Therapeutic potential of CRISPR/Cas9 genome engineering in humanized mouse

CRISPR/Cas9 基因组工程在人源化小鼠中的治疗潜力

基本信息

批准号：
10291691
负责人：
Karl-Dimiter Bissig
金额：
$ 54.97万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-20 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10291691
关键词：
Address Animal Model Apolipoproteins B Atherosclerosis Biological Assay Blood CRISPR/Cas technology Cadaver Cell Cycle Cells Cholesterol Chromatin Clinic Code Custom DNA sequencing Data Dependovirus Disease Enzymes Essential Genes Excision Exons Expression Profiling Familial Combined Hyperlipidemia Familial Hypercholesterolemia Gene Expression Genes Genome engineering Goals Guide RNA Hepatocyte Hepatotoxicity Histologic Human Human Cell Line Human Genome Hyperlipoproteinemia Immunodeficient Mouse Introns Investigational Therapies Lipids Lipoprotein (a)Lipoproteins Liver Liver diseases Mammals Measures Mediating Mendelian disorder Metabolic Metabolic Diseases Modeling Monitor Mouse Strains Mus Mutation Nodule Nonhomologous DNA End Joining Pathologic Pathology Patients Plasma Production Proteins Refractory Reporter Resistance S-Phase Fraction Safety Serotyping Squalene Synthetase System Techniques Technology Testing Therapeutic Tissues Translating Transplantation Treatment Efficacy Very low density lipoprotein Xenograft Model Xenograft procedure apolipoprotein Lp(a+)compliance behavior design efficacy testing exome sequencing experience gene therapy genome editing human disease humanized mouse hypercholesterolemia in vivo innovation lipid disorder liver cell proliferation mouse model novel particle pre-clinical repaired therapeutic genome editing transcriptome sequencing transplant model

项目摘要

PROJECT SUMMARY Liver-directed gene editing has the potential to correct numerous severe monogenic disorders. The two major hurdles of translating this therapy to the clinic are efficacy and safety. We will address both concerns using an innovative therapeutic genome editing approach in a novel humanized mouse model. CRISPR/Cas9 genome editing technology has proven extremely efficient in introducing double strand brakes (DSB) in many cellular systems. In mammals, DSB are preferentially repaired by the error prone non-homologous end joining (NHEJ) and to a lesser extent by homology-directed repair (HDR). Here, we propose to treat refractory lipid disorders with compensatory deletion of a whole exon (exon-excision) from lipid genes that increase the cholesterol levels in blood or contribute to slower clearance of blood cholesterol. Deletion will be mediated by NHEJ after introduction of two CRISPR/Cas9 induced DSBs in the two flanking introns. This repair mechanism is very efficient in non-proliferating hepatocytes and avoids the introduction of potentially harmful mutations in the coding sequence. We recently developed a mouse strain that can be repopulated with cadaveric human hepatocytes. Since genome editing is sequence dependent, it must be tested within the context of human cells of the desired target tissue. Using mice repopulated with normal human hepatocytes, we will be able to determine the safety and efficacy of liver-directed human genome editing by CRISPR/Cas9. We will then test his approach in mice repopulated with human hepatocytes from a patient with familial hypercholesterolemia (FH). This first xenograft model for FH can be used to evaluate the therapeutic efficacy and potential compensatory adaptation for this experimental therapy.

项目概要肝脏定向基因编辑有可能纠正许多严重的单基因疾病失调。将这种疗法转化为临床的两个主要障碍是功效和安全性。我们将使用创新的治疗性基因组编辑方法来解决这两个问题新型人源化小鼠模型。 CRISPR/Cas9基因组编辑技术已被证明在引入基因组方面极其有效许多蜂窝系统中的双链制动器（DSB）。在哺乳动物中，DSB 优先通过容易出错的非同源末端连接 (NHEJ) 进行修复，并在较小程度上通过同源定向修复（HDR）。在这里，我们建议用以下方法治疗难治性脂质紊乱：从脂质基因中补偿性删除整个外显子（外显子切除），从而增加血液中胆固醇水平升高或导致血液胆固醇清除速度减慢。删除将是在两侧引入两个 CRISPR/Cas9 诱导的 DSB 后，由 NHEJ 介导内含子。这种修复机制在非增殖性肝细胞中非常有效，并且避免了在编码序列中引入潜在有害的突变。我们最近开发了一种可以用人类尸体进行繁殖的小鼠品系肝细胞。由于基因组编辑依赖于序列，因此必须在上下文中进行测试所需目标组织的人体细胞。使用重新植入正常人类的小鼠肝细胞，我们将能够确定肝脏定向人类的安全性和有效性通过 CRISPR/Cas9 进行基因组编辑。然后我们将在重新植入的小鼠中测试他的方法来自家族性高胆固醇血症（FH）患者的人肝细胞。这第一个异种移植 FH 模型可用于评估治疗效果和潜在的补偿适应这种实验疗法。