Polynucleotide for hepatocyte expression of vestigial like protein 4 and method of use thereof

Abstract

Provided is a polynucleotide including a nucleotide sequence encoding a vestigial like 4 protein and a cis-regulatory element that controls hepatocyte-specific expression of the sequence encoding a vestigial like 4 protein. Also provided is a viral vector including the polynucleotide, and a cell or organism transfected by the polynucleotide. Also provided is a method of treating steatohepatitis, obesity, hyperglycemia, diabetes, insulin resistance, liver inflammation, or decreasing white adipose tissue in the subject. Also provided is a method of screening a treatment for fatty liver disease or prevention of cirrhosis, comprising feeding a diet high in fat, fructose, and / or cholesterol to a transgenic animal having an hepatocyte-specific vestigial like protein gene disruption, administering the treatment to the transgenic animal, and detecting a difference in liver morphology or liver function between the transgenic animal and a control animal.

Description

POLYNUCLEOTIDE FOR HEPATOCYTE EXPRESSION OF VESTIGIAL LIKE PROTEIN 4 AND METHOD OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority from U.S. Provisional PatentApplication No.63 / 638,809, filed April 25, 2024, the entire contents of which are incorporated herein by reference. GOVERNMENT RIGHTS STATEMENT

[0002] This invention was made with Government support under grant number 1R01HL146810 awarded by the National Institutes of Health. The Government has certain rights in the invention. SEQUENCE LISTING

[0003] The instant application contains an electronic sequence listing. The contents of the electronic sequence listing 5679.017AWO Sequence Listing XML.xml; Size: 67,387 bytes; and Date of Creation: April 24, 2025, is herein incorporated by reference in its entirety. BACKGROUND

[0004] Non-alcoholic fatty liver disease (NAFLD) is a chronic and progressive liver disease encompassing different stages, from the early stage of non-alcoholic fatty liver (NAFL), to the middle stage of non-alcoholic steatohepatitis (NASH), and ultimately reaching to the end stage of cirrhosis or hepatocellular carcinoma. Characterized by excessive lipid deposition in the hepatocytes, NAFLD is highly associated with metabolic syndromes, particularly obesity and Type II diabetes. In the past two decades, the global prevalence of NAFLD has increased from to 26% in 2005 to 38% in 2016 and beyond. In most cases, NAFLD is benign and does not progress; however, around 5% NAFLD patients do develop NASH, a condition signatured by hepatocellular ballooning degeneration, liver tissue inflammation and fibrosis deposition. A subset of NASH patients may develop cirrhosis and hepatocellular carcinoma, which eventually leads to liver failure-related mortality.

[0005] Treatment options for NAFLD and associated conditions are limited. The present disclosure is directed to overcoming these and other deficiencies in the art.SUMMARY

[0006] In one aspect, provided is a polynucleotide, including a nucleotide sequence encoding a vestigial like 4 protein and a cis-regulatory element, wherein the cis-regulatory element controls hepatocyte-specific expression of the sequence encoding a vestigial like 4 protein. In an example, the cis-regulatory element has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with, independently, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, nucleotides 6-299 of SEQ ID NO: 48, or any combination of two or more of the foregoing.

[0007] The cis-regulatory element may include one or more enhancer and the one or more enhancer has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with, independently, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or any combination of two or more of the foregoing.

[0008] The cis-regulatory element may include one or more promoter and the one or more promoter has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with, independently, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or any combination of two or more of the foregoing.

[0009] The vestigial like 4 protein may have at least 90% identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0010] The vestigial like 4 protein may have at least 90% identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, further including substitution of a TDU domain with a TDU domain variant, wherein the variant includes one or both of a TDU_1 domain variant wherein the sequence of the TDU_1 domain is DPVVEEX1X2RRSLGKNY, wherein X1 may be H or any amino acid other than H, or, independently, X2 may be F or any amino acid other than F, or a TDU_2 domain variant, wherein the sequence of the TDU_2 domain variant is TGSVDDX3X4AKALGDTW, wherein X3 may be H or any amino acid other than H, or, independently, X4may be F or any amino acid other than F, or any combination of two or more of the foregoing. Any of the foregoing vestigial like 4 protein may be substituted with a vestigial like 4 protein fragment. The vestigial like 4 protein fragment may have a sequence of SEQ ID NO: 53 or SEQ ID NO: 54.

[0011] The sequence encoding a vestigial like 4 protein may have at least 90% identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with a sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0012] The sequence encoding a vestigial like 4 protein may have at least 90% identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with a sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, optionally further including substitution of a sequence encoding a TDU domain with a sequence encoding a TDU domain variant, wherein the variant includes one or both of a TDU_1 domain variant wherein the sequence of the TDU_1 domain is DPVVEEX1X2RRSLGKNY, wherein X1 may be H or any amino acid other than H, or, independently, X2 may be F or any amino acid other than F, or a TDU_2 domain variant, wherein the sequence of the TDU_2 domain variant is TGSVDDX3X4AKALGDTW, wherein X3 may be H or any amino acid other than H, or, independently, X4 may be F or any amino acid other than F, or anycombination of two or more of the foregoing. Any of the foregoing sequence encoding a vestigial like 4 protein may be substituted with a sequence encoding a vestigial like 4 protein fragment. The sequence encoding a vestigial like 4 protein may be substituted with a sequence encoding a vestigial like protein 4 fragment having a sequence of SEQ ID NO: 53 or SEQ ID NO: 54.

[0013] The vestigial like 4 protein may have from 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, wherein the one or more substitutionis not in a TDU domain.

[0014] The polynucleotide may further include an intron between the cis-regulatory element and the nucleotide sequence encoding a vestigial like 4 protein or fragment thereof. In another further example, the intron may have at least 90% identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 42.

[0015] The nucleotide sequence may have at least 90% identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO:48.

[0016] Also provided is a viral vector including any foregoing polynucleotide. The viral vector may include an adenoviral associated vector. Also provided is a cell transfected or transduced in vitro or ex vivo with any foregoing polynucleotide or any foregoing viral vector. Also provided is a non-human organism transfected or transduced with any foregoing polynucleotide or any foregoing viral vector.

[0017] In another aspect, provided is a method, including transfecting or transducing a cell of an organism, in vivo or ex vivo, or transfecting or transducing a cell in vitro, with any foregoing polynucleotide or any foregoing viral vector. The organism may be a mammal. The organism may be a human.

[0018] Also provided is a method of treating one or more of steatohepatitis, obesity, hyperglycemia, diabetes, insulin resistance, and liver inflammation in a subject, and / or decreasing white adipose tissue in the subject, including administering any foregoingpolynucleotide to the subject. The administering may include administering any foregoing viral vector to the subject.

[0019] In another aspect, provided is a polynucleotide, including a nucleotide sequence encoding a vestigial like 4 protein and a means for controlling hepatocyte-specific expression of the sequence encoding a vestigial like 4 protein. The polynucleotide may further include an intron. The intron may have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 42.

[0020] The vestigial like 4 protein may have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0021] The vestigial like 4 protein may have at least 90% identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, optionally further including substitution of a TDU domain with a TDU domain variant, wherein the variant includes one or both of a TDU_1 domain variant wherein the sequence of the TDU_1 domain is DPVVEEX1X2RRSLGKNY, wherein X1may be H or any amino acid other than H, or, independently, X2 may be F or any amino acid other than F, or a TDU_2 domain variant, wherein the sequence of the TDU_2 domain variant is TGSVDDX3X4AKALGDTW, wherein X3 may be H or any amino acid other than H, aornd, independently, X4 may be F or any amino acid other than F, or any combination of two or more of the foregoing. Any of the foregoing vestigial like 4 protein may be substituted with a vestigial like 4 protein fragment. The vestigial like 4 protein fragment may have a sequence of SEQ ID NO: 53 or SEQ ID NO:

[0022] The sequence encoding a vestigial like 4 protein may have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with a sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0023] The sequence encoding a vestigial like 4 protein may have at least 90% identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with a sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, optionally further including substitution of a sequence encoding a TDU domain with a sequence encoding a TDU domain variant, wherein the variant includes one or both of a TDU_1 domain variant wherein the sequence of the TDU_1 domain is DPVVEEX1X2RRSLGKNY, wherein X1may be H or any amino acid other than H, or, independently, X2may be F or any amino acid other than F, or a TDU_2 domain variant, wherein the sequence of the TDU_2 domain variant is TGSVDDX3X4AKALGDTW, wherein X3may be H or any amino acid other than H, or, independently, X4 may be F or any amino acid other than F, or any combination of two or more of the foregoing. Any of the foregoing sequence encoding a vestigial like 4 protein may be substituted with a sequence encoding a vestigial like 4 protein fragment. The sequence encoding a vestigial like 4 protein may be substituted with a sequence encoding a vestigial like protein 4 fragment having a sequence of SEQ ID NO: 53 or SEQ ID NO: 54.

[0024] Also provided is a viral vector including any foregoing polynucleotide. The viral vector may include an adenoviral associated vector. Also provided is a cell transfected or transduced in vitro or ex vivo with any foregoing polynucleotide or any foregoing viral vector. Also provided is a non-human organism transfected or transduced with any foregoing polynucleotide or any foregoing viral vector.

[0025] In another aspect, provided is a method, including transfecting or transducing a cell of an organism, in vivo or ex vivo, or transfecting or transducing a cell in vitro, with any foregoing polynucleotide or any foregoing viral vector. The organism may be a mammal. The organism may be a human.

[0026] Also provided is a method of treating one or more of steatohepatitis, obesity, hyperglycemia, diabetes, insulin resistance, and / or liver inflammation in a subject, and / or decreasing white adipose tissue in the subject, including administering any foregoing polynucleotide to the subject. The administering may include administering any foregoing viral vector to the subject.

[0027] In still another aspect, provided is a method of screening a treatment for fatty liver disease or prevention of cirrhosis, including feeding a test transgenic animal a diet wherein the diet is high in one or more of fat, fructose, or cholesterol, administering the treatment to the transgenic animal, and after the administering, detecting a difference in liver morphology or liver function between the test transgenic animal and a control animal, wherein the test transgenic animal includes a disruption of the gene encoding a vestigial like 4 protein and the disruption is limited to hepatocytes. The control animal may include the disruption of the gene encoding the vestigial like 4 protein, the disruption limited to hepatocytes. The difference may include one or more of decreased liver surface nodularity, decreased hyperproliferation of liver duct cells, and decreased liver fibrosis deposition, or any combination of two or more of the foregoing, in the test transgenic animal compared to the control animal. The difference may include one or more of lower liver to body weight ratio, liver tissue surface with fewer or less irregular bumps, presence of fewer small ductular structures, presence of fewer or less growth of cells of reduced size containing less cytoplasmic volume forming fewer small ductular structures, less fibrosis deposition, less splenomegaly, lower number of cells positive for proliferative cell marker, optionally Ki67, fewer irregular shaped hepatocytes, presence of enlarged hepatocytes, presence of fewer hepatocytes occupied with macrovacuoles, less TEAD1 upregulation, or any combination of two or more of the foregoing, in the test transgenic animal. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings, wherein:

[0029] FIGs 1A-1E show hepatocyte-specific expression of VGLL4 does affect postnatal liver growth, in accordance with aspects of the present disclosure. A. pAAV.TTR.GFP and pAAV.TTR.VGLL4-GFP (SEQ ID NO: 48) construct. ITR, inverted terminal repeat. GFP or VGLL4-GFP was placed downstream of the synthetic, liver specific CRM8-TTR promoter25. B. The internal organs of a AAV9.TTR.GFP transduced mouse. S,spleen; K, kidney; H, heart; Lu, lung; Li, liver. Scar bar = 5 mm. C. Gross morphology of control and TTR.VGLL4 transduced liver. Scar bar = 5 mm. D. Liver weight and Liver to body weight ratio. N=6. B and C, mouse pups were transduced with AAV at postnatal day 2 (P2), and organs were collected at P14. E. Western blot of VGLL4. Protein extracted from liver was blotted with GFP antibody to detect the expression of VGLL4GFPfusion protein. GAPDH was used as loading control.

[0030] FIGs.2A-2F show overexpression of VGLL4 in the hepatocytes promotes lipid droplets formation, in accordance with aspects of the present disclosure. Mice were transduced with indicated AAV at P21 and the livers were collected at 4 weeks after AAV transduction. A. Gross morphology of control and TTR.VGLL4 transduced liver. B. Liver to body weight ratio. Male, TTR.GFP, n=6; TTR.VGLL4, n=5; female, TTR.GFP, n=8; TTR.VGLL4, n=8. C. Hematoxylin and eosin staining of adult liver sections. D. Oil Red staining of liver section. E. Fluorescence images of liver sections. F. Oil Red staining of HepG2 cells.200 µM palmitic acid (PA) was used to treat HepG2 cells. C, D, E, and F, scale bar = 50 µm. G. Quantification of HepG2 lipid content. One way ANOVA test, ***, p<0.001; ****, p<0.0001.

