD-fed mice, NC-fed mice injected with TSLP-AAV also showed increased sebum secretion on day 10, an early time point when TSLP-AAV-injected mice are losing white fat but are not yet visibly oily (Figure 34E-34F, 38C-38E). Histological analysis of the skin of mice injected with TSLP-AAV revealed smaller sebaceous glands with similar total lipid content compared to control mice (Figure 38F-38I). Sebum secretion occurs by programmed cell death (holocytosis) of sebocytes and the release of their intracellular contents into the hair follicle. Thus, although the smaller sebaceous glands seen in mice injected with TSLP-AAV are seemingly contradictory, this was likely the result of increased holocytosis and turnover of mature sebocytes. Indeed, an increased proportion of basal cells (the stem cells of proliferating sebocytes) lining the sebaceous glands were Ki67 positive within each gland (Figures 31F-31G, 38J-38K).

[0227] While RAG KO mice injected with TSLP-AAV did not show an increase in hair wax esters (Figures 31H, 39A), RAG KO mice reconstituted with WT T cells, but not TSLPR KO T cells, showed more hair lipid wax esters upon TSLP-AAV injection (Figures 39B-39C), indicating that the increase in sebum secretion was dependent on TSLPR signaling in T cells. Flow cytometry and histology of the skin demonstrated that CD3 + , CD4 + , and CD8 + We showed an increase in T cells (Figures 31I, 40A-40I), which clustered in or around the sebaceous glands. To test whether sebum secretion is required for TSLP-mediated white fat loss, we used asebia mice, which lack the enzyme stearoyl-CoA desaturase 1 (SCD-1) and have profound sebaceous gland dysgenesis. Acebia mice lost minimal amounts of white fat upon TSLP injection (Figure 31J), suggesting that sebum secretion is required for TSLP-induced fat loss.

[0228] Homeostatic role of TSLP in sebum secretion We next investigated whether TSLP and T cells play a physiological role in controlling sebum secretion. To this end, we examined the hair lipid composition of unmanipulated WT and TSLPR KO mice. Compared to WT mice, TSLPR KO mice showed reduced hair wax esters and a reduced percentage of Ki67+ basal cells, without differences in sebaceous gland size and lipid content (Figures 32A-32C, 41A-41E), indicating that TSLP plays a homeostatic role in sebum secretion. Similarly, unmanipulated RAG KO and E-β KO mice showed reduced hair wax esters compared to WT mice (Figures 32D, 41F-41H), indicating that T cells also play a homeostatic role in controlling sebum secretion. We investigated whether this TSLP / sebum axis could also function in humans. Examination of TSLP and a panel of sebaceous gland-related genes in publicly available data (GSE98774) revealed that TSLP expression was significantly and positively correlated with sebaceous gland gene expression in human skin (Figures 32E, 42A-42R), indicating that TSLP can also homeostatically regulate sebum production in humans.

[0229] The results presented herein support a model in which TSLP induces selective white fat loss by acting directly on T cells to induce sebum hypersecretion. The data provide a proof-of-concept for a treatment in which fat loss can be achieved by secreting calories from the skin in the form of energy-rich sebum. Physiologically, the results unraveled the unexpected biology of how the immune system, specifically TSLP-activated T cells, play a key role in sebum release. In addition to calories, sebum contains antimicrobial adipolipids, cathelicidins, defensins, and antimicrobial peptides, which form an important mechanical and immunoprotective barrier for the skin. Because TSLP expression in the skin is upregulated by inflammatory stimuli, in certain non-limiting aspects, the physiological role of TSLP-induced sebum production is not to regulate energy balance, but to promote skin barrier function. However, taken further pharmacologically, sebum hypersecretion can induce systemic fat loss via a pull mechanism (Figure 32F). That is, weight loss can be achieved by "sweating the fat."

[0230] The terms and expressions used herein are used as terms of description and not of limitation, and in the use of such terms and expressions, there is no intention to exclude the features shown and described or equivalents of some of them, but it is recognized that various modifications are possible within the scope of the aspects of this application. Thus, although this application describes certain aspects and optional features, it should be understood that modifications and variations of the compositions, methods, and concepts disclosed herein may be used by those skilled in the art, and such modifications and variations are considered to be within the scope of the aspects of this application.

