Patent Application
20180303075
This disclosure relates to the development of a genetically unique organism, namely an isogenic mouse strain that develops liver steatosis, fibrosis, steatohepatitis, cirrhosis and hepatocellular carcinoma when fed a Western diet.
This document incorporates by reference a sequence listing text file submitted with this application on CD-ROMs in ASCII text format. The text file is named 02941029TAseqlistingfinal_ST25_rev.txt, is 3.45 gigabytes, and was created on Sep. 26, 2016.
Nonalcoholic fatty liver disease (NAFLD) has emerged as the leading cause of chronic liver disease in North America and most parts of the Western world1. It has two principal phenotypes i.e. a fatty liver and steatohepatitis2. Nonalcoholic steatohepatitis (NASH) is the more aggressive form of the disease and is expected to surpass hepatitis C virus infection as the leading etiology for hepatocellular cancer (HCC) and end-stage liver disease requiring liver transplantation3. Of great concern, a large proportion of NASH-related HCC occur in the absence of cirrhosis, the traditional risk factor for HCC4,5. The growing prevalence of obesity and NASH underlies the rising incidence of HCC6. There is currently no approved therapy for NASH. Also, the initial promise of highly effective chemopreventive and therapeutic agents for HCC has not been realized.
The lack of a preclinical model for NASH that recapitulates the human disease is a barrier to therapeutic development. While a large number of models have been described7-13, their utility for preclinical identification of treatment targets and response to various interventions for drug development is limited. This is partly related to the diversity of molecular mechanisms that can induce fat accumulation in hepatocytes. Also, most models do not develop classical steatohepatitis that resembles the human disease7-13. Most models also do not develop progressive fibrosis. Some models are also not associated with insulin resistance, e.g. the methionine and choline deficient (MCD) diet7, while others require ablation of pancreatic beta cells10,11. Besides not developing typical liver lesions, the substantially different hepatobiliary physiology of rats render rat-based models suspect. While the Ossabaw pig model comes close to mimicking human disease13, this is an expensive model and does not lend itself easily to gene manipulation to study the role of specific genes in disease pathogenesis. This lack of a viable model to test drugs in a preclinical setting before launching into expensive human trials is a deterrent for therapeutic development for NASH.
Similarly, there is a paucity of animal models for HCC that closely resemble human HCC. Most models require genetic manipulation or use of specific carcinogens and are thus at variance from the human condition14. As with NASH, the shortage of animal models for HCC remains a barrier for development of preventive and therapeutic strategies.
An ideal preclinical model for NASH and NASH-related HCC should be relatively simple, triggered by the same causes as human disease (caloric excess), associated with the same risk factors as in humans (obesity, insulin-resistance and dyslipidemia), and it should match human disease with respect to metabolic features, histology, outcomes, gene expression signature, lipid accumulation and activation of pathways relevant in humans. Importantly, it should also recapitulate the various stages of human disease. The development of HCC should also be triggered by the disease state and not by administration of a chemical carcinogen. Thus, a diet-induced animal model of nonalcoholic fatty liver disease was designed and invented to address this need for a better research tool that met all of the above criteria.
The invention provides a murine animal model in which the development of NASH, NASH-related HCC, and other related disorders, is successfully mimicked. Significantly, the development of disease in the mouse does not rely on administering a chemical agent, but rather on providing the animal with a “Western style” diet that is high in fat and/or sugars. The mouse develops obesity, insulin-resistance and dyslipidemia and is a good match for human disease with respect to metabolic features, histology, outcomes, gene expression signature, lipid accumulation and disease activation of pathways related to disease development. Significantly, the mouse reproduces normally and is isogenic, i.e. the offspring consistently and reproducibly exhibit the same phenotype and disease profiles as the parents.
