WO2020079246A1

WO2020079246A1 - Non human mammal models for non-alcoholic fatty liver disease

Info

Publication number: WO2020079246A1
Application number: PCT/EP2019/078419
Authority: WO
Inventors: Dan Holmberg
Original assignee: Dan Holmberg
Priority date: 2018-10-19
Filing date: 2019-10-18
Publication date: 2020-04-23

Abstract

The invention provides a method of producing a non-human mammal model, comprising providing a 24αβΝΟD non-human mammal comprising at least one functioning RAG2 allele; and exposing the 24αβΝΟD non-human mammal comprising at least one functioning RAG2 allele to metabolic stress by maintaining the non-human mammal on a high fat diet, thereby generating the non-human mammal model. The model shows a pathology of non-alcoholic fatty liver disease (NAFLD) similar to that of the corresponding human condition. With the model mammal of the invention, it is possible to efficiently screen for substances for treating or preventing NAFLD, and to effectively evaluate the efficacy of medicinal substances. The invention also relates to the methods of using the non-human mammal model for screening and evaluation of therapeutic substances.

Description

NON HUMAN MAMMAL MODELS FOR NON-ALCOHOLIC FATTY LIVER DISEASE

Technical field

The present disclosure relates a non-human mammal model for non-alcoholic fatty liver disease (NAFLD). More specifically, the disclosure relates to a mouse exhibiting NAFLD pathology suitable for acting as a murine model of NAFLD, and to methods for producing said mouse and murine model. The disclosure further comprises methods of screening for and identifying agents capable of treating or preventing NAFLD using the non-human mammal model, as well as evaluating the efficacy of an agent in treating or preventing NAFLD using the model. Also, use of the model for screening for and identifying agents, and evaluating the ability of agents in treating or

preventing NAFLD is disclosed.

Background

Development of fibrous connective tissue as a reparative response to injury or damage is referred to as fibrosis. Fibrosis may both refer to the connective tissue deposition that occurs as part of normal healing, and to the excess tissue deposition that occurs as a pathological process. Fibrosis represents a large therapeutic area because an estimated >45% of all natural deaths in the western world are caused by an underlying fibrotic condition. Some of the main types of fibrosis occurring in the body is pulmonary fibrosis, cardiac fibrosis and hepatic (liver) fibrosis. When scar tissue builds up and takes over most of the liver, this is a more serious problem called hepatic cirrhosis.

Cirrhosis refers to the scar tissue and nodules that replace liver tissue and disrupt liver function. The condition is usually caused by alcoholism, fatty liver disease, hepatitis B or hepatitis C. Patients with fatty liver disease exhibit excess fat deposits in the liver, seen both in patients who are alcoholics as well as in patients who drink little to no alcohol. This non-alcohol induced fatty liver disease is referred to as non-alcoholic fatty liver disease (NAFLD), and is also known as simple steatosis. If NAFLD progresses it may lead to more severe stages such as cirrhosis and non-alcoholic steatohepatitis (NASH).

Fibrosis is defined by the overgrowth, hardening, and/or scarring of various tissues and is attributed to excess deposition of extracellular matrix components, including collagen, which can be detrimental to health and lead to organ failure. It is a common pathological response to tissue insults such as hyperglycemia, dyslipidemia and hypertension, hence often present in patients with diabetes. In long-standing diabetes, structural and functional defects in the vasculature may lead to diabetes complications including retinopathy, nephropathy, myocardiopathy, and atherosclerosis. An increasing focus has been on the excessive accumulation of hepatic fat in diabetic patients, which plays an important pathogenic role leading to an altered liver metabolism and the accumulation and activation of various inflammatory cells. This development of NAFLD may be present in up to 70% of patients with diabetes. Studies have shown that the co-existence of NAFLD and type 2 diabetes can act synergistically to drive adverse outcomes that increase the likelihood of diabetes complications, as well as the risk of more severe NAFLD, including hepatocellular carcinoma, cirrhosis and NASH.

NASH pathophysiology involves fat accumulation (steatosis), inflammation, and fibrosis. Steatosis results from hepatic triglyceride accumulation. Possible mechanisms for steatosis include reduced synthesis of very low density lipoprotein (VLDL) and increased hepatic triglyceride synthesis, possibly due to decreased oxidation of fatty acids or increased free fatty acids being delivered to the liver. Inflammation may result from lipid peroxidative damage to cell membranes. These changes can stimulate hepatic stellate cells, resulting in fibrosis. If advanced, NASH can cause cirrhosis and portal hypertension.

If NAFLD is detected and diagnosed during the early stages of the disease, further damage to the liver may be minimized, with the overall goal to slow disease progression and relieve symptoms. When NAFLD has advanced into for example NASH or liver cirrhosis, a liver transplant using a donor organ may be the only option for some patients. As a consequence, NASH is projected to become the leading indication for liver transplantation within developing countries by 2050. The only widely accepted treatment goal is to eliminate potential causes and risk factors, which may include discontinuation of drugs or toxins, weight loss, and treatment for dyslipidemia or treatment for hyperglycemia. To date, no evidence-based drug therapy has been approved for NASH management, and because therapeutic advances have been slow, NASH is classified as a medical condition with high unmet therapeutic need. Thus, there remains a need in the art for new improved treatments and methods for the development of new treatments for NAFLD, including severe NAFLD such as NASH.

Summary of the invention

An object of the present disclosure is to provide methods and devices which seek to mitigate, alleviate, or eliminate the above-identified deficiencies in the art and disadvantages singly or in any combination. The present disclosure aims to provide new methods for identifying and evaluating agents, for screening agents and evaluating the efficacy of agents, in the treatment or prevention of NAFLD and NASH. This object is obtained by NAFLD model mammals useful in the development of therapeutic and preventive therapies for NAFLD and NASH.

Accordingly, in a first aspect, the invention provides a method of producing a non-human mammal model, said method comprising providing a 24abNOϋ non-human mammal comprising at least one functioning (normally

functioning) RAG2 allele, and exposing the 24abNOϋ non-human mammal to metabolic stress, for example by maintaining the non-human mammal on a diet, such as a high fat diet (HFD), thereby generating the non-human mammal model. By maintaining the non-human mammal on a high fat diet, the non-human mammal is exposed to metabolic stress, which induces a pathological condition in the non-human mammal, hence making the non human mammal a suitable model mammal of said pathological condition. In particular, the model shows a pathology of non-alcoholic fatty liver disease (NAFLD) similar to that of the corresponding human condition.

In the following, mammals according to the invention are sometimes referred to as“mammals of the present invention” or "model mammals of the present invention".

The 24abNOϋ non-human mammal comprising at least one functioning RAG2 could be a 24abNOϋ non-human mammal which is either homozygote for the RAG2 allele, i.e. a 24abNOϋ or 24c^NOD.RAG2^+/+ non-human mammal, or a RAG2 heterozygote, i.e. a 24c^NOD.RAG2^+/ non-human mammal. It could not contain a null-mutated RAG2 locus, i.e. not be a 24abN0ϋ.RA02 non-human mammal.

In one embodiment, the 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele is a 24abNOϋ non-human mammal, and providing a 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele comprises generating a 24abNOϋ non-human mammal by providing fertilized first and second oocytes from a non-obese diabetic (NOD) non-human mammal, introducing a first genetic construct comprising a T-cell receptor b (TORb) gene into said first oocyte and introducing a second genetic construct comprising a TCRa gene into said second oocyte, implanting said first and second oocytes in one or more surrogate non-human mammals, and thereby generating at least one TCRa expressing non-human mammal and one TORb expressing non-human mammal from the surrogate non-human mammal(s), and breeding the TCRa expressing non-human mammal with the ^Rb expressing non-human mammal to generate a 24abNOϋ non-human mammal. Generating at least one TCRa expressing non-human mammal and one ^Rb expressing non human mammal from the surrogate non-human mammals may comprise generating the non-human mammals by allowing the surrogate mammals to go through gestation and birth of the implanted transgenic oocytes to generate the transgenic non-human mammals.

