Transforming Genes to Protect Against Liver Disease: Will siRNA-based Therapeutics Reduce the Need for Liver Transplantation?

Nonalcoholic fatty liver disease (NAFLD) affects over 25% of the global population, reflecting rising rates of diabetes, obesity, and metabolic syndrome.¹ Nonalcoholic steatohepatitis (NASH) is the fasting growing indication for liver transplantation, and the top cause of liver transplantation among women in North America.² The progression of NAFLD is heterogeneous, with only around 40% of those affected developing fibrosis and <5% dying from liver-related causes. A significant proportion of this variability (estimated 50%) is genetic (rather than environmental), with the precise underlying mechanisms still being characterized.³

The development of NASH therapeutics based on genetic modifiers has been slow. The best-characterized genetic variant associated with NAFLD is a single-nucleotide polymorphism in the PNPLA3 gene, which affects 17% to 49% of people. Loss of function of this hydrolase enzyme traps triglycerides and retinyl esters in lipid droplets of hepatocytes and hepatic stellate cells. PNPLA3 variants are associated with increased risks of NAFLD, NASH, fibrosis, and hepatocellular carcinoma.⁴ An antisense oligonucleotide has been designed to knockdown mRNA expression of PNPLA3, with promising results in mouse models,⁵ currently undergoing early phase clinical trials among PNPLA3 heterozygotes. Other genetic variants that may alter disease susceptibility involved in lipid metabolism and secretion, insulin signaling, and immunity have been less robustly identified in multiple analyses.⁶ One recent genome-wide analysis identified a common loss of function variant in HSD17B13, which led to protection from liver disease.⁷ Early phase clinical trials are currently underway for inhibitory small interfering RNAs (siRNA) to replicate this effect.

Identifying causative loci of disease within large databases is biased toward common variants, as rare variants can only be detected when there is adequate case identification in high numbers. Rare mutations may have greater effect sizes than common variants. In the New England Journal of Medicine, Verweij et al⁸ recently reported novel findings from a large genetic sequencing study focused on the discovery of rare coding variants using whole exome sequencing. The authors identified and characterized loss of function and missense variants in CIDEB, a gene that encodes a structural protein in hepatocytes, which allows smaller lipid droplets to fuse together. They demonstrated that loss-of-function variants in CIDEB provided protection against the development of liver disease and demonstrated in vitro efficacy of CIDEB inhibition in preventing large droplet formation in hepatoma cell lines.

Verweij et al conducted a multistage analysis, initially involving whole exome sequencing for over 500 000 participants from 2 biobanks (UK Biobank and Geisinger Health System MyCode cohorts). After controlling for multiple factors, including ALT-associated common genetic variants in the same persons, they identified 5 rare coding variants associated with ALT levels, AST levels, and rates of database-recorded liver disease, comparing 25 000 cases with 490 000 controls. Variants in 4 genes (APOB, ABCB4, SLC30A10, and TM6SF2) led to increased risk of liver disease as described previously. The novel finding was that rare coding variants in CIDEB were associated with lower ALT/AST levels and reduced rates of liver disease. These variants occurred in approximately 1 in 150 people in their study population. People with these CIDEB variants had 31% to 54% lower odds of developing any liver disease, liver disease because of NASH or viral hepatitis, any liver cirrhosis, or hepatocellular carcinoma. Among 3599 subjects with liver biopsies, CIDEB variants were associated with 66% lower odds of histological steatosis, NASH, or fibrosis, as well as lower NAFLD activity scores. Among 36 000 subjects with magnetic resonance imaging data, patients carrying CIDEB coding variants had lower liver fat percentages than noncarriers.

The authors then investigated the possibility of silencing normal CIDEB expression to modify the natural history of NASH. They developed CIDEB-targeting siRNA and showed that silencing CIDEB in human hepatocyte cell lines prevented increases in the size of lipid droplets when the cells were treated with oleic acid. Previous studies showed that CIDEB knockout mouse models had smaller hepatocytes lipid droplets, favorable metabolic profiles, and protection against hepatic steatosis on high-fat diets.⁹ Large lipid droplets are seen when CIDEB is overexpressed. This finding provides a plausible physiological effect and points to a potential therapy if the knockdown effect can be replicated in human studies. Given the protective association of CIDEB mutations with the development of any liver disease, CIDEB knockdown could potentially prevent disease in a broad range of liver diseases.

siRNAs bind to target mRNA and mark them for destruction before translation to proteins can occur. Although mechanisms of RNA interference were described over 20 y ago, only 5 siRNA drugs have been FDA approved to date for diseases including acute hepatic porphyria and transthyretin-mediated amyloidosis.¹⁰ There have been significant barriers to commercialization of siRNA.¹¹ Optimal siRNA sequences bind specifically to target mRNA while avoiding off-target effects or immunogenicity (siRNA can stimulate innate immunity via toll-like receptors). Bespoke delivery systems (bioconjugates or nanoparticles) are often required to allow siRNA to pass into cells and cytoplasm of the organ of interest while avoiding degradation. As such challenges are being overcome, more siRNA therapeutics are expected in clinical trials.

Indeed, there are several hurdles to cross for CIDEB-targeting siRNA therapeutics. The findings here need replication, particularly in more diverse cohorts. Fewer than 5% of the original cohort studied were of non-European ancestry, limiting generalizability, although the authors verified the findings in secondary cohorts of 33 000 non-Europeans. Careful preclinical and clinical studies will be required to establish safety and efficacy in NASH or other liver diseases. Whether changes in CIDEB expression late in life will impact hepatic steatosis, fibrosis, or avoid end-stage cirrhosis remains a significant outstanding question. Nonetheless, the potential for disease-modifying therapies that may reduce the need for liver transplantation is an exciting avenue of exploration.