[0231] Enumerated Aspects The following illustrative aspects are provided, the numbering of which should not be construed as designating a level of importance. Aspect 1 is 1. A method for treating obesity or an obesity-related disorder, comprising locally administering to a subject a pharma- tically effective amount of a vitamin D3 analog. to provide. Aspect 2 is The method of embodiment 1, wherein TSLP levels are systemically increased in the subject. to provide. Aspect 3 is 3. The method of any one of aspects 1-2, wherein the TSLP level is increased by about 5% to about 40% compared to a control subject. to provide. Aspect 4 is The method of any one of embodiments 1-3, wherein the increased TSLP level results in about a 5% to about 30% reduction in white adipose tissue in the subject compared to a control subject. to provide. Aspect 5 is The method of any one of embodiments 1 to 4, wherein the subject experiences about 5% to about 30% weight loss after a given period of time. to provide. Aspect 6 is 6. The method of any one of aspects 1 to 5, wherein the given period of time is from about 1 week to about 12 weeks. to provide. Aspect 7 is The method of any one of embodiments 1-6, wherein the obesity-related disorder is at least one disorder selected from non-alcoholic steatohepatitis (NASH), metabolic disease, type I diabetes, type II diabetes, hypertension, dyslipidemia, coronary heart disease, stroke, gallbladder disease, kidney disease, osteoarthritis, sleep apnea and respiratory disorders, and cancer. to provide. Aspect 8 is The method of any one of aspects 1 to 7, wherein the obesity related disorder is NASH. to provide. Aspect 9 is Vitamin D3 analogues include 1α,18,25-(OH)3D3; 23-(m-(dimethylhydroxymethyl)-22-yn-24,25,26,27(tetranor)-1α-OH)2D3; 1α,25-dihydroxy-trans-isotachysterol (1,25-trans-iso-T); (1S,3R,6S)-7,19-retro-1,25-(OH)2D3; (1S,3R,6R)-7,19-retro-1,25-(OH)2D3; 22-(p-(hydroxyphenyl)-23,24,25,26,27-pentanor-D3; 22-(m-(hydroxyphenyl)-23,24,25,26,27-pentanor-D3; 26,27-cyclo-22-ene-1α,24S-dihy 9. The method of any one of embodiments 1-8, wherein the vitamin D3 is selected from the group consisting of: 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3'(E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089); 1α,25-(OH)-20-epi-22-oxa-24,26,27-trichomovitamin D (KH1060); 22-oxa-1α,25(OH)2D3 (OCT or 22-OXA); 1R,25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D3, and combinations thereof. to provide. Aspect 10 is a method for producing a semiconductor device comprising the steps of: The method of any one of embodiments 1 to 9, wherein the vitamin D3 analog is MC903. to provide. Aspect 11 is a 11. The method of any one of embodiments 1-10, wherein the analog is administered locally to the subject on a dosing schedule of a treatment week followed by a treatment-free week. to provide. Aspect 12 is a method for producing a 12. The method of any one of embodiments 1-11, wherein during the treatment week, the subject is administered the analog locally at a frequency selected from the group consisting of daily and every other day. to provide. Aspect 13 is The method of any one of embodiments 1 to 12, wherein the vitamin D3 analog is the only biologically active agent administered to the subject. to provide. Aspect 14 is a method for producing a 14. The method of any one of embodiments 1 to 13, wherein the administration causes secretion of lipids from the skin of the subject. to provide. Aspect 15 is a method for producing a 1. A method for treating obesity or an obesity-related disorder, comprising administering to a subject a pharma- tically effective amount of a TSLP isoform or a viral vector expressing TSLP. to provide. Aspect 16 is a method for producing a The method of embodiment 15, wherein TSLP levels are systemically increased in the subject. to provide. Aspect 17 is a method for producing a 17. The method of any one of embodiments 15-16, wherein the TSLP isoform is of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:8. to provide. Aspect 18 is a method for producing a 18. The method of any one of embodiments 15 to 17, wherein the TSLP isoform is a stabilized isoform. to provide. Aspect 19 is a method for producing a 19. The method of any one of embodiments 15 to 18, wherein the viral vector expressing TSLP comprises an AAV8 vector comprising a TSLP expression sequence. to provide. Aspect 20 is a method for producing a 20. The method of any one of aspects 15 to 19, wherein the TSLP expression sequence is a mouse TSLP sequence or a human TSLP sequence. to provide. Aspect 21 is 21. The method of any one of embodiments 15 to 20, wherein the viral vector comprises a thyroxine-binding globulin (TBG) promoter. to provide. Aspect 22 is 22. The method of any one of embodiments 15-21, wherein the