Accordingly, provided herein is a viable and fertile mouse, and offspring thereof, whose genome comprises any or all of the chromosome sequences represented by SEQ ID NOs:1-39. In some embodiments, administration of a high-fat and high-sugar diet causes development of one or more disease conditions selected from the group consisting of steatosis, fibrosis, steatohepatitis, cirrhosis, hepatocellular carcinoma, obesity, insulin resistance, and dyslipidemia.
Another aspect of the invention provides a method of inducing development of hepatic steatosis, progressive hepatic fibrosis, steatohepatitis, hepatocellular carcinoma, or a disorder associated with metabolic syndrome in a mouse whose genome comprises any or all of the chromosome sequences represented by SEQ ID NOs:1-39 comprising the steps of:
a. administering to said mouse a diet comprising standard mouse chow and water for at least the first 8 weeks after birth,
b. discontinuing administration of the diet comprising standard mouse chow and water when the mouse is at least 8 weeks old, and,
c. further administering to the at least 8 week old mouse a diet comprising high-fat mouse chow and sugar water for a duration of at least 4 weeks.
In some embodiments, the diet administered in step (c) is administered for at least 4 weeks and hepatic steatosis is induced. In other embodiments, the diet administered in step (c) is administered for at least 16 weeks and progressive hepatic fibrosis is induced. In other embodiments, the diet administered in step (c) is administered for at least 12 weeks and steatohepatitis is induced. In other embodiments, the diet administered in step (c) is administered for at least 32 weeks and hepatocellular carcinoma is induced. In other embodiments, the diet administered in step (c) is administered for at least 4 weeks and at least one disorder associated with metabolic syndrome is induced.
The metabolic syndrome that is induced in the methods of the invention includes, but is not limited to, impaired glucose tolerance, whole body insulin resistance, hepatic insulin resistance, muscular insulin resistance, adipose tissue insulin resistance, incretin abnormalities, hyper-insulinemia, impaired glucose disposal rate, obesity, dyslipidemia, cardiovascular disease, atherosclerosis, microvascular disease, and kidney disease.
Another aspect of the invention provides a method for screening a compound for the prevention or treatment of a disorder selected from the group consisting of hepatic steatosis, progressive hepatic fibrosis, steatohepatitis, hepatocellular carcinoma, and a disorder associated with metabolic syndrome in a mouse whose genome comprises any or all of the chromosome sequences represented by SEQ ID NOs:1-39 comprising the steps of:
a. administering to said mouse a diet comprising standard mouse chow and water for at least the first 8 weeks after birth,
b. discontinuing administration of the diet comprising standard mouse chow and water when the mouse is at least 8 weeks old,
c. further administering to the at least 8 week old mouse a diet comprising high-fat mouse chow and sugar water for a duration of at least 4 weeks,
d. administering said compound concurrent with step (c) or after step (c), and
e. determining the effect of the compound on said disorder relative to a mouse not treated with the compound.
In some embodiments, the diet administered in step (c) is administered for at least 4 weeks and hepatic steatosis is induced. In other embodiments, the diet administered in step (c) is administered for at least 16 weeks and progressive hepatic fibrosis is induced. In other embodiments, the diet administered in step (c) is administered for at least 12 weeks and steatohepatitis is induced. In other embodiments, the diet administered in step (c) is administered for at least 32 weeks and hepatocellular carcinoma is induced. In other embodiments, the diet administered in step (c) is administered for at least 4 weeks and a disorder associated with metabolic syndrome is induced.
In some embodiments, the disorder associated with metabolic syndrome that is induced includes, but is not limited to, impaired glucose tolerance, whole body insulin resistance, hepatic insulin resistance, muscular insulin resistance, adipose tissue insulin resistance, incretin abnormalities, hyper-insulinemia, impaired glucose disposal rate, obesity, dyslipidemia, cardiovascular disease, atherosclerosis, microvascular disease, and kidney disease.
