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WO2025226943A1 - Polynucléotide pour l'expression d'hépatocytes de la protéine de type vestigial 4 et son procédé d'utilisation - Google Patents

Polynucléotide pour l'expression d'hépatocytes de la protéine de type vestigial 4 et son procédé d'utilisation

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Publication number
WO2025226943A1
WO2025226943A1 PCT/US2025/026195 US2025026195W WO2025226943A1 WO 2025226943 A1 WO2025226943 A1 WO 2025226943A1 US 2025026195 W US2025026195 W US 2025026195W WO 2025226943 A1 WO2025226943 A1 WO 2025226943A1
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Prior art keywords
sequence identity
seq
polynucleotide
vgll4
sequence
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PCT/US2025/026195
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English (en)
Inventor
Zhiqiang Lin
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Masonic Medical Research Laboratory
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Masonic Medical Research Laboratory
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Publication of WO2025226943A1 publication Critical patent/WO2025226943A1/fr
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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/25—Animals on a special diet
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/203—Animal model comprising inducible/conditional expression system, e.g. hormones, tet
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • Non-alcoholic fatty liver disease 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.
  • NAFLD Newcastle disease virus
  • 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.
  • Treatment options for NAFLD and associated conditions are limited. The present disclosure is directed to overcoming these and other deficiencies in the art.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 DPVVEEX 1 X 2 RRSLGKNY, 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 TGSVDDX3X4
  • the vestigial like 4 protein fragment may have a sequence of SEQ ID NO: 53 or SEQ ID NO: 54.
  • 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.
  • 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 DPVVEEX 1 X 2 RRSLGKNY, 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a viral vector including any foregoing polynucleotide.
  • the viral vector may include an adenoviral associated vector.
  • non-human organism transfected or transduced with any foregoing polynucleotide or any foregoing viral vector.
  • 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.
  • 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 foregoing polynucleotide to the subject.
  • the administering may include administering any foregoing viral vector to the subject.
  • 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.
  • 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.
  • 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 DPVVEEX 1 X 2 RRSLGKNY, wherein X 1 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 TGSVDDX 3
  • 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.
  • 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 X 1 may be H or any amino acid other than H, or, independently, X 2 may be F or any amino acid other than F, or a TDU_2 domain variant, wherein
  • 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.
  • a viral vector including any foregoing polynucleotide.
  • the viral vector may include an adenoviral associated vector.
  • non-human organism transfected or transduced with any foregoing polynucleotide or any foregoing viral vector.
  • 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.
  • the administering may include administering any foregoing viral vector to the subject.
  • 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.
  • 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 promoter 25 .
  • 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.
  • C Hematoxylin and eosin staining of adult liver sections.
  • D Oil Red staining of liver section.
  • E Fluorescence images of liver sections.
  • 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.
  • 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.
  • FIGs.4A-4I show AAV9.TTR.VGLL4 pre-treatment attenuates HFD-induced white adipose tissue expansion, in accordance with aspects of the present disclosure.
  • 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.
  • FIGs.5A-5H show AAV9.TTR.VGLL4 treatment reduces NASH mice body weight, in accordance with aspects of the present disclosure.
  • 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).
  • F Alanine transaminase (ALT) blood test. Serum was collected at 10 weeks after AAV transduction and used for ALT test.
  • FIGs.6A-6F show AAV9.TTR.VGLL4 treatment reduces body fat, in accordance with aspects of the present disclosure.
  • 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.
  • 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 ⁇ m.
  • 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 ⁇ m.
  • 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 ⁇ m.
  • FIGs.9A-9D show AAV9.TTR.VGLL4 treatment of NASH mice reduces hepatic inflammation, in accordance with aspects of the present disclosure.
  • FIGs.10A-10F show loss of hepatic VGLL4 does not affect liver growth, in accordance with aspects of the present disclosure.
  • LW Liver weight
  • BW body weight
  • N 8.
  • 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.
  • FIGs.12A-12F show HFFC diet stress impairs the homeostasis of Vgtt4hKo hepatic cells, in accordance with aspects of the present disclosure.
