Restoring DF508-CFTR trafficking and activity with epigenetic modifiers

Abnormal retention of the CFTR ΔF508 mutated protein in lung epithelial cells underlies the pathology in a large proportion of individuals with cystic fibrosis (CF). A drug discovery alliance between CFFT and Galapagos was initiated with the aim to identify novel genes which upon shRNAmediated knockdown were able to efficiently restore ΔF508 CFTR activity. The final goal of this program is to prioritize these targets for entry into drug discovery. BioFocus DPI’s proprietary adenoviral shRNA libraries totaling 11,334 viruses against the human druggable genome were screened in a highthroughput functional assay in a human cystic fibrosis bronchial epithelial 2009 Cystic Fibrosis Conference 216 cell line (CFBE41o-). A total of 354 hits were identified and confirmed in the primary assay. Validation of the hits included a lack of cytotoxicity of the shRNA, expression of target in airway epithelial cells, identification of a second shRNA against the same target, and cell surface expression by biotinylation of CFTR ΔF508 upon shRNA-mediated knock-down of the target. Most importantly, these shRNAs restored functional activity of the mutant channel in primary bronchial epithelial cells from ΔF508/ΔF508 CF patients in transepithelial CFTR mediated current assays (Table 1). There was a very strong correlation between fully glycosylated band C expression in the CFBE41o- cells and functional activity in the primary cells of CF patients. We will present an overview of the target discovery and validation program, resulting in a portfolio of 19 novel drug targets to treat CF. The identity of these novel targets also sheds light on the biology of trafficking of CFTR. For example, these data point to a role of TGF-beta signaling and inflammatory mediators in airways in the regulation of CFTR trafficking. Acknowledgments: We thank Drs. Bill Guggino, Ineke Braakman, Kevin Foskett, John Hanrahan and Hugo De Jonge for helpful discussions. We acknowledge the support of Cystic Fibrosis Foundation Therapeutics.