Leveraging a 'sliding-window' Type I CRISPR base editing platform to correct CFTR null mutations

Project Summary Cystic Fibrosis (CF) is an autosomal recessive genetic disorder affecting nearly 160,000 patients worldwide. It is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a chloride ion channel crucial for salt and water homeostasis across epithelial membranes. CFTR dysfunction leads to severe damages to the lungs, intestines, and pancreas. Recent advancements have been made in CF drug development, with 90% of CF patients able to benefit from small molecule drugs, CFTR modulators. However, these medications are expensive, can have serious side effects, and must be taken for a lifetime as they do not provide a permanent cure. Nor do they not address the unmet medical need of the remaining 10% of CF-patients with CFTR null mutations. Alternative therapeutic strategies are urgently needed to alleviate the disease burden on society. CRISPR-Cas gene editing therapy promises a permanent cure for CF by directly fixing the genetic root cause of the disease. Since only individuals with two copies of the defective CFTR gene will develop CF, correcting a single copy at the endogenous gene locus should be adequate for a cure. This proposal aims to develop a gene editing strategy for CFTR-G542X, the second most common CF mutation and the most prevalent null mutation, rendering it non-responsive to CFTR modulator drugs. We will leverage Type I CRISPR, which offers unique properties not attainable with traditional Cas9 tools derived from Type II CRISPR. The overall strategy is to install a high-efficiency, precise single base change to correct the pathogenic adenine underlying G542X into a functional CFTR variant. In Aim 1 and Aim 2, I will determine the editing efficacy and safety for strategy in a patient-derived induced pluripotent stem cell line model. In Aim 3, I will assess the therapeutic outcome by measuring CFTR channel function restoration in a preclinical human bronchial epithelial cell line model. Successful completion of these objectives will provide proof of concept for Type I CRISPR base editing therapy to treat patients with CFTR null mutations. Our long-term goal is to establish a framework for treating a variety of CFTR mutations using a unified Type I CRISPR platform with unprecedent targeting scope.