SUMMARY Alcohol-associated hepatitis (AH) is a form of subacute liver failure with high mortality, limited treatment options, and elusive pathogenesis. Emerging evidence suggests that AH is associated with a profound impairment in hepatocellular cholesterol homeostasis. Hepatocytes are the central hub for cholesterol metabolism. Meanwhile, AH hepatocytes exhibit profoundly altered FC content and pathways responsible for cholesterol esterification, transport, and conversion to bile acids. RNA sequencing studies indicate that the dysregulation of HNF4α, a master hepatocyte transcription factor, is responsible for impaired cholesterol homeostasis. Upregulation of HNF4α using small activating RNA (saRNA) could mitigate cholesterol toxicity in vitro and can be developed as a treatment for AH. Our long-term objective is to define the disease-centric pathways of AH and develop much-needed therapeutics for AH patients. We hypothesize that the HNF4a-dependent impairment of cholesterol homeostasis in hepatocytes directly contributes to hepatocellular dysfunction in AH and can be targeted therapeutically. Our approach is to map regulatory pathways of cholesterol metabolism in human AH at a single cell resolution, functionally interrogate cholesterol metabolic pathways in murine alcohol models, and test a novel therapeutic strategy by targeting HNF4a-dependent cholesterol toxicity. Aim 1 will map cholesterol metabolism in AH at single cell resolution using a multimodal approach. A three- pronged strategy will map AH-associated changes in cholesterol homeostasis using single nuclear RNA sequencing, ultra-high multiplexed imaging, and targeted spatial transcriptomics. The unbiased reference maps will guide the interrogation of cholesterol metabolic pathways in cell and murine AH models. Aim 2 will define the impact of cholesterol dysregulation on alcohol-induced liver injury in murine models. We will model cholesterol toxicity in 3 conditions relevant to AH using the chronic-plus-binge alcohol feeding: (1) acute cholesterol overload, (2) impaired bile acid synthesis, and (3) impaired cholesterol secretion in cholestasis. This effort will generate optimized murine models that recapitulate cholesterol toxicity in AH. Aim 3 will evaluate targeted manipulation of HNF4α-dependent pathways of cholesterol toxicity as a novel therapeutic strategy in AH. We use saRNA to modulate HNF4α isoform expression and cholesterol metabolism. GalNAc-conjugated HNF4α-saRNA will be tested as a hepatocyte-targeted therapeutics to mitigate cholesterol toxicity in models of AH using differentiated human hepatocytes and murine models. This project is built upon a strong team of multidisciplinary scientists combining unique expertise in lipidology, preclinical liver models, and state-of-the-art single cell and spatial technologies. The success of this project will further our understanding of AH pathogenesis and could lead to the development of a novel AH treatment ready for precli...