Kai Smith
Research Mentor: Xin Tong
Mentor Department: Molecular & Integrative Physiology, Medicine
Author(s): Johnathan Jen
Session: Session 6 (3:00 PM – 3:50 PM)
Presentation Type: Poster 32
Abstract
As the severe form of metabolic-associated steatotic liver disease (MASLD), metabolic-associated steatohepatitis (MASH) is characterized by excessive fat buildup (termed as liver steatosis), inflammation, and progressive fibrosis, ultimately leading to deadly outcomes, including cirrhosis, liver cancer, and liver failure. E4-binding protein 4 (E4BP4) is a transcription repressor involved in hepatic glucose and lipid metabolism. Our lab has shown that hepatic E4BP4 is implicated in the dysregulation of metabolic pathways that contribute to the development of MASH. Although E4BP4 plays a critical role in lipid synthesis and metabolic Homeostasis, its specific contribution to MASH progression, remains unclear. Targeted gene-editing approaches may provide insight into how alterations in E4bp4 expression in hepatocytes affect hepatic lipid accumulation, inflammation, and fibrosis. This study aims to generate an AAV virus to delete E4bp4 specifically in mouse hepatocytes based on the CRISPR-Cas gene editing technology to investigate the role and mechanisms of hepatic E4BP4 during the pathogenesis of diet-induced MASH. We will utilize an online CRISPR design platform to design gRNA sequences targeting the 3’ end of the E4bp4 genomic sequence. Two gRNAs were selected based on the on-target and off-target scores. The E4bp4-specific gRNAs will be ligated into the pX602 AAV plasmid, which drives the expression of the Sa-Ca9 enzyme in a hepatocyte-specific manner and the gRNA expression under the U6 promoter. Next, we will screen mini culture clones with restriction enzyme digestion and verify the insertion of gRNA sequences by direct sequencing. Then, we will generate AAV viruses by transfecting 293 packaging cells with the verified pX602 plasmid along with two additional plasmids and quantify the viruses by Coomassie staining and qPCR. Finally, we will inject WT mice with the AAV virus via the tail vein and determine the deletion efficiency of hepatic E4bp4 by DNA, RNA, and protein analyses. This study will generate a powerful and precise gene-editing tool to delete the mouse E4bp4 in vivo and uncover the molecular mechanism of how hepatic E4BP4 contributes to the disease progression of MASH.
