Permanent alteration of PCSK9 in vivo genome editing

项目摘要 前蛋白转化酶枯草杆菌蛋白酶/kexin 9型（PCSK 9）已成为一个有前途的治疗靶点， 预防冠心病（CHD）。在肝脏中特异性表达并分泌的基因，以及 PCSK 9被认为主要作为低密度脂蛋白受体（LDLR）的拮抗剂发挥作用， 最初被确定为常染色体显性高胆固醇血症的原因，在一些家庭中， 基因中的功能突变导致LDL胆固醇（LDL-C）水平高度升高和早发CHD。在 在随后的研究中，发现PCSK 9中具有单一功能丧失突变的个体经历了 LDL-C水平（约30%-40%）和CHD风险（88%）均显著降低。值得注意的是， PCSK 9中有两个功能缺失突变-导致LDL-C水平降低约80%-似乎患有 无不良临床后果。 永久改变人类基因组的能力已经成为可能的技术，现在通常 称为基因组编辑。最近发表的成簇规则间隔短回文重复序列 CRISPR（CRISPR）/CRISPR相关（Cas）系统使用化脓链球菌Cas9核酸酶，其靶向于 通过与合成的指导RNA复合，该指导RNA与20个核苷酸的DNA序列杂交， 在一个实施方案中，所述序列包含紧接在由Cas9识别的NGG基序（PAM，或前间区序列邻近基序）之前的前间区序列（前间区序列）。 CRISPR-Cas9产生双链断裂（DSB），通常通过非同源末端连接修复 （NHEJ），其是易错的并且有助于导致基因敲除的移码突变。较新 称为“碱基编辑器”的Cas9版本选择性地将胞嘧啶碱基编辑为胸腺嘧啶，而不需要DSB， 因此可以代表引入敲除无义突变的更安全的方法。 鉴于CRISPR-Cas9在体外哺乳动物细胞中观察到的高效率，我们试图评估CRISPR-Cas9在体外哺乳动物细胞中的作用。 CRISPR-Cas9的一次性递送是否可以用于永久性破坏人类PCSK 9基因， 如果是这样的话，最佳的CRISPR-Cas9系统用于此目的。成功 完成这个翻译项目将提供关于体内基因组可行性的关键信息， 编辑方法，最终可以产生一个一次性的，长期的治疗，永久降低血液LDL- C水平，因此可以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.