Genetic Correction of Mutant Huntingtin in Vivo

这项拟议中的研究旨在确定单基因疾病，如亨廷顿病（HD） 可以通过使用同源重组（HR）来治疗，以在遗传上校正包含等位基因的疾病， vivo.该方法将利用基因组工程系统，CRISPR（重复调控间隔 短回文重复序列）。在最近的HD-iPSC（诱导多能干细胞）研究中，我们 利用同源重组来遗传纠正含有疾病的细胞，并发现这完全 逆转HD疾病表型。我们已经改造了CRISPR技术， 校正/扩增，并已在人类细胞中发现非常高水平的同源重组 （初步结果）。总的目标是在体内使用这项技术，并进行遗传校正， 包含疾病的突变，如亨廷顿蛋白（HTT）中的CAG扩增。这项工作将作为一个证明 的概念，以证明实用性，不仅为单基因神经退行性疾病，但也为其他类型的 遗传病为了实现我们的目标，我们将实现以下目标：具体目标1。证明 使用CRISPR介导的突变HTT在人HD患者来源的神经细胞中的稳健重组 慢病毒;特异性目标2.在HD小鼠模型中证明稳健的CRISPR介导的重组 使用病毒递送表达人HTT蛋白;特异性目标3.开发一种蛋白质介导的 使用切口酶Cas9 D10 A蛋白、gRNA和DNA介导同源重组的系统， 人类患者HD神经干细胞和HD小鼠模型。这些研究将促进我们对 如何在大脑细胞中进行基因校正。成功演示了在体内 对大脑中的疾病等位基因进行遗传校正将是神经科学的一个重大飞跃。

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.