Development of miCas9 mediated gene editing therapies for cystic fibrosis

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.Project Narrative Cystic Fibrosis (CF) is a genetic disease caused by mutations in the CFTR gene, affecting multiple organs with lung disease producing most morbidity and mortality. Here we propose experiments to develop novel gene editing therapy strategies to correct different CFTR mutations, potentially with improved efficacy and safety outcome, to cure this devastating disease.