Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene6: in vitro and in vivo studies.
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, FuGene6, 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 FuGene6. Conditions for efficient gene delivery were initially studied in vitro. Human and rabbit fibroblasts were isolated from skin, cultured and transfected with phVEGF165 or pCMVbeta gal plasmids, coding for vascular endothelial growth factor (VEGF) or beta-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 FuGene6 provided high-efficiency and dose-dependent plasmid transfer both for cell-localised (beta-galactosidase) and secreted (VEGF) gene products. When analysed with an MTT assay, FuGene6 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 FuGene6 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.
Hellgren
I
I
Department of Cardiology, The Clinical Research Center, Huddinge, Stockholm, Sweden. irina.hellgren@medhs.ki.se
Drvota
V
V
Pieper
R
R
Enoksson
S
S
Blomberg
P
P
Islam
K B
KB
Sylvén
C
C
D000328:Adult; D000818:Animals; D002470:Cell Survival; D017690:Cell Transplantation / Q000379:methods; D002478:Cells, Cultured; D016228:Endothelial Growth Factors / Q000032:analysis / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology / Q000637:transplantation; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D008055:Lipids; D008222:Lymphokines / Q000032:analysis / Q000235:genetics*; D008875:Middle Aged; D018482:Muscle, Skeletal / Q000166:cytology / Q000473:pathology; D010957:Plasmids; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012867:Skin / Q000166:cytology*; D014162:Transfection / Q000379:methods*; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D016228:Endothelial Growth Factors; C411955:FuGene; D008055:Lipids; D008222:Lymphokines; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D001616:beta-Galactosidase