EP4730993A1 - A new relevant non-human animal model of progressive metabolic dysfunction-associated steatotic liver disease (masld) and atherosclerosis development - Google Patents

Abstract

Metabolic dysfunction–associated steatotic liver disease (MASLD), previously known as non- alcoholic fatty liver disease (NAFLD) is the most common liver disease with a worldwide prevalence estimated at 30% of the general population. To better understand the influence of MASLD progression on cardiovascular disease, the aim of the study was to develop a new physiopathological mouse model able to develop progressive MASLD, along with atherosclerosis development, in a reasonable period of time. LDLr -/- mice were crossed with PPARα-deficient mice in order to obtain LDLr-/- mice expressing (LDLr -/- PPARα +/+) or not (LDLr -/- PPARα -/-) PPARα. Female mice were challenged with a high-fat diet during 12 and 18 weeks. Histological liver analysis showed that, compared to control diet, LDLr -/- PPARα +/+ mice under high fat diet developed mostly steatosis at 12 weeks, and then exhibited all characteristics of human MASLD, namely the steatosis, inflammation and ballooning-triad, with presence of fibrosis at 18 weeks. This MASLD progression over time in LDLr -/- PPARα +/+ was aggravated in LDLr -/- PPARα +/+ mice. The present invenion defines a new relevant mouse model of progressive MASLD, developing all the characteristics of human MASLD (steatosis, inflammation, ballooning, fibrosis), in a relatively short time period (12-18 weeks), along with simultaneous atherosclerosis development.

Description

A NEW RELEVANT NON-HUMAN ANIMAL MODEL OF PROGRESSIVE METABOLIC DYSFUNCTION-ASSOCIATED STEATOTIC LIVER DISEASE (MASLD) AND ATHEROSCLEROSIS DEVELOPMENT FIELD OF THE INVENTION: The present invention is in the field of medicine, in particular hepatology. BACKGROUND OF THE INVENTION: Metabolic dysfunction–associated steatotic liver disease (MASLD), previously known as non- alcoholic fatty liver disease (NAFLD), is a condition characterized by the accumulation of fat in the liver of individuals with no alcohol consumption, and at least one cardiometabolic risk factor (overweight/obesity, high blood pressure, high blood sugar, high triglycerides levels, low HDL-C levels). MASLD encompasses a spectrum of liver conditions, ranging from simple fatty liver (steatosis) to a more severe form called metabolic dysfunction-associated steatohepatitis (MASH). In simple fatty liver, there is an accumulation of excess fat in the liver cells, which does not typically cause significant liver damage. However, in MASH, in addition to fat accumulation, inflammation and liver cell injury are present. Over time, MASH can progress to liver fibrosis (scarring), cirrhosis (severe scarring), and even liver failure. The exact cause of MASLD is not fully understood, but it is closely associated with certain risk factors, including obesity, insulin resistance (prediabetes or type 2 diabetes), high blood pressure, high cholesterol levels, and a sedentary lifestyle. Genetic factors and certain medications can also contribute to the development of MASLD. MASLD often does not cause noticeable symptoms in the early stages. However, as the disease progresses, individuals may experience fatigue, abdominal discomfort, and jaundice (yellowing of the skin and eyes) in severe cases. MASLD is typically detected through routine blood tests or imaging studies, such as ultrasound, computed tomography (CT), or magnetic resonance imaging (MRI). Management of MASLD involves lifestyle modifications aimed at reducing risk factors. This includes maintaining a healthy weight through diet and exercise, managing blood sugar levels, lowering cholesterol and blood pressure, and avoiding alcohol and unnecessary medications. In more advanced stages of the disease, additional interventions may be required to manage complications or treat liver fibrosis and cirrhosis. There is a growing body of evidence suggesting a link between MASLD and atherosclerosis. Both MASLD and atherosclerosis are associated with common risk factors such as obesity, insulin resistance, and metabolic syndrome. Several mechanisms have been proposed to explain the relationship between MASLD and atherosclerosis. In particular, both MASLD and atherosclerosis are characterized by chronic low-grade