BACKGROUND Mutations in low-density lipoprotein (LDL) receptor (LDLR) are one of the main causes of familial hypercholesterolemia, which induces atherosclerosis and has a high lifetime risk of cardiovascular disease. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an effective tool for gene editing to correct gene mutations and thus to ameliorate disease. METHODS The goal of this work was to determine whether in vivo somatic cell gene editing through the CRISPR/Cas9 system delivered by adeno-associated virus (AAV) could treat familial hypercholesterolemia caused by the Ldlr mutant in a mouse model. We generated a nonsense point mutation mouse line, LdlrE208X, based on a relevant familial hypercholesterolemia-related gene mutation. The AAV-CRISPR/Cas9 was designed to correct the point mutation in the Ldlr gene in hepatocytes and was delivered subcutaneously into LdlrE208X mice. RESULTS We found that homogeneous LdlrE208X mice (n=6) exhibited severe atherosclerotic phenotypes after a high-fat diet regimen and that the Ldlr mutation was corrected in a subset of hepatocytes after AAV-CRISPR/Cas9 treatment, with LDLR protein expression partially restored (n=6). Compared with the control groups (n=6 each group), the AAV-CRISPR/Cas9 with targeted single guide RNA group (n=6) had significant reductions in total cholesterol, total triglycerides, and LDL cholesterol in the serum, whereas the aorta had smaller atherosclerotic plaques and a lower degree of macrophage infiltration. CONCLUSIONS Our work shows that in vivo AAV-CRISPR/Cas9-mediated Ldlr gene correction can partially rescue LDLR expression and effectively ameliorate atherosclerosis phenotypes in Ldlr mutants, providing a potential therapeutic approach for the treatment of patients with familial hypercholesterolemia.

中文翻译：

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体内AAV-CRISPR / Cas9介导的基因编辑可改善家族性高胆固醇血症的动脉粥样硬化。

背景技术低密度脂蛋白（LDL）受体（LDLR）的突变是家族性高胆固醇血症的主要原因之一，家族性高胆固醇血症可诱发动脉粥样硬化并具有终身罹患心血管疾病的高风险。簇状规则间隔的短回文重复序列（CRISPR）/ Cas9系统是有效的工具，可用于基因编辑以纠正基因突变，从而改善疾病。方法这项工作的目的是确定通过腺相关病毒（AAV）传递的CRISPR / Cas9系统进行的体内体细胞基因编辑是否可以治疗小鼠模型中Ldlr突变体引起的家族性高胆固醇血症。我们基于相关的家族性高胆固醇血症相关基因突变产生了无意义的点突变小鼠系LdlrE208X。AAV-CRISPR / Cas9设计用于纠正肝细胞Ldlr基因中的点突变，并将其皮下递送至LdlrE208X小鼠。结果我们发现同质LdlrE208X小鼠（n = 6）在高脂饮食方案下表现出严重的动脉粥样硬化表型，并且在AAV-CRISPR / Cas9处理后一部分肝细胞中的Ldlr突变得到纠正，其中LDLR蛋白表达部分恢复（ n = 6）。与对照组（每组n = 6）相比，具有靶向单向导RNA组（n = 6）的AAV-CRISPR / Cas9血清中的总胆固醇，总甘油三酸酯和LDL胆固醇显着降低，而主动脉动脉粥样硬化斑块较小，巨噬细胞浸润程度较低。