caa a22 4500 475845633 CHVBK 20180406123913.0 cr unu---uuuuu 170329e20000801xx s 000 0 eng 10.1007/PL00000769 doi (NATIONALLICENCE)springer-10.1007/PL00000769 Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene™6: in vitro and in vivo studies [Elektronische Daten] [I. Hellgren*, V. Drvota, R. Pieper, S. Enoksson, P. Blomberg, K. B. Islam, C. Sylvén] Abstract.: The present study was undertaken to develop an efficient non-viral gene delivery system for cardiovascular gene therapy. We investigated transfection efficiency and toxic properties of the new transfection reagent, FuGene™6, and compared it with two other transfection reagents, Tfx™-50 and LipoTaxi™. For in vivo experiments, the plasmid was delivered intramuscularly via transplantation of fibroblasts transfected with plasmid and FuGene™6. Conditions for efficient gene delivery were initially studied in vitro. Human and rabbit fibroblasts were isolated from skin, cultured and transfected with phVEGF165 or pCMVβgal plasmids, coding for vascular endothelial growth factor (VEGF) or β-galactosidase, respectively. The effect of the DNA amount and the DNA:transfection reagent ratio on plasmid uptake were studied. Of the transfection reagents tested, only FuGene™6 provided high-efficiency and dose-dependent plasmid transfer both for cell-localised (β-galactosidase) and secreted (VEGF) gene products. When analysed with an MTT assay, FuGene™6 showed no toxicity at low doses. Optimised conditions were applied for in vivo reporter gene delivery. Rabbits were injected intramuscularly with ex vivo-transfected fibroblasts. As in in vitro studies, ex vivo-transfected fibroblasts showed highly efficient gene expression in vivo. Tissue sections were analysed with macrophage-specific immunostaining. No signs of inflammation were seen in the region of fibroblast injection. This study demonstrates that FuGene™6 is a highly efficient transfection reagent that may be useful for in vitro non-viral transfection of primary human and rabbit fibroblasts and for in vivo therapeutic non-viral gene delivery. Birkhäuser Verlag Basel,, 2000 Key words. Transfection nationallicence VEGF nationallicence FuGene™6 nationallicence fibroblasts nationallicence gene therapy nationallicence Hellgren* I. Department of Cardiology, The Clinical Research Center, 5th floor, Novum, 141 86 Huddinge, Stockholm (Sweden), Fax +46 8 58583810, e-mail: irina.hellgren@medhs.ki.se, SE aut Drvota V. Department of Cardiology, The Clinical Research Center, 5th floor, Novum, 141 86 Huddinge, Stockholm (Sweden), Fax +46 8 58583810, e-mail: irina.hellgren@medhs.ki.se, SE aut Pieper R. Department of Vascular Surgery, Huddinge University Hospital, Stockholm (Sweden), SE aut Enoksson S. Department of Vascular Surgery, Huddinge University Hospital, Stockholm (Sweden), SE aut Blomberg P. Gene Therapy Centre, Huddinge University Hospital, Stockholm (Sweden), SE aut Islam K. B. Gene Therapy Centre, Huddinge University Hospital, Stockholm (Sweden), SE aut Sylvén C. Department of Cardiology, The Clinical Research Center, 5th floor, Novum, 141 86 Huddinge, Stockholm (Sweden), Fax +46 8 58583810, e-mail: irina.hellgren@medhs.ki.se, SE aut https://doi.org/10.1007/PL00000769 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/PL00000769 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Hellgren* I. Department of Cardiology, The Clinical Research Center, 5th floor, Novum, 141 86 Huddinge, Stockholm (Sweden), Fax +46 8 58583810, e-mail: irina.hellgren@medhs.ki.se, SE aut NATIONALLICENCE 700 1- Drvota V. Department of Cardiology, The Clinical Research Center, 5th floor, Novum, 141 86 Huddinge, Stockholm (Sweden), Fax +46 8 58583810, e-mail: irina.hellgren@medhs.ki.se, SE aut NATIONALLICENCE 700 1- Pieper R. Department of Vascular Surgery, Huddinge University Hospital, Stockholm (Sweden), SE aut NATIONALLICENCE 700 1- Enoksson S. Department of Vascular Surgery, Huddinge University Hospital, Stockholm (Sweden), SE aut NATIONALLICENCE 700 1- Blomberg P. Gene Therapy Centre, Huddinge University Hospital, Stockholm (Sweden), SE aut NATIONALLICENCE 700 1- Islam K. B. Gene Therapy Centre, Huddinge University Hospital, Stockholm (Sweden), SE aut NATIONALLICENCE 700 1- Sylvén C. Department of Cardiology, The Clinical Research Center, 5th floor, Novum, 141 86 Huddinge, Stockholm (Sweden), Fax +46 8 58583810, e-mail: irina.hellgren@medhs.ki.se, SE aut Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer