JAX-Gladstone, SCGE Disease Models Studies Supplement

项目摘要/摘要 我们建议使用体内CRISPR/Cas9方法的体细胞基因组编辑来治疗两个小鼠模型 遗传性周围神经病(Charcot-Marie-Tooth病，CMT)的研究 格拉德斯通研究所(康克林，U01-ES032673)和杰克逊实验室(JAX，Murray和Lutz，U42- OD026635)。CMT是一组无法治愈的致残性周围神经病，总体患病率为1：2500 人民。某些CMT突变导致显性负性等位基因，可通过等位基因特异性纠正 使用CRISPR/CAS9进行静音。CMT-2E(CMT2E)是由神经丝光的显性突变引起的 链基因(NEFL)。我们已经使用等位基因特异性基因组编辑来沉默患者中严重的NEFLN98S等位基因- 衍生的IPSCs，逆转了疾病的表型。在目标1中，我们将把我们的体外研究扩展到现有的 CMT2E的NeflN98S小鼠模型。同样，CMT 2D型(CMT2D)是由显性负突变引起的 甘氨酰tRNA合成酶(GARS)。我们已经证明，AAV9递送针对等位基因特异的RNAi 突变的Gars mRNA能够有效地预防两种CMT2D小鼠模型的疾病，包括 GarsDETAQ等位基因。在目标2中，我们将在小鼠体内测试针对GarsDETAQ的等位基因特异性基因组编辑 等位基因。总之，这些研究将确定改善神经病变的基因组编辑水平 通过测量编辑效率，测试临床相关的表型结果，以及描述表型 毒理和偏离目标的影响。矢量设计和包装，以及编辑效率的评估将 在格拉德斯通完成。体内临床前研究将在日本宇宙航空研究开发机构进行。

PROJECT SUMMARY/ABSTRACT We propose to use somatic cell genome editing by CRISPR/Cas9 approaches in vivo to treat two mouse models of inherited peripheral neuropathy (Charcot-Marie-Tooth disease, CMT) in a collaboration between the Gladstone Institutes (Conklin, U01-ES032673) and The Jackson Laboratory (JAX, Murray and Lutz, U42- OD026635). CMT is a collection of incurable crippling peripheral neuropathies with overall prevalence of 1:2500 people. Certain CMT mutations result in dominant-negative alleles that could be corrected by allele-specific silencing with CRISPR/Cas9. CMT type 2E (CMT2E) is caused by dominant mutations in the neurofilament light chain gene (NEFL). We have used allele-specific genome editing to silence the severe NEFLN98S allele in patient- derived iPSCs, reversing the disease phenotype. In Aim 1 we will extend our in vitro studies to an existing NeflN98S mouse model of CMT2E. Similarly, CMT type 2D (CMT2D) is caused by dominant negative mutations in glycyl tRNA-synthetase (GARS). We have shown that AAV9 delivery of allele-specific RNAi targeting the mutant Gars mRNA was able to effectively prevent disease in two mouse models of CMT2D including the GarsDETAQ allele. In Aim 2, we will test in vivo allele-specific genome editing in mice targeting the GarsDETAQ allele. Together, these studies will establish the levels of genome editing that improve the neuropathy phenotypes by measuring the editing efficiency, testing clinically relevant phenotypic outcomes, and profiling toxicological and off-target effects. Vector design and packaging, as well as evaluation of editing efficiency will be done at Gladstone. In vivo preclinical studies will be done at JAX.