Genetic Repair of Familial Hypercholesterolemia

项目摘要 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）中两个LDLR等位基因缺失 导致血浆胆固醇水平过高、黄色瘤、过早动脉粥样硬化和死亡， 如果不治疗的话，生命的最初几十年。目前的治疗方法对HoFH基本无效， 迫切需要解决办法。使用腺相关病毒（AAV）载体的肝脏定向基因治疗是 这是一个密集研究的领域，非常接近于实现对几种遗传性疾病的有意义的纠正。 虽然正在进行的AAV临床试验显示出有希望的结果，但常规（添加剂）基因疗法 局限性包括：转基因沉默、表达水平的不精确控制、对 转基因和衣壳蛋白，以及附加型AAV基因组在细胞分裂中的损失。我们相信， 这些可以通过使用成簇的规则间隔的短回文的基因编辑方法来解决 重复序列（CRISPR）-Cas9系统。我们的长期目标是完善和优化我们的基于AAV的基因疗法 修复导致最严重脂质紊乱的致病等位基因的平台。的目的 目前的应用是进行临床前基因治疗，以纠正家族性糖尿病患者的代谢缺陷。 通过基于AAV的基因组编辑在小鼠模型中观察高胆固醇血症（FH）。我们的核心假设是， AAV介导的同源重组与CRISPR/Cas9指导的DNA切割组合，可以 以高效率有效地修复Ldlr基因的突变形式。在目标1中，我们将确定最佳 用于小鼠中AAV介导的Ldlr基因的位点特异性破坏的Cas9直系同源物和指导RNA序列 肝脏在目标2中，我们将使用这些载体，沿着携带“修复模板”的重组AAV基因组。 以递送Ldlr的功能性“野生型”外显子。编辑和更正的程度将在 遗传和表型水平-包括血脂和动脉粥样硬化易感性的变化。 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.