Development of a heat induced gene expression system in self inactivating (SIN) oncoretro- and lentiviral vectors.

F. NOYAN; D. GRIESE; CHRISTOPH KLEIN; RICARDO A. DEWEY

Edimburgh, UK

Congreso; 11th Annual Meeting of the European Society of Gene Therapy; 2003

European Society of Gene Therapy

Background: One of the challenges for the development of safe and efficient gene therapy strategies is to express therapeutic genes in an spatially and temporally regulated manner. The human heat shock 70b promoters is strong and can be activated by physical stimuli such as hyperthermia and by other stresses such as hypoxia. The potential of this promoter to be used to regulate gene expression in retroviral vectors has not been yet carefully studied. Objective: To evaluate the use of the human heat shock protein 70b promoter to regulate transgene expression in self inactivating retroviruses. Methods: The human HSP70b promoter was PCR amplified and cloned upstream the EGFP gene either in the pMMP ECT3- (SIN-Oncoretrovirus) or in the pHR’SIN-18 (SIN-Lentivirus). In all cases, the effect of the presence or absence of WPRE was also studied. A549 lung adenocarcinoma cells and GL261 mouse glioma cells were transduced with VSV-G pseudotyped SIN-vectors and two days later, cells were incubated at 43°C (heated samples) for 90 minutes in a CO2 incubator or kept at 37°C (control samples). The percentage of cells expressing EGFP was measured by FACS after heat induction. Results: In cells incubated at 43°C, the HSP70b promoter in SIN-Oncoretrovirus without WPRE increases the EGFP expression to a maximum of 17 times above basal levels observed in control samples at 37°C. This levels of inducibility could not be improved by the addition of WPRE. With respect to lentivectors, a moderate induction of gene expression was only seen in the presence of WPRE. Conclusions: In our system, the HSP70b promoter shows higher levels of heat inducible gene expression and lower levels of basal expression in the pMMP ECT3- ÄWPRE backbone compared to the pMMP ECT3-WPRE or the pHR’SIN-18 vector with and without WPRE. The development of novel self inactivating vectors with hyperthermia controlled gene expression may open new avenues to enhance control and safety in the delivery of therapeutic genes.