PROJECT SUMMARY/ABSTRACT The contribution of low density lipoproteins (LDL) and very low density lipoproteins (VLDL) to the development of cardiovascular disease (CVD) is critical in atherogenesis. Recent therapeutic advances considerably reduced the incidence of CVD. Despite the progress of clinical treatments in the past 30 years, for a significant proportion of statin-treated patients, relatively high residual risk remains, likely associated with persistent relatively high levels of triglyceride (TG), thus limiting the benefits of these therapies. This underscores the need for additional new therapies targeting lipid metabolism in CVD prevention and treatment. ASGR1 has emerged as a new promising target for dyslipidemia to control CVD risk. ASGR1 encodes asialoglycoprotein receptor type 1, which is mainly expressed on the basolateral membrane of hepatocytes, where it facilitates the uptake of circulating glycoproteins. The loss-of-function (LoF) mutations in ASGR1 were found to be associated with lowering of non-HDL-C levels and a reduced risk of CVD. The beneficial effects of ASGR1 deficiency in lowering TG and TC levels were further demonstrated in mice and pigs. In this project, we propose to develop a “One-Shot for Cure” to treat hyperlipidemia and CVD by targeting ASGR1 with a base editing approach delivered via engineered viral-like particles (eVLPs). Specifically, we will develop eVLP-BE- ASGR1 therapy 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 ASGR1 gene knockout in rabbit hepatocytes. We will determine the efficacy of eVLP-BE-ASGR1 therapy in rabbit atherosclerosis model and establish the long-term safety profile of eVLP-BE-ASGR1 therapy in rabbits. Utilizing the latest breakthroughs in base editing and non-viral eVLP technologies, the successful accomplishment of these objectives will yield compelling evidence, enabling the development of a revolutionary “One-Shot for Cure” therapy aimed at treating hyperlipidemia and CVD through the precise targeting of ASGR1.