TSLP level is increased by about 5% to about 40% compared to a control. to provide. Aspect 23 is 23. The method of any one of embodiments 15-22, wherein the subject experiences a weight loss of about 5% to about 20% over a period of about 1 week to about 12 weeks. to provide. Aspect 24 is 24. The method of any one of embodiments 15 to 23, wherein the reduction in body weight does not result in substantial loss of muscle mass. to provide. Aspect 25 is 25. The method of any one of embodiments 15 to 24, wherein the weight loss is due to loss of white adipose tissue. to provide. Aspect 26 is 26. The method of any one of embodiments 15 to 25, wherein the administration causes secretion of lipids from the skin of the subject. to provide. Aspect 27 is 27. The method of any one of embodiments 15-26, wherein the obesity-related disorder is at least one disorder selected from non-alcoholic steatohepatitis (NASH), metabolic disease, type I diabetes, type II diabetes, hypertension, dyslipidemia, coronary heart disease, stroke, gallbladder disease, kidney disease, osteoarthritis, sleep apnea and respiratory disorders, and cancer. to provide. Aspect 28 is The method of any one of aspects 15 to 27, wherein the obesity-related disorder is NASH. to provide. Aspect 29 is 29. The method of any one of embodiments 15-28, wherein the administration is by a route of administration selected from the group consisting of intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, and intrabronchial administration. to provide. Aspect 30 is a 1. A method of treating a skin disorder or improving scalp health, comprising topically administering a pharma- tically effective amount of a vitamin D3 analog to healthy areas of the skin of a subject, the subject suffering from a skin disorder or in need of improved scalp health. to provide. Aspect 31 is 31. The method of embodiment 30, wherein the improvement of skin disorders or scalp health is selected from the group consisting of eczema, atopic dermatitis, dry skin-related dermatitis, dry skin (xeroderma), ichthyosis (all forms), recurrent skin infections, wrinkles (skin aging), hair loss, and hair growth failure. to provide. Aspect 32 is The method of any one of embodiments 30-31, wherein the vitamin D3 analog is administered in a topical composition. to provide. Aspect 33 is 33. The method of any one of embodiments 30-32, wherein the vitamin D3 analog is present in an amount of about 0.0001% to about 10% by weight. to provide. Aspect 34 is Vitamin D3 analogues include 1α,18,25-(OH)3D3; 23-(m-(dimethylhydroxymethyl)-22-yn-24,25,26,27(tetranor)-1α-OH)2D3; 1α,25-dihydroxy-trans-isotachysterol (1,25-trans-iso-T); (1S,3R,6S)-7,19-retro-1,25-(OH)2D3; (1S,3R,6R)-7,19-retro-1,25-(OH)2D3; 22-(p-(hydroxyphenyl)-23,24,25,26,27-pentanor-D3; 22-(m-(hydroxyphenyl)-23,24,25,26,27-pentanor-D3; 26,27-cyclo-22-ene-1α,24S-dihydro- 34. The method of any one of aspects 30 to 33, wherein the vitamin D3 is selected from the group consisting of: 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3'(E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089); 1α,25-(OH)-20-epi-22-oxa-24,26,27-trichomovitamin D (KH1060); 22-oxa-1α,25(OH)2D3 (OCT or 22-OXA); 1R,25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D3, and combinations thereof. to provide. Aspect 35 is The method of any one of aspects 30 to 34, wherein the vitamin D3 analog is MC903. to provide. Aspect 36 is The method of any one of aspects 30 to 35, wherein the vitamin D3 analog is the only biologically active agent administered to the subject. to provide. Aspect 37 is The method of any one of embodiments 30-36, wherein the topical composition is a patch. to provide. Aspect 38 is The method of any one of aspects 30 to 37, wherein the subject is a human. to provide. Aspect 39 is 1. A method of treating an eye disorder, comprising topically administering to the skin of a subject a pharma- tically effective amount of a vitamin D3 analog, wherein the subject is suffering from an eye disorder. to provide. Aspect 40 is a method for producing a 40. The method of embodiment 39, wherein the ocular disorder is selected from the group consisting of dry eye syndrome, keratoconjunctivitis sicca, keratitis sicca, dysfunctional tears syndrome, age-related dry eye syndrome, medication-related dry eye syndrome, menopausal dry eye syndrome, contact lens-associated dry eye, environmentally induced dry eye, dysfunctional eyelid-induced dry eye, autoimmune-associated dry eye (Sjogren's syndrome, rheumatoid arthritis, systemic lupus erythematosus), and infection-associated conjunctivitis. to provide. Aspect 41 is The method of any one of embodiments 39-40, wherein the vitamin D3 analog is administered in a topical composition. to provide. Aspect 42 is 42. The method of any one of embodiments 39-41, wherein the vitamin D3 analog is present in an amount of about 0.0001% to about 10% by weight. to provide. Aspect 43 is Vitamin D3 analogues include 1α,18,25-(OH)3D3; 23-(m-(dimethylhydroxymethyl)-22-yn-24,25,26,27(tetranor)-1α-OH)2D3; 1α,25-dihydroxy-trans-isotachysterol (1,25-trans-iso-T); (1S,3R,6S)-7,19-retro-1,25-(OH)2D3; (1S,3R,6R)-7,19-retro-1,25-(OH)2D3; 22-(p-(hydroxyphenyl)-23,24,25,26,27-pentanor-D3; 22-(m-(hydroxyphenyl)-23,24,25,26,27-pentanor-D3; 26,27-cyclo-22-ene-1α,24S-dihydro- 43. The method of any one of embodiments 39-42, wherein the vitamin D3 is selected from the group consisting of: 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3'(E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089); 1α,25-(OH)-20-epi-22-oxa-24,26,27-trichomovitamin D (KH1060); 22-oxa-1α,25(OH)2D3 (OCT or 22-OXA); 1R,25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D3, and combinations thereof. to provide. Aspect 44 is The method of any one of aspects 39 to 43, wherein the vitamin D3 analog is MC903. to provide. Aspect 45 is The method of any one of embodiments 39-44, wherein the vitamin D3 analog is the only biologically active agent administered to the subject. to provide. Aspect 46 is The method of any one of embodiments 39-45, wherein the vitamin D3 analog is administered to a portion of the subject's skin without contacting the eye. to provide. Aspect 47 is 1. A method for treating, ameliorating, or preventing a skin disorder by reducing or inhibiting sebum release in the skin of a subject, comprising administering to a subject in need thereof a pharma- tically effective amount of a TSLP inhibitor. to provide. Aspect 48 is The method of embodiment 47, wherein the TSLP inhibitor inhibits sfTSLP, lfTSLP, or both sfTSLP and lfTSLP. to provide. Aspect 49 is 49. The method of any one of embodiments 47 to 48, wherein the TSLP is human TSLP. to provide. Embodiment 50 is 50. The method of any one of embodiments 47-49, wherein the TSLP inhibitor is selected from the group consisting of an antibody, a small molecule, an siRNA, an shRNA, and an miRNA. to provide. Aspect 51 is The method of any one of aspects 47 to 50, wherein the antibody is tezepelumab. to provide. Aspect 52 is 52. The method of any one of aspects 47-51, wherein the skin disorder is acne vulgaris, hidradenitis suppurativa, or seborrheic dermatitis. to provide. Aspect 53 is 1. A method of treating, ameliorating, and / or preventing alopecia in a subject, comprising topically administering to the skin of a subject a pharma- tically effective amount of a vitamin D3 analog, wherein the subject is suffering from or in need of amelioration of alopecia. to provide. Aspect 54 is 54. The method of embodiment 53, wherein the alopecia comprises male pattern baldness. to provide. Aspect 55 is a method for producing a 55. The method of any one of embodiments 53-54, wherein the administration is to an area of ​​the skin affected by alopecia. to provide. Aspect 56 is a method for producing a 55. The method of any one of embodiments 47 to 54, wherein the administration is to an area of ​​skin not affected by alopecia. to provide. Aspect 57 is 57. The method of any one of embodiments 53 to 56, wherein the vitamin D3 analog is administered in a topical composition. to provide. Aspect 58 is a method for producing a 58. The method of any one of embodiments 53-57, wherein the vitamin D3 analog is present in an amount of about 0.0001% to about 10% by weight. to provide. Aspect 59 is Vitamin D3 analogues include 1α,18,25-(OH)3D3; 23-(m-(dimethylhydroxymethyl)-22-yn-24,25,26,27(tetranor)-1α-OH)2D3; 1α,25-dihydroxy-trans-isotachysterol (1,25-trans-iso-T); (1S,3R,6S)-7,19-retro-1,25-(OH)2D3; (1S,3R,6R)-7,19-retro-1,25-(OH)2D3; 22-(p-(hydroxyphenyl)-23,24,25,26,27-pentanor-D3; 22-(m-(hydroxyphenyl)-23,24,25,26,27-pentanor-D3; 26,27-cyclo-22-ene-1α,24S-dihydro- 59. The method of any one of embodiments 53-58, wherein the vitamin D3 is selected from the group consisting of: 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3'(E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089); 1α,25-(OH)-20-epi-22-oxa-24,26,27-trichomovitamin D (KH1060); 22-oxa-1α,25(OH)2D3 (OCT or 22-OXA); 1R,25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D3, and combinations thereof. to provide. Aspect 60 is 60. The method of any one of aspects 53-59, wherein the vitamin D3 analog is MC903. to provide. Aspect 61 is The method of any one of aspects 53 to 60, wherein the vitamin D3 analog is the only biologically active agent administered to the subject. to provide. Aspect 62 is The method of any one of embodiments 53-61, wherein the topical composition is a patch. to provide. Aspect 63 is The method of any one of embodiments 53 to 62, wherein the subject is a human. to provide.

[0232] Sequence information SEQUENCE LISTING <110> The Trustees of The University of Pennsylvania <120> Methods and Compositions for Treating Obesity and / or Skin Disorders <150> US 62 / 849,656 <151> 2019-05-17 <150> US 62 / 972,462 <151> 2020-02-10 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 159 <212> PRT <213> Homo sapiens <400> 1 Met Phe Pro Phe Ala Leu Leu Tyr Val Leu Ser Val Ser Phe Arg Lys 1 5 10 15 Ile Phe Ile Leu Gln Leu Val Gly Leu Val Leu Thr Tyr Asp Phe Thr 20 25 30 Asn Cys Asp Phe Glu Lys Ile Lys Ala Ala Tyr Leu Ser Thr Ile Ser 35 40 45 Lys Asp Leu Ile Thr Tyr Met Ser Gly Thr Lys Ser Thr Glu Phe Asn 50 55 60 Asn Thr Val Ser Cys Ser Asn Arg Pro His Cys Leu Thr Glu Ile Gln 65 70 75 80 Ser Leu Thr Phe Asn Pro Thr Ala Gly Cys Ala Ser Leu Ala Lys Glu 85 90 95 Met Phe Ala Met Lys Thr Lys Ala Ala Leu Ala Ile Trp Cys Pro Gly 100 105 110 Tyr Ser Glu Thr Gln Ile Asn Ala Thr Gln Ala Met Lys Lys Arg Arg 115 120 125 Lys Arg Lys Val Thr Thr Asn Lys Cys Leu Glu Gln Val Ser Gln Leu 130 135 140 Gln Gly Leu Trp Arg Arg Phe Asn Arg Pro Leu Leu Lys Gln Gln 145 150 155 <210> 2 <211> 63 <212> PRT <213> Homo sapiens <400> 2 Met Phe Ala Met Lys Thr Lys Ala Ala Leu Ala Ile Trp Cys Pro Gly 1 5 10 15 Tyr Ser Glu Thr Gln Ile Asn Ala Thr Gln Ala Met Lys Lys Arg Arg 20 25 30 Lys Arg Lys Val Thr Thr Asn Lys Cys Leu Glu Gln Val Ser Gln Leu 35 40 45 Gln Gly Leu Trp Arg Arg Phe Asn Arg Pro Leu Leu Lys Gln Gln 50 55 60 <210> 3 <211> 480 <212> DNA <213> Homo sapiens <400> 3 atgttccct ttgccttact atatgttctg tcagtttctt tcaggaaaat cttcatctta 60 caacttgtag ggctggtgtt aacttacgac ttcactaact gtgactttga gaagattaaa 120 gcagcctatc tcagtactat ttctaaagac ctgattacat atatgagtgg gaccaaaagt 180 accgagttca acaacaccgt ctcttgtagc aatcggccac attgccttac tgaaatccag 240 agcctaacct tcaatcccac cgccggctgc gcgtcgctcg ccaaagaaat gttcgccatg 300 aaaactaagg ctgccttagc tatctggtgc ccaggctatt cggaaactca gataaatgct 360 actcaggcaa tgagagag gagaaaaagg aaagtcacaa ccaataatg tctggaacaa 420 gtgtcacaat tacaggatt gtggcgtcgc ttcaatcgac ctttactgaa acacagtaa 480 <210> 4 <211> 159 <212> PRT <213> Homo sapiens <400> 4 Met Phe Pro Phe Ala Leu Leu Tyr Val Leu Ser Val Ser Phe Arg Lys 1 5 10 15 Ile Phe Ile Leu Gln Leu Val Gly Leu Val Leu Thr Tyr Asp Phe Thr 20 25 30 Asn Cys Asp Phe Glu Lys Ile Lys Ala Ala Tyr Leu Ser Thr Ile Ser 35 40 45 Lys Asp Leu Ile Thr Tyr Met Ser Gly Thr Lys Ser Thr Glu Phe Asn 50 55 60 The Asn Thr Will Be The Asn Arg Pro His Cys Leu Thr Glu Ile Gln 65 70 75 80 Ser Leu Thr Phe Asn Pro Thr Ala Gly Cys Ala Ser Leu Ala Lys Glu 85 90 95 Met Phe Ala Met Lys Thr Lys Ala Leu Ala Ile Trp Cys Pro Gly 100 105 110 Tyr Ser Glu Thr Gln With Asn Only Three Gln Ala Met Lys Lys Arg Arg 115 120 125 Lys Arg Lys Will Thr Asn Lys Cys Leu Glu Gln Val Ser Gln Leu 130 135 140 Gln Gly Leu Trp Arg Phe Asn Arg Pro Leu Lys Gln Gln 145 150 155 <210> 5 <211> 192 <212> DNA <213> Homo sapiens <400> 5 atgttcgcca tgaaaactaa ggctgcctta gctatctggt gcccaggcta ttcggaact 60 cagaataatg ctactcaggc aatgaagaag aggaaaaa ggaagtcac aaccaataa 120 tgtctggaac aagtgtcaca attacaagga tgtggcgtc gcttcaatcg acctttactg 180 chapter aa 192 <210> 6 <211> 63 <212> PRT <213> Homo sapiens <400> 6 Met Phe Ala Met Lys Thr Lys Ala Leu Ala Ile Trp Cys Pro Gly 1 5 10 15 Tyr Ser Glu Thr Gln With Asn Only Three Gln Ala Met Lys Lys Arg Arg 20 25 30 Lys Arg Lys Val Thr Thr Asn Lys Cys Leu Glu Gln Val Ser Gln Leu 35 40 45 Gln Gly Leu Trp Arg Arg Phe Asn Arg Pro Leu Leu Lys Gln Gln 50 55 60 <210> 7 <211> 423 <212> DNA <213> Homo sapiens <400> 7 atggttcttc tcaggagcct cttcatcctg caagtactag tacggatggg gctaacttac 60 aacttttcta actgcaactt cacgtcaatt acgaaaatat attgtaacat aatttttcat 120 gacctgactg gagatttgaa aggggctaag ttcgagcaaa tcgaggactg tgagagcaag 180 ccagcttgtc tcctgaaaat cgagtactat actctcaatc ctatccctgg ctgcccttca 240 ctccccgaca aaacatttgc ccggagaaca agagaagccc tcaatgacca ctgcccaggc 300 taccctgaaa ctgagagaaa tgacggtact caggaaatgg cacaagaagt ccaaaacatc 360 tgcctgaatc aaacctcaca aattctaaga ttgtggtatt ccttcatgca atctccagaa 420 city ​​423 <210> 8 <211> 140 <212> PRT <213> Homo sapiens <400> 8 Met Val Leu Leu Arg Ser Leu Phe Ile Leu Gln Val Leu Val Arg Met 1 5 10 15 Gly Leu Thr Tyr Asn Phe Ser Asn Cys Asn Phe Thr Ser Ile Thr Lys 20 25 30 Ile Tyr Cys Asn Ile Ile Phe His Asp Leu Thr Gly Asp Leu Lys Gly 35 40 45 Ala Lys Phe Glu Gln Ile Glu Asp Cys Glu Ser Lys Pro Ala Cys Leu 50 55 60 Leu Lys Ile Glu Tyr Tyr Thr Leu Asn Pro Ile Pro Gly Cys Pro Ser 65 70 75 80 Leu Pro Asp Lys Thr Phe Ala Arg Arg Thr Arg Glu Ala Leu Asn Asp 85 90 95 His Cys Pro Gly Tyr Pro Glu Thr Glu Arg Asn Asp Gly Thr Gln Glu 100 105 110 Met Ala Gln Glu Val Gln Asn Ile Cys Leu Asn Gln Thr Ser Gln Ile 115 120 125 Leu Arg Leu Trp Tyr Ser Phe Met Gln Ser Pro Glu 130 135 140

