Manipulating UHRF1 expression in hepatocytes

Abstract

Provided is a method that involves inhibiting expression of UHRF1 in liver cells of an individual. Inhibiting the expression of UHRF1 promotes an inhibition of reduction of bone density or an increase in bone density, or inhibition of weight gain or a reduction in adipose mass, or inhibition of development of gray hair or an increase in activity level, or a decrease in biological age or enhanced liver regeneration, or increased survival following partial hepatectomy, or a combination of such effects.

Description

[0001] MANIPULATING UHRF1 EXPRESSION IN HEPATOCYTES

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to U.S. provisional application no. 63 / 689,057, filed August 30, 2024, the entire disclosure of which is incorporated herein by reference.

[0004] FEDERAL FUNDING

[0005] This invention was made with government support under R01 DK080789 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] RELATED INFORMATION

[0007] The remarkable regenerative capacity of the young mammalian liver relies on a well-coordinated gene program, and this diminishes with age. During the aging process, the epigenetic code in the liver and other organs undergoes repatterning. There is a need for new approaches to altering this repatterning to provide previously unknown benefits. The present disclosure is related to this need.

[0008] BRIEF SUMMARY

[0009] This disclosure demonstrates that inhibition of expression of liver UHRF1 promotes favorable traits in individuals in which expression of the UHRF1 is inhibited. The traits include but are not necessarily limited to: improved activity levels; inhibition of bone density reduction and reversal of bone density loss; liver regeneration; survival after hepatectomy; reduction in weight gain and adipose mass; reduction or inhibition of development of greying hair; reduction of biological age; and combinations thereof.

[0010] BRIEF DESCRIPTION OF FIGURES

[0011] FIG. 1. Uhrf1HepKOmice show reduced grey fur with age.

[0012] Panel A. Representative picture of a 2-month-old WT male mice (Young)

[0013] Panel B. Representative picture of a 24-month-old WT male mice (Geriatric) with a zoom-in showing the grey fur. Panel C. Representative picture of a 24-month-old UhrflHepKOmale mice (Geriatric) with a zoom-in showing the absence of grey fur.

[0014] FIG. 2: UhrflHepKOmice do not have age associated weight gain.

[0015] Data from weight assessment between 2-10 months are from different cohorts of mice, after 10 months all weights were obtained from the same cohort.

[0016] Body Weight curves of WT and UhrflHepKOmale mice over time.

[0017] * Indicates p<0.05; ** 0.01 ; *** 0.001 via one way ANOVA.

[0018] FIG. 3: Reduced accumulation of adipose tissue in aged UhrflHepKOmice.

[0019] Panel A. Representative thoracic CT-scan images of 2-month-old WT, 16-month-old WT and UhrflHepKOmale mice. Fat tissue is shown in red.

[0020] Panel B. Percentage of thoracic adipose tissue in all mouse groups. n=1 -3 mice per group.

[0021] FIG. 4: Increased activity in aged UhrflHepKOmice.

[0022] Panel A. Representative screenshot of the Open Field test (OFT) arena for 16-month- old WT and UhrflHepKOmale mice.

[0023] Panel B. Total distance (mm) covered by mice in the OFT arena within 5 minutes.

[0024] Panel C. Average locomotor speed (mm / s) in the OFT arena (5-minute test). n=5-15 mice per group. * Indicates p<0.05; ** 0.01 ; *** 0.001 via one way ANOVA.

[0025] FIG. 5: Increased bone density in aged UhrflHepKOmice.

[0026] Panel A. Representative CT-scan images of 2-month-old WT and 28-month-old WT male mice from Nam et al., Sc. Report, 2018.

[0027] Panel B. Bone mass density (g / cm3) of all mouse groups. n=1 -3 mice per groups.

[0028] FIG. 6: Enhanced liver regeneration in UHRF1HepKOmice at all ages. Panel A. Liver weight to body weight ratio at baseline and at different timepoints after Partial hepatectomy (PH).

[0029] Panel B. Percentage of liver weight growth following PH at different timepoints. n=20-44 mice per group.

[0030] For FIG. 6, panels A and B, greyscale bars are from wild type mice and pink bars are from Uhrfl -HepKO mice, with the shades reflecting the different time points (2 months for the darkest, 16 months for the middle, 24 months for the lightest).

