PROJECT SUMMARY/ABSTRACT Recent therapeutic advances considerably reduced the incidence of cardiovascular disease (CVD). The contribution of low-density (LDL) and very low density (VLDL) lipoproteins is critical in atherogenesis. Regardless the progress of clinical treatments in the past 30 years, for a significant proportion of statin-treated patients, with or without combination therapy, insufficient LDL-C reduction and relatively high residual risk still remain, likely associated with persistent relatively high triglyceride (TG) levels, thus limiting the benefits of these therapies. This underscores the need for additional new therapies targeting lipid metabolism in CVD prevention and treatment. In this proposal, we propose to develop genetic deficiency of ApoC3 through an adeno-associated virus (AAV) mediated in vivo silencing of ApoC3 by Cas9 base editor (Cas9-BE) strategy (AAV-Cas9-BE-ApoC3) to render protection against high cholesterol diet-induced hypercholesterolemia and atherosclerosis in rabbits. Specifically, we will 1) develop AAV-Cas9-BE-ApoC3 in a preclinical model species, the New Zealand White rabbits. We will determine the optimal targeting strategies using in vitro cultured rabbit cells, followed by experiments to determine the optimal delivery parameters to achieve effective ApoC3 gene knockout in rabbit hepatocytes; 2) evaluate the efficacy of AAV-Cas9-BE-ApoC3 using optimal conditions determined in Aim 1 to knockout ApoC3 in rabbits; 3) conduct multiple-year evaluation of the safety of AAV- Cas9-BE-ApoC3 in rabbits. Completion of these aims by leveraging new CRISPR/Cas9 technology to target ApoC3 will provide compelling evidence to establish ApoC3 as a novel feasible target for gene editing-based therapy for hyperlipidemia and CVD.