Identification and Optimization of a Minor Allele-Specific siRNA to Prevent PNPLA3 I148M-Driven Nonalcoholic Fatty Liver Disease

Abstract

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 (<i>PNPLA3</i>) with nonalcoholic fatty liver disease (NAFLD); the presence of the resulting mutant PNPLA3 I148M protein is a driver of nonalcoholic steatohepatitis (NASH). While <i>Pnpla3</i>-deficient mice do not display an adverse phenotype, the safety of knocking down endogenous wild type <i>PNPLA3</i> in humans remains unknown. To expand the scope of a potential targeted NAFLD therapeutic to both homozygous and heterozygous <i>PNPLA3</i> 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 <i>in vitro</i> for activity and selectivity toward <i>PNPLA3</i> rs738409 mRNA. Conjugation of the siRNA to a triantennary <i>N</i>-acetylgalactosamine (GalNAc) ligand enabled <i>in vivo</i> screening using adeno-associated virus to overexpress human <i>PNPLA3^I148M</i> versus human <i>PNPLA3^I148I</i> 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 <i>PNPLA3^I148M</i> but not <i>PNPLA3^I148I</i>. Testing of the minor allele-specific siRNA in <i>PNPLA3^I148M</i>-expressing mice fed a NASH-inducing diet prevented <i>PNPLA3^I148M</i>-driven disease phenotypes, thus demonstrating the potential of a precision medicine approach to treating NAFLD.