[0031] FIG. 7: Improved survival following partial hepatectomy in UHRF1HepKOmice at all ages. Percent survival following PH across all mouse groups. n=20-44 mice per group.

[0032] DETAILED DESCRIPTION

[0033] Unless defined otherwise herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0034] Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.

[0035] As used in the specification and the appended claims, the singular forms “a” "and” and “the" include plural referents unless the context clearly dictates otherwise.

[0036] Ranges and other values may be expressed herein as from “about” or “approximately” one particular value, and / or to “about” or “approximately” another particular value. When values are expressed as approximations by the use of the antecedent “about” or “approximately” it will be understood that the particular value forms another example. The term “about” and “approximately” in relation to a numerical value encompasses variations of + / -10%, to + / - 1 %.

[0037] The present disclosure relates to the discovery that repatterning the epigenetic code in the liver is a cause of the diminished regenerative capacity in the aging liver, as it prevents the activation of pro-regenerative genes and derepresses transposable elements (TEs), leading to inflammation. This discovery was made at least in part by evaluating the transcriptome and regenerative capacity of middle-aged (16-month) and geriatric (24-month) mice.

[0038] The disclosure demonstrates that aged mice exhibit a dramatic increase in TE expression in the liver and impaired regeneration capacity. To further investigate the role of the hepatic epigenome in aging, we examined mice with hepatocyte-specific deletion of UHRF1 (l / HRF7hepKO). UHRF1hepKOmice had reduced signs of aging, including increased mobility and reduced grey fur and weight gain. Gene expression analysis of WT and UHRF1hepKOlivers revealed age associated dysregulation in hundreds of genes implicated in metabolic processes, inflammation, and cell development pathways in geriatric WT mice, which were absent in UHRF1hepKOmice. These results indicate that the hepatic loss of UHRF1 helps maintain youthful physiology and gene expression. Thus, the disclosure demonstrates that inhibiting expression of UHRF1 results in improved activity levels; inhibition of bone density reduction and reversal of bone density loss; liver regeneration; survival after hepatectomy; reduction in weight gain and adipose mass; reduction or inhibition of development of greying hair; reduction of biological age; and combinations thereof.

[0039] Methods of determining biological age are known in the art. (See, for examples, Bortz, J., et al. Biological age estimation using circulating blood biomarkers. Commun Biol 6, 1089 (2023) I doi.org / 10.1038 / s42003-023-05456-z); and Jia L, et al. Common methods of biological age estimation. Clin Interv Aging. 2017 May 11 ;12:759-772. doi: 10.2147 / CIA.S134921 ).

[0040] Activity levels can be measured using established techniques and devices, such as pedometers, accelerometers, determining a Rate of Perceived Exertion (RPE), and determining Metabolic Equivalents (METs) which measure the energy expenditure of physical activity, where 1 MET is the resting metabolic rate (equivalent to 3.5 ml O2 / kg / min or 1 kcal / kg / hr); and known exercising techniques such as swimming, stationary bicycle riding, and treadmill walking. In an example, physical activity intensity is measured on a scale of 0 to 10, where 0 is the level of effort required to sit and 10 is maximal effort. The nucleotide sequences of murine and human UHRF1 genes are known in the art. In an example, expression of the human gene includes inhibiting the expression of a protein as in GenBank accession no. NM_001048201.3, from which the sequence as it exists in the database on the effective filing date of this application is hereby incorporated by reference. The disclosure includes inhibiting expression of all splice variants associated with the stated UHRF1 GenBank sequence. The disclosure includes inhibiting expression proteins comprising all amino acid sequences that are 80.00%- 99.99% identical with the with the stated UHRF1 GenBank sequence. The disclosure includes all polynucleotides encoding the described protein sequences, including DNA and mRNA sequences.

[0041] Examples of the disclosure include knockouts of the murine human UHRF1 gene, where a representative amino acid sequence encoded by the murine gene is available under GenBank accession no. NM_010931 , from which the sequence as it exists in the database on the effective filing date of this application is hereby incorporated by reference. In an example, the mouse UHRF1 gene is disrupted as described in Epigenetic Compensation Promotes Liver Regeneration; Developmental Cell, Volume 50, Issue 1 , p43-56.e6July 01 , 2019, from which the description of disrupting the murine UHRF1 gene is incorporated herein by reference.

