Comparison of Wild-Type and High-risk PNPLA3 variants in a Human Biomimetic Liver Microphysiology System for Metabolic Dysfunction-associated Steatotic Liver Disease Precision Therapy

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a worldwide health epidemic with a global occurrence of approximately 30%. The pathogenesis of MASLD is a complex, multisystem disorder driven by multiple factors including genetics, lifestyle, and the environment. Patient heterogeneity presents challenges for developing MASLD therapeutics, creation of patient cohorts for clinical trials and optimization of therapeutic strategies for specific patient cohorts. Implementing pre-clinical experimental models for drug development creates a significant challenge as simple in vitro systems and animal models do not fully recapitulate critical steps in the pathogenesis and the complexity of MASLD progression. To address this, we implemented a precision medicine strategy that couples the use of our liver acinus microphysiology system (LAMPS) constructed with patient-derived primary cells. We investigated the MASLD-associated genetic variant PNPLA3 rs738409 (I148M variant) in primary hepatocytes, as it is associated with MASLD progression. We constructed LAMPS with genotyped wild type and variant PNPLA3 hepatocytes together with key non-parenchymal cells and quantified the reproducibility of the model. We altered media components to mimic blood chemistries, including insulin, glucose, free fatty acids, and immune activating molecules to reflect normal fasting (NF), early metabolic syndrome (EMS) and late metabolic syndrome (LMS) conditions. Finally, we investigated the response to treatment with resmetirom, an approved drug for metabolic syndrome-associated steatohepatitis (MASH), the progressive form of MASLD. This study using primary cells serves as a benchmark for studies using "patient biomimetic twins" constructed with patient iPSC-derived liver cells using a panel of reproducible metrics. We observed increased steatosis, immune activation, stellate cell activation and secretion of pro-fibrotic markers in the PNPLA3 GG variant compared to wild type CC LAMPS, consistent with the clinical characterization of this variant. We also observed greater resmetirom efficacy in PNPLA3 wild type CC LAMPS compared to the GG variant in multiple MASLD metrics including steatosis, stellate cell activation and the secretion of pro-fibrotic markers. In conclusion, our study demonstrates the capability of the LAMPS platform for the development of MASLD precision therapeutics, enrichment of patient cohorts for clinical trials, and optimization of therapeutic strategies for patient subgroups with different clinical traits and disease stages.