A novel human hepatocyte cell line to study PNPLA3-associated steatotic liver disease

Patatin-like phospholipase domain-containing protein 3 (<i>PNPLA3</i>) p.I148M is a well-established variant associated with metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH). Conflicting in vitro and in vivo data about the impact of the variant suggest that the <i>PNPLA3</i> p.I148M variant could be gain- or loss-of-function, or neomorphic. Most in vitro models used to study MASLD are cancer-derived hepatoma cell lines such as HepG2 and Huh7, which already endogenously express the homozygous <i>PNPLA3</i> p.I148M variant. This highlights the need to develop models that better reflect disease and allow comparisons with wild-type cells. Clustered regularly interspaced short palindromic repeats (CRISPR) prime editing was used to introduce the <i>PNPLA3</i> p.I148M gene variant into a healthy-derived immortalized human hepatocyte (IHH) cell line to generate a new in vitro model of MASLD that would better reflect PNPLA3-associated MASLD/MASH. Heterozygous and homozygous <i>PNPLA3</i> p.I148M IHH cell lines were generated and validated with Sanger sequencing. Mutant cell lines exhibited lipid accumulation, increased cluster of differentiation 36 (<i>CD36</i>) gene expression and a decline in carnitine palmitoyltransferase 1 alpha (<i>CPT1A</i>) gene expression compared with the wild-type control, basally or in the presence of free fatty acid (FFA)-induced steatosis. The homozygous <i>PNPLA3</i> p.I148M IHH cell line also demonstrated reduced <i>PNPLA3</i> gene and protein expression compared with the wild-type control. We have developed a new human hepatocyte cell line and in vitro model to help understand PNPLA3-associated steatotic liver disease and provide a new resource for developing potential therapeutics.<b>NEW & NOTEWORTHY</b> We have developed a novel in vitro model for studying the <i>PNPLA3</i> p.I148M variant in steatotic liver disease using a normal, healthy-derived hepatocyte cell line, which does not endogenously express the variant. We show that carrying the homozygous <i>PNPLA3</i> p.I148M variant results in reduced PNPLA3 gene and protein expression, more lipid accumulation, increased lipid uptake, and reduced mitochondrial lipid oxidation-associated gene expressions and altered expression of genes associated with lipid synthesis and transport.