[0031] FIGs.3A-3F show expressing VGLL4 in the liver mitigates HFD-induced body weight gain, in accordance with aspects of the present disclosure. AAV9.TTR.VGLL4 was retro-orbitally injected into 8-weeks-old C57 / BL6 mice. AAV9.TTR.GFP was used as control. HFD treatment started one week after AAV injection and lasted for 11 weeks. A. Body weight measurements. B. Accumulated body weight gain measurements.11 weeks after HFD treatment, the glycemia of AAV9.TTR.GFP or AAV9.TTR.VGLL4 transduced mice were analyzed. C-D. Glucose tolerance test (GTT). C, glycemia values of different time points. D, area under the curve (AUC) of glycemia. E-F. Insulin tolerance test (ITT). E, glycemia values of different time points. F, area under the curve (AUC) of glycemia. Student's t test, P<0.05; **, P<0.01. TTR.GFP, n=10; TTR.VGLL4, n=10.

[0032] FIGs.4A-4I show AAV9.TTR.VGLL4 pre-treatment attenuates HFD-induced white adipose tissue expansion, in accordance with aspects of the present disclosure. A. Experimental design.11 weeks after HFD treatment, AAV transduced mice were analyzed. B. Representative 3D images generated by micro CT scan. Subcutaneous WAT was labeled with light green, and visceral WAT was labeled with brown color. Bones were labeled as grey. C. Quantification of non-fat and non-bone tissue volume (lean mass). D-F. Quantification of total WAT volume (D), visceral WAT volume (E), and subcutaneous WAT volume (F). Student's t test, *P<0.05. TTR.GFP, n=10; TTR.VGLL4, n=10. G. Liver weight.H. qRT-PCR measurement of human VGLL4 mRNA level. Gene expression level was normalized to 36B4. D and F, Student's t test, *P<0.05; ***, P<0.001. I. Correlation between body weight and VGLL4 transgene expression level. Pearson's correlation coefficient analysis, R=-0.77, p=0.0096.

[0033] FIGs.5A-5H show AAV9.TTR.VGLL4 treatment reduces NASH mice body weight, in accordance with aspects of the present disclosure. A. Therapeutic experiment design. NASH mice were transduced with AAV9.TTR.VGLL4 (treatment) or AAV9.TTR.GFP (Control). B. Body weight measurements. C. Accumulated body weight gain measurements. The glycemia of AAV9.TTR.GFP or AAV9.TTR.VGLL4 transduced mice were analyzed at 5 weeks and 7 weeks after AAV transduction, respectively. D. Glucose tolerance test (GTT). E. Insulin tolerance test (ITT). F. Alanine transaminase (ALT) blood test. Serum was collected at 10 weeks after AAV transduction and used for ALT test. B, C, D, E and F, chew diet control, n=5; AAV9.TTR.GFP, n=9; AAV9.TTR.VGLL4, n=7. B and C, Student t-test comparison between AAV9.TTR.GFP and AAV9.TTR.VGLL4 treated NASH mice. *, p<0.05, **, p<0.01. F, one-way ANOVA test. **, p<0.01; ****, p<0.0001. G-H. Measurement of food uptake (G) and feces excretion (H). N=6 for each group.

[0034] FIGs.6A-6F show AAV9.TTR.VGLL4 treatment reduces body fat, in accordance with aspects of the present disclosure. A. The appearance of NASH mice receiving vehicle (GFP) or VGLL4 treatment. B. In situ image of the liver and visceral white adipose tissue (WAT). Yellow arrows indicate peri-gonadal white adipose tissue (pgWAT). A and B, scale bar = 1cm. C. pgWAT weight. D. Inguinal WAT (iWAT) weight. E. liver wight. F. Heart weight. C to F, NASH+GFP, n=14; NASH+VGLL4, n=12. Student t-test, *, P<0.05; **, P<0.01.

[0035] FIGs.7A-7F show AAV9.TTR.VGLL4 treatment reduces hepatocytes steatosis, in accordance with aspects of the present disclosure. A. Hematoxylin and eosin staining shows the liver pathohistology. Livers from the same age, chew-diet fed mice were used as normal control. Scale bar = 50 µm. B. Liver section immunofluorescence images displaying the size of hepatocytes and lipid droplets. Phalloidin (red) and BODIPY (green) were used to label cell cortex and lipid droplets, respectively. Scale bar = 50 µm. C. Quantification of hepatocyte size. D. Quantification of lipid droplet size. C and D, for each group, 300 hepatocytes (C) and lipid droplets (D) from 3 mice were measured. Mann- Whitney test, ****, P<0.0001. E and F. qRT-PCR measurements of fatty acid binding genes (E) and lipid storage related genes (F). For each group, n=6. Student t-test, *, P<0.05.

[0036] FIGs.8A-8E sow AAV9.TTR.VGLL4 treatment reduces the expression of TAZ / YAP-TEAD target genes, in accordance with aspects of the present disclosure. A. Picrosirius red staining of the liver sections. Scale bar = 50 µm. B to E, qRT-PCR measurement of YAP / TEAD target genes (B), ColA1 (C), matrix metalloproteinases genes (D), and a metallopeptidase inhibitor gene (E). B. Student t-test, *, P<0.05. For each group, n=6. C and D, One way-ANOVA test, *, P<0.05; **, P<0.01. Chew diet control, n=5; NASH+GFP, n=8; NASH+VGLL4, n=6. NS, no significant difference.

[0037] FIGs.9A-9D show AAV9.TTR.VGLL4 treatment of NASH mice reduces hepatic inflammation, in accordance with aspects of the present disclosure. A. CD68 immunofluorescence staining of liver sections. Yellow arrowheads indicate crown-like structures and inflammatory cell aggregates. Scale bar = 50 µm. B-D, qRT-PCR measurement of pro-inflammatory cytokine genes (B), chemokine genes (C), and Toll like receptor genes (D). Chew diet control, n=4; NASH+GFP, n=8; NASH+VGLL4, n=7. One- way ANOVA test, *, P<0.05. NS, no significant difference.

[0038] FIGs.10A-10F show loss of hepatic VGLL4 does not affect liver growth, in accordance with aspects of the present disclosure. A. Breeding strategy to deplete VGLL4 in the liver. B. Liver weight (LW) to body weight (BW) ratio. N=8. C. Livers from age-matched control and Vgll4hkOmice. Scale bar = 5 mm. D. Hematoxylin and Eosin (H&E) stained liver sections.50 μm. E. YAP and VGLL4 western blots. GAPDH was used as loading control. F. Quantitative RT-PCR. The relative (Rel.) mRNA levels of Cyr61 and Ctgf were normalized to Gapdh. N=4. Student t-test, *p<0.05. B, C, D, and E, livers were collected from 8-month-old mice fed with chow diet.

[0039] FIGs.11A-11G show loss of hepatic VGLL4 predisposes the liver to develop cirrhosis under HFFC stress, in accordance with aspects of the present disclosure.6-8 weeks- old male Vgll4hKoand Vgll4fl / flmice were fed with HFFC diet for 8 weeks before being sacrificed for tissue collection. A. Body weight. B. Accumulated body weight gain. C. Liver weight to body weight ratio. A, B and C, Vgll4fl / fl, n=6; Vgll4hKo, n=8. Student t-test, *p<0.05; **, P<0.01. D. Phosphate saline buffer (PBS)-perfused livers. E. Spleens from HFFC-fed mice. D and E, scale bar = 1mm. F. Hematoxylin and Eosin (H&E) stained liver sections. Yellow Arrows indicate biliary ducts-like structures. G. Masson's Trichrome (MT) staining of liver sections. F and G, livers were fixed with 4% PFA and embedded in paraffin. Scale bar = 50 μm.

[0040] FIGs.12A-12F show HFFC diet stress impairs the homeostasis of Vgtt4hKo hepatic cells, in accordance with aspects of the present disclosure. A. Liver sectionimmunofluorescence images. Livers were collected from mice treated with HFFC diet for 8 weeks. Yellow arrowheads indicate proliferating cells; white aster indicate enlarged hepatocytes loaded with macrovacuoles. Scale bar = 50 μm. B. YAP and TEAD1 western blots. Total protein from the livers of NASH mice (on HFFC diet for 40 weeks) and the same age chow-diet control mice were used for analysis. C. Densitometry quantification of YAP and TEAD1. D. Western blots of YAP, TEAD1 and VGLL4. Total proteins from the livers of HFFC-diet stressed Vgll4fl / fland Vgll4hKomice were used tested. Yellow aster indicates a non- specific ban; arrow indicates VGLL4 protein. E. Densitometry quantification of YAP and TEAD1. B and D, TEAD1 and YAP protein levels were normalized to GAPDH. N=3-5. Student t-test, *, P<0.05. F. Schematic summary of the current findings.

[0041] FIGs.13A-13F show AAV.VGLL4HF4Atreatment does not reduce NASH mice body weight. NASH mice were transduced with AAV.VGLL4HF4Aor AAV.GFP. A. Body weight. B. Liver weight to Tibia bone (TB) length ratio. C. Perigonadal white adipose tissue (pgWAT) weight to TB length ratio. D. Inguinal white adipose tissue (iWAT) to TB length ratio. E. Food uptake. A-E, n=6 for each group. NS, not significant. F. Western blot of VGLL4HF4A-GFP.10 weeks after AAV treatment, the total protein isolated from NASH livers were applied for western blot. GFP antibody was used to detect the expression of VGLL4HF4A-GFP merge protein. DETAILED DESCRIPTION

[0042] This disclosure relates to a polynucleotide including a sequence encoding a vestigial like 4 (Vgll4) protein and a cis-regulatory element, wherein the cis-regulatory element controls hepatocyte-specific expression of the encoded protein. The polynucleotide may be included in a vector for promoting cellular transfection, such as a viral vector. As disclosed herein, administering such a polynucleotide or vector to an organism is a treatment for NAFLD, NAFL, and NASH, as well as related conditions such as steatohepatitis, hyperglycemia, insulin resistance, type II diabetes, and obesity.

[0043] The cis-regulatory element may include sequences known to drive expression of an associate coding sequence in hepatocytes but not in other cell types. Controlling hepatocyte-specific expression means causing, permitting, or stimulating expression in hepatocytes differentially from in other types of cells when transfected with the polynucleotide. A protein encoded by a polynucleotide regulated by a cis-regulatory element that controls hepatocyte-specific expression, for example, may be expressed in hepatocytes in a detectable amount but substantially not other cell types. Protein expression substantially notin other cell types may be not detectably expressed at all in one or more other cell type or, if detectably expressed at all in another cell type, detectably expressed at a substantially lower level than in hepatocytes, such as at 10% or less than in hepatocytes, 5% or less than in hepatocytes, 1% or less than in hepatocytes, or 0.1% or less than in hepatocytes, 0.01% or less than in hepatocytes, or at 0.001% or less than in hepatocytes, or below a level of detection which detection detects expression of the protein in hepatocytes. “Other cell types” may be any one or more of heart cells, liver cells, kidney cells, lung cells, spleen cells, and adipocytes.

[0044] Vgll4 is a transcription co-factor known to interact with cellular signaling molecules and transcription factors to influence cell survival and cell function. Vgll4 is particularly known for promoting cellular death by inhibiting YAP-TEAD1 complex. Several isoforms of Vgll4 have been identified, arising from splice variants to the Vgll4 gene. These include Vgll4A, Vgll4B, Vgll4C Vgll4D, Vgll4E, and Vgll4F. Amino acid sequences of these Vgll4 proteins (referred to collectively here as Vgll4), and examples of polynucleotides encoding them encoding them, are given in Tables 1 and 2, respectively. Vgll4 has been linked with an anticancer effect in several types of cancer, where lower levels of Vgll4 correlate or correspond with or cause increased tumor cell survival and higher levels of Vgll4 correlate or correspond with or cause an anti-tumor effect including decreased metastatic processes and decreased tumor cell survival or proliferation. See Deng, Vgll4 is a transcriptional cofactor acting as a novel tumor suppressor via interacting with TEADs, Am J Cancer Res (2018), 8(6):932-943. In this respect, Vgll4 differs from other member of the vestigial like (Vgll) family (Vgll1, Vgll2, and Vgll3) of transcription co-factors, which are not known to have tumor-suppressive functions. Vgll family members other than Vgll4 are not generally understood to share functional commonalities with Vgll4.

