A helper-dependent adenoviral vector rescues CFTR to wild-type functional levels in cystic fibrosis epithelial cells harbouring class I mutations.

Cystic fibrosis (CF) is a genetic disorder affecting multiple organs, including the pancreas, hepatobiliary system and reproductive organs; however, lung disease is responsible for the majority of morbidity and mortality. Management of CF involves CF transmembrane conductance regulator (CFTR) modulator agents including corrector drugs to augment cellular trafficking of mutant CFTR as well as potentiators that open defective CFTR channels. These therapies are poised to help most individuals with CF, with the notable exception of individuals with class I mutations where full-length CFTR protein is not produced. For these mutations, gene replacement has been suggested as a potential solution.In this work, we used a helper-dependent adenoviral vector (HD-CFTR) to express CFTR in nasal epithelial cell cultures derived from CF subjects with class I CFTR mutations.CFTR function was significantly restored in CF cells by HD-CFTR and reached healthy control functional levels as detected by Ussing chamber and membrane potential (FLIPR) assay. A dose-response relationship was observed between the amount of vector used and subsequent functional outcomes; small amounts of HD-CFTR were sufficient to correct CFTR function. At higher doses, HD-CFTR did not increase CFTR function in healthy control cells above baseline values. This latter observation allowed us to use this vector to benchmark in vitro efficacy testing of CFTR-modulator drugs.In summary, we demonstrate the potential for HD-CFTR to inform in vitro testing and to restore CFTR function to healthy control levels in airway cells with class I or CFTR nonsense mutations.

In Vitro Validation of a CRISPR-Mediated CFTR Correction Strategy for Preclinical Translation in Pigs.

Early efforts in cystic fibrosis (CF) gene therapy faced major challenges in delivery efficiency and sustained therapeutic gene expression. Recent advancements in engineered site-specific endonucleases such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 make permanent CF transmembrane conductance regulator (CFTR) gene correction possible. However, because of safety concerns of the CRISPR/Cas9 system and challenges in in vivo delivery to inflamed CF airway, CRISPR-based gene correction strategies need to be tested in proper animal models. In this study, we aimed at creating vectors for testing CFTR gene correction in pig models. We constructed helper-dependent adenoviral (HD-Ad) vectors to deliver CRISPR/Cas9 and a donor template (a 6 kb LacZ or 8.7 kb human CFTR expression cassette) into cultured pig cells. We demonstrated precise integration of each donor into the GGTA1 safe harbor through Cas9-induced homology directed repair with 3 kb homology arms. In addition, we showed that both LacZ and hCFTR were persistently expressed in transduced cells. Furthermore, we created a CFTR-deficient cell line for testing CFTR correction. We detected hCFTR mRNA and protein expression in cells transduced with the hCFTR vector. We also demonstrated CFTR function in the CF cells transduced with the HD-Ad delivering the CRISPR-Cas9 system and hCFTR donor at late cellular passages using the membrane potential sensitive dye-based assay (FLIPR®). Combined with our previous report on gene delivery to pig airway basal cells, these data provide the feasibility of testing CRISPR/Cas9-mediated permanent human CFTR correction through HD-Ad vector delivery in pigs.