Assembly and Functional Analysis of an S/MAR Based Episome with the Cystic Fibrosis Transmembrane Conductance Regulator Gene.

Davide De Rocco,Maria Mariggiò,Luca Cavinato,Stefano Castellani,Simone Guarnieri,Paola Del Porto,Giuseppe Cimino,Barbara Pompili,Fiorentina Ascenzioni,Elena Morini,Massimo Conese

doi:10.3390/ijms19041220

Abstract

Improving the efficacy of gene therapy vectors is still an important goal toward the development of safe and efficient gene therapy treatments. S/MAR (scaffold/matrix attached region)-based vectors are maintained extra-chromosomally in numerous cell types, which is similar to viral-based vectors. Additionally, when established as an episome, they show a very high mitotic stability. In the present study we tested the idea that addition of an S/MAR element to a CFTR (cystic fibrosis transmembrane conductance regulator) expression vector, may allow the establishment of a CFTR episome in bronchial epithelial cells. Starting from the observation that the S/MAR vector pEPI-EGFP (enhanced green fluorescence protein) is maintained as an episome in human bronchial epithelial cells, we assembled the CFTR vector pBQ-S/MAR. This vector, transfected in bronchial epithelial cells with mutated CFTR, supported long term wt CFTR expression and activity, which in turn positively impacted on the assembly of tight junctions in polarized epithelial cells. Additionally, the recovery of intact pBQ-S/MAR, but not the parental vector lacking the S/MAR element, from transfected cells after extensive proliferation, strongly suggested that pBQ-S/MAR was established as an episome. These results add a new element, the S/MAR, that can be considered to improve the persistence and safety of gene therapy vectors for cystic fibrosis pulmonary disease.

Highlights

Since the identification of cystic fibrosis (CF) disease in 1938 and the discovery of the causative CFTR (CF transmembrane conductance regulator) gene in 1989, life expectancy of CF patients has increased progressively to more than 40 years in developed country
The meticulous work done to date, mainly by the UK CF gene therapy consortium, provided plasmid-based vectors with optimized expression cassettes both in the promoters and in the codon usage of the CFTR gene [15]
In the context of vector development for CF gene therapy, the present work originates from the idea that S/MAR elements may improve vector persistence and CFTR expression, while reducing possible genotoxic effects due to insertion in the host genome

Summary

Introduction

Since the identification of cystic fibrosis (CF) disease in 1938 and the discovery of the causative CFTR (CF transmembrane conductance regulator) gene in 1989, life expectancy of CF patients has increased progressively to more than 40 years in developed country. The combinatory therapy ivacaftor/lumacaftor was shown to induce a modest improvement of lung function in homozygous Phe508del patients [5]. This may be due to the destabilizing effect of VX-770 on CFTR, which has been observed either in non-CF and CF epithelial cells [6,7]. It appears that the development of efficient CFTR-repairing molecules is still a big task with very limited success, so far

Methods

Results

Conclusion