Claims

1. A method for treating obesity or an obesity-related disorder, comprising administering a pharmacologic effective amount of vitamin D 3 The method comprises the step of locally administering the analog to a subject.

2. The method of claim 1, wherein TSLP levels are increased systemically in the subject.

3. The method of claim 2, wherein TSLP levels are increased by about 5% to about 40% compared to a control subject.

4. The method of claim 1, wherein the increased TSLP level results in about a 5% to about 30% reduction in white adipose tissue in the subject compared to a control subject.

5. 10. The method of claim 1, wherein the subject experiences about 5% to about 30% weight loss after a given period of time.

6. 6. The method of claim 5, wherein the given period of time is from about 1 week to about 12 weeks.

7. 2. The method of claim 1, wherein the obesity-related disorder is at least one disorder selected from nonalcoholic steatohepatitis (NASH), metabolic disease, type I diabetes, type II diabetes, hypertension, dyslipidemia, coronary heart disease, stroke, gallbladder disease, kidney disease, osteoarthritis, sleep apnea and respiratory disorders, and cancer.

8. 2. The method of claim 1, wherein the obesity-related disorder is NASH.

9. Vitamin D 3 The analogue is 1α,18,25-(OH) 3 D 3 23-(m-(dimethylhydroxymethyl)-22-yne-24,25,26,27(tetranor)-1α-OH) 2 D 3 1α,25-dihydroxy-trans-isotachysterol (1,25-trans-iso-T); (1S,3R,6S)-7,19-retro-1,25-(OH) 2 D 3 (1S,3R,6R)-7,19-retro-1,25-(OH) 2 D 3 22-(p-(hydroxyphenyl)-23,24,25,26,27-pentanol-D 3 22-(m-(hydroxyphenyl)-23,24,25,26,27-pentanol-D 3 26,27-cyclo-22-ene-1α,24S-dihydroxyvitamin D 3 (MC903 or calcipotriol); 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3'(E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089); 1α,25-(OH)-20-epi-22-oxa-24,26,27-trisulfomovitamin D (KH1060); 22-oxa-1α,25(OH) 2 D 3 (OCT or 22-OXA); 1R,25-dihydroxy-21-(3-hydroxy-3-methylbutyl) vitamin D 3 and combinations thereof.

10. Vitamin D 3 2. The method of claim 1, wherein the analog is MC903.

11. 10. The method of claim 1, wherein the analog is administered locally to the subject on a dosing schedule of a treatment week followed by a treatment-free week.

