A PNPLA3^I148M gene‐edited Ossabaw swine model of Nonalcoholic steatohepatitis (NASH)

Abstract

Nonalcoholic steatohepatitis (NASH) is an advanced form of nonalcoholic fatty liver disease (NAFLD), resulting from the complex interplay of genetic and environmental risk factors, e.g. diet. The development of novel therapies has been hampered by the lack of a preclinical animal model with high physiologic fidelity to the human condition. A single mutation (I148M) in the patatin‐like phospholipase domain‐containing 3 (PNPLA3) gene is significantly associated with liver fat content and has been identified as the strongest genetic risk factor for NAFLD/NASH, cirrhosis, and hepatocellular carcinoma. We hypothesized that editing PNPLA3I148M mutation into a genome of Ossabaw, a miniature pig that already has a natural propensity to develop metabolic syndrome, will develop phenotypes that hallmark NAFLD/NASH, and improve preclinical modeling over existing rodent models. PNPLA3I148M/I148M founders were developed using TALEN‐stimulated homology directed repair in fibroblasts, followed by somatic cell nuclear transfer. Mutant pigs with age/gender‐matched wildtype controls, reared on standard (SD) and specialized western diet (WD), were clinically and histopathologically characterized. No significant differences in circulating triglyceride and liver enzyme levels was observed in our initial cohort on SD of a female mutant pig relative to the wildtype control. However, this mutant female exhibited increased ALP level and a more atherogenic lipoprotein profile with LDL particle concentration than the levels of the respective control. Histopathologically, the mutant female fed on the WD for 3 months exhibited augmented interlobular inflammation, ballooning, as well lipid and collagen buildup in the liver relative to the wildtype counterparts on the same WD or SD. Longitudinal studies in second larger cohort of barrows over six‐months period revealed significantly elevated liver enzymes (ALP, ALT, GGT), bile acids, and lipid profiles (cholesterol, triglyceride, triglyceride‐rich lipoprotein and vLDL) among the mutant versus wildtypes on western diet, substantiating liver pathology and the associated comorbidity of dyslipidemia. Liver histopathology from these barrows on WD over 6‐months did not reveal as significant difference as in the females of the initial cohort, implying gender differences and variances in western diet formulation in susceptibility to NAFLD/NASH that warrant future large‐scale studies. Levels of circulating extracellular vesicles (EVs) were significantly elevated in the sera of mutant pigs relative to wildtypes on the WD, validating this as an important biomarker in NAFLD/NASH pathology. Additionally, the mutant pigs on the WD had significantly greater coronary artery plaque burden than the control groups, in line with the predicted higher comorbidity level in mutants and reinforces the relevance to human clinical medicine. Support or Funding Information NIH (1R43OD023257‐01A1 to TM) and internal funds from Recombinetics Inc., Drs. Chalasani, Sturek, and Malhi.

Characterization and Pharmacological Validation of a Preclinical Model of NASH in Göttingen Minipigs

“… 18 To further model human disease pathophysiology, it would be interesting to introduce key gene mutations associated with the development of NASH and/or fibrosis and using the Crispr/Cas9 system as recently described by Coutts and colleagues in the Ossabaw swine model for PNPLA3. 36 Furthermore, it is even now possible to follow disease progression using noninvasive imaging techniques that are currently used in the clinic. Moreover, this model can also be used to monitor the impact of the disease or the response to a given treatment on cardiovascular readouts.…”

“…The CDAHFD-fed Göttingen minipig model captures many features of MAFLD and offers the possibility to conduct serial histological examination during longitudinal studies which are key shortcomings of rodent models [18]. To further model human disease pathophysiology, it would be interesting to introduce key gene mutations associated to the development of NASH and / or fibrosis and using the Crispr/Cas9 system as recently described by Coutts and colleagues in the Ossabaw swine model for PNPLA3 [36]. Furthermore, it is even now possible to follow disease progression using non-invasive imaging techniques which are currently used in the clinic.…”