Permanent alteration of PCSK9 in vivo genome editing

PCSK9 体内基因组编辑的永久改变

• 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
• 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

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.

项目摘要 普罗蛋白转化酶枯草蛋白/KEXIN 9型（PCSK9）已成为有前途的治疗靶标 预防冠心病（CHD）。专门从肝脏中表达并分泌的基因， 据信作为对低密度脂蛋白受体（LDLR）的拮抗剂的主要作用，PCSK9 最初被确定为某些家庭中常染色体显性高胆固醇血症的原因，并获得 驱动高度升高的LDL胆固醇（LDL-C）水平和过早冠心的基因中的功能突变。在 随后的研究，发现PCSK9中单个功能丧失突变的个体经历了A 两种LDL-C水平（约30％–40％）和CHD风险（88％）的显着降低。值得注意的是，即使是个人 PCSK9中有两个功能丧失的突变（降低了80％的LDL-C水平），表现为受苦 没有不良临床后果。 现在通常的技术使得永久改变人类基因组的能力成为可能 称为基因组编辑。最近发布的定期散布的短篇小说重复序列 （CRISPR）/CRISPR相关（CAS）系统使用链球菌CAS9核酸酶，该核酸酶针对的 通过与杂交20核苷酸DNA序列的合成指南RNA复合通过络合基因组位点 （原始的）紧接在NGG基序之前（PAM或Oteospacer-Adjacent Motif），CAS9识别。 CRISPR-CAS9产生双链断裂（DSB），通常通过非同性末端结合进行修复 （nhej），它容易出错，导电到移码突变，导致基因敲除。一个新的 CAS9的版本称为“基本编辑器”，有选择地编辑胞嘧啶碱为胸腺素，而无需DSB， 因此，可能代表了一种更安全的手段来引入淘汰赛胡说八道突变。 鉴于在体外观察到的CRISPR-CAS9效率很高，我们试图评估 是否可以使用CRISPR-CAS9的一次性传递来永久破坏人类PCSK9基因 体内有效，安全，如果是的，则可以用于此目的的最佳CRISPR-CAS9系统。成功 完成此翻译项目将提供有关体内基因组可行性的关键信息 - 编辑方法最终可以产生一弹性的长期治疗，从而永久降低血液LDL- C水平，因此是针对心血管疾病的“疫苗接种”。