Human genome wide association studies confirm the association of the rs738409 single nucleotide polymorphism (SNP) in the gene encoding protein patatin like phospholipase domain containing 3 (PNPLA3) with nonalcoholic fatty liver disease (NAFLD); the presence of the resulting mutant PNPLA3 I148M protein is a driver of nonalcoholic steatohepatitis (NASH). While Pnpla3-deficient mice do not display an adverse phenotype, the safety of knocking down endogenous wild type PNPLA3 in humans remains unknown. To expand the scope of a potential targeted NAFLD therapeutic to both homozygous and heterozygous PNPLA3 rs738409 populations, we sought to identify a minor allele-specific small interfering RNA (siRNA). Limiting our search to SNP-spanning triggers, a series of chemically modified siRNA were tested in vitro for activity and selectivity toward PNPLA3 rs738409 mRNA. Conjugation of the siRNA to a triantennary N-acetylgalactosamine (GalNAc) ligand enabled in vivo screening using adeno-associated virus to overexpress human PNPLA3^I148M versus human PNPLA3^I148I in mouse livers. Structure-activity relationship optimization yielded potent and minor allele-specific compounds that achieved high levels of mRNA and protein knockdown of human PNPLA3^I148M but not PNPLA3^I148I. Testing of the minor allele-specific siRNA in PNPLA3^I148M-expressing mice fed a NASH-inducing diet prevented PNPLA3^I148M-driven disease phenotypes, thus demonstrating the potential of a precision medicine approach to treating NAFLD.

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