inflammation. The inflammatory substances released in MASLD can promote the development of atherosclerosis by damaging the arterial walls and facilitating the deposition of cholesterol and plaque. Insulin resistance, a key feature of MASLD, is also associated with atherosclerosis. MASLD is often associated with abnormal lipid profiles, including increased levels of triglycerides and decreased levels of high-density lipoprotein (HDL) cholesterol. These lipid abnormalities contribute to the development of atherosclerosis by promoting the deposition of cholesterol in the arterial walls. Finally, MASLD and atherosclerosis share common risk factors such as obesity, type 2 diabetes, hypertension, and dyslipidemia. These risk factors contribute to both conditions and create a metabolic milieu that promotes the development of both MASLD and atherosclerosis. Several animal models are used to study MASLD. These models help researchers understand the underlying mechanisms, progression, and potential therapeutic interventions for MASLD. The commonly used animal models include: High-Fat Diet (HFD)-Induced Models: This is one of the most widely used models for MASLD. Animals, such as rodents (mice or rats), are fed a diet high in fat content, typically rich in saturated fats and/or fructose, for an extended period. The HFD leads to obesity, insulin resistance, hepatic lipid accumulation, inflammation, and liver injury similar to human MASLD. Genetic Models: These models involve genetically modified animals that mimic certain aspects of human MASLD. For example, ob/ob mice and db/db mice are models of obesity and insulin resistance due to leptin or leptin receptor deficiency, respectively. These mice exhibit features of MASLD, including hepatic steatosis and inflammation. Knockout or Knockdown Models also exist. In these models, specific genes involved in lipid metabolism, inflammation, or oxidative stress are genetically manipulated to be knocked out or downregulated. For example, knockout or knockdown of genes involved in lipid synthesis (e.g., SREBP-1c) or antioxidant defense (e.g., Nrf2) can lead to MASLD -like features. Animals with genetic mutations leading to obesity, such as Zucker fatty rats or KKAy mice, also develop obesity-associated metabolic abnormalities, including MASLD. Methionine-Choline Deficient (MCD) Diet Model: Animals, typically mice or rats, are fed a diet lacking methionine and choline. This diet induces liver injury and steatohepatitis, mimicking certain aspects of human MASH. The MCD diet model is particularly useful for studying the progression from simple steatosis to steatohepatitis. Currently, it is important to note that no single animal model completely recapitulates all aspects of human MASLD, as the disease is complex and multifactorial. Therefore, there is a need for an animal model that can recapitulate the physio pathological features of MASLD. More particularly, there is a need for having a model of progressive MASLD and atherosclerosis development. SUMMARY OF THE INVENTION: The present invention is defined by the claims. In particular, the present invention relates to a new relevant non-human animal model of progressive metabolic dysfunction–associated steatotic liver disease (MASLD) and atherosclerosis development DETAILED DESCRIPTION OF THE INVENTION: The present invention relates to a method for preparing a non-human animal model of progressive metabolic dysfunction–associated steatotic liver disease (MASLD) and atherosclerosis development comprising the steps of (a) providing a non-human transgenic animal for which the Ldlr and Ppara genes are knocked-out and (b) feeding the non-human transgenic animal of step (a) with a diet that causes development of metabolic dysfunction– associated steatotic liver disease (MASLD) and atherosclerosis. As used herein, the term “animal” includes all vertebrate and invertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. According to the present invention, the non-human animal of the present invention is a transgenic animal. As used herein, the term “transgenic animal” refers to a non- human animal, such as a mammal, generally a rodent such as a rat or mouse, in which one or more (preferably all) of the cells of the animal includes a transgene as described herein. A “transgene” is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and