Targeted Integration of a Super-Exon into the CFTR Locus Leads to Functional Correction of a Cystic Fibrosis Cell Line Model.

In vitro disease models have enabled insights into the pathophysiology of human disease as well as the functional evaluation of new therapies, such as novel genome engineering strategies. In the context of cystic fibrosis (CF), various cellular disease models have been established in recent years, including organoids based on induced pluripotent stem cell technology that allowed for functional readouts of CFTR activity. Yet, many of these in vitro CF models require complex and expensive culturing protocols that are difficult to implement and may not be amenable for high throughput screens. Here, we show that a simple cellular CF disease model based on the bronchial epithelial ΔF508 cell line CFBE41o- can be used to validate functional CFTR correction. We used an engineered nuclease to target the integration of a super-exon, encompassing the sequences of CFTR exons 11 to 27, into exon 11 and re-activated endogenous CFTR expression by treating CFBE41o- cells with a demethylating agent. We demonstrate that the integration of this super-exon resulted in expression of a corrected mRNA from the endogenous CFTR promoter and used short-circuit current measurements in Ussing chambers to corroborate restored ion transport of the repaired CFTR channels. In conclusion, this study proves that the targeted integration of a large super-exon in CFTR exon 11 leads to functional correction of CFTR, suggesting that this strategy can be used to functionally correct all CFTR mutations located downstream of the 5' end of exon 11.

Targeting ENaC as a Molecular Suspect in Cystic Fibrosis.

Cystic fibrosis (CF) is the most common life shortening autosomal inherited disorder, affecting 1 in 2500 newborns in the Caucasian population. In CF the lung pathology is associated with dehydration of the airways epithelial surface which in part results from Na(+) hyperabsorption via the epithelial sodium channel (ENaC). The molecular mechanisms of this Na(+) hyperabsorption and its correlation with the underlying genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) are not fully understood. However, it is obvious that a reduced Cl(-) secretion by CFTR and an enhanced Na+ absorption through ENaC lead to the so far incurable disease. Therefore, it could be indicated to pursue a double-tracked strategy in that way enabling Cl(-) secretion by a reconstitution of the defect CFTR as well as blocking ENaC to prevent Na(+) hyperabsorption. Since the cloning of CFTR great efforts have been done in delivery of CFTR for the correction of the reduced Cl(-) secretion. Positive benefits for the inhibition of the CF related Na(+) hyperabsorption offer technologies using small molecule inhibitors like ASOs or siRNA, which target translation and knockdown of ENaC, respectively. In this review we discuss possible CFTR/ENaC interactions in the context of CF, describe ENaC structure as well as some of the numerous attempts that were performed to prevent the Na(+) hyperabsorption in CF related lung disease. Thus, we give a short summary of e.g. amiloride therapy approaches and focus on inventive blocking efforts using ASOs and siRNA.

Cystic fibrosis transmembrane conductance regulator-mRNA delivery: a novel alternative for cystic fibrosis gene therapy.

BACKGROUND: Cystic fibrosis (CF) is the most frequent lethal genetic disease in the Caucasian population. CF is caused by a defective gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP- and ATP-dependent Cl(-) channel and central regulatory protein in epithelia. CFTR influences the fluid composition of the mucus in the respiratory tract. The most common mutation inducing CF, ΔF508, impairs CFTR processing within the cell and thus prevents functional CFTR expression in the apical membrane. The present study aimed to investigate the functional restoration of CFTR in human CF airway epithelia after transfection with optimized wild-type (wt)CFTR-mRNA. METHODS: We used primary cultured human nasal epithelial (HNE) cells and the human bronchial epithelial cell line CFBE41o(-) that stably expresses ΔF508-CFTR and carried out transepithelial Ussing chamber measurements after transfection with optimized wtCFTR-mRNA. We confirmed the data obtained using immunofluorescence and protein biochemical approaches. RESULTS: Transfection of the CFBE41o(-) cells with wtCFTR-mRNA restored cAMP-induced CFTR currents similar to the values seen in control cells (16HBE14o(-)). Using immunofluorescence approaches, we demonstrated that a considerable amount of CFTR is located at the apical surface in the CF cells after transfection. Western blot analyses of wtCFTR-mRNA transfected CFBE41o(-) cells confirmed these findings. Furthermore, we demonstrated physiological relevance by using primary cultured HNE cells and showed an almost two-fold increase in the cAMP-stimulated CFTR current after transfection. CONCLUSIONS: From these data, we conclude that CFTR-mRNA transfection could comprise a novel alternative for gene therapy to restore impaired CFTR function.