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.

项目概要/摘要 我们建议使用体内 CRISPR/Cas9 方法进行体细胞基因组编辑来治疗两种小鼠模型 遗传性周围神经病（Charcot-Marie-Tooth 病，CMT）的合作 格拉德斯通研究所（Conklin，U01-ES032673）和杰克逊实验室（JAX，Murray 和 Lutz，U42- OD026635）。 CMT 是一系列无法​​治愈的致残性周围神经病，总体患病率为 1:2500 人们。某些 CMT 突变会导致显性失活等位基因，可以通过等位基因特异性来纠正 使用 CRISPR/Cas9 进行沉默。 2E 型 CMT (CMT2E) 是由神经丝光显性突变引起的 链基因（NEFL）。我们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 iPSC，逆转疾病表型。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，2D 型 CMT (CMT2D) 是由显性失活突变引起的 甘氨酰 tRNA 合成酶 (GARS)。我们已经证明 AAV9 可以针对等位基因特异性 RNAi 进行递送 突变的 Gars mRNA 能够有效预防两种 CMT2D 小鼠模型的疾病，包括 GarsDETAQ 等位基因。在目标 2 中，我们将在针对 GarsDETAQ 的小鼠体内测试等位基因特异性基因组编辑 等位基因。这些研究将共同​​确定改善神经病变的基因组编辑水平 通过测量编辑效率、测试临床相关表型结果和分析来确定表型 毒理学和脱靶效应。载体设计和包装以及编辑效率评估将 在格拉德斯通完成。体内临床前研究将在 JAX 进行。