  • 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 Vgll4 fl/fl and Vgll4 hKo mice were used tested.
  • FIGs.13A-13F show AAV.VGLL4 HF4A treatment does not reduce NASH mice body weight. NASH mice were transduced with AAV.VGLL4 HF4A or AAV.GFP.
  • E. Food uptake. A-E, n 6 for each group. NS, not significant.
  • 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.
  • 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.
  • 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 not in 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.
  • 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.
  • Vgll4 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 (2016), 8(6):932-943.
  • 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.
  • a cis-regulatory element may include a promotor, an enhancer, or both.
  • 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 than 200 nucleotides from a transcription start site, fewer than 225 nucle
  • 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.
  • promotors many genes have enhances that are involved in cell- or tissue-specific or cell- or tissue-enhanced expression.
  • 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.
  • cis-regulatory elements including enhancers, promoters, or both, that control hepatocyte-specific expression that may be included in a polynucleotide as disclosed 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.
  • 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).
  • 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
  • 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 the foregoing 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.
  • Vgll4A 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.
  • SEQ ID NO: 1 amino acid sequences
  • SEQ ID NO: 2 amino acid sequences
  • SEQ ID NO: 3 amino acid sequences
  • SEQ ID NO: 5 SEQ ID NO: 6
  • 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.
  • 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.
  • a Vgll4 peptide may include one or more amino acid substitution (relative to the examples disclosed in Table 1).
  • 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).
  • 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
  • TDU_2 has the amino acid sequence DPVVEEHFRRSLGKNY (SEQ ID NO: 49).
  • 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.
  • 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.
  • 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 DPVVEEX 1 X 2 RRSLGKNY (SEQ ID NO: 51), wherein X 1 may 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 X 3 may be H or any amino acid other than H, and, independently, X 4 may be F or any amino acid other than F.
  • X1, X2, X3, and X4 in any combination, including all of X 1 , X 2 , X 3 , and X 4 , 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.
  • X1, X2, X3, and X4 in any combination, including all of X 1 , X 2 , X 3 , and X 4 , 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 X 1 and X 3 is not H, one or both of X 2 and X 4 is 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.
  • 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 known correspondences 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 X 3 but not an F for X 4 , an F for X 4 but not an H for X 3 , or neither an H for X 3
  • 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.
  • a cis-regulatory element including without limitation and enhancer and/or promoter disclosed herein
  • any vector disclosed herein including such a construct such as any viral vector disclosed herein including any AAV viral vector disclosed herein.
  • 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.
  • a Vgll4 fragment includes a portion of full Vgll4 protein that retains at least some structural and functional features of full Vgll4 protein.
  • 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 domain may 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.
  • Vgll4 fragment 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.
  • 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.
  • 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.
  • 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.
  • a polynucleotide disclosed herein may include a polynucleotide sequence encoding for a Vgll4 protein wherein the Vgll4 protein may have any Vgll4 amino acid sequence 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.
  • 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.
  • 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.
  • a non-limiting list of introns 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.
  • 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.
  • 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.
  • viral vectors examples include retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAV).
  • AAV adeno-associated viruses
  • 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.).
  • scAAV self-complementary AAV vectors
  • scAAV self-complementary polynucleotide sequences of constructs are packaged.
  • AAV vectors made using combinations of genetic sequences from different AAV serotypes for improving expression in hepatocytes are 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.
  • 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.
  • ITRs inverted terminal repeats
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • GFP green fluorescent protein
  • Other reporters may also be included. In other examples, no reporter is included.
  • a construct may include any nucleotide sequence for encoding any reporter protein.
  • 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.
  • a cell- or tissue-specific cis-regulatory element 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.
  • 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.
  • 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.
  • 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).
  • NAFL non-alcoholic fatty liver
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • administering a polynucleotide as disclosed herein may be a treatment for NAFL, NASFLD, and/or NASH, or prevent the development of any of the foregoing.
  • a vector such as a viral vector such as an AAV vector
  • administering may be a treatment for NAFL, NASFLD, and/or NASH, or prevent the development of any of the foregoing.
  • 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.
  • 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.
  • treatment 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.