In a further embodiment, the 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele is a 24c^NOD.RAG2^+/ non-human mammal, and providing a 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele further comprises providing a NOD.RAG2^7· non human mammal; and generating the 24c^NOD.RAG2^+/ non-human mammal by breeding the 24abNOϋ non-human mammal with the NOD.RAG2 ^/_ non human mammal. In a further embodiment, the 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele is a

24c^NOD.RAG2^+/+ non-human mammal, and providing a 24abNOϋ non human mammal comprising at least one functioning RAG2 allele further comprises generating a 24c^NOD.RAG2^+/+ non-human mammal by breeding a male and female 24c^NOD.RAG2^+/ non-human mammal. Since the

24abNOϋ non-human mammal and the 24c^NOD.RAG2^+/+ non-human mammal should be genetically identical, if the RAG2 homozygote is used for generating the MEL mouse the 24abNOϋ non-human mammal could be preferred over the 24c^NOD.RAG2^+/+ non-human mammal since the extra breeding steps may be avoided. For other reasons, the 24c^NOD.RAG2^+/+ non-human mammal obtained via the NOD. RAG2 ^/_ breeding could be preferred. The 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele could also provided by, for example, buying an existing 24abNOϋ non-human mammal.

The metabolic stress caused by exposure to high fat diet may trigger the development of non-alcoholic fatty liver disease (NAFLD) pathology in the 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele. Hence, in one aspect, the the non-human mammal model is a model of NAFLD pathology. In a further aspect, exposing the 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele to metabolic stress comprises maintaining the non-human mammal on a high fat diet until it develops NAFLD pathology. In some embodiments, the non-human mammal is maintained on a high fat diet for at least 12 weeks. Maintaining the non human mammal on a high fat diet for a longer time period further worsens the pathological condition. The NAFLD pathology or NAFLD-like

pathology/symptoms may include one or more of impaired glucose tolerance, steatotis, chronic liver fibrosis with porto-portal and porto-central bridging fibrosis, non-alcoholic steatohepatitis (NASFI), cirrhosis, and hepatocellular carcinoma. For instance, NAFLD pathology or NAFLD-like

pathology/symptoms may include two or more of obesity, impaired glucose tolerance, steatotis, chronic liver fibrosis with porto-portal and porto-central bridging fibrosis, NASH, cirrhosis, and hepatocellular carcinoma. In particular, NAFLD may comprise one or more of NASH, cirrhosis and hepatocellular carcinoma.

In one embodiment, the non-human mammal is a rodent, preferably a rat or mouse. The high fat diet may be a rodent diet with 60% of the kcal from fat, such as diet D12492 provided by company Research Diets Inc.

(www.researchdiets.com).

In another aspect, the invention provides a non-human mammal model obtainable by the production methods above. In yet another aspect is provided a model of NAFLD comprising the non-human mammal model. In a further aspect is provided the model for use in the screening or evaluation of agents for treatment or prevention of said disease.

In another aspect, the resultant non-human mammal model is used for evaluating an agent for its suitability for treating or preventing NAFLD. In a further embodiment, evaluating an agent for its suitability for treating or preventing NAFLD comprises evaluating the efficacy of an agent in the treatment or prevention of NAFLD. According to another aspect, the resultant non-human mammal model is used in screening for an agent suitable for the treatment or prevention of NAFLD. In one embodiment, the screening identifies an agent as suitable for the treatment or prevention of NAFLD upon detecting an ameliorating effect of the agent of the NAFLD pathology of the non-human mammal model.

In a further embodiment, the resultant non-human mammal model is used for identifying biomarkers useful in diagnosing NAFLD.

In another aspect, the invention provides use of a 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele, such as

a24c^NOD.RAG2^+/ non-human mammal, for generating a non-human model mammal exhibiting NAFLD pathology. The NAFLD pathology may have been induced by consumption of a high fat diet by the non-human mammal.

In another aspect, the invention provides a 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele and exhibiting NAFLD pathology. In an embodiment, the NAFLD pathology has been induced by consumption of a high fat diet by the non-human mammal.

In another aspect, use of the non-human mammal model as a model for NAFLD is provided. The model may be studied as such, or may be

administered, e.g. by ingestion or injection, an agent to be evaluated. A further aspect relates to the use of the non-human mammal model for screening for an agent suitable for treating or preventing NAFLD or NAFLD pathology/symptoms, or the use of the non-human mammal model for evaluating an agent for its ability to treat or prevent NAFLD or NAFLD pathology/symptoms.

In some aspects of the invention is provided a method of evaluating an agent for the ability to treat or prevent NAFLD, the method comprising administering an agent to a24c^NOD.RAG2^+/ non-human mammal exhibiting NAFLD pathology, and monitoring an effect of the administered agent on the NAFLD pathology of the non-human mammal. Optionally, the method also includes providing a 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele; and maintaining the non-human mammal on a high fat diet to induce metabolic stress until the non-human mammal develops NAFLD pathology, before administering the agent to be evaluated.

In a further embodiment is provided a method of screening for an agent capable of treating or preventing NAFLD, the method comprising

administering an agent to a24c^NOD.RAG2^+/_ non-human mammal exhibiting NAFLD pathology, and monitoring an eventual effect of the administered agent on the NAFLD pathology of the non-human mammal, and, if detecting an ameliorating effect of the administered agent on the NAFLD pathology, identifying the agent as capable of treating or preventing NAFLD. Optionally, the method also includes providing a 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele; and maintaining the non human mammal on a high fat diet to induce metabolic stress until the non human mammal develops NAFLD pathology, before administering the agent(s) to be screened.

It is noted that the invention relates to all possible combinations of features recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.

Brief description of the drawings

Figure 1 is an illustration of an exemplary process for generating a 24abNOϋ mouse. Figure 2 an illustration of an exemplary process for generating

24abNOϋ. RAG2^+/ and 24c^NOD.RAG2^+/+ mice.

Figure 3 is a weight curve of a 24c^NOD.RAG2^+/ mouse on high fat diet (HFD) or normal diet (ND), respectively.

Figure 4 is an illustration of the glucose tolerance of the 24c^NOD.RAG2^+/ mouse on HFD or ND after 12 weeks (4A) and after 24 weeks (4B).

Figure 5 illustrates the presence of steatosis and inflammation in a

24c^NOD.RAG2^+/ mouse after 12 weeks on HFD (5B, D) compared to healthy control on ND (5A, C).

Figure 6 illustrates the presence of fibrosis in a 24c^NOD.RAG2^+/ mouse after 12 weeks on HFD (6B, 6D) compared to healthy control on ND (6A, 6C).

Figure 7 is a flowchart of an exemplary process for generating a 24abNOϋ non-human mammal.

Figure 8 is a flowchart of an exemplary process for producing a non-human mammal model of the invention.

Figure 9 is a flowchart of an exemplary process for screening for and identifying an agent capable of treating or preventing NAFLD, or evaluating an agent for the ability to treat or prevent NAFLD.

