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 a/ß-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 promising therapeutic lead for late-stage pre-
clinical and the first-in-human studies.