[0042] In examples a chromosome within a liver cell is modified to decrease expression of the UHRF1 gene. In examples, a genome of liver cells as described herein may be modified to disrupt or delete the UHRF1 gene. This approach can be performed using any designer nuclease, such as those that are functional in any CRISPR system. In examples, the nuclease is an RNA-guided CRISPR nuclease. A variety of suitable CRISPR nucleases (e.g., Cas nucleases) are known in the art. In non-limiting examples, the Cas comprises a Cas9, such as Streptococcus pyogenes (SpCas9). Derivatives of Cas9 are known in the art and may also be used. Such derivatives may be, for example, smaller enzymes than Cas9, and / or have different proto adjacent motif (PAM) requirements. In a non-limiting examples, the Cas enzyme may be Cas12a, also known as Cpf1 , or SpCas9-HF1. In examples a Cas3 enzyme or a transposon coupled to a nuclease may be used. In example, all of some of the gene is deleted. In examples, one or more mutations may be made such that a functional protein encoded by the UHRF1 gene is not produced. In examples, a gene is replaced using homologous recombination with a DNA template that replaces the UHRF1 gene with a sequence that does not encode a functional UHRF1 protein.

[0043] In examples, a nuclease may be administered directly to the liver cells. For example, a Cas protein and suitable guide RNA(s) may be administered to the cells as ribonucleoproteins (RNPs) using any suitable technique. Alternatively, the Cas protein may be introduced into the cells separately from the guide RNAs. In examples, a viral expression vector may be used to introduce sequences encoding one or more of the described nucleases and / or related effector complex proteins into the liver cells, or to express a described miRNA. In examples, and adeno viral vector or adeno-associated viral vector may be used. As an alternative to a Cas system, a zinc finger nuclease, or a transcription activator-like effector nuclease (TALEN), or a transposon-based DNA editing system can be used to modify liver cells as described herein.

[0044] In examples, RNAi-mediated silencing of a UHRF1 gene is performed by delivery of any suitable RNAi agent. In examples, an siRNA-based approach is used. This can be performed by introducing and / or expressing one or more suitable short hairpin RNAs (shRNA) in the cells. shRNA is an RNA molecule that contains a sense strand, antisense strand, and a short loop sequence between the sense and antisense fragments. shRNA is exported into the cytoplasm where it is processed by dicer into short interfering RNA (siRNA). siRNA are 21 -23 nucleotide double-stranded RNA molecules that are recognized by the RNA-induced silencing complex (RISC). Once incorporated into RISC, siRNA facilitate cleavage and degradation of targeted mRNA. Thus, for use in RNAi mediated silencing or downregulation of expression of a described protein, siRNA, shRNA, or miRNA can be used. In alternative examples, a functional RNA, such as a ribozyme is used. In examples, the ribozyme comprises a hammerhead ribozyme, a hairpin ribozyme, or a Hepatitis Delta Virus ribozyme. In examples, the RNAi agent may be modified to improve its efficacy, such as by being resistant to nuclease digestion.

[0045] “Agents” as used herein include but are not necessarily limited RNAi agents, gene editing systems, binding partners, and small molecule drugs. In examples one or more agents described herein may be combined with other agents that are intended for prophylaxis or treatment of a described disorder or any condition or symptom for which an individual is in need of prophylaxis or therapy.

[0046] In an example, a prophylactic or therapeutic agent, such as RNA or other described polynucleotides or derivatives thereof, is provided in combination with any suitable nucleic acid delivery agent, non-limiting examples of which are described in Mitchell, M.J., et al.. Nat Rev Drug Discov. 20, 101-124 (2021 ), the description of which is incorporated herein by reference. In non-limiting embodiments a polynucleotide of this disclosure is combined with one or more unilamellar and / or multilamellar vesicular structures such as liposomes or lipid nanoparticles, cationic polymers, lipoplexes, polyplexes, or inorganic nanoparticles. Any delivery agent described herein may comprise polyethylene glycol (PEG) and thus may be PEGylated.