Detailed description

Aspects of the present disclosure will be described more fully hereinafter with reference to the accompanying drawings. The products and method disclosed herein can, however, be realized in many different forms and should not be construed as being limited to the aspects set forth herein. Like numbers in the drawings refer to like elements throughout. The term“non-human mammal” refers to any mammal, other than humans, that may be used as a model for NAFLD according to the current disclosure. Preferably, the mammal is a rodent, such as a rat or mouse.

A model animal, such as a non-human mammal in this case, is an animal with a disease either the same as or similar to a disease in humans. Animal models are used to study the development and progression of diseases and to test new treatments before being applied to humans. Hence, by the term “model” as in, for instance,“non-human mammal model”,“model mammal” or more generic“animal model”, is meant an animal, in this case a non-human mammal, that exhibits symptoms of a certain pathology, such as NAFLD pathology, which is the same or similar to the corresponding human pathology, and which therefore can be used a model for said pathology, and as a model for testing therapeutic agents, by being administered agents and evaluated for disease progress/amelioration. Thus, a non-human mammal model of NAFLD/NAFLD pathology according to the invention is a non-human mammal exhibiting NAFLD pathology similar to the NAFLD pathology seen in humans, wherein the NAFLD pathology has been induced by metabolic stress.

By the term“functioning RAG2 allele” or“normally functioning RAG2 allele” is meant any genotype that comprises at least one allele that can generate a functioning RAG2 protein, hence comprising a functional RAG2 gene, i.e. that does not contain two null mutated alleles.

Hence, a“24abNOϋ non-human mammal comprising a functioning RAG2 allele” or a“24abNOϋ non-human mammal comprising a normally functioning RAG2 allele” is a non-obese diabetic (NOD) non-human mammal comprising the complete transgenic TCR (24ab) having at least one functioning RAG2 allele, i.e. a functioning RAG2 gene. The non-obese diabetic (NOD) non human mammal comprising the complete transgenic TCR (24ab) might be a double transgenic 24abNOϋ non-human mammal, or a 24abNOϋ non-human mammal that has been bred with a NOD.RAG2⁷ non-human mammal but that still contains one functional RAG2 allele, i.e. a 24c^NOD.RAG2^+/ (RAG2 heterozygote) or a 24c^NOD.RAG2^+/+ (RAG2 + homozygote) non-human mammal. A 24abNOϋ non-human mammal should be genetically identical with a 24c^NOD.RAG2^+/+ non-human mammal in that they both contain two functioning RAG2 alleles. However, for the sake of clarity, in this disclosure the 24abNOϋ mice that has undergone the procedure of being bred with the RAG2 knock (NOD.RAG2^7·) and then selected from the crossing of the RAG2 heterozygote, are referred to as 24c^NOD.RAG2^+/+.

In some embodiments a non-limiting term“high fat diet” or“HFD” is used. The high fat diet herein can be any type of diet with a high fat/calorie content that has the capacity to metabolically induce a NAFLD like pathology in the 24abNOϋ non-human mammals of the invention (24abNOϋ,

24c^NOD.RAG2^+/ or 24c^NOD.RAG2^+/+). It could be a high fat diet with at least 30% of total calories derived from fat/saturated fatty acids such as 30% -75%, or 40-65%, preferably 60%, and/or it could be a high fat diet with lower methionine and choline content, and/or a high fat diet with >20% fructose.

The high fat diet used in the experiments was the D12492 diet provided by Research Diets (New Brunswick, NJ, USA), which is a Rodent Diet with 60% kcal from fat. As an alternative, a GUBRA or Diamond diet may be used.

NAFLD pathology refers to a pathological condition where the subject suffering from the condition shows symptoms of NAFLD, these symptoms including one or more of impaired glucose tolerance, steatotis, chronic liver fibrosis with porto-portal and porto-central bridging fibrosis, NASH, cirrhosis, and hepatocellular carcinoma. Hence, by maintaining a non-human mammal on a high fat diet until it develops NAFLD pathology, or signs of NAFLD pathology, or a pathological condition similar to NAFLD, is meant to maintain the mammal on the high fat diet until it develops one or more symptoms of NAFLD, the symptoms including impaired glucose tolerance, steatotis, chronic liver fibrosis with porto-portal and porto-central bridging fibrosis, NASH, cirrhosis, and hepatocellular carcinoma.

Also in some embodiments generic terminology“agent”, is used. It can be any kind of agent, such as a substance, compound or drug, or a combination of substances, compounds or drugs, which may be used for prevention or therapeutic treatment of a condition.

Herein, "amelioration" means that the symptoms, such as at least one symptom, of NAFLD/NASH are alleviated or restored to normal. By using as an indicator the pathological findings described herein, those skilled in the art can appropriately evaluate whether the symptoms of hepatic fibrosis are ameliorated in the model animals.

The non-human mammals of the current disclosure may be defined as a transgenic animal, a transgenic non-human mammal. As used herein, “transgenic” is to be understood as referring to a specific desired genetic modification being introduced into the genome of the animal. A modification may be represented by the introduction of a gene or a part thereof, or the removal or silencing of a gene, or a modification that alters the expression of a gene. The origin of the modification (e.g., a newly introduced gene) may be the same species as the animal in question, or it may be a different species.

Excessive accumulation of hepatic fat and development of NAFLD is an increasing problem, especially for patients with diabetes. NAFLD

encompasses a spectrum of disease states, from steatosis (fatty liver) to NASH (steatosis with inflammatory changes) followed by progression to fibrosis, cirrhosis and hepatocellular carcinoma. A progressing simple steatosis NAFLD may thus lead to NASH including liver steatosis, liver inflammation and hepatocellular degeneration. Today, no evidence-based drug therapy has been approved for NASH management, and because therapeutic advances have been slow, NASH is classified as a medical condition with high unmet therapeutic need.

Accordingly, the lack of a preclinical model of progressive NASH that recapitulates human disease is a barrier to therapeutic development. To facilitate the development of novel diagnostic and therapeutic interventions in NASH, a plethora of animal models have been used to identify molecular targets, but a lack of relevant, robust and translatable preclinical models to test the new drugs have hampered this development. Thus, there remains a need in the art for improved methods and models for the development of and treatments for NAFLD/NASH.

The current disclosure provides solutions to the above-mentioned problems and drawbacks by providing a new non-human mammal exhibiting NAFLD and/or NASH pathology hence acting as a new non-human mammal model for NAFLD and NASH. The non-human mammal/non-human mammal model may be used for studying NAFLD/NASH pathology and development, for identifying agent that may be used as diagnostics for NAFLD and NASH, to screen agents to identify an agent or agents which may be used in the treatment or prevention of NAFLD or NASH, as well as to test the efficacy of an agent for use in the treatment and prevention of NAFLD and/or NASH. The non-human mammal model of NAFLD and/or NASH pathology may be used for the development of therapeutic and preventive treatments for

NAFLD/NASH. The present disclosure hence relates to new non-human mammal models for human NAFLD and NASH, and their use as in vivo models for testing and evaluating potential preventive measures, and therapeutic modalities for the intervention in the progression of human NAFLD and/or NASH.

Previously published journal paper Fransen-Pettersson et al. PlosOne 1 1 0159850, 201 ., relates to a non-human animal model of fibrosis and inflammation. The paper discloses a mouse that spontaneously develops fibrosis and inflammation, and thus may act as a murine model for hepatic and renal fibrosis. The mouse constituting this murine model, referred to as the N-IF mouse, was obtained by first producing a double transgenic

24abNOϋ mouse strain and then crossing (breeding) it with a double knock out NOD.RAG2^_/· mouse strain to obtain the N-IF mouse strain, a null-mutated RAG-2 locus mice strain (24a NOD.RAG2 ^/ ). The N-IF mouse overproduces a monoclonal natural killer T cell (NKT cell) population and spontaneously develops an inflammatory syndrome most evident in the liver but also affecting other organ systems such as the kidney. Flepatic fibrosis in the N-IF mouse occurs spontaneously and is preceded by the infiltration of

inflammatory cells (extensive cellular infiltration dominated by granulocytes, particularly eosinophils, macrophages, mast cells and multinucleated giant cells can be observed already at three weeks of age), which mainly occurs in the portal regions and around the central vein. The development of fibrosis in this model is similar to what is seen in many human fibrotic conditions.

