Biochemical Interrogation of Rare Cystic Fibrosis Mutations Informs Strategies for Future Therapeutic Intervention

There are over 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene associated with Cystic Fibrosis (CF) disease, and to date, the two recently FDA-approved drugs KalydecoTM and OrkambiTM have been effective in rescuing functional expression of the two most common CFTR mutations, G551D and ΔF508, respectively, representing approximately 90% of the patient population worldwide. However, potential efficacy of these therapies for the remaining ~2000 mutations is unknown. Thus, biochemical characterization of each rare variant is needed to assess potential clinical benefit. My thesis attempts to understand molecular defects associated with less common CFTR mutations, mainly c.3700A>G (causing abberant splicing and subsequent translation of ΔI1234_R1239-CFTR) and c.2052_2053insA (Q685TfsX4-CFTR), with the goal of determining whether current CF therapies (specifically designed for G551D and ΔF508) could be used for these rare variants. Additionally, my work aims to identify unique molecular aberrations for these rare, mutant CFTR proteins in order to facilitate development of mutation-specific therapeutics. Through a collaborative initiative involving clinicians, biochemists and biophysicists, we found that ΔI1234_R1239-CFTR shared certain biochemical attributes with ΔF508-CFTR, however contained intrinsic defects which differed significantly (i.e. poorer interdomain assembly between the amino- and carboxy-termini, and decreased functional responses to cAMP agonists, CFTR correctors and potentiators). When CFTR activity was evaluated in nasal epithelial cultures from siblings homozygous for c.3700A>G, we likewise found that ΔI1234_R1239-CFTR responded poorly to small molecules. For Q685TfsX4-CFTR, we found that this variant was prematurely truncated, and therefore did not respond to CFTR-specific therapies. However, a proof-of-concept in vitro strategy to ‘skip’ the exon containing the c.2052_2053insA mutation led to an incomplete, yet partially mature CFTR molecule which exhibited channel activity, albeit this function was unregulated. In parallel to these studies, we generated and functionally characterized several other rare CFTR mutations using novel mutagenesis and high-throughput functional screening methods, allowing for rapid identification and stratification of genotypes that could potentially benefit from current CF therapies. Furthermore, our efforts described here aim to facilitate future mutation-specific CF drug discovery, as well as a ‘personalized medicine’ approach to assist in the development of novel strategies to combat this fatal genetic disease.Ph.D.2017-07-08 00:00:0