Development of miCas9 mediated gene editing therapies for cystic fibrosis

项目摘要 囊性纤维化是一种由囊性纤维化跨膜突变引起的遗传病。 电导调节因子(CFTR)基因。本病累及多个器官，伴有肺部疾病 产生了最多的CF发病率和死亡率。在过去的十年里取得了重大进展 在慢性阻塞性肺疾病药物开发方面。超过90%的患者现在受益于单核细胞或联合细胞 几种CFTR型增强器和矫正器的治疗。然而，这些化合物既不是 既不能根治这种疾病，也不能解决10%零突变携带者未得到满足的需求。 基因编辑疗法(GETx)代表了一种有望永久治愈这种疾病的策略。 2019年，我们报道了在人类诱导的多能干细胞中主要CFTR基因座的高效基因编辑 (IPS)细胞。2020年，我们报道了具有非凡同源性的mi-spCas9的发展。 定向修复能力。在这两大工作线的基础上，我们在这里建议开发和 优化一种适合体内GETx的新型mi-saCas9突变体。在目标1中，我们将开发 MI-saCas9-KKH-A，对纠正CFR引起的突变特别有用。在AIM 2 我们将测试两种针对CF的GETX策略：(I)适用于 纠正所有cftr突变；以及(Ii)适合纠正的无供体策略 复合杂合cftr突变。圆满完成拟议的第一阶段工作 将为第二阶段奠定坚实的基础，在这一阶段，我们计划评估 基于MI-saCas9-KKH-A的GETx策略在临床前CF动物模型中的应用

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.