12. 12. The method of claim 11, wherein during the treatment week, the subject receives topical administration of the analog at a frequency selected from the group consisting of daily and every other day.

13. Vitamin D 3 10. The method of claim 1, wherein the analog is the only biologically active agent administered to the subject.

14. 10. The method of claim 1, wherein administration causes secretion of lipids from the skin of the subject.

15. A method for treating obesity or an obesity-related disorder, comprising administering to a subject a pharma- tically effective amount of a TSLP isoform or a viral vector expressing TSLP.

16. The method of claim 15, wherein TSLP levels are increased systemically in the subject.

17. The method of claim 15, wherein the TSLP isoform is of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:

8.

18. The method of claim 15, wherein the TSLP isoform is a stabilized isoform.

19. The method of claim 15, wherein the viral vector expressing TSLP comprises an AAV8 vector comprising a TSLP expression sequence.

20. 20. The method of claim 19, wherein the TSLP expression sequence is a mouse TSLP sequence or a human TSLP sequence.

21. 20. The method of claim 19, wherein the viral vector comprises a thyroxine-binding globulin (TBG) promoter.

22. The method of claim 15, wherein TSLP levels are increased by about 5% to about 40% compared to a control.

23. 16. The method of claim 15, wherein the subject experiences about 5% to about 20% weight loss over a period of about 1 week to 12 weeks.

24. 24. The method of claim 23, wherein the weight loss does not result in substantial loss of muscle mass.

25. 24. The method of claim 23, wherein the weight loss is due to loss of white adipose tissue.

26. 16. The method of claim 15, wherein administration causes secretion of lipids from the skin of the subject.

27. 16. The method of claim 15, wherein the obesity-related disorder is at least one disorder selected from nonalcoholic steatohepatitis (NASH), metabolic disease, type I diabetes, type II diabetes, hypertension, dyslipidemia, coronary heart disease, stroke, gallbladder disease, kidney disease, osteoarthritis, sleep apnea and respiratory disorders, and cancer.

28. 16. The method of claim 15, wherein the obesity-related disorder is NASH.

29. 16. The method of claim 15, wherein the administration is by a route of administration selected from the group consisting of intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, and intrabronchial administration.

30. 1. A method for treating a skin disorder or improving scalp health, comprising: A pharmacologic dose of vitamin D 3 topically administering the analog to healthy areas of the skin of a subject; The subject is suffering from a skin disorder or is in need of improved scalp health; method.

31. 31. The method of claim 30, wherein the improvement of skin disorders or scalp health is selected from the group consisting of eczema, atopic dermatitis, dry skin-related dermatitis, dry skin (xeroderma), ichthyosis (all forms), recurrent skin infections, wrinkles (skin aging), hair loss, and hair growth failure.

32. Vitamin D 3 31. The method of claim 30, wherein the analog is administered in a topical composition.

33. Vitamin D 3 33. The method of claim 32, wherein the analog is present in an amount of about 0.0001% to about 10% by weight.

34. Vitamin D 3 The analogue is 1α,18,25-(OH) 3 D 3 23-(m-(dimethylhydroxymethyl)-22-yne-24,25,26,27(tetranor)-1α-OH) 2 D 3 1α,25-dihydroxy-trans-isotachysterol (1,25-trans-iso-T); (1S,3R,6S)-7,19-retro-1,25-(OH) 2 D 3 (1S,3R,6R)-7,19-retro-1,25-(OH) 2 D 3 22-(p-(hydroxyphenyl)-23,24,25,26,27-pentanol-D 3 22-(m-(hydroxyphenyl)-23,24,25,26,27-pentanol-D 3 26,27-cyclo-22-ene-1α,24S-dihydroxyvitamin D 3 (MC903 or calcipotriol); 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3'(E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089); 1α,25-(OH)-20-epi-22-oxa-24,26,27-trisulfomovitamin D (KH1060); 22-oxa-1α,25(OH) 2 D 3 (OCT or 22-OXA); 1R,25-dihydroxy-21-(3-hydroxy-3-methylbutyl) vitamin D 3 and combinations thereof.

35. Vitamin D 3 35. The method of claim 34, wherein the analog is MC903.

36. Vitamin D 3 31. The method of claim 30, wherein the analog is the only biologically active agent administered to the subject.

37. 33. The method of claim 32, wherein the topical composition is a patch.

38. 31. The method of claim 30, wherein the subject is a human.

39. 1. A method of treating an eye disorder, comprising: A pharmacologic dose of vitamin D 3 topically administering an analog to the skin of a subject; the subject is suffering from an eye disorder; method.

40. 40. The method of claim 39, wherein the ocular disorder is selected from the group consisting of dry eye syndrome, keratoconjunctivitis sicca, keratitis sicca, dysfunctional tear syndrome, age-related dry eye syndrome, medication-related dry eye syndrome, menopausal dry eye syndrome, contact lens-associated dry eye, environmentally induced dry eye, dysfunctional eyelid-induced dry eye, autoimmune-associated dry eye, and infection-associated conjunctivitis.

