Discovery Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Pepparedsleden 1, Mölndal, 43 183, Gothenburg, Sweden.
Present Address: Department of Molecular and Clinical Medicine, University of Gothenburg, The Wallenberg Laboratory, Sahlgrenska University Hospital, Gothenburg, Sweden.
BMC Biol. 2019 Jan 15;17(1):4. doi: 10.1186/s12915-018-0624-2.
Plasma concentration of low-density lipoprotein (LDL) cholesterol is a well-established risk factor for cardiovascular disease. Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates cholesterol homeostasis, has recently emerged as an approach to reduce cholesterol levels. The development of humanized animal models is an important step to validate and study human drug targets, and use of genome and base editing has been proposed as a mean to target disease alleles.
To address the lack of validated models to test the safety and efficacy of techniques to target human PCSK9, we generated a liver-specific human PCSK9 knock-in mouse model (hPCSK9-KI). We showed that plasma concentrations of total cholesterol were higher in hPCSK9-KI than in wildtype mice and increased with age. Treatment with evolocumab, a monoclonal antibody that targets human PCSK9, reduced cholesterol levels in hPCSK9-KI but not in wildtype mice, showing that the hypercholesterolemic phenotype was driven by overexpression of human PCSK9. CRISPR-Cas9-mediated genome editing of human PCSK9 reduced plasma levels of human and not mouse PCSK9, and in parallel reduced plasma concentrations of total cholesterol; genome editing of mouse Pcsk9 did not reduce cholesterol levels. Base editing using a guide RNA that targeted human and mouse PCSK9 reduced plasma levels of human and mouse PCSK9 and total cholesterol. In our mouse model, base editing was more precise than genome editing, and no off-target editing nor chromosomal translocations were identified.
Here, we describe a humanized mouse model with liver-specific expression of human PCSK9 and a human-like hypercholesterolemia phenotype, and demonstrate that this mouse can be used to evaluate antibody and gene editing-based (genome and base editing) therapies to modulate the expression of human PCSK9 and reduce cholesterol levels. We predict that this mouse model will be used in the future to understand the efficacy and safety of novel therapeutic approaches for hypercholesterolemia.
低密度脂蛋白（LDL）胆固醇的血浆浓度是心血管疾病的一个明确的危险因素。 最近，作为降低胆固醇水平的一种方法，对调节胆固醇稳态的前蛋白转化酶枯草溶菌素/柯萨奇蛋白酶 9（PCSK9）的抑制作用已经出现。 人源化动物模型的开发是验证和研究人类药物靶点的重要步骤，并且已经提出使用基因组和碱基编辑作为靶向疾病等位基因的手段。
为了解决缺乏验证模型来测试针对人类 PCSK9 的技术的安全性和有效性的问题，我们生成了一种肝脏特异性人 PCSK9 敲入小鼠模型（hPCSK9-KI）。 我们表明，hPCSK9-KI 小鼠的总胆固醇血浆浓度高于野生型小鼠，并且随年龄增长而增加。 针对人 PCSK9 的单克隆抗体依洛尤单抗治疗降低了 hPCSK9-KI 但不降低野生型小鼠的胆固醇水平，表明高胆固醇血症表型是由人 PCSK9 的过表达驱动的。 CRISPR-Cas9 介导的人 PCSK9 的基因组编辑降低了人而非鼠 PCSK9 的血浆水平，并平行降低了总胆固醇的血浆浓度； 鼠 Pcsk9 的基因组编辑不能降低胆固醇水平。 使用靶向人 PCSK9 和鼠 PCSK9 的向导 RNA 的碱基编辑降低了人 PCSK9 和鼠 PCSK9 的血浆水平以及总胆固醇的血浆浓度。 在我们的小鼠模型中，碱基编辑比基因组编辑更精确，并且没有发现脱靶编辑或染色体易位。
在这里，我们描述了一种肝脏特异性表达人 PCSK9 和人样高胆固醇血症表型的人源化小鼠模型，并证明该小鼠可用于评估抗体和基于基因编辑（基因组和碱基编辑）的疗法来调节人 PCSK9 的表达并降低胆固醇水平。 我们预测，这种小鼠模型将在未来用于了解新型治疗高胆固醇血症方法的疗效和安全性。