[0045] A cis-regulatory element may include a promotor, an enhancer, or both. In some cases, a sequence for a cis-regulatory element may be located within fewer than 10 nucleotides from a transcription start site, fewer than 20 nucleotides from a transcription start site, fewer than 30 nucleotides from a transcription start site, fewer than 40 nucleotides from a transcription start site, fewer than 50 nucleotides from a transcription start site, fewer than 60 nucleotides from a transcription start site, fewer than 70 nucleotides from a transcription start site, fewer than 80 nucleotides from a transcription start site, fewer than 90 nucleotides from a transcription start site, fewer than 100 nucleotides from a transcription start site, fewer than 125 nucleotides from a transcription start site, fewer than 150 nucleotides from a transcription start site, fewer than 175 nucleotides from a transcription start site, fewer than200 nucleotides from a transcription start site, fewer than 225 nucleotides from a transcription start site, fewer than 250 nucleotides from a transcription start site, fewer than 275 nucleotides from a transcription start site, fewer than 300 nucleotides from a transcription start site, fewer than 325 nucleotides from a transcription start site, fewer than 35 nucleotides from a transcription start site, fewer than 375 nucleotides from a transcription start site, fewer than 400 nucleotides from a transcription start site, fewer than 425 nucleotides from a transcription start site, fewer than 450 nucleotides from a transcription start site, fewer than 475 nucleotides from a transcription start site, fewer than 500 nucleotides from a transcription start site, or between 500 and 1,000 nucleotides from a transcription start site

[0046] A promoter is a nucleotide sequence to which RNA polymerizing enzymes bind for initiation of transcription of a downstream gene sequence. Many genes that show tissue- or cell-type specific expression including a promotor upstream of the DNA sequence that codes for the RNA that is particularly active in cells where the gene is expressed. A promoter may be more active in some cells than other, such as being active only in specific cell- or tissue-types, or highly active in certain cell- or tissue-types relative to others. Promoters include a sequence where transcription is initiated. Eukaryotic promoters may and typically do include features such as a TATA box, a transcription factor IIB recognition site, and a core promotor sequence (or an initiator). Transcription factors bind and RNA polymerase bind to a promoter for transcription initiation.

[0047] Also included in a cis-regulatory element may be one or more enhancer sequence. An enhancer may be part of a cis-regulatory element that enhances transcription initiated in or by the promotor. An enhancer may serve to promote an initiation of transcription at a promoter, for example, such as through binding of additional transcription factors to the enhancer that facilitate or enhance recruitment of other factors and transcriptional machinery to the promotor. As with promotors, many genes have enhances that are involved in cell- or tissue-specific or cell- or tissue-enhanced expression.

[0048] Cis-regulatory elements that control hepatocyte-specific expression of an associated polynucleotide coding sequence are known. Some non-limiting examples of proteins expressed in liver whose genes include cis-regulatory elements, including enhancers, promoters, or both, that have been identified as useful for controlling hepatocyte-specific expression of recombinant transgenes include, without limitation, albumin, alpha 1- antitrypsin, hepatitis B virus core protein, hemopexin, thyroglobulin, and transthyretin.

[0049] Examples of cis-regulatory elements, including enhancers, promoters, or both, that control hepatocyte-specific expression that may be included in a polynucleotide asdisclosed herein are described in references known to skilled persons, including Chuah at al, Liver-specific transcriptional modules identified by genome-wide in silico analysis enable efficient gene therapy in mice and non-human primates. Mol Ther.2014 Sep;22(9):1605-13; Yan et al., 1990, Distinct positive and negative elements control the limited hepatocyte and choroid plexus expression of transthyretin in transgenic mice. EMBO J., 9(3):869-78; Vanrell et al., Development of a liver-specific Tet-on inducible system for AAV vectors and its application in the treatment of liver cancer. Mol Ther.2011 Jul;19(7):1245-53; Kramer et al., In vitro and in vivo comparative study of chimeric liver-specific promoters. Mol Ther.2003 Mar;7(3):375-85; Tanigawa et al., Expression of cholesteryl ester transfer protein in mice promotes macrophage reverse cholesterol transport. Circulation.2007 Sep 11;116(11):1267- 73; Minghetti et al., Molecular structure of the human albumin gene is revealed by nucleotide sequence within q11-22 of chromosome 4. J Biol Chem.1986 May 25;261(15):6747-57; De Simone et al., Cis- and trans-acting elements responsible for the cell-specific expression of the human alpha 1-antitrypsin gene. EMBO J.1987 Sep;6(9):2759-66; Poli et al., The analysis of the human hemopexin promoter defines a new class of liver-specific genes. Nucleic Acids Res.1989 Nov 25;17(22):9351-65; Guo et al., Hepatocyte-specific expression of the hepatitis B virus core promoter depends on both positive and negative regulation. Mol Cell Biol.1993 Jan;13(1):443-8; Postic et al., Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem.1999 Jan 1;274(1):305-15; Lee et al. The Hippo-Salvador pathway restrains hepatic oval cell proliferation, liver size, and liver tumorigenesis. Proc Natl Acad Sci U S A.2010 May 4;107(18):8248-53; Nathwani et al., Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med.2011 Dec 22;365(25):2357- 65; Nathwani et al., Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver. Blood.2006 Apr 1;107(7):2653-61; Nathwani et al., Safe and efficient transduction of the liver after peripheral vein infusion of self- complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates. Blood.2007 Feb 15;109(4):1414-21; Nathwani et al., Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med.2014 Nov 20;371(21):1994-2004; Nathwani et al., Long-term safety and efficacy following systemic administration of a self-complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins. Mol Ther.2011 May;19(5):876-85; Cai et al., YAP-VGLL4 antagonism defines the major physiological function of the Hippo signaling effector YAP.Genes Dev.2022 Nov-Dec 1;36(21-24):1119-1128; Nair et al., 2014, Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy, Blood, 123(20):3195-9; Domenger et al., 2019, AAV vectors-do not judge a virus (only) by its cover, Hum Mol Genet, 28(R1):R3-R14; Wu et al., 2008, Optimization of self-complementary AAV vectors for liver-directed expression results in sustained correction of hemophilia B at low vector dose, Mol Ther, 16(2):280-9; WO2021128692A1; US 2023 / 0045478 A1; US 11,191,847B2; WO2009130208; US 2011 / 0184049 A1; WO2014064277; US 2015 / 0283267 A1; WO2017074526; and US 2023 / 0119850 A1, the entireties of all of which references are hereby incorporated by reference herein.

[0050] Non-limiting examples of enhancers of cis-regulatory elements that control hepatocyte-specific expression are presented in Table 3. A cis-regulatory element controlling hepatocyte-specific expression of an associated polynucleotide in accordance with the present disclosure may include, as a non-limiting example, any one or more, in any combination, of an enhancer for liver expression disclosed in any of the foregoing references, an enhancer for albumin, alpha 1-antitrypsin, hepatitis B virus core protein, hemopexin, thyroglobulin, or transthyretin, or an enhancer having a sequence listed in Table 3 (e.g., SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and / or SEQ ID NO: 23).

[0051] Non-limiting examples of promoters of cis-regulatory elements that control hepatocyte-specific expression are presented in Table 4. A cis-regulatory element controlling hepatocyte-specific expression of an associated polynucleotide in accordance with the present disclosure may include, as a non-limiting example, any one or more, in any combination, of a promoter for liver expression disclosed in any of the foregoing references, a promoter for albumin, alpha 1-antitrypsin, hepatitis B virus core protein, hemopexin, thyroglobulin, or transthyretin, or a promoter having a sequence listed in Table 4 (e.g., SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, and / or SEQ ID NO: 37).

[0052] Non-limiting examples of cis-regulatory elements that control hepatocyte- specific expression are presented in Table 6 (e.g., SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and / or SEQ ID NO: 47). A cis-regulatory element controlling hepatocyte-specific expression of an associated polynucleotide in accordance with the present disclosure may include, as a non-limiting example, any one or more, in any combination, of a cis-regulatory element for controlling hepatocyte-specific expression disclosed in any of theforegoing references, a cis-regulatory element for albumin, alpha 1-antitrypsin, hepatitis B virus core protein, hemopexin, thyroglobulin, or transthyretin, or a cis-regulatory element having a sequence listed in Table 6. A cis-regulatory element controlling hepatocyte-specific expression of an associated polynucleotide in accordance with the present disclosure may include, without limitation, a combination of any one or more of the foregoing enhancers, including of the enhancers with sequences listed in Table 3, with any one or more of the foregoing promoters, including of the promoters with sequences listed in Table 4. A cis- regulatory element controlling hepatocyte-specific expression of an associated polynucleotide in accordance with the present disclosure may include combinations of any of the foregoing enhancers, including more than one of any one or more of any of the foregoing enhancers.

[0053] At least six isoforms (A-F) of Vgll4 have been identified, referred to herein as Vgll4A, Vgll4B, Vgll4C, Vgll4D, Vgll4E, and Vgll4F, having amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. These six isoforms are collectively included in the term Vgll4 as used herein. Also included herein is any nucleotide sequence that encodes any of the foregoing Vgll4 isoforms, including, without limitation, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, including one or more codon substitution to any of the foregoing nucleotide sequences that nevertheless still encodes a Vgl4 (e.g., A- F), owing to codon degeneracy. A construct as disclosed herein may include a nucleotide sequence encoding a Vgll4 peptide as disclosed herein with any cis-regulatory element controlling hepatocyte-specific expression as disclosed herein, including sequences disclosed in Table 3, Table 4, and Table 6, including any variation thereof described above.

[0054] A Vgll4 protein, in accordance with the present disclosure, may be a human Vgll4, or mouse or rat Vgll4, or a Vgll4 sequence having at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with any of the Vgll4 amino acid sequences presented in Table 1. In an example, A Vgll4 peptide may include one or more amino acid substitution (relative to the examples disclosed in Table 1). In an example, a Vgll4 peptide may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ,16 ,17 ,18 ,19, or 20 amino acid substitutions, or any range of amino acid substitutions between any two of the foregoing number of amino acid substitutions (relative to the examples disclosed in Table 1).

[0055] Vgll4 peptides include two TONDU (or TDU) domains, referred to herein as TDU_1 and TDU_2. Each TDU domain includes an HF dipeptide sequence. A Vgll4 as disclosed herein may include one or more amino acid substitution not including within one or either TDU domain, or not including within one or either TDU domain HF dipeptide. The first TDU domain, referred to as TDU_1, has the amino acid sequence DPVVEEHFRRSLGKNY (SEQ ID NO: 49). The second TDU domain, referred to as TDU_2, has the amino acid sequence TGSVDDHFAKALGDTW (SEQ ID NO: 50). The HF dipeptides within the TDU_1 and TDU_2 domains are underlined in the preceding sentences. The Vgll4 peptide sequences presented in Table 1 include SEQ ID NO: 49 and SEQ ID NO: 50, i.e., as amino acids 212-227 and 240-255, respectively, of SEQ ID NO: 1, amino acids 206-221 and 234-249, respectively, of SEQ ID NO: 2, 126-141 and 154-169, respectively, of SEQ ID NO: 3, amino acids 122-137 and 150-165, respectively, of SEQ ID NO: 4, amino acids 211-226 and 239-254, respectively, of SEQ ID NO: 5, and amino acids 147-162 and 175-190, respectively, of SEQ ID NO: 6.

[0056] One or both amino acid of an HF dipeptide of one or both TDU domains in a Vgll4 peptide as disclosed herein may be substituted, independently, with a different amino acid (e.g., an amino acid other than H substituted in place of an H in one or both HF dipeptide and / or an amino acid other than F substituted in place of an F in one or both HF dipeptide). A TDU_1 domain allowing for a substitution at one or both amino acid of the HF dipeptide has the amino acid sequence DPVVEEX1X2RRSLGKNY (SEQ ID NO: 51), wherein X1may be H or any amino acid other than H, and, independently, X2 may be F or any amino acid other than F. A TDU_2 domain allowing for a substitution at one or both amino acid of the HF dipeptide has the amino acid sequence TGSVDDX3X4AKALGDTW (SEQ ID NO: 52), wherein X3may be H or any amino acid other than H, and, independently, X4may be F or any amino acid other than F. One or more of X1, X2, X3, and X4, in any combination, including all of X1, X2, X3, and X4, may be A. A Vgll4 in accordance with the present disclosure may include one or both of, independently, SEQ ID NO: 51 and SEQ ID NO: 52 substituted for one or both of SEQ ID NO: 49 and SEQ ID NO: 50, respectively, i.e., for one or both of amino acids 212-227 and 240-255, respectively, of SEQ ID NO: 1, one or both of amino acids 206-221 and 234-249, respectively, of SEQ ID NO: 2, one or both of amino acids 126-141 and 154-169, respectively, of SEQ ID NO: 3, one or both of amino acids 122- 137 and 150-165, respectively, of SEQ ID NO: 4, one or both of amino acids 211-226 and 239-254, respectively, of SEQ ID NO: 5, and / or one or both of amino acids 147-162 and 175- 190, respectively, of SEQ ID NO: 6. One or more of X1, X2, X3, and X4, in any combination,including all of X1, X2, X3, and X4, may be an amino acid conservatively substituted for A instead of H or F, respectively, including P, G, E, D, Q, N, S, or T. Substituting amino acids for the TDU domain HF dipeptides is known to disrupt the ability of Vgll4 peptide to bind TEAD and, correspondingly, Vgll4’s ability to suppress YAP-TEAD complex formation.