Furthermore, the fibrotic areas display accumulation of activated aSMA+ hepatic stellate cells, which is also a characteristic of most human liver fibrosis. The disease develops in 100% of the N-IF mice and is evident already at three weeks of age. Treatment of the N-IF mouse with a standard anti-inflammatory drug, Rapamycin (Pfizer Inc.) has been demonstrated to inhibit the inflammation but not the fibrosis. In contrast, while the quinoline derivative Paquinimod (Active Biotech) inhibits and reverses the inflammation in a similar fashion as Rapamycin, Paquinimod also efficiently inhibits also the fibrosis in the N-IF mouse (Fransen Pettersson, N. et al. PlosOne, 13:e 0203228. 2018). The results demonstrated proof-of-principle of the N-IF mouse model as a novel and improved model for liver fibrosis.

However, even though the N-IF mouse exhibits fibrosis pathology as seen in NAFLD, it does not reflect the early stages of the disease development which are usually metabolically driven. Therefore, a model animal which mimics the whole disease progression of NAFLD and NASH is still lacking. To meet this need, a new model animal has been developed, a non-human mammal exhibiting NAFLD pathology, which also mimics the early stages of disease development. It has been found that the previously developed double transgenic 24abNOϋ mouse used for generating the N-IF mouse, or an offspring of the 24abNOϋ mouse bred with a NOD.RAG2^7· mouse strain having at least one functioning (normally functioning) RAG2 allele, i.e.

24c^NOD.RAG2^+/ or 24c^NOD.RAG2^+/+, develops signs of NAFLD pathology upon consumption of a high fat diet (HFD). This 24abNOϋ mouse having at least one functioning RAG2 allele (24abNOϋ, 24c^NOD.RAG2^+/ or 24c^NOD.RAG2^+/+) expressing NAFLD pathology upon HFD consumption has been named the MEL mouse. Compared to the N-IF mouse which develops fibrosis and inflammation spontaneously, the NAFLD pathology of the MEL mouse is metabolically driven. Hence, the MEL mouse can act as an animal model for NAFLD (including NASH), modelling both metabolic and inflammatory phases of disease development, thus including both the early and late stages of disease progression. This can be very valuable when testing potential drug candidates having a potential effect in different phases of disease progression, and when testing combination therapies where both metabolic and inflammatory targets are tested simultaneously.

It has been shown in experimental studies that metabolic stress induced by consumption of a high fat diet triggers the NKT cell population in

24abNOD.RAG2^+/_ mice leading to a pro-inflammatory and pro-fibrotic process similar to that observed in the N-IF mouse. In one example,

24abNOD.RAG2^+/_ mice were maintained on either a high-fat diet (HFD) of 12.6 kiloJoule/gram, kJ/g (5.24 kilocalories/gram, kcal/g, of diet) , 34.9 weight/weight (wt/wt) fat, 26.2 wt/wt protein, 26.2 wt/wt carbohydrate, or a normal diet (ND) of 21 .9 kJ/g (3 kcal/g), 4 wt/wt fat, 18.5 wt/wt protein, 55.7 wt/wt carbohydrate, during 1 2weeks, starting at 5 weeks of age, after which the mice were sacrificed and studied for signs of NAFLD pathology. In another experiment the mice were kept on the same diets for 24 weeks, also starting at 5 weeks of age, after which they were sacrificed. It was found that already after 12 weeks on high fat diet, the mice developed NAFLD with macrovesicular steatosis, hepatocyte ballooning and with area of lobular steatohepatitis and porto-portal and porto-central bridging fibrosis. No such pathology was observed in aged and sex matched animals that received ND.

The high fat diet used in the experiment was the D12492 diet provided by Research Diets (New Brunswick, NJ, USA), which is a Rodent Diet with 60 kcal% fat. Of the provided energy (kcal) in the diet 60% of the kcal come from fat, 20% of the kcal from proteins and the last 20% of the kcal from

carbohydrates, with a total energy density of 5.21 kcal/g. Typically, a high fat diet has a fat content of 30-75% of the kcal from fat, preferably 60%. A diet with fat content higher than 75% should also theoretically give the same result. The ND was R36 provided by Lactamin AB (Stockholm, Sweden), but any normal rodent diet would work such as provided by Special Diets, Ltd. A ND typically has a fat content of 4-7% of the total kilocalories from fat

(standard chow), preferably around 4 % of the total kilocalories from fat.

Hence, when 24a NOD.RAG2^+/ mice (which do not spontaneously develop any signs of inflammation or fibrosis) are fed a diet with a high fat and carbohydrate content (HFD) from 5 weeks of age, the mice develop steatosis, hepatitis and liver fibrosis (Fig.5 and Fig.6), as well as impaired glucose tolerance test (Fig. 4). Thus, this model, called the MEL mouse, can model both the early (metabolic/obesity-related) as well as late (inflammation driven) stages of NAFLD/NASH. Thus, these models may represent novel and better tools for analyzing the pathogenesis of this disease process and for identifying new biomarkers and preclinical testing of intervention regimes, which may be used in diagnosis of NAFLD/NASH. Together, these findings demonstrate that the 24abNOD.RAG2^+/ mice fed HFD develops a liver disease bearing the major hallmarks of NAFLD and NASH reflecting the entire spectrum of both metabolic and inflammatory phases of disease development. Thus, the model mice may be attained by exposing the 24c^NOD.RAG2^+/_ or 24ab NOD.RAG2^+/+ mice to metabolic stress, such as feeding the

24c^NOD.RAG2^+/ or 24ab NOD.RAG2^+/+ mice a HFD. As an alternative, the 24c^NOD.RAG2^+/ or 24ab NOD.RAG2^+/+ mice may exposed to metabolic stress by being fed other diets, which may also be considered as high in fat, such as a GUBRA diet or Diamond diet (Diet Induced Animal Model Of Non alcoholic fatty liver Disease). The GUBRA diet includes AMLN (Amylin liver NASH) diet (40% total fat kcal of which 18.5% were trans-fat kcal, 20% fructose, 2% cholesterol; Research Diets #D09100301 ), or a modified AMLN diet with Primex substituted by equivalent amounts of palm oil (Research Diets, #D09100310), termed Gubra Amylin NASH (GAN) diet, and the Diamod diet includes 42% kcal from fat and containing 0.1 % cholesterol (Harlan TD.88137) with a high fructose-glucose solution (SW, 23.1 g/L d- fructose + 18.9 g/L d-glucose). Accordingly, the animal model may also be attained using these or similar diets.

Advantageously, a model mammal of the present invention may naturally exhibit symptoms of NAFLD, that is, it may exhibit such symptoms upon dietary induction, without any additional induction, such as chemical or surgical induction. Further, it does not spontaneously develop signs of NAFLD pathology upon consumption of a normal diet.

The mammals described herein are the first model animals that exhibit the same course of disease progression as humans. For example, upon examination of an organ, specifically the liver, of an animal of the present invention, the exhibited the following pathological findings were noted:

(i) Obesity

(ii) Abnormal glucose tolerance test

(iii) Macrovesicular and small droplet steatosis with hepatocellular “ballooning”

(iv) Steatohepatitis

(v) Chronic fibrosis with porto-portal and porto-central bridging fibrosis These pathological findings are characteristic of human NAFLD and NASH. The NAFLD/NASH model mammals described herein differ from conventional animal models for these conditions in the following:

(i) Many conventional animal models require induction by chemical/toxic reagents or surgical manipulation while the MEL mouse similar to the human condition is only induced by diet.

(ii) Most conventional animal models display the only a section of the disease spectrum e.g. the metabolic phase or the immunological phase while the MEL mouse display the full spectrum.

As described herein, the produced model mammals for steatosis, NAFLD and NASH show similar pathological findings to those of the corresponding human conditions. By using these model mammals, it is possible to efficiently screen for substances for treating or preventing NAFLD/NASH, and to effectively evaluate the efficacy of medicinal substances.

The previously established N-IF mouse model eliminated some of the problems of currently available animal models for fibrosis including the inflammatory phase of NAFLD/NASH, not only serving as a model for efficacy tests but also enabling novel approaches to elucidate the cellular and molecular mechanisms underlying the pathogenesis of fibrotic diseases.

However, the N-IF mouse does not reflect the entire spectrum of both metabolic and inflammatory phases of disease development for

NAFLD/NASH. Thus, the MEL mouse model provides a unique tool for analyzing the complete process of NAFLD/NASH including the metabolic as well as the inflammatory phase of the disease in the same mice. This reflects better the process in the human disease and is therefore likely to have improved translational potential both for analysis of pathogenic mechanisms and as models for efficacy testing of drug candidates against this disease conditions. Hence, also drugs (agents/compositions/substances) targeting components in signaling pathways present in these early stages may be screened and evaluated according to this disease model.

Hence, in the present disclosure is described non-human mammal model, such as murine models, exhibiting NAFLD pathology, which may be used in screening of agents to identify an agent, a drug candidate or a target, which may be used for prevention or treatment of NAFLD/NASH. The model may also be used for target validation, drug lead optimization and for testing and evaluating the efficacy of an agent in the treatment or prevention of NAFLD and/or NASH.

The type of non-human mammals to be used in the present invention are not particularly limited, as long as they are useful as experimental mammals. Examples of mammals that can be used for producing model animals of the present invention specifically include mice, rats, rabbits, dogs, and monkeys (such mammals are sometimes also referred to simply as "experimental mammals"). The genetic background of the mammals to be used to produce model mammals of the present invention is not particularly limited; and it is possible to use mammals with any genetic background. In general, wild-type mammals can be preferably used.

In embodiments, the mammal may be, may be based on, or may include genetic characteristics of a non-obese diabetic (NOD) model mammal, for example a non-obese diabetic mouse. In some aspects, the invention involves a NOD mammal expressing a transgenic a,b T cell receptor. A model mammal according to the various aspects and embodiments of the invention may be obtained by feeding a high fat diet to a NOD mammal expressing a transgenic a,b T cell receptor, such as a 24abNOϋ mouse strain having a functioning RAG2 gene, i.e. at least one functioning RAG2 allele. The

24abNOϋ mouse strain having a functioning RAG2 allele may be a 24abNOϋ mouse strain, or may be a 24c^NOD.RAG2^+/- mouse strain obtained by crossing the 24abNOϋ mouse strain with a NOD.RAG2 ^/_ mouse strain, or may be a 24a NOD.RAG2^+/+ mouse strain obtained by crossing a male and a female of the 24a NOD.RAG2^+/_ mouse strain.

Hence, here is also provided a method of making a genetically modified mouse comprising the steps of:

a) providing fertilized first and second oocytes from a NOD mouse;

b) introducing a first genetic construct comprising cDNA of a TCRa gene into said first oocyte, and introducing a second genetic construct comprising cDNA of a TCR gene into said second oocyte;

c) implanting said first and second oocytes in one or more surrogate animals to generate at least to two single transgenic mice;

d) breeding said at least two mice together to generate a double transgenic 24abNOϋ mouse expressing the TCRa and the TCR genes; and optionally, if the 24abNOϋ mouse strain having a functioning RAG2 allele is a 24ab NOD.RAG2^+/ or a 24ab NOD.RAG2^+/+ mouse strain,

e) (optional) breeding said double transgenic 24abNOϋ mouse strain with a NOD.RAG2^7· mouse strain to obtain a 24ab NOD.RAG2^+/_ mouse strain, and further optionally

f) (optional) breeding a female and a male of the 24abN0ϋ.HA02⁺ mouse strain

to obtain a 24abN0ϋ.HA02^+/+ mouse strain.

The breeding in step f) will also generate the 24abN0ϋ.HA02 (N-IF) mouse strain. However, this mouse strain is not suitable for use in the current invention since it spontaneously develops fibrosis. Upon feeding the

24abNOϋ mouse strain having a functioning RAG2 allele (24abNOϋ, 24ab NOD.RAG2^+/_ or a 24ab NOD.RAG2^+/+) a high fat diet, the MEL mouse is produced, a mouse that exhibits NAFLD pathology upon dietary induced metabolic stress. Hence, exhibiting NAFLD pathology as a result of metabolic stress, induced by a HFD consumption, makes the MEL mouse suitable as a murine model for NAFLD and NASH, especially since it mimics the whole disease progression including the early metabolically driven phase of the corresponding human diseases. Even though the experiments were performed on the N-IF mouse and a MEL mouse generated from a

24c^NOD.RAG2^+/ mouse strain, the phenotype of the 24abNOϋ and the 24a i\IOD.RAG2^+/- are the same, all containing the TCR 24ab transgene in a NOD mouse with a functioning RAG2 gene, i.e. having an evolved adaptive immune system, hence it is considered that the TCR 24ab transgene in a NOD mouse comprising at least one functioning RAG2 allele is the common feature needed for the mouse strain used to generate the MEL mouse.

Further, the genotype of the 24abNOϋ and the 24c^NOD.RAG2^+/+ mouse (generated by breeding the 24abNOϋ with the NOD.RAG2^7· to generate the RAG2 heterozygous 24c^NOD.RAG2^+/ and then breeding the heterozygous 24c^NOD.RAG2^+/ with itself to generate the 24c^NOD.RAG2^+/+ homozygote and further heterozygous 24c^NOD.RAG2^+/ , and the RAG2 null mutated 24c^NOD.RAG2 ^/_), should be identical, since the RAG2^+/+ indicates two functioning RAG2 alleles, which is the case for the NOD“wild type” mice. I.e. genetically the 24abNOϋ mouse is a 24c^NOD.RAG2^+/+ mouse. However, in this disclosure, the 24abNOϋ mice that has undergone the procedure of being bred with the RAG2 knock and then selected from the crossing of the RAG2 heterozygote, are referred to as 24c^NOD.RAG2^+/+.

NKT cells constitute a population of unconventional T lymphocytes that express the ab T cell receptor (TCR) together with several NK surface markers and recognize glycolipids presented by the MHC class I like CD1 d molecule. The NKT cell population is heterogeneous where the majority, referred to as type I NKT cells, express an invariant TCR and display specificity for glycolipids presented by CD1 d, with the prototype antigen being a- GalCer. Type II NKT cells resemble type I NKT cells in their restriction to CD1 d, but use a diverse set of TCR and have a less well-defined range of antigen specificities. NKT cells are highly enriched in the liver and have been shown to be able to promote as well as to protect from inflammation and fibrosis development, suggesting that the net effect of the NKT cells depends on the balance between these properties. In line with this activated NKT cells are known to be able to produce large amounts of both anti-fibrotic (e.g.

interferon (I FN)-Y) and profibrotic (e.g. interleukin-4 (IL-4), IL-13) cytokines.

