VP22-mediated intercellular transport for suicide gene therapy under oxic and hypoxic conditions

Abstract

During herpes simplex virus type 1 (HSV 1) infection, the tegument protein VP22 is exported from infected cells to the nuclei of surrounding uninfected cells. These intercellular transport characteristics have prompted the exploitation of VP22 fusion proteins for cancer gene therapy, with the goal of maximizing the bystander effect. Since solid tumors contain hypoxic cell populations that are often refractive to therapy, for efficient targeting, it would be optimal if VP22 functioned even at reduced oxygen concentrations. In the present work, VP22 activity under hypoxic conditions was examined for the first time. Plasmid-transfected human glioma U87-MG and U373-MG cells expressing VP22 fused to the green fluorescent protein (GFP) showed protein export to untransfected cells under tumor oxygenation conditions (0-5% O 2 ). For suicide gene therapy, VP22 activity was demonstrated under hypoxia by coupling VP22 to the HSV thymidine kinase (HSVtk). In the presence of the prodrug ganciclovir, cell cultures expressing VP22-HSVtk showed a significant increase in toxicity compared with cells transfected with a construct containing HSVtk only, under all tested conditions. To allow effective suicide gene therapy and simultaneous visualization of therapeutic enzyme localization, a triple fusion protein GFP-HSVtk-VP22 was engineered. Functionality of all components was demonstrated under oxia and hypoxia. Gene Therapy (2005) 12, 974-979.

Viral-based gene delivery and regulated gene expression for targeted cancer therapy

“…Another approach used hypoxia-and radiation-activated Cre/loxP 'molecular switch' to turn on the otherwise muted HSV-TK gene for specific targeting of breast cancer and glioma cells under hypoxic conditions and/or radiation therapy [68]. Therefore, by combining tissue/tumor-specific promoters and GDEPT strategies as described above, the transcriptionally regulated, cancer-targeted gene therapy should provide an effective and specific therapy against cancer.…”

Radiation to control transgene expression in tumors

“…38 The E9 and E9ns2 constructs were also found to be responsive to clinically relevant neutron doses (1.3 Gy) to a similar extend than that obtained after x-ray doses of 2 Gy, or even 4 Gy. 39 These findings are of great interest as neutrons have a higher relative biological effectiveness than photons, making them more effective at cell killing. Neutrons also present the advantage of a reduced oxygen effect, making them more effective at treating hypoxic tumors.…”

“…This was higher than that obtained with a 2 Gy x-rays dose (48%) and similar to that obtained with the strong constitutive CMV-IE promoter. 39 These promoters were also activated by exposure to the drugs cisplatin (5 and 50 mM) and doxorubicin (0.1 and 1 mM). After drug incubation, transfected MCF-7 cells were efficiently and selectively sensitised to GCV treatment.…”

“…Addition of gene therapy resulted in an increase in growth inhibition of around 15% when compared with radiation alone. 39 When this expression cassette was developed into the Cre/loxP recombination system, GCV-mediated toxicity became higher and more selective. 49 Retroviral delivery of the Epo/E9 promoter linked to the mammary-specific minimal promoter of the murine whey acidic protein (WAP) encoding gene resulted is tissue-selective induction in response to both radiation and hypoxia.…”

“…Growth inhibition of 65% and 67% were reported for each E9HSVtk and E9ns2HSVtk transfected cells, respectively, making the technique as efficient as that obtained with radiation. 39 Cre/Lox molecular switch. The Cre/Lox molecular switch system has been tested in a gene therapy setting, using the HSVtk suicide gene.…”