Genetic Correction of Mutant Huntingtin in Vivo

这项拟议的研究旨在确定一种单基因疾病，如亨廷顿病(HD) 可以通过使用同源重组(HR)从基因上纠正含有等位基因的疾病来治疗 活着。该方法将利用基因组工程系统CRISPR(集群监管间隔 短回文重复)体内传递。在最近对HD-IPSCs(诱导多能干细胞)的研究中，我们 利用同源重组对含有疾病的细胞进行遗传纠正，并发现完全 逆转HD疾病的表型。我们采用了CRISPR技术来进行遗传 并在人类细胞中发现了非常高水平的同源重组 (初步结果)。总体目标是在体内使用这项技术，并进行遗传纠正 包含疾病的突变，如亨廷顿蛋白(HTT)中的CAG扩张。这项工作将作为证据 概念证明不仅对单基因神经退行性疾病有效，而且对其他类型的 遗传病。为实现我们的目标，我们将实现以下目标：具体目标1.示范 CRISPR介导的突变型HTT在人HD患者来源神经细胞中的重组 慢病毒；特异性目的2.在HD小鼠模型中展示CRISPR介导的重组 利用病毒传递表达人HTT蛋白；特定目的3.建立一种蛋白质介导的传递 利用尼克酶Cas9 D10A蛋白、gRNA和DNA介导的同源重组系统 人-患者HD神经干细胞和HD小鼠模型。这些研究将增进我们对 如何对来自大脑的细胞进行基因纠正。在体内的成功演示 大脑中疾病等位基因的遗传纠正将是神经科学的重大飞跃。

The proposed research is intended to determine if a monogenetic disease such as Huntington's disease (HD) can be treated by using homologous recombination (HR) to genetically correct the disease containing allele in vivo. The approach will utilize a genome engineering system, CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) delivered in vivo. In recent work in HD-iPSCs (induced pluripotent stem cells), we utilized homologous recombination to genetically correct the disease containing cells and found this completely reverses HD disease phenotypes. We have adapted the CRISPR technology to carry out genetic correction/expansion and have found very high levels of homologous recombination in human cells (Preliminary Results). The overall goal is to use this technology in vivo and carry out genetic correction of disease-containing mutations such as the CAG expansion in huntingtin (HTT). This work will serve as a proof of concept to demonstrate utility not only for monogenic neurodegenerative diseases but also for other types of genetic diseases. To achieve our goals we will carry out the following aims: Specific Aim 1. To demonstrate robust CRISPR-mediated recombination of the mutant HTT in human HD patient-derived neural cells using lentivirus; Specific Aim 2. To demonstrate robust CRISPR-mediated recombination in mouse models of HD expressing the human HTT protein using viral delivery; Specific Aim 3. To develop a protein-mediated delivery system using the nickase Cas9 D10A protein, gRNA and DNA to mediate homologous recombination in human-patient HD neural stem cells and HD mouse models. These studies will advance our understanding of how to perform genetic correction in cells derived from the brain. The successful demonstration of in vivo genetic correction of the disease allele in the brain would be a major leap forward in neuroscience.