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

International Journal of Molecular Sciences

Barbara Pompili¹  Paola Del Porto¹  Fiorentina Ascenzioni¹  Davide De Rocco¹  Elena Morini¹  Luca Cavinato¹  Giuseppe Cimino²  Massimo Conese³  Stefano Castellani³  MariaA Mariggiò⁴  Simone Guarnieri⁴ 

[1] Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, 00185 Rome, Italy;Department of Pediatrics and Infant Neuropsychiatry, Centro di Riferimento Fibrosi Cistica Regione Lazio, Sapienza University, 00165 Rome, Italy;Laboratory of Experimental and Regenerative Medicine, Department of Medical and Surgical Sciences, University of Foggia, 71122 Foggia, Italy;Laboratory of Functional Biotechnology, Center of Sciences on Aging and Translational Medicine (CeSI-MeT), Department of Neuroscience, Imaging and Clinical Sciences, University G. d’Annunzio of Chieti-Pescara, 66013 Chieti, Italy;

关键词: episome;    S/MAR;    cystic fibrosis;    CFTR;    gene therapy;   

DOI  :  10.3390/ijms19041220

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown