Successful genetic transduction in vivo into synovium by means of electroporation.
This present study aims at establishing a novel in vivo gene delivery system for intra-articular tissues. Plasmid DNA (pDNA) carrying the firefly luciferase or enhanced green fluorescent protein (EGFP) genes as markers was injected into a joint space and electric stimuli were given percutaneously with a pair of electrodes. Injection with naked pDNA alone did not induce any detectable level of luciferase activity, whereas electroporation at 25-500 V/0.7 cm resulted in a significant expression of the marker gene in the synovium. The expression level depended on the voltage, the optimum transfection being achieved at 150 V/0.7 cm. When the Epstein-Barr virus (EBV)-based plasmid vectors harboring the EBV nuclear antigen 1 (EBNA1) gene and oriP sequence were substituted for conventional pDNA, the transfection efficiency was increased approximately 5-10 times. Histological examination of the EGFP gene-transfected joints revealed that the marker gene was expressed in the synovial membrane while other intra-articular tissues such as articular cartilage were negative for the transgene product. Transgene-specific mRNA was demonstrated in synovium but not in other organs as estimated by RT-PCR analysis. The present results strongly suggest that in vivo electroporation is a quite simple, safe, and effective gene delivery method that could be applicable to gene therapy against articular diseases.
Ohashi
Suzuyo
S
Department of Orthopaedic Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Kubo
Toshikazu
T
Kishida
Tsunao
T
Ikeda
Takumi
T
Takahashi
Kenji
K
Arai
Yuji
Y
Terauchi
Ryu
R
Asada
Hidetsugu
H
Imanishi
Jiro
J
Mazda
Osam
O
D000818:Animals; D002412:Cations / Q000378:metabolism; D019902:Chondrocytes / Q000378:metabolism; D018274:Electroporation; D004926:Escherichia coli / Q000378:metabolism; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D050356:Lipid Metabolism; D008156:Luciferases / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D010957:Plasmids / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000378:metabolism*; D013997:Time Factors; D014161:Transduction, Genetic; D014162:Transfection
D002412:Cations; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases