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

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