Genetic Repair of Familial Hypercholesterolemia

项目总结 家族性高胆固醇血症是一种常染色体显性遗传疾病，最常见的原因是功能丧失。 低密度脂蛋白受体(LDLR)突变。纯合子FH(HoFH)两个LDLR等位基因缺失 导致血浆胆固醇水平过高、黄瘤、过早动脉粥样硬化和死亡 生命的头几十年如果不治疗的话。目前的治疗方法对HoFH很大程度上无效，而且更持久 迫切需要解决方案。用腺相关病毒(AAV)载体进行肝脏导向基因治疗 这是一个非常接近实现对几种遗传性疾病进行有意义的纠正的紧张研究领域。 虽然正在进行的AAV临床试验显示出有希望的结果，但传统的(添加剂)基因疗法已经 限制包括：转基因沉默，表达水平的不精确控制，免疫反应 转基因和衣壳蛋白，以及由于细胞分裂而失去的副体AAV基因组。我们相信许多人 这些可以通过使用成簇的规则间隔短回文的基因编辑方法来解决 重复(CRISPR)-CAS9系统。我们的长期目标是改进和优化我们基于AAV的基因治疗 修复导致最严重的血脂紊乱的致病等位基因的平台。该计划的目标是 目前的应用是进行临床前基因治疗以纠正家族性代谢缺陷 通过基于AAV的基因组编辑研究小鼠模型中的高胆固醇血症(FH)。我们的中心假设是 AAV介导的同源重组结合CRISPR/Cas9介导的DNA切割，可以 高效修复突变的Ldlr基因。在目标1中，我们将确定最优 AAV介导的小鼠Ldlr基因定点特异性破坏的Cas9同源和引导RNA序列 肝脏。在目标2中，我们将使用这些载体，以及含有“修复模板”的重组AAV基因组。 为Ldlr提供功能性的“野生型”外显子。编辑和更正的程度将在 遗传和表型水平--包括血脂的变化和动脉粥样硬化的易感性。 这些目标的完成将产生一个强大的基于AAV的系统，用于体细胞基因的敲除和修复 肝脏，可广泛应用于基础科学和基因治疗应用。

PROJECT SUMMARY Familial hypercholesterolemia is an autosomal dominant disease most often caused by loss of function mutations in the low density lipoprotein receptor (LDLR). Loss of both LDLR alleles in homozygous FH (HoFH) results in excessively high levels of plasma cholesterol, xanthomas, premature atherosclerosis, and death in the first decades of life if untreated. Current treatments are largely ineffective for HoFH and more permanent solutions are desperately needed. Liver-directed gene therapy using Adeno-Associated Viral (AAV) vectors is an area of intense research that is very near to achieving meaningful correction of several inherited diseases. While ongoing clinical trials with AAV are showing promising results, conventional (additive) gene therapy has limitations including: transgene silencing, imprecise control of expression levels, immune responses to transgene and capsid protein, and the loss of episomal AAV genomes to cell division. We believe many of these may be solved by a gene editing approach using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system. Our long-term goal is to refine and optimize our AAV-based gene therapy platform for repair of disease-causing alleles underlying the most severe lipid disorders. The objective of the current application is to perform preclinical gene therapy to correct the metabolic defect in Familial Hypercholesterolemia (FH) in a mouse model via AAV-based genome editing. Our central hypothesis is that AAV-mediated homologous recombination, in combination with CRISPR/Cas9 directed DNA cleavage, can effectively repair a mutant version of the Ldlr gene with high efficiency. In Aim 1 we will determine the optimal Cas9 ortholog and guide RNA sequence for AAV-mediated site specific disruption of the Ldlr gene in mouse liver. In Aim 2, we will use these vectors, along with a recombinant AAV genome harboring a “repair template” to deliver the functional “wild type” exon for Ldlr. The extent of editing and correction will be assessed at the genetic as well as phenotypic levels- include changes in plasma lipids and susceptibility to atherosclerosis. Completion of the aims will produce a powerful AAV-based system for somatic gene knockdown and repair in the liver, which can be broadly applied for both basic science and gene therapy applications.