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 (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 I148Mdriven disease phenotypes, thus demonstrating the potential of a precision medicine approach to treating NAFLD.

The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics

“…In human induced pluripotent stem cells and organoid systems, PNPLA3-I148M elicits an intermediate phenotype between the wild-type and complete PNPLA3 knockout (15,16). Reducing PNPLA3-I148M protein levels in the liver, including with I148M-specific siRNA, reduces fatty liver disease phenotypes, supporting a specific role for PNPLA3-I148M in driving MASLD (17)(18)(19). Purified wild-type PNPLA3 has been shown to possess triglyceride hydrolase or lysophosphatidic acid acyltransferase (LPAAT) activity, with the mutant variant losing the hydrolase activity or showing elevated LPAAT activity (20)(21)(22)(23)(24)(25).…”

Precision medicine in nonalcoholic fatty liver disease

“…The correction of the determinants of NAFLD and NASH that are specific to the individual patient has fundamental therapeutic importance. This could be accomplished, for example, through finely modulating either the gut microbiota; or the activity of those genes involved in the development and progression of disease 34–37 . However, it should be highlighted that the safe and effective use of various therapeutics is hampered by individual variability in response to treatment 38 .…”

“…This could be accomplished, for example, through finely modulating either the gut microbiota; or the activity of those genes involved in the development and progression of disease. [34][35][36][37] However, it should be highlighted that the safe and effective use of various therapeutics is hampered by individual variability in response to treatment. 38 This, in its turn, will result from variations due to genetics, ethnicity, age, sex, hepatic and renal detoxifying capacity, diet, environmental chemicals, alcohol, drug-drug interactions, drug dynamics, and kinetics.…”

Metabolic dysfunction-associated fatty liver disease: advances in genetic and epigenetic implications

“…The available information on NAFLD and NASH genomics to design targeted strategies shows remarkable results. For example, a recent study using specific small interfering RNA (siRNA) demonstrates the potential of targeting PNPLA3 I148M - rs738409 G-risk allele testing a series of chemically modified siRNA toward the PNPLA3 rs738409 mRNA [55 ▪▪ ]. Although progress has been made, unanswered questions remain, but this area of research has a bright future.…”