thus remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. Knock-out animals, which include a gene deletion or mutation as described herein, are included in the definition of transgenic animals. The term “knockout” of a gene means an alteration in the sequence of the gene that results in a decrease in the expression and/or function of the target gene, preferably the target gene expression is undetectable or insignificant. A knockout of an endogenous gene means that the expression and/or function of the gene has been substantially decreased so that it is not detectable or only present at insignificant levels. “Knockout” transgenics can be transgenic animals having a heterozygous knockout of a gene or a homozygous knockout of a gene. “Knockouts” also include conditional knockouts, where alteration of the target gene can occur upon satisfaction of certain conditions, such as, exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g., Cre in the Cre-lox system), or other conditions that direct the target gene alteration postnatally. As used herein, the term “Ldlr” has its general meaning in the art and refers to the gene that encodes for the low-density lipoprotein receptor. An exemplary amino acid sequence for LDLr is show as SEQ ID NO:1. The term “Ldr - indicates that both copies of the Ldlr gene have been knocked out, in other words this gene is no longer functional on either homologous chromosome. MSTADLMRRWVIALLLAAAGVAAEDSCSRNEFQCRDGKCIASKWVCDGSPECPDGSDESP ETCMSVTCQSNQFSCGGRVSRCIPDSWRCDGQVDCENDSDEQGCPPKTCSQDDFRCQDGK CISPQFVCDGDRDCLDGSDEAHCQATTCGPAHFRCNSSICIPSLWACDGDVDCVDGSDEW PQNCQGRDTASKGVSSPCSSLEFHCGSSECIHRSWVCDGEADCKDKSDEEHCAVATCRPD EFQCADGSCIHGSRQCDREHDCKDMSDELGCVNVTQCDGPNKFKCHSGECISLDKVCDSA RDCQDWSDEPIKECKTNECLDNNGGCSHICKDLKIGSECLCPSGFRLVDLHRCEDIDECQ EPDTCSQLCVNLEGSYKCECQAGFHMDPHTRVCKAVGSIGYLLFTNRHEVRKMTLDRSEY TSLLPNLKNVVALDTEVTNNRIYWSDLSQKKIYSALMDQAPNLSYDTIISEDLHAPDGLA VDWIHRNIYWTDSVPGSVSVADTKGVKRRTLFQEAGSRPRAIVVDPVHGFMYWTDWGTPA KIKKGGLNGVDIHSLVTENIQWPNGITLDLSSGRLYWVDSKLHSISSIDVNGGNRKTILE DENRLAHPFSLAIYEDKVYWTDVINEAIFSANRLTGSDVNLVAENLLSPEDIVLFHKVTQ PRGVNWCETTALLPNGGCQYLCLPAPQIGPHSPKFTCACPDGMLLAKDMRSCLTEVDTVL TTQGTSAVRPVVTASATRPPKHSEDLSAPSTPRQPVDTPGLSTVASVTVSHQVQGDMAGR GNEEQPHGMRFLSIFFPIALVALLVLGAVLLWRNWRLKNINSINFDNPVYQKTTEDELHI CRSQDGYTYPSRQMVSLEDDVA As used herein, the term “Ppara” has its general meaning of the art and refers to the gene encoding for peroxisome proliferator-activated receptor alpha. An exemplary amino acid sequence for PPARα is show as SEQ ID NO:2. The term “Ppara - indicates that both copies of the Ppara gene have been knocked out, in other words this gene is no longer functional on either homologous chromosome. SEQ ID NO:2 >sp|P23204|PPARA_MOUSE Peroxisome proliferator-activated receptor alpha OS=Mus musculus OX=10090 GN=Ppara PE=1 SV=2 MVDTESPICPLSPLEADDLESPLSEEFLQEMGNIQEISQSIGEESSGSFGFADYQYLGSC PGSEGSVITDTLSPASSPSSVSCPVIPASTDESPGSALNIECRICGDKASGYHYGVHACE GCKGFFRRTIRLKLVYDKCDRSCKIQKKNRNKCQYCRFHKCLSVGMSHNAIRFGRMPRSE KAKLKAEILTCEHDLKDSETADLKSLGKRIHEAYLKNFNMNKVKARVILAGKTSNNPPFV IHDMETLCMAEKTLVAKMVANGVEDKEAEVRFFHCCQCMSVETVTELTEFAKAIPGFANL DLNDQVTLLKYGVYEAIFTMLSSLMNKDGMLIAYGNGFITREFLKNLRKPFCDIMEPKFD FAMKFNALELDDSDISLFVAAIICCGDRPGLLNIGYIEKLQEGIVHVLKLHLQSNHPDDT FLFPKLLQKMVDLRQLVTEHAQLVQVIKKTESDAALHPLLQEIYRDMY According to the present invention, the non-human transgenic animal is Ldlr^-/- Ppara^-/-. Typically, said non-human transgenic animal results from the cross-breeding of LDLr^-/- animals and Ppara^-/- animals. Methods for generating transgenic animals, particularly animals such as mice, via embryo manipulation and electroporation or microinjection of pluripotent stem cells or oocytes, are known in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No.4,873,191, U.S. Ser. No.10/006,611, “Transgenic Mouse Methods and Protocols (Methods in Molecular Biology),” Hofker and van Deursen, Editors (Humana Press, Totowa, N.J., 2002); and in “Manipulating the Mouse Embryo,” Nagy et al., Editors (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2002), which are incorporated herein by reference in their entirety. Methods similar to those used to create transgenic mice can be used for production of other transgenic animals. In general, in the present methods, a transgenic mouse as described herein is made by injecting a vector made as described herein into the pronucleus of a fertilized mouse oocyte and used for generation of a transgenic mouse with all of the LDLr or PPARa genes knocked out in all cells, using standard transgenic techniques, e.g., as described in “Transgenic Mouse Methods and Protocols (Methods in Molecular Biology),” Hofker and van Deursen, Editors (Humana Press, Totowa, N.J., 2002); U.S. Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. Nos. 4,873,191 and 6,791,006, and in Hogan, “Manipulating the Mouse Embryo,” Nagy et al., Editors (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2002). Knockout may be achieved by a variety of mechanisms well known in the art, including but not limited to, introducing a disruption of the coding sequence, e.g., insertion of one or more stop codons, insertion of a DNA fragment, etc., deleting a portion of the coding sequence, substituting stop codons for the coding sequence, etc. In some embodiments, a chromosomal deletion of all or part of the target gene (e.g., Ldlr or Ppara) may be achieved by deletions of all or part of the coding region or deletions of the non-coding regions, which include the promoter region, 3’ regulatory sequences, and/or enhancers, etc. In some embodiments, knockout of a target gene (e.g., Ldlr or Ppara) can be achieved by deletions of gene that is necessary for activating expression of the target gene (e.g., Ldlr or Ppara). In other embodiments, a functional knockout can be achieved by introducing an antisense construct that blocks expression of the target gene (e.g., Ldlr or Ppara). See Li and Cohen, Cell 1996, 85:319- 329. In some embodiments, “knockouts” also include conditional knockouts, for example, where alteration of the target gene (e.g., Ldlr or Ppara) occurs when certain conditions are satisfied, including but not limited to, exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g., Cre in the Cre-lox system), or other method for directing the target gene alteration postnatally. Methods for disrupting genes are known in the art. See, e.g., U.S. Pat. No. 7,022,893 to Takeda et al. and U.S. Pat. No.6,218,595 to Giros et al., as well as U.S. Pat. No. 6,344,596 to W. Velander et al. (American Grey Cross); U.S. Pat. No.6,339,183 to T. T. Sun (New York University); U.S. Pat. No. 6,331,658 to D. Cooper and E. Koren; U.S. Pat. No. 6,255,554 to H. Lubon et al. (American National Grey Cross; Virginia Polytechnic Institute); U.S. Pat. No.6,204,431 to P. Prieto et al. (Abbott Laboratories); U.S. Pat. No.6,166,288 to L. Diamond et al. (Nextran Inc., Princeton, N.J.); U.S. Pat. No.5,959,171 to J. M. Hyttinin et al. (Pharming BV); U.S. Pat. No.5,880,327 to H. Lubon et al. (American Grey Cross); U.S. Pat. No.5,639,457 to G. Brem; U.S. Pat. No.5,639,940 to I. Garner et al. (Pharmaceutical Proteins Ltd.; Zymogenetics Inc); U.S. Pat. No.5,589,604 to W. Drohan et al. (American Grey Cross); U.S. Pat. No.5,602,306 to Townes et al. (UAB Research Foundation); U.S. Pat. No.4,736,866 to Leder and Stewart (Harvard); and U.S. Pat. No.4,873,316 to Meade and Lonberg (Biogen). In some embodiments, CRISPR/Cas9 gene editing methods are used to disrupt each of the five Ldlr or Ppara genes in the mice. See, e.g., Wang et al., Cell.2013 May 9; 153(4):910-8 In some embodiments, the non-human transgenic animal for which the Ldlr and Ppara genes are knocking-out is fed with a high fat diet. As used herein, the term "high fat diet" is used herein to refer to diets that typically have a higher percentage of calories (obtained from the fat in the diet) compared to a normal diet. The fat in the diet may include all types of dietary fat, whether animal or vegetable and whether monounsaturated, polyunsaturated, saturated, etc. Thus, the high fat diet has a higher caloric content than a normal diet. In some embodiments, the non-human transgenic animal is fed with a diet having equal to or greater than 30% of total energy from fat. In some embodiments, the non-human transgenic animal is fed with a diet having equal to or greater than 35%, 40%, 45%, 50%, 55%, 60%, or 65% of total energy from fat. For instance, the high fat diet contains 414.0 kcal/100 g with 43% as carbohydrate, 17% as protein, and 40% as fat. Another example of diet contains 23% of fat (e.g. butter), 17% as protein, and 0,2% of cholesterol. In some embodiments, the composition of the diet is as follows: In some