JAX-Gladstone, SCGE Disease Models Studies Supplement

JAX-Gladstone，SCGE 疾病模型研究补充材料

• 批准号: 10620067
• 负责人: Bruce R Conklin
• 金额: $ 20万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-25 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10620067
• 关键词: Alleles; Animals; Axon; Axonal Neuropathy; Base Pairing; Behavior; Behavioral; Biological Assay; CRISPR/Cas technology; Cells; Charcot-Marie-Tooth Disease; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Collection; Cytoskeleton; DNA Repair; Disease; Disease model; Dominant-Negative Mutation; Dose; Evaluation; GARS gene; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Genome; Goals; Hereditary Motor and Sensory-Neuropathy Type II; Histopathology; In Vitro; Inherited; Institutes; Intermediate Filament Proteins; Knock-in; Light; Measures; Messenger RNA; Mitotic; Motor; Motor Neurons; Mus; Mutation; Nature; Neurons; Neuropathy; Outcome; Pathway interactions; Patients; Peripheral Nervous System Diseases; Persons; Phenotype; Prevalence; Proteins; RNA Interference; Research; Sampling; Somatic Cell; Specificity; Spinal Cord; Spinal Ganglia; Study models; Testing; The Jackson Laboratory; Tissues; Toxicology; Translating; Work; axonal degeneration; base; cell type; clinically relevant; design; disease phenotype; gain of function mutation; gene therapy; genome editing; improved; in vivo; in vivo evaluation; induced pluripotent stem cell; insertion/deletion mutation; mouse model; mutant; mutation correction; myelination; neurofilament; neurophysiology; preclinical study; prevent; protein expression; repaired; sensory neuropathy; somatic cell gene editing; therapeutic gene; trafficking; transcriptome; translational potential; vector

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.

项目摘要/摘要 我们建议使用CRISPR/CAS9方法在体内使用体细胞基因组编辑来处理两种鼠标模型 遗传性周围神经病（Charcot-Marie-Tooth疾病，CMT） Gladstone Institutes（Conklin，U01-ES032673）和杰克逊实验室（Jax，Murray和Lutz，U42-- OD026635）。 CMT是无法治愈的残破周围神经病的集合，总体患病率为1：2500 人们。某些CMT突变会导致可通过等位基因特异性校正的显性阴性等位基因 用CRISPR/CAS9沉默。 CMT 2e型（CMT2E）是由神经丝光中的显性突变引起的 链基因（NEFL）。我们已经使用了等位基因特异性基因组编辑来使患者的严重NEFLN98S等位基因沉默 - 衍生的IPSC，逆转了疾病表型。在AIM 1中，我们将将我们的体外研究扩展到现有 CMT2E的NEFLN98S小鼠模型。同样，CMT型2D（CMT2D）是由主要的负突变引起的 在糖基tRNA-合成酶（GARS）中。我们已经表明，AAV9的特定等位基因特异性RNAi靶向 突变的GARS mRNA能够有效预防CMT2D的两种小鼠模型，包括 GARSDETAQ等位基因。在AIM 2中，我们将在针对GARSDETAQ的小鼠中测试体内等位基因特异性基因组编辑 等位基因。这些研究将共同​​确定改善神经病的基因组编辑水平 通过测量编辑效率，测试临床相关的表型结果和分析来表型 毒理学和脱靶效应。向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JAX进行。