RP3: Characterization of Hepatic Stellate Cells (HSCs) in AALD SUMMARY Alcohol associated liver disease (AALD) is a leading cause of cirrhosis, liver failure, hepatocellular carcinoma, and liver transplantation. The activation of Hepatic Stellate Cells (HSCs) in AALD produce myofibroblasts that drive liver fibrosis. There are no therapies for AALD induced liver fibrosis. Because of animal models of AALD, such as the intragastric ethanol model developed at our Center, we have some insights into HSC phenotypes in mouse models. However, we currently know very little about the phenotypes of HSCs in human AALD. Nor do we know if HSCs differ between AALD and NASH, two metabolic diseases that induce liver fibrosis. This study is designed to identify genes expressed in human HSCs that specifically drive liver fibrosis in AALD compared to NAFLD by analyzing HSCs from patients with these two diagnoses. Phenotypic changes in HSCs occur without a change in the DNA sequence but are regulated on an epigenetic level, e.g. specific modifications in the chromatin structure, which affect DNA accessibility of the regulatory transcription factors (TFs), causing transcriptional activation or repression of their target genes. We will analyze human HSCs from normal, AALD, NAFL, and NASH human livers that have been declined for liver transplantation. To gain insight into gene expression and chromatin remodeling, we will analyze these livers by snRNA-seq and snATAC-seq. We will identify genes that are expressed in HSCs at a higher level in AALD than in normal, NAFL, and NASH. We have developed a rational three step approach to assess the functional consequences of this increased gene expression in specific genes. 1) Knockdown the expression of the specific gene in human HSCs using dsiRNA and assess their activation in 2D cultures. 2) Knockdown the expression of the specific gene in human HSCs using dsiRNA in the context of a 3D human liver spheroid model of AALD. 3) Genetic deletion of the specific genes in the mouse intragastric ethanol model of AALD. A complimentary approach will be used to identify potential small molecules to target specific genes to suppress alcohol-specific HSC activation and halt fibrosis. For this purpose, two Specific AIMs were developed. AIM1. To assess the effect of deleting genes (identified in AIM1) specifically in HSCs. AIM2. To assess the effect of blocking the expression of genes (identified in AIM1) specifically in HSCs. We anticipate that our analysis will provide new insights into the regulation of alcohol-specific activation of human and mouse HSCs and identify new targets for anti-fibrotic therapy.