Modulating the fibrinolytic system of peripheral blood mononuclear cells with adenovirus.
Gene therapy utilizing leukocytes is an unexplored therapeutic strategy for targeting tissue-type plasminogen activator (t-PA) to fibrin and sites of inflammation. In this study, five cationic lipids were observed to enhance the adenovirus (Ad)-mediated expression of t-PA in human peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner between 1000 and 15,000 lipid molecules per Ad particle (efficiency:LipofectAMINE > GenePORTER > Effectene > SuperFect > DMRIE-C). PBMCs treated with Ad/t-PA * LipofectAMINE complexes displayed elevated t-PA expression over a 4-day period and the t-PA-expressing cells facilitated the lysis of plasma clots in vitro. Functional and immunologic assays revealed that the Ad * LipofectAMINE infection protocol did not affect monocyte adhesion in vitro or elevate the expression of procoagulant activity, interleukin 8, or tumor necrosis factor alpha. The potential of this system was documented with an in vivo rat model system that involved the injection of lipopolysaccharide into the peritoneal cavity to induce an inflammatory response. Infusion of Ad/t-PA-infected rat PBMCs into the vasculature of lipopolysaccharide-treated animals was found to increase local fibrinolytic activity by 4-fold. These data provide a framework for utilizing adenovirus to transfer genes into PBMCs.
Department of Vascular Biology, Scripps Research Institute, La Jolla, CA 92037, USA. rschleef@scripps.edu
R R
RR
Schleef
M A
MA
Olman
L A
LA
Miles
J L
JL
Chuang
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005342:Fibrinolysis / Q000502:physiology*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D009000:Monocytes / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016209:Interleukin-8; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D010959:Tissue Plasminogen Activator