In embodiments of the present invention, a transgenic modification may comprise a TCRo gene and/or a TCR[ 3 gene(s) introduced into a mammal. In embodiments, a modification may also comprise a mutation of the RAG2 locus, as long as one of the alleles are still functioning. The modification may be introduced into a mammal that is an ancestor of an animal according to embodiments of the invention. Thus, a genetic modification is not necessarily introduced via molecular genetic engineering directly into the animals according to the invention, but may be introduced into an ancestor animal which is further bred using conventional methods to produce a descendant animal according to the invention having the desired genotype. In

embodiments, the TCRo and/or the TCR[ 3 gene(s) may originate from the same species. In embodiments where the animal of the invention is a mouse, the TCRo and/or the TCR[ 3 gene(s) may be of murine origin.

To produce mice that develop pathological conditions similar to those of humans, fertilized oocytes from NOD mice were injected with constructs containing cDNA from a TCRa or TCR gene, respectively, to generate two single transgenic mice. The 24ab TCR-transgenic mice were made directly on a non-obese diabetic (NOD) genetic background using TCR expression constructs encoding a CD1 d-reactive TCR. Each TCR chain construct (containing rearranged TCR Va3.2 and TCR nb9 regions, respectively) was microinjected into fertilized embryos of NOD origin alone to create single chain transgenic mice. The two mouse strains, mice positive for the transgenic TCRa and b chains, were then intercrossed (bred together) to generate a double transgenic mouse expressing the TCRa and TΰRb genes, i.e. to obtain 24abNOϋ mice expressing the complete transgenic TCR. The process of producing the 24abNOϋ mice has previously been described in Duarte, N et al. J. Immunol. 173:31 12-31 18, 2004, which is incorporated herein by reference. Optionally, to obtain 24abNOϋ mice having at least one functional RAG2 allele, the 24abNOϋ mouse strain and a NOD.RAG2 ^/_ mouse strain were intercrossed (bred) to generate 24c^NOD.Rag2^+/ rmice. The NOD.RAG2 ^/_ mouse strain used in the invention is a well-known strain, known from for example Soderstrom et al. Scan. J. Immunol. 43: 525-530, 1996. Then, by crossing male and female 24c^NOD.RAG2^+/ mice together,

24abNOϋ. RAG2^+/+ mice (25%), 24c^NOD.RAG2^+/- mice (50%)

and 24c^NOD.RAG2^_/· mice (25%) were generated. This process has also been previously described in Fransen-Pettersson N. et at. PlosOne 1 1 ,

0159850, 2016, when generating the N-IF mouse.

The mice of embodiments of the present invention were fed a high fat diet, starting from 5 weeks of age, and were sacrificed at different ages. Organs (mainly liver, skin, kidney) were analyzed histopathologically (FIE staining, immune staining for macrophages, NKT cells, hepatic stellate cells and fibroblasts) and immunohistochemically (hepatic stellate cells, bile ducts). Already after 12 weeks on high fat diet a NAFLD pathology were seen in the mice, which got worse at 24 weeks.

Specifically, in embodiments the present invention provides methods of screening for substances for treating or preventing an NAFLD or NAFLD pathology, especially NASH, which comprise:

a) Administering a test substance (agent) to a model animal of the present invention; and

b) Evaluating an ameliorating effect on the NAFLD pathology

In embodiments of the invention, substances that produce the ameliorating effect in the step of (b) above can be selected as substances for treating or preventing NAFLD, such as NASH. The agents or test substances to be used in these methods are not particularly limited. For example, such substances include single compounds such as natural compounds, synthetic compounds, organic compounds, inorganic compounds, proteins, and peptides, as well as compound libraries, expression products of gene libraries, cell extracts, cell culture supernatants, products of fermenting microorganisms, extracts of marine organisms, and plant extracts, but are not limited thereto.

Furthermore, medicinal substances (therapeutic agents) can be assessed for their efficacy in ameliorating a NAFLD condition, such as NASH, by using model mammals of the present invention. Specifically, the present invention provides methods for evaluating the efficacy of medicinal substances in ameliorating NAFLD pathology, which comprise the steps of:

a) Administering a test medicinal substance to a NAFLD model mammal according to embodiments of the present invention; and

b) Evaluating an ameliorating effect on the NAFLD pathology.

The type of medicinal substances that can be evaluated for efficacy by the above-described methods is not particularly limited; and such medicinal substances include, for example, various known pharmaceutical agents (low- molecular-weight compounds, proteins, nucleic acids, and the like).

When a test medicinal substance exerts an ameliorating effect on NAFLD and or NASH, as determined by observation of pathological findings as described above or by other methods of assessment of the degree or severity of NAFLD pathology/symptoms appreciated by a person of skill in the art, the medicinal substance is judged to have therapeutic effect on NAFLD/NASH.

Methods for administering agents, such as test substances or medicinal substances, of the present invention are not particularly limited; however, they can be administered, for example, by injection. When such a test substance is a protein, for example, a viral vector carrying a gene encoding the protein may be constructed and can be introduced into model animals of the present invention using their infectability.

Examples

The methods of producing the non-human mammals and non-human mammal models of the invention will now be described in more detail referring to the illustrations in Figures 1 and 2, and the corresponding flowcharts of Figures 7 and 8. It should be appreciated that flowcharts in Figures 7, 8 and 9 comprise some operations and modules which are illustrated with a solid border and some operations and modules which are illustrated with a dashed border. The operations and modules which are illustrated with solid border are operations which are comprised in the broadest example embodiment. The operations and modules which are illustrated with dashed border are example embodiments which may be comprised in, or a part of, or are, further embodiments which may be taken in addition to the operations and modules of the broader example embodiments. It should be appreciated that not all of the operations need to be performed.

Figures 1 and 7 show an illustration and a flowchart, respectively, of a method of generating S1 a a 24abNOϋ non-human mammal by providing S1 a (i) fertilized first 102 and second 101 oocytes from a non-obese diabetic (NOD) non-human mammal, and then introducing S1 a (ii) a first genetic construct comprising a T-cell receptor a (TCRa) gene into said first oocyte 102, generating a TCRa comprising oocyte 102^' and introducing S1 a (iii) a second genetic construct comprising a TCR gene into said second oocyte 101 generating a TCR comprising oocyte 101 ^', and then implanting S1 a (iv) the first 102^' and second 101 ^' oocytes in one or more surrogate non-human mammals 104, 103, thereby generating S1 a (v) at least one TCRa expressing non-human mammal 106 and one TCR expressing non-human mammal 105 from the surrogate non-human mammals 104, 103, and then breeding S1 a (vi) the TCRa expressing non-human mammal 106 with the TCR expressing non-human mammal 105 to generate S1 a a 24abNOϋ non-human mammal 107.

In one embodiment, the non-human mammals 105, 106 are generated from the surrogate non-human mammals 103, 104 as offspring from the surrogate non-human mammals 103, 1 04. I.e., the fertilized transgenic oocytes 101 ^', 102^' give rise non-human mammals 105, 106 as offspring of the surrogate non-human mammals 103, 1 04 upon implantation, gestation and birth.

Figure 2 shows an illustration of a method of providing S1 c, S1 d a non-human mammal comprising at least one functioning RAG2 allele and Figure 8 is a flowchart of a method of producing a non-human mammal model by providing S1 a 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele, and exposing S2 the non-human mammal to metabolic stress by maintaining the non-human mammal on a high fat diet (HFD), thereby generating the non-human mammal model. Thus, the exposure to metabolic stress induces an expression in the non-human mammal which turns the non human mammal into a non-human mammal model. I.e., maintaining the 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele on a high fat diet induces a pathological condition in the non-human mammal turning the non-human mammal into a model mammal of said condition, hence thereby generating the non-human mammal model. In embodiments the pathological condition is NAFLD or NASH, and the method is a method of producing a non-human mammal model of NAFLD and/or NASH.

In one embodiment, the 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele is a 24abNOϋ non-human mammal 107, and providing S1 a 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele comprises generating S1 a a 24abNOϋ non-human mammal 107 according to the above mention method. Alternatively, the 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele may be a 24c^NOD.RAG2^+/_ non-human mammal 109 and providing S1 further comprises providing S1 b a NOD.RAG2^7· non-human mammal 108 and generating Sl c the 24c^NOD.RAG2^+/ non-human mammal 109 by breeding the 24abNOϋ non-human mammal 107 with the NOD.RAG2⁷ non-human mammal 108. In other embodiments, the 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele is a 24c^NOD.RAG2^+/+ non human mammal 1 10 and providing S1 further comprises generating S1 d a 24c^NOD.RAG2^+/+ non-human mammal 1 10 by breeding a male and female 24c^NOD.RAG2^+/ non-human mammal 109. Breeding the male and female 24c^NOD.RAG2^+/ non-human mammals 109 generates mammals of three genotypes, the 24c^NOD.RAG2^+/ non-human mammal 109 itself, the

24c^NOD.RAG2^+/+ non-human mammal 1 10 and the 24abNOϋ.RA02 non human mammal 1 1 1 , also known as the N-IF mouse. This mouse 1 1 1 is not a part of the present invention, while both the double transgenic 24abNOϋ non human mammal 107, the heterozygous 24c^NOD.RAG2^+/ non-human mammal 109 and the homozygous 24c^NOD.RAG2^+/+ non-human mammal 1 10 can be used for producing the model mammals of the invention, such as the MEL mouse. The mammal 107 and the mammal 1 10 should be

genetically idenentical with the only difference that the mammal 1 10 has undergone some more breeding and selection in the production process. Hence, either the RAG2 homozygote 107, 1 10 or heterozygote 109, may be used for generating the MEL mice.

In yet further embodiments, exposing S2 the 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele to metabolic stress comprises maintaining S2^' the non-human mammal on a high fat diet until it develops NAFLD pathology.

Figure 3 shows a weight gain curve for 24abNOϋ non-human mammals comprising at least one functioning RAG2 allele, in this case

24c^NOD.RAG2^+/ mice, fed HFD (D12495) from 5 weeks of age compared with 24c^NOD.RAG2^+/ mice fed ND (low fat diet, LFD, chow) for the whole period. The mice were maintained on either a high-fat diet (5.24 kcal/g, 34.9 % (wt/wt) fat, 26.2 % (wt/wt) protein, 26.2 % (wt/wt) carbohydrate; D12492, Research Diets, New Brunswick, NJ, USA) or a LFD chow (3 kcal/g, 4 % (wt/wt) fat, 18.5 % (wt/wt) protein, 55.7 % (wt/wt) carbohydrate; R36,

Lactamin AB, Stockholm, Sweden) for 18 weeks, starting at 5 weeks of age. The body weight (in grams) of the mice on HFD or ND were recorded weekly from 5 weeks of age until 40 weeks of age, and for endpoint analysis. It is clear that the high fat diet has a large impact on the body weight of the mice, inducing obesity in the HFD fed mice.

Figure 4 shows the result of oral glucose tolerance tests, showing blood glucose levels (mmol/l) minutes after oral glucose stimulation of

24a NOD.RAG2^+/_ mice fed HFD (D12495) from 5 weeks of age for either 12 weeks (Fig. 4A) or 24 weeks (Fig. 4B), compared with 24a NOD.RAG2^+/_ mice fed ND (LFD chow) for the whole 17 (A) or 29 (B) week period. The animals were fasted for 6 hours followed by an oral glucose tolerance test (OGTT), where blood glucose was measured at various time points before and after oral glucose administration (2g/kg). Cheek blood samples were collected before and after glucose administration for hormone analysis. The figures show the blood glucose levels of the mice (mmol/l) at different timepoints, in minutes, following oral glucose stimulation at time 0 minutes. 45 minutes after glucose stimulation, a significant difference in the blood glucose levels is seen in the mice fed a NFD for 12 weeks (Fig. 4A) compared to ND, and in the mice fed a HFD for 24 weeks (Fig. 4B), a significant difference is seen even sooner, within 15-45 minutes after glucose stimulation, compared to mice fed ND (control). Hence, these result indicates that the MEL mouse has developed an impaired glucose tolerance compared to control mice.

Figure 5 illustrates the presence of steatosis and inflammation in a

24a NOD.RAG2^+/ mouse after 12 weeks on ND (FIG. 5A, 5C) and HFD (FIG. 5B, 5D). Figure 5 is based on photographs of the result of

Eosin/hematoxylin staining of hepatic tissue from a 17-week-old NFL mouse fed ND (A, C) or fed HFD for the last 12 weeks (B, D), where A and B show 5X enlargement of the tissue and C and D show 20X enlargement of the tissue (500pm and 100 pm respectively). Liver tissues were fixed in 4% neutral buffered formalin, embedded in paraffin and sectioned. Sections (5 pm) were stained with hematoxylin and eosin (H&E). The figures demonstrate that the mice fed high fat diet (MEL mice) has gained NAFLD pathological symptoms of steatosis and inflammation, compared to the mice fed a ND (control).

Figure 6 illustrates an example the presence of NASH and fibrosis in a 24c^NOD.RAG2^+/ mouse after 12 weeks on ND (6A, 6C) and HFD (6B, 6D). Figure 6 is based on photographs of the result of Picorna Sirius red staining of hepatic tissue from a 17-week-old NFL mouse fed ND (A, C) or fed HFD for the last 12 weeks (B, D), where A and B show 5X enlargement of the tissue and C and D show 20X enlargement of the tissue. Liver tissues were fixed in 4% neutral buffered formalin, embedded in paraffin and sectioned. Sections (5 pm) were stained with Sirius red and were evaluated microscopically. The figures demonstrate that the mice fed HFD (MEL mice) has gained NAFLD pathological symptoms of NASH and fibrosis, compared to the mice fed a ND (control).

Figure 9 is a flowchart of exemplary methods of screening for and identifying agents capable or suitable of treating and/or preventing NAFLD or symptoms of NAFLD pathology, as well as evaluating the efficacy of an agent, which may already be identified as a potential candidate, in treating and/or preventing NAFLD and/or symptoms of NAFLD pathology. The method comprises, optionally, providing S10 a 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele, and optionally maintaining S11 the non-human mammal on a high fat diet until it develops NAFLD pathology. The methods further comprise administering S12 an agent to a 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele. Where the method is a method of evaluating an agent for the ability to treat or prevent NAFLD/NAFLD symptoms, and/or to evaluate the efficacy of an agent in treating or preventing NAFLD, the method further comprises monitoring S13a an effect of the administered agent on the NAFLD

pathology/symptoms of the non-human mammal. If very little or no

ameliorating effect is detected, the agent’s ability to treat or prevent NAFLD is considered lacking. If moderate or high ameliorating effect is detected, then the agent’s ability to treat or prevent NAFLD is considered

present/substantial. The efficacy of the agent in treating or preventing NAFLD may be related to the degree of ameliorating effect of the agent. Typically, the higher degree of ameliorating effect, the higher the expected efficacy of the agent. If the effect of the agent is a worsening of the NAFLD pathology or symptoms, it is considered to be unable to treat or prevent NAFLD.

