SCGE Disease Models Studies Supplement: Allele-specific Genome Editing in Two Mouse Models of Charcot-Marie-Tooth Disease

摘要 我们提出通过CRISPR/Cas9方法在体内使用体细胞基因组编辑来治疗两种小鼠， 遗传性周围神经病变（Charcot-Marie-Tooth病，CMT）的模型， 格莱斯顿研究所（康克林，U 01-ES 032673）和杰克逊实验室（JAX，默里和卢茨，U42- OD 026635）。CMT是一组无法治愈的致残性周围神经病变，总体患病率为 1：2500人。某些CMT突变导致显性阴性等位基因，可以通过等位基因- 使用CRISPR/Cas9进行特异性沉默。CMT 2 E型（CMT 2 E）是由CMT 2 E基因的显性突变引起的。 神经丝轻链基因（NEFL）。我们已经使用等位基因特异性基因组编辑来沉默严重的 NEFLN 98 S等位基因，逆转疾病表型。在目标1中，我们将扩展我们的体外 研究了CMT 2 E的现有NeflN 98 S小鼠模型。类似地，CMT 2D型（CMT 2D）由以下原因引起： 甘氨酰tRNA合成酶（加尔斯）的显性负突变。我们已经证明，AAV 9递送等位基因- 靶向突变型加尔斯mRNA的特异性RNAi能够有效地预防两种小鼠模型中的疾病。 CMT 2D，包括GarsoviQ等位基因。在目标2中，我们将在小鼠中测试体内等位基因特异性基因组编辑 针对的是GarsoviQ等位基因总之，这些研究将建立基因组编辑的水平， 通过测量编辑效率，测试临床相关的表型结果， 并分析毒理学和脱靶效应。矢量设计和包装，以及编辑评价 效率将在格莱斯顿完成。体内临床前研究将在JAX进行。

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.