Evaluation of CFTR mRNA stability and dosage

Abstract

Cystic fibrosis (CF) is an autosomal recessive disorder caused by more than 2000 variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR modulators have completely revolutionized the treatment of CF, with over 90% of individuals with CF being eligible for treatment. However, nonsense variants in CFTR have presented a particular challenge for therapeutic treatment. Nonsense mediated mRNA decay (NMD) targets premature termination codon (PTC)-bearing transcripts to reduce the potentially damaging effects of truncated proteins. CFTR modulators specifically target CFTR to aid in protein processing and function. Since NMD results in absence of CFTR protein, this prevents modulators from restoring CFTR function. mRNA stability is a critical consideration when evaluating therapeutic strategies for CF. In Chapter 2 of my thesis work, I investigate mRNA stability for the sixth most common CF- causing mutation, W1282X. I establish that this variant generates a PTC, targeting the transcript for degradation by NMD and resulting in low levels of steady state mRNA. This finding suggests that W1282X-bearing transcripts will not generate any protein, explaining the lack of CFTR functional response to modulators. In Chapter 3, I investigated several nonsense variants within the N- terminal region of CFTR and show that downstream translation initiation at M265 allows these variants to escape NMD. Treatment of the variant L88X with readthrough compounds combined with CFTR modulators restores full-length, functional CFTR protein. This work indicates that N-terminal variants that evade NMD are ideal candidates for combination treatment of readthrough compounds and modulator therapy. In Chapter 4, I establish a cell-based model system that enables independent expression of two copies of CFTR, which will aid in assessment of rare variants that are difficult to obtain through primary cell collection. Additionally, this system enables us to assess the fitness of the predicted model that each CFTR gene contributes to total CFTR function in an additive manner