Genetic Repair of Familial Hypercholesterolemia

这是一个竞争性的更新R01申请，旨在开发新的基因编辑方法来治疗家族性高胆固醇血症（FH）。FH是一种常染色体显性疾病，最常由低密度脂蛋白受体（LDLR）的功能丧失突变引起，低密度脂蛋白受体负责由肝脏摄取含载脂蛋白b的脂蛋白颗粒（如LDL）。纯合子FH （HoFH）中两个LDLR等位基因的缺失会导致血浆胆固醇过高（~1000 mg/dl）、黄斑瘤、动脉粥样硬化，如果不治疗，在生命的最初几十年就会死亡。目前，治疗HoFH的唯一方法是肝移植，这使得这种疾病成为基因治疗的重要优先事项。通过基因治疗实现对HoFH的永久矫正需要修改患者自身的DNA，以使表达LDLR的细胞在肝脏中持续存在。在这个应用中，我们将进一步扩展我们之前的工作，通过开发策略将全长LDLR转基因整合到基因组安全港位点。我们的目标是确保这种疗法可以推广到HoFH患者，而不是针对特定的突变或蛋白质区域。其次，我们将使用FH小鼠模型，通过基因校正肝细胞的选择扩展来测试肝脏定向基因治疗的新策略。这种方法利用肝脏的再生能力，并使用内源性必需基因作为选择性标记。随着时间的推移，必需基因的抑制可以用来选择性地扩增基因靶向肝细胞，确保可以实现全肝范围的校正。我们提出了两个具体目标：1)确定Ldlr和Apoa1位点的靶向整合是否可以纠正FH， 2)开发一种新的系统来选择性扩增基因纠正的肝细胞。这项工作的完成将为HoFH提供一种新的基因治疗方案，以及通过体内选择对其他肝脏疾病进行基因校正的通用系统。

This is a competing renewal R01 application that seeks to develop new gene editing approaches to treat Familial Hypercholesterolemia (FH). FH is an autosomal dominant disease most often caused by loss-of-function mutations in the low density lipoprotein receptor (LDLR), the protein responsible for uptake of ApoB-containing lipoprotein particles (such as LDL) by the liver. Loss of both LDLR alleles in homozygous FH (HoFH) causes excessively high plasma cholesterol (~1000 mg/dl), xanthomas, atherosclerosis, and death in the first few decades of life if untreated. Currently, the only curative treatment for HoFH is a liver transplant, making this disease an important priority for gene therapy. Achieving permanent correction of HoFH through gene therapy will require modifying the patient’s own DNA, so that cells expressing LDLR will persist in the liver. In this application we will further expand on our previous work, by developing strategies to integrate a full length LDLR transgene into genomic safe harbor sites. The goal is to ensure that the therapy will be generalizable to HoFH patients, and not specific to a particular mutation or region of the protein. Secondly, we will use FH mouse models to test a new strategy for liver-directed gene therapy through selection expansion of gene-corrected hepatocytes. This approach capitalizes on the regenerative capacity of the liver, and uses endogenous essential gene as a selectable marker. Over time, inhibition of the essential gene can be used to selectively expand gene-targeted hepatocytes, ensuring that liver-wide correction can be achieved. We propose two specific Aims: 1) Determine if targeted integration at the Ldlr and Apoa1 loci can correct FH, and 2) Develop a novel system for selective expansion of gene- corrected hepatocytes. Completion of this work will produce a new gene therapy solutions for HoFH, as well as a universal system for genetic correction of other liver disorders through in vivo selection.