[0047] In examples, a therapeutically effective amount of one or more described agents is delivered to an individual. The term “therapeutically effective amount” as used herein refers to an amount of a described miRNA or other described agent, in a single dose or multiple doses, to achieve the intended purpose of treatment. The amount desired or required may vary depending its mode of administration, patient specifics and the like. Appropriate effective amounts can be determined by one of ordinary skill in the art informed by the instant disclosure using routine experimentation. In examples, a therapeutically effective amount of a described agent is administered to an individual in need thereof. In examples, a described agent is administered to an individual using an expression vector such as a viral vector. In examples, a combination of described agents is administered to an individual. In examples, a described agent alone or in combination with at least one other described agent is administered to an individual and is sufficient to achieve a prophylactic of therapeutic effect.

[0048] In examples, the cells into which a described polynucleotide or other agent as described herein is introduced comprise human liver cells. In examples, the cells are within a human individual. The disclosure includes the proviso that an individual diagnosed with cancer, including liver cancer, may be excluded from the claims. In examples, use of a described polynucleotide or other agent that inhibits the expression of UHRF1 results in preservation or improvement of at least one of: a biological age that is less than the time period during which the individual has been alive; a frailty index; endurance or vitality or mobility; inhibition, or reversal of liver cellular senescence; regenerative capacity and / or function of liver. In examples, use of a described agent improves or preserves the structure of bone. In examples, the individual is in need of a therapeutic or prophylactic treatment for any aging-related diseases, including but not necessarily limited to Alzheimer’s Disease; Parkinson’s disease; aging-related macular degeneration; canities / achromotrichia (graying of hair whether premature or not); metabolic dysfunction-associated steatotic liver disease (MASLD); steatohepatitis (MASH); osteoarthritis; osteoporosis; osteopenia; Osteogenesis Imperfecta; aging-associated immunosenescence; genomic instability; a hepatitis virus infection; or any combination of the foregoing. In examples, the individual is in need of treatment for undesirable weight gain, obesity, or obesity-related conditions. In examples, the individual has Obesity class I (BMI 30 to 34.9 kg / mA2); or Obesity class II (BMI 35 to 39.9 kg / mA2) or Obesity class III (BMI greater than or equal to 40 kg / mA2), which may also be referred to as severe, extreme, or massive obesity, respectively. Thus, in examples, administration of a described agent results in a reduction of adipose tissue in the individual. In examples, administration of a described agent improves survival after hepatectomy, including but not necessarily limited to partial hepatectomy. In an example, administration of a described agent is to a human individual that is above 30, 40, 50, 60, 70, or 80 years old. In examples, a described agent is administered to a non-human mammal, such as a canine, feline, or equine animal, with adjustments for any UHRF1 gene sequences in these mammals which can be readily determined by those skilled in the art.

[0049] Non-limiting description and data are presented on the accompanying figures. In particular, the figures demonstrate that by inhibiting express of UHRF1 in a relevant mouse model, mice show reduced grey fur with age (FIG. 1); UhrflHepKOmice do not have age associated weight gain (FIG. 2); there is reduced accumulation of adipose tissue in aged UhrflHepKOmice (FIG. 3); aged UhrflHepKOmice exhibit improved activity levels (FIG. 4) and increased bone density (FIG. 6). UHRF1HepKOmice at all ages show enhanced liver regeneration (FIG. 6). UHRF1HepKOmice at all ages show improved survival following partial hepatectomy in UHRF1HepKO(FIG. 7).

[0050] Data presented on the figures is not meant to be limiting. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only.

Claims

What is claimed is:1 . A method comprising inhibiting expression of UHRF1 in liver cells of an individual to thereby promote one or a combination of the following effects: a) inhibition of reduction of bone density or an increase in bone density; b) inhibition of weight gain or a reduction in adipose mass; c) inhibition of development of gray hair; d) an increase in activity level; e) a decrease in biological age; f) enhanced liver regeneration; g) increased survival following partial hepatectomy; or h) a combination of a) - g).

2. The method of claim 1 , wherein the inhibition of reduction of bone density or the increase in bone density occurs.

3. The method of claim 1 , wherein the inhibition of weight gain or the reduction in adipose mass occurs.

4. The method of claim 1 , wherein the inhibition of development of gray hair occurs.

5. The method of claim 1 , wherein the increase in activity level occurs.

6. The method of claim 1 , wherein the decrease in biological age occurs.

7. The method of claim 1 , wherein the enhanced liver regeneration occurs.

8. The method of claim 1 , wherein the increased survival following partial hepatectomy occurs.

9. The method or any one of claims 1-8, wherein expression of the UHRF1 gene is inhibited only in hepatocytes.

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