Heat shock activates transgene CFTR Cl channels

Abstract

Loss of transgene expression is a common problem in gene therapy and has limited the effective correction of the cystic fibrosis‐linked CFTR anion channel. Transgene CFTR expression was measured in response to heat shock (BHT, 41–43°C for 60 min) or exposure to small molecule inhibitors of DNA methyltransferases (DNMTi) using CF bronchial epithelial cells (CFBE41o‐) stably expressing wild‐type CFTR under the regulation of the cytomegalovirus (CMV) immediate‐early gene promoter. After 6 hours, BHT induced a significant increase in CFTR mRNA (2.4‐fold), CFTR‐positive cells (5‐fold), and CFTR Cl currents (ICl, 3.6‐fold). Although CFTR mRNA returned to baseline after 24 h, CFTR‐positive cells and ICl remained elevated (3.9 and 2.5‐fold, respectively). Treatment with actinomycin D blunted BHT response suggesting effects on CFTR transcription. 5‐Aza‐dC, a DNMTi, increased CFTR‐positive cells and time‐dependently activated ICl similar to that observed by BHT. A structurally unrelated DNMTi, RG‐108, mimicked effects, whereas native ICl was not affected, suggesting specific regulatory effects on vector‐induced CFTR expression. These studies imply that methylation of DNA CpG sites (likely in the CMV promoter) is a key and reversible factor modulating transgene CFTR expression.