Abstract The ultimate goal of this project is to explore the feasibility and mechanism of an allele-specific, RNAi-based genetic medicine approach to treating nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). NAFLD/NASH is the most common chronic liver disease worldwide. However, no proven drug for NAFLD/NASH is available. There is an urgent need to identify novel targets and develop promising drugs for both NASH prevention and treatment. Recent GWAS has identified that a genetic mutation (148I>M) in the patatin-like phospholipase domain containing 3 (PNPLA3) gene is the single largest genetic risk factor contributing to the progression of NAFLD to NASH. Our and others’ studies demonstrated that the PNPLA3148M- associated NASH should be classified as a unique disease. Mechanistically, our team recently demonstrated that accumulated PNPLA3148M protein isoform on lipid droplets competes with PNPLA2 (also known as ATGL) for α/β-hydrolase-domain-containing 5 (ABHD5, or CGI-58), an important activator for PNPLA2, blocking the ABHD5-mediated, PNPLA2-driving lipolysis and leading to triglycerides accumulation in lipid droplets. We thus hypothesize that transcriptional downregulation of the PNPLA3148M isoform is a cause-targeting strategy for the treatment of NAFLD/NASH among patients carrying the PNPLA3148M allele. To this end, we have developed and patented an allele-specific small interfering RNA (siRNA) that possesses excellent potency and specificity for downregulating the PNPLA3148M mutant isoform but not its wild-type isoform (148I). We have further discovered two more modified oligonucleotides with excellent pharmacological properties. To facilitate the preclinical evaluation of these agents, we have developed a humanized PNPLA3148I/M mouse model, which recapitulates the typical NASH phenotype and related comorbidities. Delivering our siRNA into this mouse model with N- Acetylgalactosamine (GalNAc)-conjugation, a well-established, clinically validated, and liver-specific RNAi drug delivery strategy effectively knocked down hepatic hPNPLA3148M mRNA and ameliorated the liver histology. With this success, we propose to advance our drug development by evaluating the efficacy, toxicity, and mechanism of action of our therapeutics for both NASH prevention and treatment, with the final goal to generate the first precision genetic medicine for PNPLA3148M-specific NASH. We aim in this study 1) to evaluate and compare the efficacy of our 3 hPNPLA3148M-targeting, GalNAc-conjugated oligonucleotide therapeutics; 2) to evaluate and compare the safety and toxicity of our therapeutic candidates; and 3) to explore the mechanism of action of our therapeutics. We expect that via this study, we will eventually develop the first genetic medicine for NAFLD/NASH prevention/treatment, which could also serve as a new prototype for drug development for other common human diseases. The collected data in this study will generate the most prom...