[0057] Skilled persons would envisage polynucleotide sequences that encode any permissible amino acid sequence included within SEQ ID NO: 51 and SEQ ID NO: 52. A skilled person would likewise envision a substitution of tri-nucleotides corresponding to codons in a nucleotide sequence encoding a Vgll4 having a TDU_1 domain having a sequence of SEQ ID NO: 51, a TDU_2 domain having a sequence of SEQ ID NO: 52, or both, wherein one or both of X1and X3is not H, one or both of X2and X4is not F, or both, and including it in a nucleotide sequence encoding a Vgll4 in a polynucleotide as otherwise disclosed herein, including with a cis-regulatory element controlling hepatocyte-specific expression. See Table 7 for amino acid sequences of TDU domains of Vgll4 proteins, with or without possible amino acid substitutions for H and / or F of the relevant TDU HF dipeptide sequences.

[0058] For a Vgll4 peptide encoded by a nucleotide sequence of any of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, nucleotides corresponding to nucleotides 652-657, nucleotides 634-639, nucleotides 394-399, nucleotides 382-387, nucleotides 649-654, or nucleotides 457-462, respectively (which encode the HF dipeptides of the TDU_1 domains), could be modified, according to known correspondences of nucleotide codons to amino acids encoded thereby, for a dipeptide sequence of the encoded Vgll4 wherein X1X2(as per SEQ ID NO: 51) encodes either an H for X1 but not an F for X2, an F for X2 but not an H for X1, or neither an H for X1 nor an F for X2. All possible substitutions for H and F in X1and X2described above, including A or conservative substitutions for A (such as P, G, E, D, Q, N, S, or T), or other substitution for H and / or F, and any corresponding nucleotide substitution to include codon(s) for any of the foregoing, is included in the present disclosure. See Table 8 for polynucleotide sequences encoding amino acid sequences of TDU domains of Vgll4 proteins, with or without possible amino acid substitutions for H and / or F of the relevant dipeptide sequences.

[0059] For a Vgll4 peptide encoded by a nucleotide sequence of any of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, nucleotides corresponding to nucleotides 736-741, nucleotides 718-723, nucleotides 478-483, nucleotides 466-471, nucleotides 733-738, or nucleotides 541-546, respectively (which encode the HF dipeptides of the TDU_2 domains), could be modified, according to knowncorrespondences of nucleotide codons to amino acids encoded thereby, for a dipeptide sequence of the encoded Vgll4 wherein X3X4 (as per SEQ ID NO: 52) encodes either an H for X3but not an F for X4, an F for X4but not an H for X3, or neither an H for X3nor an F for X4. All possible substitutions for H and F in X1 and X2 described above, including A or conservative substitutions for A (such as P, G, E, D, Q, N, S, or T), or other substitution for H and / or F, and any corresponding nucleotide substitution to include codon(s) for any of the foregoing, is included in the present disclosure. See Table 8 for polynucleotide sequences encoding amino acid sequences of TDU domains of Vgll4 proteins, with or without possible amino acid substitutions for H and / or F of the relevant dipeptide sequences.

[0060] Also disclosed herein are a Vgll4 fragment, polynucleotide encoding such a fragment with a cis-regulatory element (including without limitation and enhancer and / or promoter disclosed herein) controlling hepatocyte specific expression thereof and any intron including any as disclosed herein, and any vector disclosed herein including such a construct such as any viral vector disclosed herein including any AAV viral vector disclosed herein. All of the foregoing and following examples given in respect of a Vgll4 protein is likewise applicable in respect of a Vgll4 fragment as disclosed herein, substituting a Vgll4 fragment sequence or polynucleotide encoding such fragment for any foregoing or following example of Vgll4 protein sequence or polynucleotide encoding such protein, expressly and without limitation. Likewise any cell or non-human organism transfected with any such polynucleotide or vector, or any use of any such polynucleotide or vector, including for transfecting any cell or organism, or for treatment of steatohepatitis, treatment of obesity, treatment of hyperglycemia, method of treating diabetes, method of treating insulin resistance, and method of decreasing adipose tissue, as disclosed herein, without limitation, is expressly included in the present disclosure, without limitation. All combinations of all such features disclosed herein are expressly included within the present disclosure.

[0061] A Vgll4 fragment includes a portion of full Vgll4 protein that retains at least some structural and functional features of full Vgll4 protein. For example, Vgll4 fragments including one or both of the TDU domains are known to retain functions of full-length Vgll4. Examples of Vgll4 fragments disclosed herein are given in Table 8 and include the TDU_1 domain (SEQ ID NO: 49), the TDU_2 domain (SEQ ID NO: 50), and a tandem sequence including the TDU_1 domain, the TDU_2 domain, and a linker connecting them including as a non-limiting example the intervening Vgll413 amino acid sequence connecting them, with or without additional N- or C-terminal amino acids (SEQ ID NO: 53 and SEQ ID NO: 54). The sequence of linker amino acids connecting the TDU_1 domain to the TDU_2 domainmay differ in length and / or amino acid composition from the 13 Vgll4 amino acids included in linker sequence in SEQ ID NO: 53 and SEQ ID NO: 54. It may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 1, 18, 29, 20, 21, 22, 23, 24, 25, or 26 amino acids in length, or have a range between any two of the foregoing lengths, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the13 Vgll4 amino acids included in linker sequence in SEQ ID NO: 53 and SEQ ID NO: 54 may be substituted with another amino acid, such as by a conservative amino acid substitution as disclosed herein. Any such fragment that retains all or some functionality of full-length Vgll4 peptide (e.g. of Vgll4 disclosed in Table 1) is included as a Vgll4 fragment as disclosed herein. See, for example, Jiao et al., 2014, A peptide mimicking VGLL4 function acts as a YAP antagonist therapy against gastric cancer, Cancer Cell, 25(2):166-80, incorporated by reference herein in its entirety, disclosing that such fragments of Vgll4 inhibiting YAP-stimulated cancer cell viability and clonogenicity and TEAD4 reporter gene expression.

[0062] An amino acid of one type of class may be substituted by another amino acid in the same class, or having similar chemical or physical properties, as would be understood by skilled persons, in what is referred to as a conservative substitution. A conservative substitution is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. In general, a substitution of one amino acid within the following groups for another amino acid within the following groups represents a conservative substitution: (1) Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr; (2) Cys, Ser, Try, Thr; (3) Val, Ile, Leu, Met, Ala, Phe; (4) Lys, Arg, His; and (5) Phe, Tyr, Trp, His. As skilled persons would appreciate, a conservative amino acid substitution, which is a substitution of one amino acid within a foregoing group (1), (2), (3), (4), or (5) for another within the same group, would not be expected to disturb Vgll4 function. A Vgll4 peptide in accordance with the present disclosure may include conservative amino acid substitutions for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of a Vgll4 amino acid sequence, or any range of conservative amino acid substitutions between any two of the foregoing number of amino acid substitutions (relative to the examples disclosed in Table 1). A Vgll4 as disclosed herein may include one or more conservative amino acid substitution not including within one or either TDU domain, or not including within one or either TDU domain HF dipeptide.

[0063] A polynucleotide disclosed herein may include a polynucleotide sequence encoding for a Vgll4 protein wherein the Vgll4 protein may have any Vgll4 amino acidsequence as disclosed herein, including without limitation a Vgll4 amino acid sequence disclosed in Table 1 or any of the variations thereto described in the foregoing paragraphs. Non-limiting examples of polynucleotide sequences encoding Vgll4 protein amino acid sequences in accordance with the present disclosure are given in Table 2, including sequences encoding a Vgl4A protein, a Vgll4B protein, a Vgll4C protein, a Vgll4D protein, a Vgll4E protein, and a Vgll4F protein. Also included herein is any nucleotide sequence that encodes any of the foregoing examples of Vgll4 isoforms, including, without limitation, including one or more codon substitution to any of the foregoing nucleotide sequences that nevertheless still encodes a Vgll4 (e.g., relative to any sequence disclosed in Table 2), owing to codon degeneracy whereby more than one codon may be used to encode for a given amino acid in a protein’s amino acid sequence.

[0064] Any polynucleotide disclosed herein may include an intron, including between a cis-regulatory element and a coding sequence for a transgene. Transgene expression may be enhanced by including an intron between a cis-regulatory element controlling expression of the transgene and the coding sequence of the transgene. Numerous introns are known to favor transgene expression in this matter, including in liver and including when included in a polynucleotide sequence carried in a viral vector such as an AAV vector. Examples of references disclosing such introns and their sequences include Chuah et al., 2014, Liver- specific transcriptional modules identified by genome-wide in silico analysis enable efficient gene therapy in mice and non-human primates. Molecular Therapy 22, 1605-1613; Yan et al., 1990, Distinct positive and negative elements control the limited hepatocyte and choroid plexus expression of transthyretin in transgenic mice. EMBO J., 9(3):869-78; Nathwani et al., 2006, Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver, Blood, 107(7):2653-61; Nair et al., 2014, Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy, Blood, 123(20):3195-9; Wu et al., 2008, Optimization of self-complementary AAV vectors for liver-directed expression results in sustained correction of hemophilia B at low vector dose, Mol Ther, 16(2):280-9; Greig et al., 2023, Integrated vector genomes may contribute to long-term expression in primate liver after AAV administration. Nat Biotechnol. 2023 Nov 6, PMID: 37932420; Domenger et al., 2019, AAV vectors-do not judge a virus (only) by its cover, Hum Mol Genet, 28(R1):R3-R14; US 2023 / 0045478A1; US 2023 / 0119850A1; US 10,471,157B2; US 11,191,847B2; and US 11,419,950B2; the entireties of all of which references are hereby incorporated by reference herein. A non-limiting list ofintrons of the present disclosure for inclusion in a polynucleotide as disclosed here are given in Table 5 and include, without limitation, those with nucleotide sequences as set out in SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42.

[0065] A cell may be transfected with a construct as disclosed above by various methods, such as chemical transfection, electroporation, impalefaction, gene gun transfection, or viral vector mediated gene transfer, or any other method known to skilled persons in the relevant field. A Vgll4 coding sequence with associated cis-regulatory element controlling hepatocyte-specific expression may be packaged in a viral vector for cellular transfection. Viral vector in this case refers to a viral-like particle that contains or includes a payload gene construct or cassette capable of attaching to a cell and delivering the payload into the cell. In some examples, a viral vector may be of a type wherein a payload, once introduced into a transfected cell, integrates into the cell’s genomic DNA, though such genomic integration is not an essential feature of a viral vector as disclosed herein. Viral vector may also refer to a gene sequence including a gene construct or cassette structured for inclusion in a viral-like particle. Examples of viral vectors include retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAV). Several serotypes of AAV vectors are useful for cellular transfection, including any of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Anc80, rh10, or subtypes thereof. Recombinant AAV pseudotypes have also been engineered, including a genotype of one AAV serotype and capsid proteins of another or different AAV serotype, for liver expression (e.g., AAV 2 / 6, AAV 2 / 8, etc.). Also included are self-complementary AAV vectors (scAAV), in which self-complementary polynucleotide sequences of constructs are packaged. Including double-stranded, self-complementary sequences in some examples may improve expression after transfection with such an AAV vector. Also included are AAV vectors made using combinations of genetic sequences from different AAV serotypes for improving expression in hepatocytes. AAV-DJ is one such example, but all such examples are included within an AAV vector as described herein. Genetic sequences and methods of making any of the aforementioned AAV vectors are known and may be found in publicly accessible databases, as are methods of packaging a construct of interest in viral vector particles for cellular transfection and promotion of construct expression in transfected cells.

[0066] Examples are described in references known to skilled persons, including for example Greig et al., 2023, Integrated vector genomes may contribute to long-term expression in primate liver after AAV administration. Nat Biotechnol.2023 Nov 6, PMID: 37932420; Nathwani et al., Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med.2011 Dec 22;365(25):2357-65; Nathwani et al., Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver. Blood.2006 Apr 1;107(7):2653-61; Nathwani et al., Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates. Blood.2007 Feb 15;109(4):1414-21; Nathwani et al., 2014, Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med., 371(21):1994-2004; Nathwani et al., Long-term safety and efficacy following systemic administration of a self-complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins. Mol Ther.2011 May;19(5):876-85; Domenger et al., 2019, AAV vectors-do not judge a virus (only) by its cover, Hum Mol Genet, 28(R1):R3-R14; Wu et al., 2008, Optimization of self- complementary AAV vectors for liver-directed expression results in sustained correction of hemophilia B at low vector dose, Mol Ther, 16(2):280-9; Vanrell et al., 2011, Development of a liver-specific Tet-on inducible system for AAV vectors and its application in the treatment of liver cancer. Mol Ther., 19(7):1245-53; US 11,319,354B2; Bartel et al., 2012, Directed evolution of novel adeno-associated viruses for therapeutic gene delivery. Gene Ther., 19(6):694-700; Gray et al., 2010, Directed evolution of a novel adeno-associated virus (AAV) vector that crosses the seizure-compromised blood-brain barrier (BBB), Mol Ther., 18(3):570-8; Pei et al., 2020, Development of AAV Variants with Human Hepatocyte Tropism and Neutralizing Antibody Escape Capacity, Mol Ther Methods Clin Dev., 18:259- 268; Chuah at al, 2014, Liver-specific transcriptional modules identified by genome-wide in silico analysis enable efficient gene therapy in mice and non-human primates, Mol Ther, 22(9):1605-13; Li et al., 2008, Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles, Mol Ther, 16(7):1252-60; Tanigawa et al., 2007, Expression of cholesteryl ester transfer protein in mice promotes macrophage reverse cholesterol transport, Circulation, 116(11):1267-73; Sands, 2011, AAV-mediated liver-directed gene therapy, Methods Mol Biol, 807:141-57; Mao et al., 2016, Single point mutation in adeno-associated viral vectors -DJ capsid leads to improvement for gene delivery in vivo, BMC Biotechnol, 16, 1; Paulk et al., 2018, Bioengineered AAV Capsids with Combined High Human Liver Transduction In Vivo and Unique Humoral Seroreactivity, Mol Ther, 26(1):289-303; Liu et al., 2016, Simple Purification of Adeno-Associated Virus-DJ for Liver-Specific Gene Expression, Yonsei Med J, 57(3):790-4; Zinn, 2015, In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector, Cell Rep, 12(6):1056-68; Nair et al., 2014, Computationally designed liver-specific transcriptionalmodules and hyperactive factor IX improve hepatic gene therapy, Blood, 123(20):3195-9; Mooring et al., 2020, Hepatocyte Stress Increases Expression of Yes-Associated Protein and Transcriptional Coactivator With PDZ-Binding Motif in Hepatocytes to Promote Parenchymal Inflammation and Fibrosis, Hepatology, 71(5):1813-1830; Wang et al., 2016, Hepatocyte TAZ / WWTR1 Promotes Inflammation and Fibrosis in Nonalcoholic Steatohepatitis, Cell Metab, 24(6), 848-862; Lisowski et al., 2014, Selection and evaluation of clinically relevant AAV variants in a xenograft liver model, Nature, 506(7488):382-6; US2023 / 0119850A1; US 11,419,950 B2; US2023 / 0045478A; and US9,617,548B2, the entireties of all of which references are hereby incorporated by reference herein.

[0067] An AAV vector includes sequences bounding a payload construct referred to as inverted terminal repeats (ITRs). ITR sequences are involved in transcription of AAV genome, encapsulation of payload in a vector particle, genome multiplication for particle generation, and integration into host genome. A cassette, construct, transgene, payload, etc., placed between ITRs of an AAV vector may promote production of an AAV vector and / or expression of transfected gene within cells. In an example, a Ucp1 cis-regulatory element neighboring a nucleotide sequence encoding a Vgll4 peptide may be placed between ITRs and used for generation of an AAV particle, wherein said particle may be contacted with cells of an organism to transfect them with such construct.

[0068] Viral vectors other than AAV vectors for transfection with a polynucleotide as disclosed are also included in the present disclosure. Viral vectors other than AAV vectors for transfecting liver tissue are known, including for example lentiviral vectors. Included in the present disclosure are lentiviral vectors including as a payload a polynucleotide encoding a Vgll4 peptide or fragment thereof with a cis-regulatory element for controlling hepatocyte- specific expression, optionally including an intron. Appropriate lentiviral vectors and methods of their manufacture are known and included herein. Some examples of references disclosing such vectors include Dalsgaard et al., 2018, Improved Lentiviral Gene Delivery to Mouse Liver by Hydrodynamic Vector Injection through Tail Vein, Mol Ther Nucleic Acids, 12:672-683; Milani et al., 2022, Liver-directed lentiviral gene therapy corrects hemophilia A mice and achieves normal-range factor VIII activity in non-human primates, Nat Commun 13, 2454; US20210038744A1; US7745179B2; and US20200199626A1, the entireties of which references are hereby incorporated by reference herein.

[0069] A polynucleotide as disclosed herein may include nucleotide sequences within a coding sequence that lead to inclusion of amino acid sequences in addition to those of a Vgll4 protein or fragment thereof as disclosed herein, including at one or both of the amino-terminus and carboxyl terminus of the encoded protein. In some cases a reporter gene may be used or included in aVgll4 construct as disclosed herein to verify expression of a construct gene included in a vector or for testing tissue- or cell-type specific expression of a gene under control of a given cis-regulatory element. Numerous reporter genes are known and have been widely used in the relevant field. A non-limiting list of examples includes a green fluorescent protein (GFP), a yellow fluorescent protein, a red fluorescent protein, a blue fluorescent protein, a luciferase protein, a beta-galactosidase protein, a glutathione S-transferase protein, a chloramphenicol acetyltransferase protein, and any combination of two or more of the foregoing. Other reporters may also be included. In other examples, no reporter is included. By detecting expression of a reporter protein, the ability of a given cis-regulatory element, or viral vector or other vector including a polynucleotide having the cis-regulatory element, to promote transfection and / or expression in various cell and tissue types, e.g. hepatocytes, can be evaluated. A construct may include any nucleotide sequence for encoding any reporter protein.

[0070] A viral vector or viral-like particle, such as an AAV vector, can be injected into an organism, such as subcutaneously, intramuscularly, intravenously, intraperitoneally, or by other methods for introduction of the vector into the organism for contact with cells thereof. A vector may contact various different cell and tissue types and transfect them. However, inclusion of a cell- or tissue-specific cis-regulatory element (enhancer, promotor, or both) may restrict expression of the transfected gene to a given cell or tissue type or types, wherein the construct is not transcribed or is otherwise dormant or at most barely or minimally expressed in other cell types. As explained above, a cis-regulatory element may include elements that are known or believed to drive expression in hepatocytes, or liver cells, specifically. However, it is not necessary that expression be limited absolutely to a given cell type, including only in hepatocytes, even under control of a cis-regulatory element.

[0071] As disclosed herein, a subject having a disorder, syndrome, or condition, or susceptibility for any of the foregoing, related to excess accumulation of fat in the liver may be treated by administration of a polynucleotide as disclosed herein. Examples of a syndrome, condition, or disorder, or susceptibility therefor, a subject may have that such administration may treat include non-alcoholic fatty liver (NAFL), non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH). As disclosed herein, administering a polynucleotide as disclosed herein (including a polynucleotide encoding a Vgll4 protein, or fragment thereof, and a cis-regulatory element controlling hepatocyte- specific expression, and optionally including an intron), such as in a vector such as a viralvector such as an AAV vector, may be a treatment for NAFL, NASFLD, and / or NASH, or prevent the development of any of the foregoing. Similarly, it may also be used as a treatment of other conditions sometimes attendant with NAFL, NAFLD, and / or NASH. These include obesity, diabetes, insulin resistance, cirrhosis, hyperglycemia, and any combination of any two or more of the foregoing. As disclosed herein, driving hepatocyte-specific expression of Vgll4 or fragment thereof as disclosed herein is a treatment for one or more of NAFL, NAFLD, NASH, cirrhosis of the liver, obesity, diabetes, insulin resistance, cirrhosis, and hyperglycemia. Administering a polynucleotide, such as via a vector such as a viral vector such as an AAV vector, can prevent. reduce, or minimize, any and all of the foregoing conditions, syndromes, and diseases.

[0072] As used herein, “treatment” or “treating” refers to an approach for obtaining a therapeutic benefit in the form of eradication or amelioration of the condition, syndrome, disease, or disorder being treated, and also preventing the occurrence or worsening thereof, such as in a subject at risk for development or worsening thereof. A therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the condition, syndrome, disease, or disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the condition, syndrome, disease, or disorder. For example, obesity, type II diabetes, and hyperglycemia are risk factors for NAFL, NAFLD, NASH, and cirrhosis of the liver, NAFL is a risk factor for NAFLD, NASH, and cirrhosis of the liver, NAFLD is a risk factor for NASH and cirrhosis of the liver, and NASH is a risk factor for cirrhosis of the liver. Obesity is also a risk factor for type II diabetes, and type II diabetes is a risk factor for hyperglycemia. Treatment or prevention of any of the foregoing risk factors may include treatment of one of the conditions, syndromes, diseases, or disorders for which it is a risk factor.

[0073] Also disclosed herein is any cell or animal transfected with a polynucleotide or vector as described herein.

[0074] Also included is a method of screening a treatment for fatty liver disease or prevention of cirrhosis in a transgenic animal. Disclosed herein is a transgenic mouse wherein Vgll4 expression is knocked-out in an organ-specific manner such that its expression is disrupted in liver but not in other tissues, such as by being excised from he genomes of liver cells but not other cell types. Such mice and the manner of making them are known to skilled persons and have previously been disclosed. See, for example, Cai et al., 2022, YAP-VGLL4 antagonism defines the major physiological function of the Hippo signaling effector YAP. Genes Dev.36(21-24):1119-1128. Liver development and function in such mice is typical ofwild-type mice when fed a normal diet. However, unexpectedly and as disclosed here, when such mice are fed a diet high in fat (about 40% of total kcal), high in fructose (about 20% total kcal) and high in cholesterol (about 2%) (HFFC diet), they develop NASH sooner than control, wild-type mice fed HFFC diet.

[0075] Accordingly, disclosed herein is a method of screening a treatment for fatty liver disease or prevention of cirrhosis. Two groups of mice with liver-specific disruption of Vgll4 expression, such as a liver-specific or hepatocyte-specific Vgll4 knockout, may be fed a HFFC diet, one group administered a known or hypothesized treatment for fatty liver disease or cirrhosis. After several weeks of being fed such a diet, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, liver morphology between the two groups can be compared. The known or hypothesized treatment for fatty liver disease of cirrhosis is effective for such treatment when steatohepatitis or morphological characteristics of cirrhosis are absent or reduced in the mice administered the known or hypothesized treatment. In an example, the known or hypothesized treatment may be a viral vector including a polynucleotide encoding Vgll4 or a fragment thereof and a cis-regulatory element controlling liver-specific expression as further disclosed herein.

[0076] Examples of such steatohepatitis or morphological characteristics of cirrhosis for observation or measurement in such a method include, without limitation, higher liver to body weight ratio, liver tissue with presence or a number of irregular bumps rather than being smooth, presence of small ductular structures, presence or growth of cells of reduced size containing less cytoplasmic volume forming small ductular structures, fibrosis deposition, splenomegaly, increased number of cells positive for proliferative cell marker (such as but not limited to Ki67), irregular shaped hepatocytes rather than similar-sized polygonal hepatocytes, presence of enlarged hepatocytes, presence of hepatocytes occupied with macrovacuoles, TEAD1 upregulation, or any combination of two or more of the foregoing.

[0077] Included in the present disclosure is a means for stimulating production of a Vgll4 protein or fragment thereof in a cell. Such means include any and all of the polynucleotide sequences disclosed herein that encode a Vgll4 protein or fragment thereof, including without limitation those encoding a full-length Vgll4 disclosed in Table 2, and those disclosing a Vgll4 fragment having sequences of SEQ ID NO: 59 or SEQ ID NO: 60 of Table 8, and those encoding full-length Vgll4 with one or more amino acid substitution in one or more TDU domain HF dipeptide constituting a Vgll4 protein that does not suppress YAP-TEAD complex formation according to SEQ ID NO: 57 or SEQ ID NO: 58, as described more fully in this application for modification of sequences disclosed in Table 2.

[0078] Included in the present disclosure is means for controlling hepatocyte-specific expression of a polynucleotide sequence encoding a VGll4 protein or fragment thereof. Such means include any and all of the enhancers, promoters, and cis-regulatory elements disclosed herein, including without limitation those provided in Table 3, Table 4, and Table 6 herein.

[0079] Included in the present disclosure is means for transfecting cells with a polynucleotide including a cis-regulatory element for controlling hepatocyte-specific expression and sequence encoding a VGll4 protein or fragment thereof. Such means include any and all of the AAV and lentiviral vectors disclosed herein. EXAMPLES

[0080] The following examples are intended to illustrate particular embodiments of the present disclosure, but are by no means intended to limit the scope thereof.

[0081] Example 1. Overexpressing VGLL4 in hepatocytes does not affect postnatal liver growth.

[0082] A liver-specific pAAV.TTR.VGLL4 construct was generated by replacing the BCE sequence of AAV9.BCE.VGLL4 with HS-CRM8-TTR (Chuah et al., 2014, Liver- specific transcriptional modules identified by genome-wide in silico analysis enable efficient gene therapy in mice and non-human primates. Molecular Therapy 22, 1605-1613), a chimera cis-regulatory element that contains a hepatocyte-specific transcriptional module and the mini promoter of mouse transthyretin (TTR) gene (Yan at al., 1990, Distinct positive and negative elements control the limited hepatocyte and choroid plexus expression of transthyretin in transgenic mice. EMBO J 9, 869-878). Fig.1A. AAV9.TTR.GFP was used to examine the liver-specificity of our AAV vectors. Postnatal day 1 mouse pups were transduced with AAV9.TTR.GFP, and the internal organs including the heart, liver, kidney, lung and spleen, were collected at P14 to exhibit GFP expression pattern, which showed that only the liver expressed GFP. Fig.1B.

[0083] VGLL4 suppresses the YAP / TEAD complex, which is a master regulator of cell proliferation and survival. Overexpression of VGLL4 in the hepatocytes may therefore impair liver development. To test this possibility, P1 mouse pups were transduced with AAV9.TTR.VGLL4GFP(TTR.VGLL4) and their livers collected for analysis at P14. Compared with their littermate controls that did not receive AAV, TTR.VGLL4-transduced mouse pups had normal liver size, liver weight and liver to body weight ratio (Fig.1C and 1D). Both fluorescence imaging (Fig.1C) and western blot (Fig.1E) confirmed that VGLL4-GFP was successfully expressed in the liver. Together, these data show that overexpressing VGLL4 in hepatocytes does not impair postnatal liver growth.

[0084] Example 2. Overexpressing VGLL4 in hepatocytes mitigates body weight gain in high fat diet-fed mice.

[0085] Two month-old male C57BL / 6J mice were transduced with 1x10^10 GC / g AAV9.TTR.VGLL4 and the same dose of AAV9.TTR.GFP, respectively, and fed high fat diet (HFD, 60% of total kcal from fat) treatment two weeks after AAV injection. Similar with AAV9.BCE.VGLL4, AAV9.TTR.VGLL4 pretreatment significantly slowed down the body weight gain (Fig.3A and 3B). Different from AAV9.BCE.VGLL4, which did not improve the animals' glucose metabolism, AAV9.TTR.VGLL4 pretreatment improved glucose metabolism (Fig.3C and 3D) and increased insulin sensitivity (Fig.3E and 3F). Overexpressing VGLL4 specifically in liver therefore improves whole-body metabolism.

[0086] Example 3. AAV9.TTR.VGLL4 pre-treatment mitigates the expansion of white adipose tissue.

[0087] White adipose tissue (WAT) overgrowth is the main driver of obesity. To demonstrate effects of AAV9.TTR.VGLL4 pre-treatment on WAT expansion in HFD-treated mice, 11 weeks post HFD treatment, VGLL4 and GFP cohort animals were scanned with micro CT, and the volumes of different depots WAT were measured (Fig.4A). Compared with GFP control mice, VGLL4 treated mice were skinnier (Fig.4B) and showed no difference for lean mass volume (Fig.4C). Furthermore, VGLL4 mice had significantly less subcutaneous WAT (Fig.4D), similar visceral WAT (Fig.4E), and significantly lower total WAT volume than GFP controls (Fig.4F).

[0088] Expressing VGLL4 in hepatocytes increased the liver weight in chew diet-fed mice (Fig.2). However, under HFD stress, VGLL4 pre-treatment did not increase liver weight (Fig.4G). Expression of AAV-coded human VGLL413 weeks after AAV transduction was confirmed using a pair of primers that specifically amplify a fragment of the human VGLL4 gene (Fig.4H). Additionally, linear correlation analysis showed that body weight had a strong negative correlation with hepatic human VGLL4 expression level (R= - 0.77, 95% confidence interval, -0.94 to -0.27) (Fig.4I). Therefore, AAV9.TTR.VGLL4 pre- treatment is beneficial for reducing WAT expansion in HFD-induced obese mice.

[0089] Example 4. AAV9.TTR.VGLL4 treatment of NASH mice decreases body weight and improves liver function.

[0090] As disclosed herein, overexpressing VGLL4 specifically in hepatocytes attenuated HFD-induced metabolism dysfunction. Thus, AAV9.TTR.VGLL4-mediatedhepatic expression of VGLL4 may mitigate the progression of NAFLD. During the progression of NAFLD, the intermediate NASH stage is more concerning than the initial NAFL stage, because NASH patients are at a higher risk of developing cirrhosis or hepatocellular carcinoma. AAV9.TTR.VGLL4 treatment was therefore tested in a diet- induced NASH model, in which mice were treated with a modified Amylin liver NASH diet that contains 40% of total kcal from fat, 20% of total kcal from fructose and 2% cholesterol (Trevaskis et al., 2012, 2012, Glucagon-like peptide-1 receptor agonism improves metabolic, biochemical, and histopathological indices of nonalcoholic steatohepatitis in mice. American Journal of Physiology-Gastrointestinal and Liver Physiology 302, G762-G772).

[0091] To induce NASH, 6-week-old male C57BL / 6N mice were fed with NASH diet for 30 weeks, at which stage the mice had already developed hepatomegaly and the liver displayed histological features of NASH including hepatosteatosis and fibrosis. In the treatment cohorts, 1x10^10 vg / g AAV9.TTR.VGLL4 was delivered into the NASH mice through the retro-orbital vasculature. Another cohort of NASH mice receiving the same dose of AAV9.TTR.GFP were used as vehicle control. To further evaluate the therapeutic effects of AAV9.TTR.VGLL4, a cohort of 36-weeks-old C57BL / 6N mice that received no AAV and fed with chew diet as healthy control was also included (Fig.5A). Body weight of these three cohort mice was monitored for 10 weeks, and GTT and ITT were performed at 5 weeks and 7 weeks, respectively (Fig.5A).

[0092] Compared with GFP vehicle control, VGLL4 treatment significantly reduced body weight at as early as two weeks after AAV transduction, and this body weight reduction effect continued throughout the whole study period (Fig.5B and 5C). During the 10-weeks monitoring period, the average accumulated body weight gain of the healthy control, NASH+GFP and NASH+VGLL4 group, were 3.5±1.88 (g), 8.65±1.22 (g), and 6.1±2.9 (g), respectively (Fig.5C). Besides reducing body weight, VGLL4 treatment also improved glucose metabolism (Fig.5D). However, the treatment did not increase insulin sensitivity (Fig.5E). NASH is a common cause of serum alanine aminotransferase (ALT) activity elevation, and decreased ALT level is associated with liver function improvement. Control NASH mice had much higher serum ALT than the healthy control mice, and this serum ALT elevation was significantly reversed by VGLL4 treatment (Fig.5F). The physiological changes induced by VGLL4 were not due to food consumption impairment because both vehicle control and treatment groups had undistinguishable food update (Fig.5H) and feces excretion rate (Fig.5H).

[0093] Example 5. AAV9.TTR.VGLL4 treatment of NASH mice decreases white adipose tissue mass.

[0094] 8 weeks after AAV delivery, VGLL4 treated NASH mice were much skinner than the GFP control NASH mice (Fig.6A). Visceral and subcutaneous WAT depots were examined to determine whether VGLL4 treatment reduced body weight by decreasing WAT mass. Perigonadal WAT (pgWAT) is one of the largest visceral adipose depots in rodents. NASH+VGLL4 mice had much smaller pgWAT volume than the NASH+GFP mice (Fig. 6B), and the pdWAT weight significantly differed between these groups (Fig.6C). Furthermore, using inguinal WAT (iWAT) depot as an example, subcutaneous WAT mass was compared between these two groups. NASH+VGLL4 mice had significantly lower iWAT weight than the NASH+GFP mice (Fig.6D).

[0095] Because AAV9.TTR.VGLL4 therapy specifically targeted the liver, liver weight was also compared between NASH+VGLL4 and NASH+GFP mice. VGLL4 treatment had a trend to reduce liver weight in these NASH mice (Fig.6E). Unlike body fat and liver, the heart was not distinguishable between these two groups of NASH mice (Fig. 6F), indicating that expressing VGLL4 in the hepatocytes does not affect cardiac homeostasis.

[0096] Example 6. AAV9.TTR.VGLL4 treatment attenuates hepatocytes ballooning degeneration.

[0097] Hepatocyte ballooning, fibrosis deposition, and immune cells infiltration are histological signatures of NASH.As disclosed herein, VGLL4 treated NASH liver had much less hepatocytes ballooning than the NASH+GFP livers (Fig.7A). Furthermore, liver was stained with BODIPY, a fluorescence dye labeling lipid droplets, and Phalloidin, which binds to filament actin, to visualize hepatocyte lipid deposition and actin cortex, respectively. Compared with the NASH+GFP hepatocytes, VGLL4 treated NASH hepatocytes were much smaller (Fig.7B and 7C). The VGLL4 hepatocytes held many small lipid droplets, the size of which was dramatically reduced in comparison with the lipid droplets stored in the NASH+GFP hepatocytes (Fig.7B and 7D). On the molecular level, VGLL4 treatment did not change the expression of two fatty acid binding protein genes Fabp3 and Fabp4 (Fig. 7E), but decreased the expression of Cidea and Plin2 (Fig.7F), two genes positively correlated with hepatic steatosis and hepatocytes ballooning. Different from Plin2, Plin1 is mainly expressed in adipose tissue and is in charge of lipolysis. Compared with vehicle control NASH mice, VGLL4 treated NASH mice had decreased increased Plin1 expression (Fig.7F). Together, these data indicate that AAV9.TTR.VGLL4 treatment of NASH miceattenuates hepatocytes ballooning degeneration through improving hepatocytes lipid metabolism.

[0098] Example 7. AAV9.TTR.VGLL4 treatment decreases the expression of YAP / TEAD target genes.

[0099] Besides hepatocellular ballooning degeneration, hepatic fibrosis is another pathophysiological signature of NASH disease. Thus, liver sections were examined for fibrosis deposition. Compared with the same age chew diet-fed control mice that had a low degree of hepatic fibrosis primarily distributed around the perivascular regions, both the NASH+GFP and the NASH+VGLL4 mice developed wide spread pericellular fibrosis, and no obvious fibrosis deposition difference was noticed between these two groups of NASH mice (Fig.8A).

[0100] VGLL4 is a suppressor of YAP / TAZ-TEAD complex. Therefore, VGLL4 expression-induced decrease in YAP / TAZ target gene expression was assessed. Indian hedgehog signaling molecule (Ihh), communication network factor 1 (Ccn1, also known as Cyr61), and Ccn2 (also known as Ctgf) are three genes directly regulated by YAP / TAZ- TEAD complex, and increased expression of Ihh and Ccn1 is positively correlated with hepatic inflammation / fibrosis. VGLL4 treatment of NASH mice decreased the expression of TEAD target genes (Ihh and Ccn1), with the exception of Ctgf (Fig.8B).

[0101] In NASH liver, the expression of Col1a1 gene is correlated directly with hepatic matrix deposition. Compared with chew-diet fed mice, NASH+GFP mice had more than ten times higher hepatic Col1a1 mRNA level, which was significantly reversed in the NASH+VGLL4 mice (Fig.8C). This Col1a1 expression result was not consistent with histological data (Fig.8A), which did not show an obvious fibrosis deposition difference between NASH+GFP and NASH+VGLL4 liver. Because the extracellular matrix deposition is regulated by both collagen protein synthesis and collagen fiber degradation, and the latter is controlled by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), we examined the expression of Mmp8, Mmp13, Mmp2 and Timp1 in the liver was examined. NASH diet increased the expression of Mmp8, Mmp13, Mmp2, and VGLL4 treatment of the NASH mice reversed Mmp8 and Mmp13 expression (Fig.8D). The expression of Timp1 was neither affected by NASH diet nor by VGLL4 treatment (Fig.8E). These data indicate that, at least in part, VGLL4 may regulate liver fibrosis by repressing Col1a1 and crucial matrix metalloproteinase genes, which are all positively correlated with the progression of NASH disease.

[0102] Example 8. AAV9.TTR.VGLL4 treatment of NASH mice attenuates hepatitis.

[0103] During NASH, blood monocytes are recruited into the stressed liver and locally differentiate into monocyte-derived macrophages, which aggregate around dead / dying cells or surround hepatocytes with large lipid vacuoles to form hepatic crown-like structure (hCLS), a unique histological feature of steatohepatitis. To test whether expressing VGLL4 in the hepatocytes decreased steatohepatitis, liver sections were stained with CD68 antibody, a molecular maker that labels both residential Kupffer cells and blood-derived macrophages. In NASH+GFP liver, CD68+ macrophages formed hCLSs were densely distributed, whereas they were robustly decreased in the NASH+VGLL4 liver (Fig.9A). Second, the expression of two cytokine genes (Il-1b and Il-6) and two chemokine genes (Icam-1 and Ccl-2), the increase of which is associated with a higher risk of having non-alcoholic fatty liver disease, were examined. Compared with chew-died fed controls, the expression of hepatic Il-6 was not significantly changed in NASH mice; however, expression of hepatic Il-1b, Icam-1 and Ccl-2 were increased in NASH+GFP animals, which was largely reversed in the NASH+VGLL4 cohort (Fig.9B and 9C). Expression of two Toll like receptor genes, Tlr2 and Tlr4, both of which contribute to the progression of NASH, was also examined. In comparison with the chew-died fed controls, expression of Tlr2 and Tlr4 was increased in the NASH+GFP animals, although the Tlr2 expression varied among this cohort (Fig.9D). Similar with the cytokine genes, Tlr2 and Tlr4 were both decreased in the VGLL4 treated NASH mice (Fig.9D). Together, these data indicate that AAV9.TTR.VGLL4 treatment of NASH mice decreases hepatic inflammation.

[0104] Example 9. Liver-specific depletion of VGLL4 does not affect liver growth.

[0105] Germ-line loss of VGLL4 caused perinatal lethality. Sheldon et al., 2022, Depletion of VGLL4 Causes Perinatal Lethality without Affecting Myocardial Development. Cells 11, 283. To test whether VGLL4 regulates liver growth under physiological conditions, transgenic mice with a floxed Vgll4 allele (i.e., Vgll4 allele a portion of which is flanked by Cre recombinase recognition sequences such that excision of the sequence therebetween prevents normal expression thereof) were crossed with transgenic Alb-Cre mice, which express Cre recombinase specifically in hepatocytes and not other cell types (Postic et al, 1999, Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem 274, 305- 315) to deplete VGLL4 in liver (Fig.10A). In such transgenic animals, Cre recombinase, which excises regions of the genome flanked by flox sequences, is therefore restricted to hepatocytes, causing disruption of Vgll4 gene by excising the floxed region but only in hepatocytes, where Cre is expressed. Presence of the flox sequences in the absence Creexpression does not constitute disruption of the Vgll4 gene in this example. Compared with the Vgll4fl / fllittermates, Alb-Cre+ / -;Vgll4fl / fl(Vgll4hKO) mice had a similar liver to body weight ratio (Fig.10B), and displayed normal liver morphology (Fig.10C) and histology (Fig.10D). Depletion of VGLL4 in Vgll4hKOliver was confirmed (Fig.10E). VGLL4 is a YAP suppressor. Expression of YAP protein and its target genes (Ctgf and Cyr61) was therefore compared between Vgll4fl / fland Vgll4hKOlivers to verify whether loss of VGLL4 affected YAP expression and activity. In Vgll4hKOliver, although YAP expression was not affected (Fig.10E), one of its two target genes was mildly increased (Fig.10F), suggesting that loss of VGLL4 partially activates YAP without affecting its expression. Altogether, these data suggest that depletion of VGLL4 in the liver does not disrupt liver homeostasis under physiological conditions.

[0106] Example 10. Short-term HFFC diet stress causes cirrhosis in Vgll4hKOmice.

[0107] To investigate the pathophysiological role VGLL4 in NASH, 6-8 week old Vgll4fl / fland Vgll4hKOmice were fed with HFFC diet, a standard diet formula to induce NASH (Kristiansen et al., 2016, Obese diet-induced mouse models of nonalcoholic steatohepatitis- tracking disease by liver biopsy. World J Hepatol 8, 673-684.). During 8 weeks of HFFC diet treatment, Vgll4hKOmice showed slower body weight gain than their Vgll4fl / fllittermates (Fig. 11A and 11B). At the end of HFFC stress, the Vgll4hKOmice displayed significantly higher liver to body weight ratio than the Vgll4fl / flcontrols (Fig.11C). Interestingly, a fraction of the Vgll4hKOmice (~25%) had disrupted liver morphology, with irregular bumps replacing the smooth liver tissue (Fig.11D), and these mice also displayed splenomegaly (Fig.11E). Histological analysis of the malformed Vgll4hKOlivers revealed the extensive growth of small cells with scant cytoplasm, which formed small ductular structures that spread out the liver sections (Fig.11F). Mason's Trichrome staining to was also performed to examine fibrosis deposition, showing substantial fibrosis deposition in the HFFC-stressed Vgll4hKOmice (Fig. 11G). These data indicate that short term HFFC stress caused Vgll4hKOmice to develop cirrhosis, characterized by liver surface nodularity (Colli et al, 2003, Severe liver fibrosis or cirrhosis: accuracy of US for detection—analysis of 300 cases. Radiology 227, 89-94), advanced hepatic fibrosis (Bataller et al., 2005, Liver fibrosis. The Journal of clinical investigation 115, 209-218), and increased congestion in the portal vein system that leads to splenomegaly (Orlando et al., 2011, Splenomegaly as risk factor of liver cirrhosis. A retrospective cohort study of 2,525 patients who underwent laparoscopy. in vivo 25, 1009- 1012).

[0108] Example 11. HFFC stress induces excessive cell proliferation in the Vgll4hKOliver.

[0109] To further define the pathology of Vgll4hKOcirrhosis, we stained the liver sections with Ki67 antibody, a cell proliferation marker, and found that the Vgll4hKOlivers had much more Ki67 positive cells than the control counterparts (Fig.12A). Additionally, different from the HFFC+Vgll4fl / fllivers that contain similar size polygonal shape hepatocytes, the Vgll4hKOcirrhosis livers were inhabited with different size and irregular shape cells, some of which were enlarged and occupied with macrovacuoles (Fig.12A). TEAD1 protein levels were compared between normal and NASH mouse livers, and TEAD1 protein was robustly increased in NASH livers (Fig.12B and 12C). Loss of VGLL4 is known to increase abundance of YAP / TEAD1 complex in the heart, and activation of YAP is able to form a YAP-TEAD1 feed-forward loop that enhances Tead1 gene expression. Thus, VGLL4 may suppresses TEAD1 expression by inhibiting the formation of YAP / TEAD1 complex. In support of this possibility, as disclosed herein, hepatic TEAD1 but not YAP was upregulated in HFFC-stressed Vgll4hKOmice (Fig.12D and 12E). These data indicate that Vgll4-null hepatic cells are vulnerable to short-term HFFC stress, perhaps because, at least, these cells my be prone to undergo pathological remodeling due to the presence of excessive YAP / TEAD complex (Fig.12F).

[0110] Example 12. AAV.VGLL4HF4Atreatment does not reduce NASH mice body weight.

[0111] VGLL4 interacts with TEAD proteins through its two Tondu (TDU) domains, and mutating the TDU1 and TDU2 domains to replace the HA sequences in each with AA (HF4A) minimize the interaction between VGLL4 and TEADs, thereby abolishing VGLL4’s inhibition of YAP. An AAV construct was generated express VGLL4HF4A(AAV.VGLL4HF4A) in the liver. To test whether VGLL4 mitigates NASH progression through TEADs, NASH mice were treated with 1x1010vg / g AAV.VGLL4HF4Aand the same dose AAV.GFP, respectively. Compared with AAV.GFP, AAV.VGLL4HF4Atreatment neither reduced NASH mice body weight (FIG.13A), liver weight (FIG.13B), or white adipose tissue weight (FIGs.13C and 13D), nor affected food uptake (FIG.13E). Hepatic VGLL4HF4Aexpression was confirmed by western blot (FIG.13F). These data indicate that VGLL4 needs to interact with TEADs to regulate liver metabolism. In striking contrast, as previously disclosed in U.S. Patent No.11,319,354, which is hereby incorporated herein by reference in its entirety for all purposes, administering an AAV.VGLL4HF4Avector driving transduction of cells with a polynucleotide having a cis-regulatory element that controlsadipocyte-specific expression driving expression of VGLL4HF4Aincreases brown adipose tissue mitochondrial gene expression, mitigates body weight gain, reduces serum glucose level, reduces brown adipose tissue weight, and reduces liver weight and fatty acid synthesis. Thus, unlike when expression is driven by a cis-regulatory element that controls adipocyte- specific expression, VGLL4 expression driven by a cis-regulatory element that controls hepatocyte-specific expression does not regulate liver metabolism or body weight unless the VGLL4 can interact with TEADs.

[0112] Example 13. Discussion.

[0113] As disclosed herein, AAV9.TTR.VGLL4 treatment of NASH mice decreased hepatocytes ballooning degeneration, reduced body fat, and reduced hepatic inflammation (i.e., hepatitis), but did not change hepatic fibrosis deposition.

[0114] VGLL4 regulates hepatocytes lipid metabolism. Disclosed herein is an AAV9.TTR.VGLL4 that specifically expresses VGLL4 in the hepatocytes. When expressed in the hepatocytes of neonatal mice, VGLL4 did not affect liver growth. However, when VGLL4 was overexpressed in the hepatocytes of chew diet-fed adult mice, it mildly but significantly increased the liver weight of male and not female mice. Surprisingly, unlike in preadipocytes (Zhang et al, 2018, The TEA domain family transcription factor TEAD4 represses murine adipogenesis by recruiting the cofactors VGLL4 and CtBP2 into a transcriptional complex. J Biol Chem 293, 17119-17134.), as disclosed herein VGLL4 increased instead of decreased lipid droplet formation in both murine and human hepatocytes. These data indicate that VGLL4 controls the homeostasis of adult hepatocytes and that VGLL4 is a crucial regulator of hepatocytes lipid metabolism.

[0115] As disclosed herein, hepatocyte-specific VGLL4 expression reduces body weight. Compared with AAV9.TTR.GFP (a vehicle control), AAV9.TTR.VGLL4 treatment reduced body weight, improved glucose metabolism, and decreased the mass of white adipose tissue. These results suggest that activating VGLL4 specifically in hepatocytes improves whole body lipid metabolism, such as under high fat diet stress conditions.

[0116] As disclosed herein, AAV9.TTR.GFP treatment of NASH mice attenuates hepatocytes ballooning. Compared with vehicle control NASH mice, VGLL4 treated NASH mice had decreased Cidea, Plin2, and increased Plin1 expression. These data indicate that VGLL4 may attenuate hepatocytes ballooning degeneration, at least in part, by harnessing the expression of lipid metabolism genes towards the direction of lipolysis, which breaks down large lipid droplets and therefore reduces large lipid droplet accumulation-caused cellular stress.

[0117] As disclosed herein, AAV9.TTR.GFP treatment of NASH mice does not reduce hepatic fibrosis. VGLL4 is known as a suppressor of TAZ / YAP-TEAD complex, and as disclosed herein, VGLL4 treatment of NASH mice decreased hepatic Ihh expression.

[0118] As also disclosed herein, knocking out VGLL4 specifically in liver did not affect liver growth. However, liver-specific loss of VGLL4 predisposed the liver to develop cirrhosis under HFFC diet stress.

[0119] In conclusion, as disclosed herein, VGLL4 regulates hepatocytes lipid metabolism and AAV-mediated hepatocyte-specific VGLL4 overexpression improved liver function and whole body metabolism in a murine NASH model.

