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

Patatin-like phospholipase domain-containing protein 3 (PNPLA3) 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 PNPLA3 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 PNPLA3 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 PNPLA3 p.I148M gene variant into a healthy-derived immortalised human hepatocyte (IHH) cell line to generate a new in vitro model of MASLD that would better reflect PNPLA3-associated MASLD/MASH. Mutant cell lines exhibited lipid accumulation, increased CD36 (cluster of differentiation 36) gene expression and a decline in CPT1A (carnitine palmitoyltransferase 1A) and PPARA (peroxisome proliferator-activated receptor α) gene expression compared to the wild-type control. Changes in the expression of genes involved in lipid synthesis or transport suggest the involvement of PNPLA3 p.I148M in the dysregulation of these pathways and processes. The homozygous PNPLA3 p.I148M IHH cell line also demonstrated reduced PNPLA3 gene and protein expression compared to 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.