Permanent alteration of PCSK9 in vivo genome editing

PCSK9 体内基因组编辑的永久改变

基本信息

批准号：
9307483
负责人：
Kiran Musunuru
金额：
$ 40.25万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-05-01 至 2021-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9307483
关键词：
Adenoviruses Adult Antibodies Automobile Driving Blood Cardiovascular Diseases Cardiovascular system Cause of Death Cell Line Cholesterol Clinical Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Complex Coronary heart disease Cytosine DNA Sequence Dependovirus Family Frameshift Mutation Future Genes Genomics Guide RNA Hepatocyte Human Human Genetics Human Genome Hybrids In Vitro Individual Injection of therapeutic agent Knock-out LDL Cholesterol Lipoproteins Light Liver Low Density Lipoprotein Receptor Mammalian Cell Mus Mutation Nonhomologous DNA End Joining Nonsense Mutation Nucleotides Patients Pharmaceutical Preparations Pharmacology Prevention Preventive therapy Production Proprotein Convertases Proteins Publishing Residual state Risk Risk Reduction Safety Sampling Site Streptococcus pyogenes Subtilisins System Technology Testing Thymine Time Transplantation Vaccination Work base cardiovascular risk factor deep sequencing disorder risk experience gain of function mutation genome editing genome-wide analysis humanized mouse hypercholesterolemia in vivo loss of function mutation mouse model new technology nuclease premature repaired success therapeutic target

项目摘要

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.

项目概要前蛋白转化酶枯草杆菌蛋白酶/kexin 9 型 (PCSK9) 已成为一种有前途的治疗靶点预防冠心病（CHD）。在肝脏中特异性表达和分泌的基因，以及据认为，PCSK9 的主要功能是作为低密度脂蛋白受体 (LDLR) 的拮抗剂，最初被确定为某些家族中常染色体显性高胆固醇血症的原因，导致低密度脂蛋白胆固醇（LDL-C）水平升高和过早冠心病的基因功能突变。随后的研究发现，PCSK9 具有单一功能丧失突变的个体会经历显着降低 LDL-C 水平（约 30%–40%）以及 CHD 风险（88%）。 PCSK9 中存在两个功能丧失突变，导致 LDL-C 水平降低约 80%，似乎受到影响无不良临床后果。现在普遍使用的技术使永久改变人类基因组的能力成为可能最近发表的聚类规则间隔短回文重复序列。 (CRISPR)/CRISPR 相关 (Cas) 系统使用化脓性链球菌 Cas9 核酸酶，该酶靶向通过与与 20 核苷酸 DNA 序列杂交的合成引导 RNA 复合而形成基因组位点（原型间隔子）紧接在 Cas9 识别的 NGG 基序（PAM 或原型间隔子相邻基序）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），它容易出错并且有利于移码突变，导致基因敲除。 Cas9 版本被称为“碱基编辑器”，选择性地将胞嘧啶碱基编辑为胸腺嘧啶，无需 DSB，因此可能代表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，我们寻求评估一次性递送 CRISPR-Cas9 是否可用于永久破坏人类 PCSK9 基因体内高效且安全，如果是的话，用于此目的的 CRISPR-Cas9 系统就成功了。这个完成的转化项目将提供有关体内基因组可行性的关键信息编辑方法最终可能产生一种一次性长期疗法，永久降低血液中的低密度脂蛋白- C水平，因此可以作为预防心血管疾病的“疫苗”。