Coexpression of Herpesviral Thymidine Kinase Reporter Gene for Nonivasive Monitoring of Therapeutic Gene Transfer - In Vivo- and Initial In Vivo-Evaluation

Background: Coexpression of a reporter gene and therapeutic gene may allow for non-invasive 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 VEGF₁₂₁ in independent expression cassettes(Ad4tk).
Methods and Results: Accumulation of [¹⁴C]FIAU and [¹⁸F] FHBG as reporter probes, and VEGF secretion into medium were determined for Ad4tk-infected H9c2 rat cardiac cells in vitro. Tracer uptake increased with increasing vector concentration and over time, and 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 uninfected cells or 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 uptake (r=.98). In an initial in vivo experiment, Ad4tk and control vector were injected directly into two separate myocardial areas of a healthy pig following thoracotomy. Two days later, PET imaging was performed using [¹²⁴I]FIAU, allowing for specific visualization of the area of Ad4tk injection. Identification of regional reporter gene expression was accompanied by significant increase of plasma VEGF concentration.
Conclusion: The usefulness of a vector coexpressing HSV1-tk and VEGF for noninvasive imaging of the expression of a therapeutic transgene has been demonstrated for the first time. This approach may allow for in vivo monitoring of cardiac angiogenesis gene therapy in the future.
Condensed Abstract: Usefulness of an adenoviral vector(Ad4tk) expressing mutant herpesviral thymidine kinase reporter gene and VEGF₁₂₁ was evaluated. Accumulation of radiolabelled reporter probes increased for Ad4tk-infected rat cardiac cells with increading vector concentration and over time, and was comparabel to cells expressing reporter gene alone. No significant uptake was observed in cells expressing VEGF alone. VEGF production after Ad4tk-infecion correlated significantly with reporter probe uptake in vitro. In vivo, PET yielded specific visualization of Ad4tk injection area in a pig. This approach may allow for noninvasive monitoring of cardiac angiogenesis gene therapy.