Where the method is screening for and identifying one or more agents capable or suitable for treating or preventing NAFLD or NAFLD-like

pathology, the method further comprises monitoring S13b the presence of an eventual effect of the administered agent on the NAFLD pathology or NAFLD symptoms of the non-human mammal, and, if detecting an ameliorating effect of the administered agent on the NAFLD pathology/symptoms, identifying S14b the agent as capable of treating or preventing NAFLD. Hence, the presence of an ameliorating effect on the NAFLD pathology/symptoms upon administration of the agent to the mammal model indicates that the agent may be a suitable candidate for the treatment or prevention of NAFLD.

The content of this disclosure thus provides new non-human mammal models of NAFLD and NASH. The use of such model mammals facilitates the analysis of the pathogenesis and the pathological condition of human NAFLD and NASH, and facilitates the development of techniques and agents for treating human NAFLD, including human NASH.

In the drawings and specification, there have been disclosed exemplary aspects and embodiments of the disclosure. However, many variations and modifications can be made without substantially departing from the principles of the present disclosure. Thus, the disclosure should be regarded as illustrative rather than restrictive, and not as being limited to the particular aspects and embodiments discussed above. Accordingly, although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation. The features of the embodiments described herein may be combined in all possible combinations of methods. It should be appreciated that the example embodiments presented herein may be practiced in any combination with each other. It should be noted that the word “comprising” does not necessarily exclude the presence of other elements or steps than those listed and the words“a” or“an” preceding an element do not exclude the presence of a plurality of such elements. It should further be noted that any reference signs do not limit the scope of the claims, that the example embodiments may be realized in the broadest sense of the claims.

All references cited herein are incorporated by reference to the extent allowed.

Claims

1. A method of producing a non-human mammal model, said method comprising:

providing (S1 ) a 24abNOϋ non-human mammal (107, 109, 110) comprising at least one functioning RAG2 allele; and

exposing (S2) the 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele to metabolic stress by maintaining the non-human mammal on a high fat diet (HFD), thereby generating the non-human mammal model.

2. A method of producing a non-human mammal model, said method comprising:

providing (S1 ) a 24abNOϋ non-human mammal (107, 109, 110) comprising at least one functioning RAG2 allele; wherein the providing (S1 ) comprises:

generating (S1 a) a 24abNOϋ non-human mammal (107) by providing (i) fertilized first (102) and second (101 ) oocytes from a non-obese diabetic (NOD) non-human mammal; introducing (ii) a first genetic construct comprising a T-cell receptor a (TCRa) gene into said first oocyte;

introducing (iii) a second genetic construct comprising a TORb gene into said second oocyte;

implanting (iv) said first and second oocytes in one or more surrogate non-human mammals (103, 104);

generating (v) at least one TCRa expressing non-human mammal (106) and one TΰRb expressing non-human mammal (105) from the surrogate non-human mammal(s); and breeding (vi) the TCRa expressing non-human mammal with the TORb expressing non-human mammal to generate a 24abNOϋ non-human mammal (107); and exposing (S2) the 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele to metabolic stress by maintaining the non-human mammal on a high fat diet (HFD), thereby generating the non-human mammal model.

3. The method of claim 1 or 2, wherein the 24abNOϋ non-human

mammal comprising at least one functioning RAG2 allele is a

24c^NOD.RAG2^+/ non-human mammal (109), and wherein providing (S1 ) further comprises:

providing (S1 b) a NOD.RAG2 ^/ non-human mammal (108); and generating (S1 c) the 24c^NOD.RAG2^+/ non-human mammal by breeding the 24abNOϋ non-human mammal with the

NOD.RAG2^7· non-human mammal.

4. The method of any one of the claims 1 -3, wherein the metabolic stress triggers the development of non-alcoholic fatty liver disease (NAFLD) pathology in the non-human mammal.

5. The method of any one of the preceding claims, wherein the non

human mammal model is a model of NAFLD pathology.

6. The method of any one of the preceding claims, wherein exposing (S2) the 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele to metabolic stress comprises maintaining (S2^') the non human mammal on a high fat diet until it develops NAFLD pathology.

7. The method of any one of claims 4-6, wherein the NAFLD pathology includes one or more of impaired glucose tolerance, steatotis, chronic liver fibrosis with porto-portal and porto-central bridging fibrosis, non alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma.

8. The method of any one of claims 1 -7, wherein the non-human mammal is a rodent, preferably a mouse.

9. The method of any one of the preceding claims, wherein the resultant non-human mammal model is used for evaluating an agent for its suitability for treating or preventing non-alcoholic fatty liver disease (NAFLD).

10.The method of claim 9, wherein evaluating an agent for its suitability for treating or preventing NAFLD comprises evaluating the efficacy of an agent in the treatment or prevention of NAFLD.

1 1 .The method of any one of claims 1 -8, wherein the resultant non-human mammal model is used in screening for an agent suitable for the treatment or prevention of NAFLD.

12. The method of any one of claims 4-1 1 , wherein the NAFLD comprises one or more of impaired glucose tolerance, steatotis, chronic liver fibrosis with porto-portal and porto-central bridging fibrosis, non alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma.

13. A non-human mammal model obtainable by the method of any one of claims 1 -8.

14. Use of the non-human mammal model of claim 13 as a model for non alcoholic fatty liver disease (NAFLD).

15. The use of claim 14, further comprising using the non-human mammal model for evaluating an agent for its ability to treat or prevent NAFLD and/or for screening for an agent suitable for the treatment or prevention of NAFLD.

16. Use of a 24abNOϋ non-human mammal comprising at least one

functioning RAG2 allele for generating a non-human mammal model exhibiting NAFLD pathology.

17. The use of claim 16, wherein the NAFLD pathology is attained by

exposing the non-human mammal to metabolic stress.

18. The use of claim 17, wherein the metabolic stress is induced by

comsuption of a diet, such as a HFD.

19. The use of any one of claim 16-18, further comprising using the

generated non-human mammal model exhibiting NAFLD pathology for screening for an agent suitable for treating or preventing NAFLD and/or for evaluating an agent for its ability to treat or prevent NAFLD.

20. The use of any one of claims 14-19, wherein the NAFLD is non

alcoholic steatohepatitis (NASH), steatosis, cirrhosis, chronic liver fibrosis or hepatocellular carcinoma.

21.A method of evaluating an agent for the ability to treat or prevent non alcoholic fatty liver disease (NAFLD), the method comprising:

administering (S12) an agent to a 24a NOD non-human mammal comprising at least one functioning RAG2 allele and exhibiting NAFLD pathology; and

monitoring (S13a) an effect of the administered agent on the NAFLD pathology of the non-human mammal.

22. A method of screening for an agent capable of treating or preventing non-alcoholic fatty liver disease (NAFLD), the method comprising: administering (S12) an agent to a 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele and exhibiting

NAFLD pathology;

monitoring (S13b) the presence of an eventual effect of the administered agent on the NAFLD pathology of the non-human mammal; and,

if detecting an ameliorating effect of the administered agent on the NAFLD pathology,

identifying (S14b) the agent as capable of treating or preventing NAFLD.

23. The method of claims 21 or 22 further comprising:

providing (S10) a 24abNOϋ non-human mammal comprising at least one functioning RAG2 allele; and

maintaining (S1 1 ) the non-human mammal on a high fat diet until the non-human mammal develops NAFLD pathology.