Identification and Optimization of a Minor Allele-Specific siRNA to Prevent PNPLA3 I148M-Driven Nonalcoholic Fatty Liver Disease
Justin K. Murray, Jason Long, Lei Liu, Shivani Singh, Danielle Pruitt, Michael Ollmann, Elissa Swearingen, Miki Hardy, Oliver Homann, Bin Wu, Jerry Ryan Holder, Kelvin Sham, Brad Herberich, Mei-Chu Lo, Hui Dou, Artem Shkumatov, Mónica Florio, Ingrid C. Rulifson
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 ( 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.