embodiments, the non-human animal model of the present invention is a male. In some embodiments, the non-human animal model of the present invention is a female. In some embodiments, the non-human animal model of the present invention develops dyslipidaemia within 12-18 weeks of being fed with the diet of the present invention. In some embodiments, the non-human animal model of the present invention develops MASLD within 12-18 weeks of being fed with the diet of the present invention. In some embodiments, the non-human animal model of the present invention exhibits at least one symptom of human metabolic dysfunction–associated steatotic liver disease selected from the group consisting of lipid droplet deposition, ballooning, inflammatory cell infiltration and fibrosis in the hepatic tissue. In some embodiments, the non-human animal model of the present invention exhibits all characteristics of human MASLD, namely the steatosis, inflammation and ballooning-triad, with presence of fibrosis at 18 weeks. As used herein, the term “metabolic dysfunction–associated steatotic liver disease” (MASLD), previously known as non-alcoholic fatty liver disease (NAFLD), is a condition characterized by the accumulation of fat in the liver of individuals with no alcohol consumption, and at least one cardiometabolic risk factor (overweight/obesity, high blood pressure, high blood sugar, high triglycerides levels, low HDL-C levels). The term metabolic dysfunction– associated steatotic liver disease (MASLD) encompasses the term non-alcoholic fatty liver disease (NAFLD). As used herein, the term “metabolic dysfunction-associated steatohepatitis” (MASH) previously known as non-alcoholic steatohepatitis (NASH). In simple fatty liver, there is an accumulation of excess fat in the liver cells, which does not typically cause significant liver damage. However, in MASH, in addition to fat accumulation, inflammation and liver cell injury are present. The term metabolic dysfunction-associated steatohepatitis (MASH) encompasses the term non-alcoholic steatohepatitis (NASH). In some embodiments, the non-human animal model of the present invention develops atherosclerosis within 12-18 weeks of being fed with the diet of the present invention. The non-human animal model of the present invention is particularly suitable for screening agents (e.g. for the treatment of MASLD and/or atherosclerosis. The non-human animal model of the present invention is also particularly suitable for research purposes. Thus a further object of the present invention relates to a method of screening for a prophylactic and/or therapeutic agent for preventing or ameliorating MASLD and/or atherosclerosis comprising administering a test compound to the non-human animal model of the present invention , or a tissue or cell thereof, and evaluating the effect of the test compound on MASLD and/or atherosclerosis in the transgenic mouse, or tissue or cell thereof, in order to screen for a prophylactic and/or therapeutic agent for preventing or ameliorating MASLD and/or atherosclerosis. A further object of the present invention relates to a method of studying molecular and cellular aspects associated with MASLD and/or atherosclerosis comprising administering a test compound to the non-human animal model of the present invention, or a tissue or cell thereof, and evaluating the effect of the test compound on MASLD and/or atherosclerosis in the transgenic mouse, or tissue or cell thereof, in order to study the molecular and cellular aspects associated with MASLD and/or atherosclerosis. In some embodiments, the agent is diet or exercise. In some embodiments, the agent is a drug. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES: Figure 1: TD diet-induced metabolic profile in female and male LDLRKO mice deficient or not in PPARα. Body weight (A), perigonadal (B) and subcutaneous (C) white adipose tissue weight normalized to body weight. Fasting plasma glucose (D) and insulin (E) and homeostatic model for assessment of insulin resistance (HOMA-IR) calculated from fasting blood glucose and insulin level (F). Data are expressed as mean ± SEM. (n=4-11 mice/group in chow diet, n=9-14 mice/group in TD diet). Diet effect *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Genotype effect ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001. Figure 2: TD