Tupper, Joanna; (2004) Use of horseradish peroxidase for gene directed enzyme prodrug therapy. Doctoral thesis (Ph.D.), University College London.

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Abstract

Gene directed enzyme prodrug therapy (GDEPT) is a form of targeted cancer therapy, where an enzyme is used to create a cytotoxin from a prodrug specifically within the tumour. The treatment of solid tumours is often hampered by the presence of hypoxia, which can limit the effectiveness of conventional therapies. Horseradish peroxidase (HRP)-directed GDEPT has been shown to target normoxic and hypoxic cells in monolayers. This study further evaluated HRP for gene therapy of cancer. The HRP/indole-3-acetic acid (HRP/IAA) combination was shown to retain its activity under the tumour-simulated conditions seen in 3-dimensional tumour cell spheroids. In particular, the halogenated derivative of IAA, 5-Br-IAA, showed specificity against larger spheroids, which contain significant regions of hypoxia. The use of alternative prodrugs was investigated using paracetamol. The HRP/paracetamol combination was effective in monolayers, even under the severe tumour-associated condition of anoxia. Following the successful in vitro investigations in the FaDu squamous carcinoma cell line, the HRP-GDEPT system was analysed in vivo. Characterisation of solid tumours grown from stable FaDu transfectants showed that the indole prodrugs had satisfactory pharmacokinetic profiles, and millimolar concentrations could be achieved in tumour tissue at non-toxic doses. The potential for the HRP/IAA system to cause delayed growth was determined using indoles, alone, and in combination with radiation. Unfortunately the HRP/IAA combination did not appear to have any reproducible growth delay effects. The immune response to HRP was assessed by generating murine carcinoma cells expressing HRP or the green fluorescent protein (GFP). However, passaging of the syngeneic cell line in vitro was sufficient to increase immunogenicity and decrease tumourigenicity even in the absence of transgene expression. Gene delivery in vivo was attempted in order to circumvent the use of stable transfectants. Electroporation could significantly increase the expression of GFP delivered intratumourally, although expression as a percentage of tumour area remained low. The data presented demonstrates the continued promise of the HRP-GDEPT system in vitro, but more work is required to improve the in vivo efficacy.

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