Permanent alteration of PCSK9 in vivo genome editing

项目总结 枯草杆菌前蛋白转换酶9(PCSK9)已成为一种很有前途的治疗靶点。 预防冠心病。在肝脏中特异表达和分泌的基因，以及 PCSK9被认为主要是低密度脂蛋白受体(LDLR)的拮抗剂，它是 最初在一些家庭中被确定为常染色体显性高胆固醇血症的原因，具有- 导致低密度脂蛋白(LDL-C)水平升高和过早冠心病的基因功能突变。在……里面 随后的研究发现，PCSK9基因单一功能缺失突变的个体经历了 显著降低低密度脂蛋白水平(~30%-40%)和冠心病风险(88%)。值得注意的是，即使是个人 PCSK9的两个功能缺失突变--导致低密度脂蛋白-C水平下降约80%--似乎受到了 没有不良的临床后果。 永久改变人类基因组的能力已经成为可能，这是现在普遍使用的技术 也就是基因组编辑。最近发表的成簇规则间隔的短回文重复 (CRISPR)/CRISPR相关(CAS)系统使用目标为化脓性链球菌Cas9核酸酶 通过与合成的与20个核苷酸的DNA序列杂交的引导RNA络合而形成的基因组位置 (Protspacer)紧接在Cas9识别的NGG基序(PAM或Protspacer相邻基序)之前。 CRISPR-Cas9产生双链断裂(DSB)，通常通过非同源末端连接修复 (NHEJ)，这是容易出错的，并有助于移码突变导致基因敲除。一台较新的 称为“碱基编辑”的Cas9版本选择性地将胞嘧啶碱基编辑成胸腺嘧啶，而不需要DSB， 因此可能代表了一种更安全的引入敲除无义突变的方法。 鉴于在体外哺乳动物细胞中观察到的CRISPR-Cas9的高效率，我们试图评估 一次性交付CRISPR-Cas9是否可用于永久干扰人PCSK9基因 VIVO高效和安全，如果是的话，则是用于这一目的的最佳CRISPR-CAS9系统。在以下方面取得成功 完成这一翻译项目将为体内基因组的可行性提供关键信息- 编辑方法，最终可能产生一种一次性的长期疗法，永久降低血液低密度脂蛋白- C水平，因此可作为预防心血管疾病的“疫苗”。

PROJECT SUMMARY Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising therapeutic target for the prevention of coronary heart disease (CHD). A gene specifically expressed in and secreted from the liver, and believed to function primarily as an antagonist to the low-density lipoprotein receptor (LDLR), PCSK9 was originally identified as the cause of autosomal dominant hypercholesterolemia in some families, with gain-of- function mutations in the gene driving highly elevated LDL cholesterol (LDL-C) levels and premature CHD. In subsequent studies, individuals with single loss-of-function mutations in PCSK9 were found to experience a significant reduction of both LDL-C levels (~30%–40%) as well as CHD risk (88%). Notably, even individuals with two loss-of-function mutations in PCSK9—resulting in ~80% reduction in LDL-C levels—appear to suffer no adverse clinical consequences. The ability to permanently alter the human genome has been made possible by the technology now commonly known as genome editing. Recently published clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated (Cas) systems use Streptococcus pyogenes Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes a 20-nucleotide DNA sequence (protospacer) immediately preceding an NGG motif (PAM, or protospacer-adjacent motif) recognized by Cas9. CRISPR-Cas9 generates a double-strand break (DSB) that is usually repaired by non-homologous end-joining (NHEJ), which is error-prone and conducive to frameshift mutations resulting in gene knock-out. A newer version of Cas9 termed a “base editor” selectively edits cytosine bases to thymine, without the need for DSBs, and thus may represent a safer means by which to introduce knock-out nonsense mutations. In light of the observed high efficiencies of CRISPR-Cas9 in mammalian cells in vitro, we seek to assess whether a one-time delivery of CRISPR-Cas9 can be used to permanently disrupt the human PCSK9 gene in vivo efficiently and safely and, if so, the optimal CRISPR-Cas9 system to use for this purpose. Success in completing this translational project will provide critical information on the feasibility of an in vivo genome- editing approach that could ultimately yield a one-shot, long-term therapy that permanently reduces blood LDL- C levels and thus serves as a “vaccination” against cardiovascular disease.