Development of gene editing based therapy for cardiovascular diseases

项目摘要/摘要 最近的治疗进展大大降低了心血管疾病(CVD)的发生率。这个 低密度脂蛋白(LDL)和极低密度脂蛋白(VLDL)在动脉粥样硬化形成中起重要作用。 不管过去30年的临床治疗进展如何，对于接受他汀类药物治疗的患者来说 接受或不接受联合治疗的患者，低密度脂蛋白降低不足和相对较高的残留风险仍然存在 仍然存在，可能与持续相对较高的甘油三酯(TG)水平有关，因此限制了 这些疗法。这强调了针对心血管疾病脂代谢的额外新疗法的必要性。 预防和治疗。在这项建议中，我们建议通过一种 腺相关病毒(AAV)通过Cas9碱基编辑(Cas9-BE)策略介导APOC3的体内沉默 (AAV-Cas9-BE-APOC3)对高胆固醇饮食诱导的高胆固醇血症具有保护作用 兔动脉粥样硬化。具体地说，我们将1)在临床前模式物种中开发AAV-Cas9-Be-APOC3， 新西兰白兔。我们将确定使用体外培养的兔的最佳靶向策略 细胞，然后进行实验以确定获得有效APOC3基因的最佳递送参数 在兔肝细胞中进行基因敲除；2)用优化条件评价AAV-Cas9-Be-APOC3的效果 目的1确定在兔体内敲除APOC3；3)对AAV的安全性进行多年评估- 兔CAS9-BE-APOC3。通过利用CRISPR/CAS9新技术实现这些目标 APOC3将提供令人信服的证据，将APOC3确立为基于基因编辑的新的可行目标 治疗高脂血症和心血管疾病。

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.