ADENOVIRUS-ASSISTED LIPOFECTION - EFFICIENT IN-VITRO GENE-TRANSFER OFLUCIFERASE AND CYTOSINE DEAMINASE TO HUMAN SMOOTH-MUSCLE CELLS

Smooth muscle cells (SMC) are a central cell type involved in multiple processes of coronary artery diseases including restenosis and theref ore are major target cells for different aspects of gene transfer. Pre vious attempts to transfect primary arterial cells using different tec hniques like liposomes, CaPO, and electroporation resulted in only low transfection efficiency. The development of recombinant adenoviruses dramatically improved the delivery of foreign genes into different cel l types including SMC. However, cloning and identification of recombin ants remain difficult and time-consuming techniques. The present study demonstrates that a complex consisting of. reporter plasmid encoding firefly luciferase (pLUC), polycationic liposomes and replication-defi cient adenovirus was able to yield very high in vitro transfection of primary human smooth muscle cells under optimized conditions. The tech nique of adenovirus-assisted lipofection (AAL) increases transfer and expression of plasmid DNA in human smooth muscle cells in vitro up to 1000-fold compared to lipofection. To verify the applicability of AAL for gene transfer into human smooth muscle cells we studied a gene the rapy approach to suppress proliferation of SMC in vitro, using the pro karyotic cytosine deaminase gene (CD) which enables transfected mammal ian cells to deaminate 5-fluorocytosine (5-FC) to the highly toxic 5-f luorouracil (5-FU). The effect of a transient CD expression on RNA syn thesis was investigated by means of a cotransfection with a RSV-CD exp ression plasmid and the luciferase reporter plasmid. Western blot anal ysis demonstrated high expression of CD protein in transfected SMC. Co transfected SMC demonstrated two-fold less luciferase activity in the presence of 5-FC (5 mmol/l) after 48 h compared to cells transfected w ith a non-CD coding plasmid. The data demonstrate that a transient exp ression of CD could be sufficient to reduce the capacity of protein sy nthesis in human SMC. This simple and effective in vitro transfection method may also be applicable to in vivo delivery of target genes to t he vascular wall to inhibit SMC proliferation.