Another aspect of the invention provides an in vivo model system for at least one condition selected from the group consisting of hepatic steatosis, progressive hepatic fibrosis, steatohepatitis, hepatocellular carcinoma, and a condition associated with metabolic syndrome comprising a viable and fertile mouse whose genome comprises any or all of the chromosome sequences represented by SEQ ID NOs:1-39, wherein said at least one condition is induced by administering to said mouse a diet comprising standard mouse chow and water for at least the first 8 weeks after birth, discontinuing administration of the diet comprising standard mouse chow and water when the mouse is at least 8 weeks old, and further administering to the at least 8 week old mouse a diet comprising high-fat mouse chow and sugar water for a duration of at least 4 weeks.
A further aspect of the invention provides a use of an in vivo model system according to claim 17 for: the study of at least one condition selected from the group consisting of hepatic steatosis, progressive hepatic fibrosis, steatohepatitis, hepatocellular carcinoma, and a condition associated with metabolic syndrome, or in vivo screening or testing of the efficacy of candidate drugs for the treatment of a condition recited in a).
Described herein is a novel murine animal model with utility for the study of the development and treatment of disease conditions resulting from Western diet, and particularly liver disorders arising in humans. This new Diet-Induced Animal Model of Nonalcoholic fatty liver Disease (DIAMOND™) mouse model comprises an isogenic strain derived from a cross of two common mouse strains, 129S1/SvImJ and C57BI/6J where a simple high fat diet accompanied by ad lib consumption of water with a high fructose and glucose content (Western Diet sugar water (WD SW)) sequentially induces steatosis, steatohepatitis, progressive fibrosis and HCC. The mouse genome has the genetic sequence comprising any or all of the chromosome sequences represented by SEQ ID NOs:1-39. In exemplary embodiments, the mouse has a genetic sequence that comprises or consists of a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to any or all of the chromosome sequences represented by SEQ ID NOs:1-39.
SEQ ID NOs 1 and 2 together comprise a representative sequence of chromosome 10. SEQ ID NOs 3 and 4 together comprise a representative sequence of chromosome 11. SEQ ID NOs 5 and 6 together comprise a representative sequence of chromosome 12. SEQ ID NOs 7 and 8 together comprise a representative sequence of chromosome 13. SEQ ID NOs 9 and 10 together comprise a representative sequence of chromosome 14. SEQ ID NOs 11 and 12 together comprise a representative sequence of chromosome 15. SEQ ID NO: 13 comprises a representative sequence of chromosome 16. SEQ ID NO: 14 comprises a representative sequence of chromosome 17. SEQ ID NO: 15 comprises a representative sequence of chromosome 18. SEQ ID NO: 16 comprises a representative sequence of chromosome 19. SEQ ID NOs 17, 18, and 19 together comprise a representative sequence of chromosome 1. SEQ ID NOs 20 and 21 together comprise a representative sequence of chromosome 2. SEQ ID NOs 22 and 23 together comprise a representative sequence of chromosome 3. SEQ ID NOs 24 and 25 together comprise a representative sequence of chromosome 4. SEQ ID NOs 26 and 27 together comprise a representative sequence of chromosome 5. SEQ ID NOs 28 and 29 together comprise a representative sequence of chromosome 6. SEQ ID NOs 30 and 31 together comprise a representative sequence of chromosome 7. SEQ ID NOs 32 and 33 together comprise a representative sequence of chromosome 8. SEQ ID NOs 34 and 35 together comprise a representative sequence of chromosome 9. SEQ ID NO: 36 comprises a representative sequence of chromosome M. SEQ ID NOs 37 and 38 together comprise a representative sequence of chromosome X. SEQ ID NO: 39 comprises a representative sequence of chromosome Y.
The inventors have deposited a hepatocyte cell culture having the genomic DNA sequence of a mouse as described herein at the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, Va. 20110), in accordance with the terms of Budapest Treaty on Oct. 11, 2016. The deposited cell culture has the ATCC deposit number PTA-123551.
The inventors observed that, in the original non-isogenic cross-bred mice, 12951/SvImJ:C57BI/6J cross mice (termed 129/B6), a small percentage developed insulin resistance, obesity and liver pathology similar to humans when fed a Western diet. However, these heterogenous 129/B6 progeny do not have utility as a model of human disease because they are not isogenic and all of the mice do not consistently develop the pathology in response to the trigger (being fed a Western diet). Therefore, the inventors selectively bred the progeny by crossing mice that did develop the disease conditions to one another. After many generations were crossed over a period of years, a strain was obtained in which the histological phenotype of steatohepatitis was obtained consistently when the mice were fed the trigger diet. The mice were confirmed to be isogenic and the genetic sequence of the genome was determined (SEQ ID NOs:1-39).
In some aspects, the mice of the invention are fed a high fat, sugar water diet in order to induce one or more diseases or conditions of interest. As used herein, “ ”high fat” refers to food (e.g. rat chow) with a fat content of at least 42 energy % and 0.2% cholesterol. The chow diet typically contains about 5.8% fat. The Western diet may contain about 12.8% saturated fat. The presence of saturated FFA and cholesterol is important for disease progression into steatohepatitis. As used herein, “sugar water” refers to an aqueous solution comprising at least about 23.1 g/L d-fructose+18.9 g/L d-glucose of a “sugar” or sugar source such as glucose, fructose, sucrose, high fructose corn syrup.
The DIAMOND™ mouse has great utility as a research tool. The pattern of gene and protein expression can be studied during development and progression of the diseases from steatosis, to steatohepatitis, progressive fibrosis, cirrhosis, and hepatocellular carcinoma. Other comorbidities other than liver disease can be studied using this model, including various components of metabolic syndrome. Metabolic syndrome is recognized in the art as a constellation of associated disorders or conditions which are characteristic of metabolic syndrome. Non-limiting examples of disorders included in this group include impaired glucose tolerance, whole body insulin resistance, hepatic insulin resistance, muscular insulin resistance, adipose tissue insulin resistance, incretin abnormalities, hyper-insulinemia, impaired glucose disposal rate, obesity, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, cardiovascular disease, macrovascular disease, atherosclerosis, microvascular disease, and kidney disease.
Thus the model may be exploited to discover the metabolic pathways that are actively involved in the development of the constellation of diseases discussed above. Experiments can be designed using the new mouse model to discover treatments that interrupt the disease process and halt disease development, and/or to treat or reverse already developed diseases. The model is particularly useful as a screening tool for new compounds in pre-clinical drug development. Specifically, mice may be dosed with compounds or drugs at any point during the development of any of the disease conditions to determine if the compound or drug has therapeutic value. In particular, if symptoms of disease or other disease markers increase or stay the same after administration of the compound or drug as compared to a suitable control mouse (e.g. an isogenic mouse that is fed the same diet but does not receive the compound or drug), then the compound or drug is determined to be ineffective. Alternatively, if symptoms of disease or other disease markers decrease after administration of the compound or drug as compared to a suitable control mouse, then the compound or drug is determined to be therapeutically effective. Because the physiology and pathology of the DIAMOND™ mouse closely resembles the physiology and pathology of humans during disease development, compounds or drugs that prevent or treat disease in the mice are more likely to be effective in human clinical trials. Thus, use of the mice as a screening tool will speed up the pre-clinical drug development process and enable better decision-making regarding which compounds or drugs should advance to human clinical trials.
It will be appreciated that although the methods in this application are directed primarily towards dosing the DIAMOND™ mice with compounds or drugs to prevent or treat disease, these are non-limiting examples and the utility of the mice extends beyond pharmaceutical screening purposes. Additionally, while the dietary regime described herein comprises allowing the mice to “grow up” on a normal chow diet before being switched to a “Western” diet at around 8 weeks of age, this is not the only useful schedule. For instance, in some embodiments, the female breeders are not fed a Western diet. However, epigenetic changes in organisms may result due to diets of mothers before and during pregnancy, or in immature individuals. Therefore, in some embodiments, the mice begin the Western diet prior to 8 weeks, e.g. prior to sexual maturity. In some embodiments, the Western diet is fed to breeders of either sex for various durations prior to mating and/or during pregnancy. Such alterations in protocols yield useful information regarding epigenetics, fertility, etc.