41. Vitamin D 3 40. The method of claim 39, wherein the analog is administered in a topical composition.

42. Vitamin D 3 42. The method of claim 41, wherein the analog is present in an amount of about 0.0001% to about 10% by weight.

43. Vitamin D 3 The analogue is 1α,18,25-(OH) 3 D 3 23-(m-(dimethylhydroxymethyl)-22-yne-24,25,26,27(tetranor)-1α-OH) 2 D 3 1α,25-dihydroxy-trans-isotachysterol (1,25-trans-iso-T); (1S,3R,6S)-7,19-retro-1,25-(OH) 2 D 3 (1S,3R,6R)-7,19-retro-1,25-(OH) 2 D 3 22-(p-(hydroxyphenyl)-23,24,25,26,27-pentanol-D 3 22-(m-(hydroxyphenyl)-23,24,25,26,27-pentanol-D 3 26,27-cyclo-22-ene-1α,24S-dihydroxyvitamin D 3 (MC903 or calcipotriol); 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3'(E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089); 1α,25-(OH)-20-epi-22-oxa-24,26,27-trisulfomovitamin D (KH1060); 22-oxa-1α,25(OH) 2 D 3 (OCT or 22-OXA); 1R,25-dihydroxy-21-(3-hydroxy-3-methylbutyl) vitamin D 3 and combinations thereof.

44. Vitamin D 3 44. The method of claim 43, wherein the analog is MC903.

45. Vitamin D 3 40. The method of claim 39, wherein the analog is the only biologically active agent administered to the subject.

46. Vitamin D 3 40. The method of claim 39, wherein the analog is administered to a portion of the subject's skin without contacting the eye.

47. 1. A method for treating, ameliorating, or preventing a skin disorder by reducing or inhibiting sebum release in the skin of a subject, comprising administering to a subject in need thereof a pharma- tically effective amount of a TSLP inhibitor.

48. The method of claim 47, wherein the TSLP inhibitor inhibits sfTSLP, lfTSLP, or both sfTSLP and lfTSLP.

49. The method of claim 48, wherein the TSLP is human TSLP.

50. 48. The method of claim 47, wherein the TSLP inhibitor is selected from the group consisting of an antibody, a small molecule, an siRNA, an shRNA, and an miRNA.

51. 48. The method of claim 47, wherein the antibody is tezepelumab.

52. 48. The method of claim 47, wherein the skin disorder is acne vulgaris, hidradenitis suppurativa, or seborrheic dermatitis.

53. 1. A method of treating, ameliorating, and / or preventing alopecia in a subject, comprising: A pharmacologic dose of vitamin D 3 topically administering an analog to the skin of a subject; The subject is suffering from alopecia or in need of amelioration of alopecia. method.

54. 54. The method of claim 53, wherein the alopecia comprises male pattern baldness.

55. 54. The method of claim 53, wherein administration is to an area of ​​skin affected by alopecia.

56. 54. The method of claim 53, wherein administration is to an area of ​​skin not affected by alopecia.

57. Vitamin D 3 54. The method of claim 53, wherein the analog is administered in a topical composition.

58. Vitamin D 3 54. The method of claim 53, wherein the analog is present in an amount of about 0.0001% to about 10% by weight.

59. Vitamin D 3 The analogue is 1α,18,25-(OH) 3 D 3 23-(m-(dimethylhydroxymethyl)-22-yne-24,25,26,27(tetranor)-1α-OH) 2 D 3 1α,25-dihydroxy-trans-isotachysterol (1,25-trans-iso-T); (1S,3R,6S)-7,19-retro-1,25-(OH) 2 D 3 (1S,3R,6R)-7,19-retro-1,25-(OH) 2 D 3 22-(p-(hydroxyphenyl)-23,24,25,26,27-pentanol-D 3 22-(m-(hydroxyphenyl)-23,24,25,26,27-pentanol-D 3 26,27-cyclo-22-ene-1α,24S-dihydroxyvitamin D 3 (MC903 or calcipotriol); 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3'(E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089); 1α,25-(OH)-20-epi-22-oxa-24,26,27-trisulfomovitamin D (KH1060); 22-oxa-1α,25(OH) 2 D 3 (OCT or 22-OXA); 1R,25-dihydroxy-21-(3-hydroxy-3-methylbutyl) vitamin D 3 and combinations thereof.

60. Vitamin D 3 54. The method of claim 53, wherein the analog is MC903.

61. Vitamin D 3 54. The method of claim 53, wherein the analog is the only biologically active agent administered to the subject.

62. 54. The method of claim 53, wherein the topical composition is a patch.

63. 54. The method of claim 53, wherein the subject is a human.

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