142 Development of an S/MAR based episomal vector for the CFTR gene delivery

Abstract

Objectives The recent use of mutation-specific correctors in Cystic Fibrosis, reinforces the concept of gene therapy as a therapeutic treatment for all CF patients. Non-viral vectors are preferable to viral ones, particularly in treatment of chronic diseases, for their simpler composition, lack of immunogenicity and genotoxicity. Episomal vectors based on the S/MAR (scaffold/matrix attachment region) sequence have self-replicating capability and episomal persistence in different cell types, both in vitro and in vivo. To obtain long-term and episomal maintenance of the CFTR gene in target cells, we constructed a vector containing the S/MAR, GFP and the CFTR cDNA (pBQ-S/MAR). Methods pBQ-S/MAR was transfected in CF(DF508/DF508) airway epithelial cells and the cells were grown in non-selective conditions up to 14 days after transfection. The cells were evaluated for the episome maintenance and persistence as well as for CFTR gene expression (RT-PCR, immunofluorescence, western blot). Results The resulting data demonstrate that pBQ-S/MAR is maintained as an episome, with unchanged structure respect to the input vector, for at least 14 days after transfection, corresponding to about six cell duplications. CFTR expression analyses confirmed the presence of the wt transcript and the mature form of the protein. Additionally, the expression of a functional CFTR in pBQ-S/MAR-transfected cells was associated with an increase of Trans Epithelial Resistance respect to parental cells. Conclusion Our results suggest that the pBQ-S/MAR is stably maintained as an episome in airway epithelial cells and support expression of a functional CFTR.