diet-induced dyslipidemia and liver injury in female and male LDLRKO mice deficient or not in PPARα. Fasting plasma total cholesterol (A), triglyceride (B) and free fatty acid (C) levels. Liver weight normalized to body weight ratio (D). Plasma ALT (E) and AST (F). Data are expressed as mean ± SEM. (n=4-11 mice/group in chow diet, n=9-14 mice/group in TD diet). Diet effect *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Genotype effect ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 Figure 3: TD diet-induced metabolic dysfunction-associated steatohepatitis (MASH) in female and male LDLRKO mice deficient or not in PPARα. Representative microscopic images of hematoxylin and eosin (H&E)-stained liver sections. Original magnification x100 (A). Score of steatosis (0-3) (B), inflammation (0-2) (C), ballooning (0-2) (D) according to Bedossa et al. MASLD Activity score (0-7) (E) and percentage of mice with MASH (F). Data are expressed as mean ± SEM. (n=4-11 mice/group in chow diet, n=9-14 mice/group in TD diet). Diet effect *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Genotype effect ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 Figure 4: TD diet-induced effects on hepatic expression of lipid and inflammatory-related genes in female and male LDLRKO mice deficient or not in PPARα. Hepatic mRNA expression levels of PPARα (A), ACO (B), BIEN (C), VLCAD (D), CD36 (E), F480 (F), CD68 (G), CD11b (H) and IL1b (I) genes by qPCR analysis. Data are expressed as mean ± SEM. (n=4-11 mice/group in chow diet, n=9-14 mice/group in TD diet). Diet effect *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Genotype effect ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001. Figure 5: TD diet-induced fibrosis in female and male LDLRKO mice deficient or not in PPARα. Representative microscopic images of Sirius Red-stained liver sections. Original magnification x40 (A). Quantification of Sirius Red-positive area (B). Hepatic mRNA expression levels of Col1A1 (C), TGFβ (D), αSMA (E) and BNC2 (F) genes by qPCR analysis. Data are expressed as mean ± SEM. (n=4-11 mice/group in chow diet, n=9-14 mice/group in TD diet). Diet effect *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Genotype effect ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 Figure 6: TD diet-induced atherosclerosis in female and male LDLRKO mice deficient or not in PPARα. Representative microscopic images showing Oil Red O-staining in the atherosclerotic lesions. Original magnification x 40 (A). Mean area of lesions of the analysed sections by quantification of Oil Red O-positive area (B). Data are expressed as mean ± SEM. (n=4-11 mice/group in chow diet, n=9-14 mice/group in TD diet). Diet effect ***p < 0.001, ****p < 0.0001. Genotype effect ^#p < 0.05. EXAMPLE: Background: Metabolic dysfunction–associated steatotic liver disease (MASLD) also known as Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease with a worldwide prevalence estimated at 30% of the general population. During the progression of MASLD, simple steatosis may evolve into metabolic dysfunction-associated steatohepatitis (MASH) previously known as non-alcoholic steatohepatitis (NASH), diagnosed as a combination of steatosis, inflammation and ballooning of hepatocytes, then leading to fibrosis and cirrhosis. Recently, MASH has been proposed as a new risk factor of cardiovascular diseases (CVD), independently of classical cardiovascular risk factors. There is growing evidence to support the link between these two pathologies, but the mechanisms involved remain unexplained. Objectives: To better understand the influence of MASLD progression on CVD, the aim of the study was to develop a new physiopathological mouse model able to develop progressive MASLD, along with atherosclerosis development, in a reasonable period of time. Methods and results: Figures 1A-1F, figures 2A-2F, figures 3A-3F, figures 4A-4I, figures 5A- 5F and figures 6A-6B. LDLr^-/- mice were crossed with PPARα-deficient mice in order to obtain LDLr^-/- mice expressing (LDLr^-/- PPARα^+/+) or not (LDLr^-/- PPARα^-/-) PPARα. Female mice were challenged with a control diet or with a high-fat diet during 12 and 18 weeks to establish the kinetics of evolution for MASLD and atherosclerosis development. Histological liver analysis showed that, compared to control diet, LDLr^-/- PPARα^+/+ mice under high fat diet developed mostly steatosis at 12 weeks, and then exhibited all characteristics of human MASLD, namely the steatosis, inflammation and ballooning-triad, with presence of fibrosis at 18 weeks. This MASLD progression over time in LDLr^-/- PPARα^+/+ was aggravated in LDLr^-/- PPARα^-/- mice, with presence of strong inflammation, ballooning and fibrosis already after 12 weeks of high fat diet. In addition, quantification of lesions near the aortic sinus also revealed progressive atherosclerosis development over time under high fat diet in LDLr^-/- PPARα^+/+ mice, a progression which was also more severe with PPARα deficiency. Conclusion: The present study defines a new relevant mouse model of progressive MASLD, developing all the characteristics of human MASLD (steatosis, inflammation, ballooning, fibrosis), in a relatively short time period (12-18 weeks), along with simultaneous atherosclerosis development. This model suggests a contribution of MASLD to atherosclerosis development and can be relevant to study the involved mechanisms and test new-targeted anti- MASH therapies with potential CVD benefits. TABLES Table 1 : summary of the main characteristics of female and male LDLRKO mice deficient or not in PPARα after 12 and 18 weeks of TD diet Minus and plus signs indicate the absence or presence of each alteration, with the relative degree of the alteration

REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS: 1. A method for preparing a non-human animal model of progressive metabolic dysfunction–associated steatotic liver disease (MASLD) and atherosclerosis development comprising the steps of (a) providing a non-human transgenic animal for which the Ldlr and Ppara genes are knocked-out and (b) feeding the non-human transgenic animal of step (a) with a diet that causes development of metabolic dysfunction–associated steatotic liver disease (MASLD) and atherosclerosis. 2. The method of claim 1 wherein the non-human transgenic animal is a rodent, in particular a mouse. 3. The method of claim 1 wherein the non-human transgenic animal is Ldlr^-/- Ppara^-/-. 4. The method of claim 1 wherein the non-human transgenic animal is fed with a high fat diet. 5. The method of claim 4 wherein the diet contains 414.0 kcal/100 g with 43% as carbohydrate, 17% as protein, and 40% as fat. 6. The method of claim 4 wherein the diet contains 23% of fat (e.g. butter), 17% as protein, and 0,2% of cholesterol. 7. The method according to any one of claims 1 to 6 wherein the non-human transgenic animal is fed with a the high fat diet for at least 12 weeks, preferably 18 weeks. 8. The non-human animal model obtainable by the method according to any one of claims 1 to 7. 9. The non-human animal model of claim 8 that is a male. 10. The non-human animal model of claim 8 that is a female. 11. The non-human animal model of claim 8 that develops MASLD within 12-18 weeks of being fed with the high fat diet. 12. The non-human animal model of claim 8 that exhibits at least one symptom of human metabolic dysfunction–associated steatotic liver disease selected from the group consisting of lipid droplet deposition, ballooning, inflammatory cell infiltration and fibrosis in the hepatic tissue. 13. The non-human animal model of claim 8 that develops atherosclerosis within 12-18 weeks of being fed with the high fat diet. 14. A method of screening for a prophylactic and/or therapeutic agent for preventing or ameliorating MASLD and/or atherosclerosis comprising administering a test compound to the non-human animal model according to any one of claims 8 to 13 , or a tissue or cell thereof, and evaluating the effect of the test compound on MASLD and/or atherosclerosis in the transgenic mouse, or tissue or cell thereof, in order to screen for a prophylactic and/or therapeutic agent for preventing or ameliorating MASLD and/or atherosclerosis. 15. A method of studying molecular and cellular aspects associated with MASLD and/or atherosclerosis comprising administering a test compound to the non-human animal model according to any one of claims 8 to 14, or a tissue or cell thereof, and evaluating the effect of the test compound on MASLD and/or atherosclerosis in the transgenic mouse, or tissue or cell thereof, in order to study the molecular and cellular aspects associated with MASLD and/or atherosclerosis.