A novel enzyme/prodrug system for hypoxia- and radiation-regulated suicide gene therapy

We have previously proposed the plant enzyme horseradish peroxidase

( HRP ) and the nontoxic plant hormone indole - 3 - acetic acid ( IAA ) as a

novel enzyme/ prodrug combination for suicide gene therapy ( Greco et al.

Cancer Gene Ther. 7: 1414, 2000 ). Compared with other widely used

systems ( e.g. HSV TK/ GCV ), HRP/ IAA has the advantage of inducing

tumour cell killing and a strong bystander effect not only in normoxia, but

also in the tumour conditions of anoxia and hypoxia ( 0.1% O2 ). Here we

demonstrate that HRP -mediated gene therapy can selectively enhance the

tumoricidal action of ionising radiation. Incubation with IAA prior to or postX - rays induced an increase in sensitivity of HRP - expressing cells of 2.5 –

2.6 ( 0.1 mM IAA ) and 5.4 – 5.6 ( 0.5 mM IAA ). Delivery of the HRP gene

itself or exposure of HRP ?cells to IAA did not affect the response to

radiation. To specifically target the radio - and chemoresistant population in

solid tumours, the HRP gene was placed under the control of hypoxia - or

radiation - responsive promoters. Five copies of hypoxia - regulatory elements

( HREs ) from the PGK - 1 or the EPO genes were inserted upstream of

the basal cytomegalovirus promoter. After 24 - h hypoxic incubation, the

EPO and the PGK - 1 HREs induced a 30 – 40 - fold and a 5 – 6 - fold increase

in HRP expression respectively. Transfection with the EPO - HRP construct

conferred hypoxia - selective sensitivity to IAA and analogues. HRP

production in irradiated cells was achieved by using radiation - responsive

CArG elements (Marples et al. Gene Therapy. 7: 511, 2000 ). After 5 Gy X -

irradiation, a selective 2 – 3 - fold increase in transgene expression was

detected.