Furthermore, in some embodiments, animals are dosed with compounds or drugs during administration of the Western diet as disease develops, or at timepoints after diseases have developed and progressed. However, it will be appreciated that compounds or drugs may be administered to the mice at any time, including prior to initiating the Western diet, for prevention studies. In some embodiments, the DIAMOND™ mouse is used as an experimental platform for creation of “knock-ins” and “knock-outs” to investigate the effects of adding and deleting genes.
Further, agents other than “drugs” may be tested using the mice, e.g. various natural products, so-called “nutraceuticals”, herbs, dietary additives such as probiotics, vitamins, etc. may be tested to determine their effects on disease occurrence and/or progression; as may changes (variations) in diet (e.g. varying the amount or type of fats and sugars, etc.); and/or activity levels (mimicking increased or decreased exercise), etc.
While the majority of the data obtained on the DIAMOND™ mouse physiology and pathology presented herein concerns liver pathology and some blood-borne proteins and metabolites, it is expressly noted that other organ systems and biological fluids may and do show abnormalities. Thus the utility of this mouse model extends beyond liver disease and blood-based biomarkers and into all areas of physiology known to be affected by the development of metabolic syndrome, including but not limited to the cardiovascular system, the muscular system, the endocrine system, the reproductive system, the digestive system, the kidneys, and the adipose tissue.
It has been postulated that the microbiome of the gut plays a role in the development of liver disease. Another useful aspect of this model, therefore, is the ability to inoculate the gut with various organisms and determine if the changes in the gut microbiome alter disease development and progression. Thus the model has utility for the discovery and development of pro-biotics and gut flora supplements. Accordingly, in some embodiments we contemplate the use of a “clean” DIAMOND™ mouse model, and in other embodiments we contemplate the addition of one or more commensal or pathogenic bacterial, viral, or fungal species to the model.
The mouse of the invention is also useful for biomarker discovery studies. For example, studies of circulating lipids, proteins and nuclear material could be used for biomarker development. Also, changes in the microbiome and measurement of microbial metabolites and linking them to disease phenotype would guide biomarker development efforts.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.
All examples and descriptions in this application are non-limiting and shall not be construed as the only way that the invention can be practiced. Variations in methodology and protocol that are within the spirit of the invention are also claimed.
Two pure wholly genetically characterized mouse strains were cross-bred. These two parental strains are 129S1/SvImJ and C57BI/6J. The progeny that resulted from the cross-breeding are called 129/B6 mice. The first progeny were heterogeneous (not genetically identical to one another). In order to transform the heterogeneous progeny into an isogenic mouse strain in which all individuals were equally genetically susceptible to developing NAFLD, NASH and HCC in response to Western diet, further selective inbreeding was carried out for over 20 generations to yield the isogenic strain of DIAMOND™ mice that all develop disease pathology in response to dietary trigger. The isogenic status of the mice was confirmed by testing for a set of 158 single nucleotide polymorphisms (SNPs) that included both specific C57BI/6 and 129S1 SNPs in 6 randomly chosen mice from the colony (Table 1). This SNP testing demonstrated that approximately 60% of the genetic marker SNPs were of C571BI/6 origin while 40% of the genetic marker SNPs were of 129S1 origin. Importantly, all mice were genetically identical. The sequence of the entire genome of the new mouse was determined (SEQ ID NOs:1-39). The development of the new organism with unique genetic makeup and utility as a model of human disease was complete.
Chow diet normal water (CD NW)-fed mice gained a small amount of weight compared to baseline values but essentially had stable weights from 8 to 52 weeks (
This was accompanied by an increase in aspartate aminotransferase (AST) and alanine aminotransferases (ALT) in WD SW-fed mice compared to CD NW-fed mice (p<0.05 for AST and <0.001 for ALT) (
Insulin resistance was measured by an insulin tolerance test (ITT) that was performed at an early time point after initiation of the diet at 8 weeks and then at 52 weeks. This was done to assess the initial metabolic response to the diet and to determine if these changes were sustained. At 8 weeks, following insulin administration, the blood glucose drop in mice receiving WD SW was less when compared to CD NW-fed controls (
Gross morphology of the liver: Within 4-8 weeks of WD SW administration, the liver became tan and visibly lighter in color compared to CD NW-fed mice for the same duration (
Development of steatosis, steatohepatitis and progressive fibrosis: CD NW-fed mice had normal hepatic architecture and histology at all time-points studied (
Between weeks 12-16, steatohepatitis developed in mice fed WD SW (
Having established the presence of steatohepatitis and the individual components of steatohepatitis seen in humans in mice fed WD SW, the severity of the individual lesions were quantified using the NASH Clinical Research Network (CRN) criteria and calculation of the NAFLD activity score (NAS)17. All mice fed WD SW developed grade 3 macrovesicular steatosis by week 8 (
To confirm the progressive increase in fibrosis over time after administration of WD SW, quantitative morphometry was performed on Sirius Red-stained liver sections (
Female mice also developed steatosis at week 8 and steatohepatitis by week 16 (data not shown). They developed increasing fibrosis but did not develop fully established cirrhosis by week 52. The severity of the steatohepatitis was also milder than in male mice. To determine the contribution of high fat diet alone (Western Diet (WD NW)) and sugar water (CD SW) alone, independent experiments where the mice were fed WD NW or CD SW was performed.
WD NW led to similar weight gain as seen in WD SW-fed mice by week 52 (Table 2). WD NW also led to an elevation of AST and ALT relative to both CD NW and administration of CD SW alone. The AST and ALT levels following WD NW were higher than seen with WD SW administration, but this did not reach significance. More importantly, WD NW-fed mice developed severe steatosis but did not have hepatocyte ballooning by week 8 and only mild ballooning by week 52 (
Activation of Signaling Pathways Relevant for Humans with NASH:
Increased lipogenic-, inflammatory- and pro-apoptotic signaling are hallmarks of NASH in humans18-20. The activation of such signaling pathways was assessed at both early (8 week) and late (52 week) time points in CD NW- and WD SW-fed mice. At both weeks 8 and 52, there was a significant increase in expression of fatty acid synthetase (FAS) and acyl CoA carboxylase (ACC) as well as phosphorylation of acyl CoA carboxylase indicative of its activation (
WD SW-Induced NASH has a Transcriptomic Profile Similar to Humans with NASH:
The Illumina Expression BeadChip (Illumina) platform was used to interrogate the hepatic transcriptome at early (8 week) and late (52 week) stages of NAFLD in mice fed WD SW. Volcano plot and heatmap visualization demonstrated distinct differences between CD-fed and WD SW-fed mouse liver transcriptome at 8 weeks (
Hepatic gene expression profiles were also analyzed in mice fed a WD SW at 52 weeks and further compared to a known human gene expression databank of liver cirrhosis and NASH patients26,27 (
GSEA at 52 weeks identified changes in androstenedione metabolism and transcriptional regulation of lipoprotein metabolism, oxidative stress-related signaling pathways related to activation of the innate immune system and inflammatory pathways as the principal pathways that were altered (
HCC developed in 90% of male mice and 75% of female mice between weeks 32-52. There were 5 or more foci of tumors in each male mouse whereas 1-2 foci were seen in females (
There is a substantial need for models of NASH to accelerate therapeutic and biomarker development. The invention described herein provides a mouse model where fatty liver disease is induced by caloric excess as it occurs in most humans with NASH. The mice sequentially develop steatosis, steatohepatitis, progressive fibrosis and HCC. This is accompanied by weight gain, insulin resistance, hypertriglyceridemia and increased circulating levels of LDL-cholesterol. The hepatic transcriptome of these mice was also similar to that seen in human NASH from week 8 through 52. In later stages of the disease, the hepatic transcriptome from affected mice demonstrated concordance with a 186-gene signature of cirrhosis of varied etiology including NASH containing 73 genes associated with poor prognosis29. Furthermore, striking similarity of global liver transcriptome between WD SW-fed mice and human NASH was observed for 8 weeks and 52 weeks. Pathways related to lipogenesis, inflammation and apoptosis that are considered to be relevant for humans with NASH15, 19 were also activated. These mice developed progressive fibrosis with early cirrhotic remodeling and also HCC which resembled S1 or S2 human HCC28. Interestingly, the induction was relatively stronger for S1 subclass which is associated with steatohepatitic HCC variant31. The disease also developed over a 52 week time-frame which is analogous to about 25-30 years in a human, the time course of disease progression to cirrhosis and HCC in many chronic liver diseases32,33. Together, this demonstrates that this mouse model overcomes several deficiencies of other models and mimics the key aspects of human NASH. It is therefore used as a preclinical model for this disease.
