title: Adenovirus-assisted lipofection: efficient in vitro gene transfer of luciferase and cytosine deaminase to human smooth muscle cells.
abstract: Smooth muscle cells (SMC) are a central cell type involved in multiple processes of coronary artery diseases including restenosis and therefore are major target cells for different aspects of gene transfer. Previous attempts to transfect primary arterial cells using different techniques like liposomes, CaPO4 and electroporation resulted in only low transfection efficiency. The development of recombinant adenoviruses dramatically improved the delivery of foreign genes into different cell types including SMC. However, cloning and identification of recombinants 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-deficient adenovirus was able to yield very high in vitro transfection of primary human smooth muscle cells under optimized conditions. The technique 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 therapy approach to suppress proliferation of SMC in vitro, using the prokaryotic cytosine deaminase gene (CD) which enables transfected mammalian cells to deaminate 5-fluorocytosine (5-FC) to the highly toxic 5-fluorouracil (5-FU). The effect of a transient CD expression on RNA synthesis was investigated by means of a cotransfection with a RSV-CD expression plasmid and the luciferase reporter plasmid. Western blot analysis demonstrated high expression of CD protein in transfected SMC. Cotransfected SMC demonstrated two-fold less luciferase activity in the presence of 5-FC (5 mmol/l) after 48 h compared to cells transfected with a non-CD coding plasmid. The data demonstrate that a transient expression of CD could be sufficient to reduce the capacity of protein synthesis in human SMC. This simple and effective in vitro transfection method may also be applicable to in vivo delivery of target genes to the vascular wall to inhibit SMC proliferation.
authors.affiliation: Innere Medizin III, Universität Heidelberg, Germany. JOERGKREUZER@KRZMAIL.KRZ.UNI-HEIDELBERG.DE
authors.forename: J
authors.initials: J
authors.lastname: Kreuzer
authors.forename: S
authors.initials: S
authors.lastname: Denger
authors.forename: F
authors.initials: F
authors.lastname: Reifers
authors.forename: C
authors.initials: C
authors.lastname: Beisel
authors.forename: K
authors.initials: K
authors.lastname: Haack
authors.forename: J
authors.initials: J
authors.lastname: Gebert
authors.forename: W
authors.initials: W
authors.lastname: Kübler
mesh_terms: D000260:Adenoviruses, Human / Q000502:physiology*; D001011:Aorta; D002130:Calcium Phosphates / Q000494:pharmacology; D002411:Cation Exchange Resins / Q000008:administration & dosage*; D002478:Cells, Cultured; D043525:Cytosine Deaminase; D004274:DNA, Recombinant / Q000008:administration & dosage*; D003673:Defective Viruses / Q000502:physiology*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000235:genetics; D006801:Humans; D008055:Lipids / Q000008:administration & dosage*; D008081:Liposomes; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D009700:Nucleoside Deaminases / Q000096:biosynthesis / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection
chemical_list: D002130:Calcium Phosphates; D002411:Cation Exchange Resins; D004274:DNA, Recombinant; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D011993:Recombinant Fusion Proteins; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D012313:RNA; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D008156:Luciferases; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; C485829:calcium phosphate, dibasic, anhydrous