  • 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
  • NASH is a risk factor for cirrhosis of the liver.
  • Obesity is also a risk factor for type II diabetes
  • 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.
  • 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.
  • YAP-VGLL4 antagonism defines the major physiological function of the Hippo signaling effector YAP.
  • Liver development and function in such mice is typical of wild-type mice when fed a normal diet.
  • a diet high in fat about 40% of total kcal
  • high in fructose about 20% total kcal
  • high in cholesterol about 2%)
  • Two groups of mice with liver-specific disruption of Vgll4 expression may be fed a HFFC diet, one group administered a known or hypothesized treatment for fatty liver disease or cirrhosis.
  • 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.
  • 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.
  • 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
  • a means for stimulating production of a Vgll4 protein or fragment thereof in a cell 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.
  • 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.
  • Example 1 Overexpressing VGLL4 in hepatocytes does not affect postnatal liver growth.
  • 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.
  • 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.
  • TTR mouse transthyretin
  • 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.VGLL4 GFP (TTR.VGLL4) and their livers collected for analysis at P14.
  • 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.
  • AAV9.TTR.VGLL4 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).
  • VGLL4 Overexpressing VGLL4 specifically in liver therefore improves whole-body metabolism.
  • Example 3. AAV9.TTR.VGLL4 pre-treatment mitigates the expansion of white adipose tissue.
  • WAT White adipose tissue
  • 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).
  • 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).
  • Fig.4D subcutaneous WAT
  • Fig.4E similar visceral WAT
  • Fig.4F significantly lower total WAT volume than GFP controls
  • AAV9.TTR.VGLL4 pre- treatment is beneficial for reducing WAT expansion in HFD-induced obese mice.
  • Example 4. AAV9.TTR.VGLL4 treatment of NASH mice decreases body weight and improves liver function.
  • overexpressing VGLL4 specifically in hepatocytes attenuated HFD-induced metabolism dysfunction.
  • AAV9.TTR.VGLL4-mediated hepatic expression of VGLL4 may mitigate the progression of NAFLD.
  • 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.
  • 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).
  • 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).
  • VGLL4 treatment also improved glucose metabolism (Fig.5D).
  • 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.
  • VGLL4 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).
  • Example 5 AAV9.TTR.VGLL4 treatment of NASH mice decreases white adipose tissue mass.
  • 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).
  • VGLL4 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.
  • 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).
  • 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.
  • BODIPY a fluorescence dye labeling lipid droplets
  • Phalloidin which binds to filament actin
  • 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).
  • VGLL4 treatment did not change the expression of two fatty acid binding protein genes Fabp3 and Fabp4 (Fig.
  • hepatic fibrosis is another pathophysiological signature of NASH disease.
  • liver sections were examined for fibrosis deposition.
  • 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).
  • 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
  • Ccn2 also known as Ctgf
  • VGLL4 treatment of NASH mice decreased the expression of TEAD target genes (Ihh and Ccn1), with the exception of Ctgf (Fig.8B).
  • 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.
  • MMPs matrix metalloproteinases
  • TIMPs tissue inhibitors of metalloproteinases
  • 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).
  • VGLL4 may regulate liver fibrosis by repressing Col1a1 and crucial matrix metalloproteinase genes, which are all positively correlated with the progression of NASH disease.
  • hepatic crown-like structure a unique histological feature of steatohepatitis.
  • liver sections were stained with CD68 antibody, a molecular maker that labels both residential Kupffer cells and blood-derived macrophages.
  • 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
  • transgenic Alb-Cre mice which express Cre recombinase specifically in hepatocytes and not other cell types
  • Vgll4 hKO 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 Vgll4 hKO liver was confirmed (Fig.10E).
  • VGLL4 is a YAP suppressor. Expression of YAP protein and its target genes (Ctgf and Cyr61) was therefore compared between Vgll4 fl/fl and Vgll4 hKO livers to verify whether loss of VGLL4 affected YAP expression and activity.
  • Vgll4 fl/fl and Vgll4 hKO mice 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.).
  • HFFC diet a standard diet formula to induce NASH.
  • Vgll4 hKO mice showed slower body weight gain than their Vgll4 fl/fl littermates (Fig. 11A and 11B).
