Coexpression of herpesviral thymidine kinase reporter gene and VEGF gene for noninvasive monitoring of therapeutic gene transfer: an in vitro evaluation.
Coexpression of a reporter gene and a therapeutic gene may allow for noninvasive monitoring of cardiac gene therapy. We sought to evaluate the usefulness of an adenoviral vector expressing mutant herpesviral thymidine kinase reporter gene (HSV1-sr39tk) and vascular endothelial growth factor (VEGF) 121 in independent expression cassettes (Ad4tk).


METHODS
Accumulation of 14C-2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (FIAU) and 9-(4-18F-fluoro-3-hydroxymethylbutyl)guanine (FHBG) as reporter probes, and secretion of VEGF into medium, were determined for Ad4tk-infected H9c2 rat cardiac cells in vitro.


RESULTS
In vitro tracer uptake increased with increasing vector concentration and over time. It was comparable to cells infected with adenovirus expressing only wild-type HSV1-tk (reporter probe: 14C-FIAU) or mutant HSV1-sr39tk (reporter probe: 18F-FHBG). No significant uptake was observed in cells infected with adenovirus expressing VEGF alone. With increasing vector concentration, Ad4tk-infected cells increasingly released VEGF into medium. VEGF production correlated significantly with cellular reporter probe uptake (r = 0.93; P = 0.0003).


CONCLUSIONS
The usefulness of a vector coexpressing HSV1-tk and VEGF for noninvasive imaging of expression of a therapeutic transgene has been demonstrated in vitro. This approach may allow for future in vivo monitoring of cardiac angiogenesis gene therapy.
Anton
Martina
M
Institut für Experimentelle Onkologie und Therapieforschung, Technische Universität München, Munich, Germany.
Wittermann
Constanze
C
Haubner
Roland
R
Simoes
Marcus
M
Reder
Sybille
S
Essien
Bryan
B
Wagner
Bettina
B
Henke
Julia
J
Erhardt
Wolf
W
Noll
Steffi
S
Hackett
Neil R
NR
Crystal
Ronald G
RG
Schwaiger
Markus
M
Gansbacher
Bernd
B
Bengel
Frank M
FM
D000818:Animals; D002460:Cell Line; D005240:Feasibility Studies; D020869:Gene Expression Profiling / Q000379:methods; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D032342:Muscle Cells / Q000000981:diagnostic imaging* / Q000187:drug effects / Q000378:metabolism*; D011877:Radionuclide Imaging; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000493:pharmacokinetics; D013937:Thymidine Kinase / Q000008:administration & dosage / Q000493:pharmacokinetics*; D042461:Vascular Endothelial Growth Factor A / Q000008:administration & dosage / Q000493:pharmacokinetics*; D014764:Viral Proteins / Q000008:administration & dosage / Q000493:pharmacokinetics*
D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D014764:Viral Proteins; C099241:thymidine kinase, Canid herpesvirus 1; D013937:Thymidine Kinase