The current model impacts three important areas of liver disease for which there is an unmet need for more therapeutic development. These include NASH, hepatic fibrosis and HCC. In addition to mimicking human NASH, this model demonstrates progressive fibrosis with marked perisinusoidal fibrosis, the distinctive pattern of fibrosis in human NASH, and in some cases, bridging fibrosis and even early cirrhotic changes. Most prior approaches for anti-fibrotic drug development have utilized carbon tetrachloride, thioacetamide administration, or bile duct ligation as models of hepatic fibrosis for preclinical testing of potential therapies. These models are not representative of the human disease state and thus are not ideal for testing anti-fibrotic therapies. In the current model, fibrosis develops as a consequence of a disease process that resembles human disease and is better suited for the preclinical assessment of drugs targeting hepatic fibrosis to prevent disease progression or induce regression of advanced fibrosis. Given the high incidence of HCC in this model, it also has utility for testing preventive and therapeutic approaches against HCC.
It is interesting to note the development of hepatic adenomas as well as HCC within hepatic adenomas in our model. There are case reports of HCC arising in a hepatic adenoma in subjects with metabolic syndrome without cirrhosis34. Therefore, the DIAMOND™ model also recapitulates this phenomenon and provides further opportunities to dissect the molecular basis for this observation.
It is important to note that a high fat diet alone can induce the phenotype but with lesser severity. However, sugar water administration alone did not have any impact on liver histology. This could be related to the amount of sugar consumed in this protocol which allowed ad lib consumption of sugar water. Also, female mice developed a less pronounced phenotype and a lower incidence of HCC. These gender-based differences should also permit studies that may shed novel insights on why HCC affects males with NASH more than females35,36. The model is also relatively specific since mice with a pure background did not develop the phenotype.
In summary, this diet-induced animal model of nonalcoholic fatty liver disease recapitulates the various phenotypes of the disease and their associated metabolic and underlying molecular characteristics. It serves as a relevant model to identify therapeutic targets, model disease progression and test preventive and therapeutic approaches against NASH, hepatic fibrosis and HCC.
The detailed descriptions and photographs, graphs, figures, and genetic data are supportive and descriptive of the embodiments of the invention described herein, and while some of the preferred embodiments of this invention have been described in detail, various alternative designs and embodiments exist for practicing the methods disclosed herein. It is expressly noted that the present invention is not limited to the embodiments described in detail herein; rather, modifications and additions to what has been expressly described herein are also included within the scope of the invention. Moreover, it will be understood that the features of the various embodiments described herein are not generally mutually exclusive and can exist in various combinations and permutations without departing from the spirit and the scope of the invention.
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.
This application claims priority to and the benefit of the filing date of U.S. Provisional Application No. 62/240,223 filed Oct. 12, 2015, the disclosure of which is hereby incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US16/56506 | 10/12/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62240223 | Oct 2015 | US |