  • Vgll4 hKO mice displayed significantly higher liver to body weight ratio than the Vgll4 fl/fl controls (Fig.11C).
  • Fig.11D a fraction of the Vgll4 hKO mice ( ⁇ 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 Vgll4 hKO livers revealed the extensive growth of small cells with scant cytoplasm, which formed small ductular structures that spread out the liver sections (Fig.11F).
  • Vgll4 hKO cirrhosis 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.
  • VGLL4 may suppresses TEAD1 expression by inhibiting the formation of YAP/TEAD1 complex.
  • hepatic TEAD1 but not YAP was upregulated in HFFC-stressed Vgll4 hKO mice (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).
  • Example 12 AAV.VGLL4 HF4A treatment does not reduce NASH mice body weight.
  • 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 VGLL4 HF4A (AAV.VGLL4 HF4A ) in the liver. To test whether VGLL4 mitigates NASH progression through TEADs, NASH mice were treated with 1x10 10 vg/g AAV.VGLL4 HF4A and the same dose AAV.GFP, respectively.
  • AAV.VGLL4 HF4A treatment 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 VGLL4 HF4A expression 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.
  • 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.
  • 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.
  • VGLL4 regulates hepatocytes lipid metabolism.
  • 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.
  • VGLL4 increased instead of decreased lipid droplet formation in both murine and human hepatocytes.
  • VGLL4 controls the homeostasis of adult hepatocytes and that VGLL4 is a crucial regulator of hepatocytes lipid metabolism.
  • hepatocyte-specific VGLL4 expression reduces body weight.
  • AAV9.TTR.GFP a vehicle control
  • AAV9.TTR.VGLL4 treatment reduced body weight, improved glucose metabolism, and decreased the mass of white adipose tissue.
  • AAV9.TTR.GFP treatment of NASH mice attenuates hepatocytes ballooning.
  • VGLL4 treated NASH mice had decreased Cidea, Plin2, and increased Plin1 expression.
  • Plin2 and 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.
  • 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.
  • 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 K NSLDASRPAGLSPTLTPGERQQNRPSVITCASAGARNCNLSHCPIAHS GCAAPGPASYRRPPSAATTCDPVVEEHFRRSLGKNYKEPEPAPNSVSI TGSVDDHFAKALGDTWL IKAAKDGASSSPESASRRG PASPSAHM E P T disclosure include the following: [0124] Table 2: Polynucleotide sequences encoding a Vgll4 SEQ Identity Sequence ID C G T C G C T G C T G T A C C C ACTGCCCCATCGCGCACAGCGGCTGTGCCGCGCCCGGGCCTGCCA GCTACCGGAGGCCACCGAGCGCTGCCACCACCTGTGACCCCGTGG

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Abstract

L'invention concerne un polynucléotide comprenant une séquence nucléotidique codant pour une protéine de type vestigial 4 et un élément cis-régulateur qui commande l'expression spécifique d'hépatocytes de la séquence codant pour une protéine vestigiale de type 4. L'invention concerne également un vecteur viral comprenant le polynucléotide, ainsi qu'une cellule ou un organisme transfecté par le polynucléotide. L'invention concerne également une méthode de traitement de la stéatohépatite, de l'obésité, de l'hyperglycémie, du diabète, de la résistance à l'insuline, de l'inflammation hépatique ou de la diminution du tissu adipeux blanc chez le sujet. L'invention concerne également une méthode de dépistage d'un traitement de la stéatose hépatique ou de prévention de la cirrhose, consistant à donner un régime alimentaire riche en graisse, fructose et/ou cholestérol à un animal transgénique présentant une interruption du gène de la protéine vestigiale spécifique des hépatocytes, à administrer le traitement à l'animal transgénique et à détecter une différence de morphologie hépatique ou de fonction hépatique entre l'animal transgénique et un animal témoin.
PCT/US2025/026195 2024-04-25 2025-04-24 Polynucléotide pour l'expression d'hépatocytes de la protéine de type vestigial 4 et son procédé d'utilisation Pending WO2025226943A1 (fr)

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