A dual function fusion protein of Herpes simplex virus type 1 thymidine kinase and firefly luciferase for noninvasive in vivoimaging of gene therapy in malignant gliomaReport as inadecuate




A dual function fusion protein of Herpes simplex virus type 1 thymidine kinase and firefly luciferase for noninvasive in vivoimaging of gene therapy in malignant glioma - Download this document for free, or read online. Document in PDF available to download.

Genetic Vaccines and Therapy

, 2:7

First Online: 04 August 2004Received: 05 April 2004Accepted: 04 August 2004

Abstract

BackgroundSuicide gene therapy employing the prodrug activating system Herpes simplex virus type 1 thymidine kinase HSV-TK- ganciclovir GCV has proven to be effective in killing experimental brain tumors. In contrast, glioma patients treated with HSV-TK- GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive in vivo monitoring of the activity of a therapeutic gene in brain tumor cells.

MethodsThe HSV-TK gene was fused to the firefly luciferase Luc gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper.

ResultsTransient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK- GCV mediated cell kill in vivo.

ConclusionThis approach may represent a valuable tool for the in vivo evaluation of gene therapy strategies for treatment of malignant disease.

Keywordsglioma bioluminescence imaging gene therapy herpes simplex virus type 1 thymidine kinase luciferase List of abbreviationsCCDcharged coupled device

CMVcytomegalovirus

cpscounts per second

EGFPenhanced green fluorescent protein

GCVganciclovir

HSV-TKherpes simplex virus type 1 thymidine kinase

Lucluciferase

mRFPmonomeric red fluorescent protein, PET, positron emission tomography

ROIregion of interest

SDstandard deviation, SEM, standard error of the mean.

Electronic supplementary materialThe online version of this article doi:10.1186-1479-0556-2-7 contains supplementary material, which is available to authorized users.

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Author: Ariane Söling - Christian Theiß - Stephanie Jungmichel - Nikolai G Rainov

Source: https://link.springer.com/



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