Development of miCas9 mediated gene editing therapies for cystic fibrosis

项目摘要 囊性纤维化（CF）是一种遗传性疾病，由囊性纤维化跨膜蛋白突变引起。 电导调节因子（CFTR）基因。该疾病影响多个器官，伴有肺部疾病 导致大多数CF发病率和死亡率。在过去十年中取得了重大进展 CF药物开发。超过90%的患者现在受益于单药或联合用药 几种CFTR增效剂和校正剂的治疗。然而，这些化合物既 永久治愈疾病，也不能解决10%无效突变携带者未满足的需求。 基因编辑疗法（GETx）是永久治愈这种疾病的一种有前途的策略。 在2019年，我们报告了对人类诱导多能干细胞中主要CFTR基因座的有效基因编辑 (iPS)细胞在2020年，我们报道了具有非凡同源性的mi-spCas 9的开发 定向修复能力。在这两个主要工作领域的基础上，我们建议发展和 优化适合于CF的体内GETx的新型mi-saCas 9变体。在目标1中，我们将开发 mi-saCas 9-KKH-A特别适用于校正CF引起的CFTR突变。在目标2中 我们将测试两种用于CF的GETx策略：（i）适合于CF的大尺寸基因敲入策略， 校正所有CFTR突变;和（ii）适用于校正所有CFTR突变的无供体策略， 复合杂合CFTR突变。顺利完成拟议的第一阶段工作 将为第二阶段奠定坚实的基础，我们计划在第二阶段评估 在临床前CF动物模型中基于mi-saCas 9-KKH-A的GETx策略。

Project Summary Cystic Fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The disease affects multiple organs, with lung disease producing most CF morbidity and mortality. Major progress has been made over the past decade in CF drug development. Greater than 90% patients now benefit from mono or combination therapy of several CFTR potentiators and correctors. However, these compounds neither permanently cure the disease nor address the unmet need of the 10% null mutation carriers. Gene editing therapy (GETx) represents a promising strategy to permanently cure the disease. In 2019 we reported efficient gene editing on major CFTR loci in human induced pluripotent stem (iPS) cells. In 2020, we reported the development of mi-spCas9 that has extraordinary homology directed repair capacity. Built on these two major lines of work, here we propose to develop and optimize a novel mi-saCas9 variant that is suitable for in vivo GETx of CF. In Aim 1, we will develop mi-saCas9-KKH-A that is particular useful for correcting CF-causing CFTR mutations. In Aim 2 we will test two GETx strategies for CF: (i) a large size gene knock-in strategy that is suitable for correcting all CFTR mutations; and (ii) a donor free strategy that is suitable for correcting compound heterozygous CFTR mutations. Successful completion of the proposed Phase I work will set a solid foundation for Phase II, in which we plan to evaluate the efficacy and safety of the mi-saCas9-KKH-A based GETx strategies in preclinical CF animal models.