[0120] Example 13. Sequences

[0121] Amino acid sequences of a Vgll4 encoded by polynucleotides included within the present disclosure include the following:

[0122] Table 1: Vgll4 amino acid sequences SEQ Identity Sequence ID A T T H V H G A P D H C Q H A S S KNSLDASRPAGLSPTLTPGERQQNRPSVITCASAGARNCNLSHCPIAHS GCAAPGPASYRRPPSAATTCDPVVEEHFRRSLGKNYKEPEPAPNSVSI TGSVDDHFAKALGDTWL IKAAKDGASSSPESASRRG PASPSAHM E P Tdisclosure include the following:

[0124] Table 2: Polynucleotide sequences encoding a Vgll4 SEQ Identity Sequence ID C G T C G C T T G C T G G T A C CACTGCCCCATCGCGCACAGCGGCTGTGCCGCGCCCGGGCCTGCCA GCTACCGGAGGCCACCGAGCGCTGCCACCACCTGTGACCCCGTGG TGGAGGAGCATTTCCGCAGGAGCCTGGGCAAGAATTACAAGGAG C A C T G C C G G A C A C A T A G C G C G C A T C G A T C C A CGCCCGGGCCTGCCAGCTACCGGAGGCCACCGAGCGCTGCCACCA CCTGTGACCCCGTGGTGGAGGAGCATTTCCGCAGGAGCCTGGGCA AGAATTACAAGGAGCCCGAGCCGGCACCCAACTCCGTGTCCATCA T T C T C C C C T G G A C

[0005] oynuceo de sequences o an enancer o a cs-reguaory eemen hepatocyte-specific expression included within the present disclosure include the following:

[0126] Table 3: Polynucleotide sequences of enhancers: SEQ Identity Sequence ID18 Serpin1 GTCACCACAGTTATTGGTAGAGCAAACAGGGGCTATGTCC enhancer i t 6 C C G T Ty q p g y controlling hepatocyte-specific expression included within the present disclosure include the following:

[0128] Table 4: Polynucleotide sequences of promoters: SEQ Identity Sequence ID T G C C T C G C G G CChimeric GGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCC promoter 1 TCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGGCAAGGTT CATATTTGTGTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAAT C G T G C T G G C C G C G T T T C T G G C T34 Thyro- TGCATGTATAATTTCTACAGAACCTATTAGAAAGGATCACCCAGC globulin CTCTGCTTTTGTACAACTTTCCCTTAAAAAACTGCCAATTCCACTG t CTGTTTGGCCCAATAGTGAGAACTTTTTCCTGCTGCCTCTTGGTGC G G A A A A T A C T A A C T C C G G

[0129] Polynucleotide sequences of an intron between the cis-regulatory element and sequence encoding a vestigial like 4 protein included within the present disclosure include the following:

[0130] Table 5: Polynucleotide sequences of introns: SEQ Identity Sequence C39 Minute AAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGCATTAAT virus of GTTTAATTACCTGAACGACGCGCCACTAATCACTTTTTTTCAGGTT i GG T G T Cspecific expression included within the present disclosure include the following:

[0132] Table 6: Polynucleotide sequences of cis-regulatory elements: SEQ Identity Sequence ID G T A C A A T T C G T46 Minute AAGAGGTAAGGGTTTAAGTTATCGTTAGTTCGTGCACCATTAATG virus of TTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTTTCAGGT i TGG C_ 48): AATTCGGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGG AGCAAACAGGGGCTAAGTCCACGTCTGTCTGCACATTTCGTAGAGCGAGTGTT CCGATACTCTAATCTCCCTAGGCAAGGTTCATATTTGTGTAGGTTACTTATT CTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCT TGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTC ACCAGGAGAAGCCGTCACACAGATCCACAAGCTCCTGCTGCAGAAGTTGGTC GTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCA ATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCT ATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTT CAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTCACTATAGGCTAGCATG CTATTTATGAAGATGGACCTGTTGAACTATCAGTACTTGGACAAGATGAACA ACAATATCGGCATTCTGTGCTACGAAGGCGAAGCTGCTCTCAGGGGAGAAC CCAGAATGCAGACCCTGCCGGTGGCCTCTGCCCTCAGCAGTCACCGCACCG GCCCTCCCCCAATCAGCCCCAGCAAGAGGAAGTTCAGCATGGAGCCAGGTG ACGAGGACCTAGACTGTGACAACGACCACGTCTCCAAAATGAGTCGCATCTT CAACCCCCATCTGAACAAGACTGCCAATGGAGACTGCCGCAGAGACCCCCG GGAGCGGAGCCGCAGCCCCATCGAGCGCGCTGTGGCCCCCACCATGAGCCT GCACGGCAGCCACCTGTACACCTCCCTCCCCAGCCTTGGCCTGGAGCAGCC CCTCGCACTGACCAAGAACAGCCTGGACGCCAGCAGGCCAGCCGGCCTCTC GCCCACACTGACCCCGGGGGAGCGGCAGCAGAACCGGCCCTCCGTGATCAC CTGTGCCTCGGCTGGCGCCCGCAACTGCAACCTCTCGCACTGCCCCATCGC GCACAGCGGCTGTGCCGCGCCCGGGCCTGCCAGCTACCGGAGGCCACCGA GCGCTGCCACCACCTGTGACCCCGTGGTGGAGGAGCATTTCCGCAGGAGCC TGGGCAAGAATTACAAGGAGCCCGAGCCGGCACCCAACTCCGTGTCCATCA CGGGCTCCGTGGACGACCACTTTGCCAAAGCTCTGGGTGACACGTGGCTCC AGATCAAAGCGGCCAAGGACGGAGCATCCAGCAGCCCTGAGTCCGCCTCTC GCAGGGGCCAGCCCGCCAGCCCCTCTGCCCACATGGTCAGCCACAGTCACT CCCCCTCTGTGGTCTCCGCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGG GTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGC GTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTC ATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGA CCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTT CTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAG ACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCG GGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

[0134] Amino acid sequences of a Vgll4 fragment encoded by polynucleotides included within the present disclosure include the following:

[0135] Table 7: Vgll4 fragment amino acid sequences SEQ ID NO Identity Sequence 49 Vgll4 TDU_1 DPVVEEHFRRSLGKNY r r

[0136] Polynucleotide sequences encoding a Vgll4 fragment included within the present disclosure include the following:

[0137] Table 8: Polynucleotide sequences encoding a Vgll4 fragment SEQ ID NO Identity Sequence 55 V ll4 TDU 1 GACCCCGTGGTGGAGGAGCATTTCCGCAGGAGCCT G G or y A T A TTTGCCAAAGCTCTGGGTGACACGTGGCTCCAGATCA AAGCGGCCdetail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the present disclosure and these are therefore considered to be within the scope of the present disclosure as defined in the claims that follow.

[0139] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail herein (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits and advantages described herein. The term “about” as used herein in reference to a numeric value means a range of numeric values from 90% to 110% of the numeric value. For example, “about 1 mg” means “from 0.9 mg to 1.1 mg,” etc.

Claims

WHAT IS CLAIMED IS:

1. A polynucleotide, comprising a nucleotide sequence encoding a vestigial like 4 protein and a cis-regulatory element, wherein the cis-regulatory element controls hepatocyte-specific expression of the sequence encoding a vestigial like 4 protein.

2. The polynucleotide of claim 1, wherein the cis-regulatory element has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with, independently, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, nucleotides 6-299 of SEQ ID NO: 48, or any combination of two or more of the foregoing.

3. The polynucleotide of claim 1 or 2, wherein the cis-regulatory element comprises one or more enhancer and the one or more enhancer has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with, independently, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or any combination of two or more of the foregoing.

4. The polynucleotide of any one of claims 1 through 3, wherein the cis- regulatory element comprises one or more promoter and the one or more promoter has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with, independently, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or any combination of two or more of the foregoing.

5. The polynucleotide of any one of claims 1 through 4, wherein the vestigial like 4 protein has at least 90% identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95%sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO:

6.

6. The polynucleotide of any one of claims 1 through 5, wherein the sequence encoding a vestigial like 4 protein has at least 90% identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with a sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:

12.

7. The polynucleotide of any one of claims 1 through 6, wherein the vestigial like 4 protein has from 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, wherein the substitutions are not in a TDU domain.

8. The polynucleotide of any one of claims 1 through 7, further comprising an intron between the cis-regulatory element and the nucleotide sequence encoding a vestigial like 4 protein.

9. The polynucleotide of claim 8, wherein the intron has at least 90% identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or any combination of two or more of the foregoing.

10. The polynucleotide of any one of claims 1 through 16, comprising a nucleotide sequence having at least 90% identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO:

48.

11. A viral vector comprising the polynucleotide of any one of claims 1 through 10.

12. The viral vector of claim 11, wherein the viral vector comprises an adenoviral associated vector.

13. An cell transfected in vitro or ex vivo with the polynucleotide of any one of claims 1 through 10 or the viral vector of claim 11 or 12.

14. The cell of claim 13 wherein the cell was contacted with the viral vector of claim 11 or 12.

15. A non-human organism transfected or transduced with the polynucleotide of any one of claims 1 through 10 or the viral vector of claim 11 or 12.

16. A method, comprising transfecting or transducing a cell of an organism, in vivo or ex vivo, or transfecting or transducing a cell in vitro, with the polynucleotide of any one of claims 1 through 10 or the viral vector of claim 11 or 12.

17. The method of claim 16, wherein the organism is a mammal.

18. The method of any one of claims 16 or 17, wherein the organism is a human.

19. A method of treating steatohepatitis in a subject, comprising administering the polynucleotide of any one of claims 1 through 10 or the viral vector of claim 11 or 12 to the subject.

20. A method of treating obesity in a subject, comprising administering the polynucleotide of any one of claims 1 through 10 or the viral vector of claim 11 or 12 to the subject.

21. A method of decreasing white adipose tissue in a subject, comprising administering the polynucleotide of any one of claims 1 through 10 or the viral vector of claim 11 or 12 to the subject.

22. A method of treating hyperglycemia in a subject, comprising administering the polynucleotide of any one of claims 1 through 10 or the viral vector of claim 11 or 12 to the subject.

23. A method of treating diabetes or insulin resistance in a subject, comprising administering the polynucleotide of any one of claims 1 through 10 or the viral vector of claim 11 or 12 to the subject.

24. The method of any one of claims 19 through 23, wherein the administering comprises administering the viral vector of clam 11 or 12 to the subject.

25. A polynucleotide, comprising a nucleotide sequence encoding a vestigial like 4 protein and a means for controlling hepatocyte-specific expression of the sequence encoding a vestigial like 4 protein.

26. The polynucleotide of claim 25, further comprising an intron.

27. The polynucleotide of claim 26, wherein the intron has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequenceat least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or any combination of two or more of the foregoing.

28. The polynucleotide of any one of claims 25 through 27, wherein the vestigial like 4 protein has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or any combination of two or more of the foregoing.

29. The polynucleotide of any one of claims 25 through 28, wherein the sequence encoding a vestigial like 4 protein has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with a sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or any combination of two or more of the foregoing.

30. A viral vector comprising the polynucleotide of any one of claims 25 through 29.

31. The viral vector of claim 30, wherein the viral vector comprises an adenoviral associated vector.

32. A cell transfected in vitro or ex vivo with the polynucleotide of any one of claims 25 through 29 or the viral vector of claim 30 or 31.

33. The cell of claim 32 wherein the cell was contacted with the viral vector of claim 30 or 31.

34. A non-human organism transfected with the polynucleotide of any one of claims 25 through 29 or transduced with the viral vector of claim 30 or 31.

35. A method, comprising transfecting or transducing a cell of an organism, in vivo or ex vivo, or transfecting a cell in vitro, with the polynucleotide of any one of claims 1 through 10 or the viral vector of claim 30 or 31.

36. The method of claim 35, wherein the organism is a mammal.

37. The method of claim any one of claims 35 or 36, wherein the organism is a human.

38. A method of treating steatohepatitis in a subject, comprising administering the polynucleotide of any one of claims 25 through 29 to the subject.

39. A method of treating obesity in a subject, comprising administering the polynucleotide of any one of claims 25 through 29 to the subject.

40. A method of decreasing white adipose tissue in a subject, comprising administering the polynucleotide of any one of claims 25 through 29 to the subject.

41. A method of treating hyperglycemia in a subject, comprising administering the polynucleotide of any one of claims 25 through 29 to the subject.

42. A method of treating diabetes or insulin resistance in a subject, comprising administering the polynucleotide of any one of claims 25 through 29 to the subject.

43. The method of any one of claims 25 through 42, wherein the administering comprises administering the viral vector of clam 30 or 31 to the subject.

44. A method of screening a treatment for fatty liver disease or prevention of cirrhosis, comprising feeding a test transgenic animal a diet wherein the diet is high in one or more of fat, fructose, and cholesterol, administering the treatment to the transgenic animal, and after the administering, detecting a difference in liver morphology or liver function between the test transgenic animal and a control animal, wherein the test transgenic animal comprises a disruption of the gene encoding a vestigial like 4 protein and the disruption is limited to hepatocytes.

45. The method of claim 44, wherein the control animal comprises the disruption of the gene encoding the vestigial like 4 protein and the disruption is limited to hepatocytes.

46. The method of claim 44 or 45, wherein the difference comprises one or more of decreased liver surface nodularity, decreased hyperproliferation of liver duct cells,and decreased liver fibrosis deposition, or any combination of two or more of the foregoing, in the test transgenic animal compared to the control animal.

47. The method of cany one of claims 44 through 46, wherein the difference comprises one or more of lower liver to body weight ratio, liver tissue surface with fewer or less irregular bumps, presence of fewer small ductular structures, presence of fewer or less growth of cells of reduced size containing less cytoplasmic volume forming fewer small ductular structures, less fibrosis deposition, less splenomegaly, lower number of cells positive for proliferative cell marker, optionally Ki67, fewer irregular shaped hepatocytes, presence of enlarged hepatocytes, presence of fewer hepatocytes occupied with macrovacuoles, less TEAD1 upregulation, or any combination of two or more of the foregoing, in the test transgenic animal.

Images