Preclinical evaluation of a prostate-targeted gene-directed enzyme prodrug therapy delivered by ovine atadenovirus

Abstract

Gene-directed enzyme prodrug therapy (GDEPT) based on the Escherichia coli enzyme, purine nucleoside phosphorylase (PNP), provides a novel strategy for treating slowly growing tumors like prostate cancer (CaP). PNP converts systemically administered prodrug, fludarabine phosphate, to a toxic metabolite, 2-fluoroadenine, that kills PNP-expressing and nearby cells by inhibiting DNA, RNA and protein synthesis. Reporter gene expression directed by a hybrid prostatedirected promoter and enhancer, PSMEPb, was assayed after plasmid transfection or viral transduction of prostate and nonCaP cell lines. Androgen-sensitive (AS) LNCaP-LN3 and androgen-independent (AI) PC3 human CaP xenografts in nude mice were injected intratumorally with an ovine atadenovirus vector, OAdV623, that carries the PNP gene under PSMEPb, formulated with cationic lipid for enhanced infectivity. Fludarabine phosphate was then given intraperitoneally for 5 days at 75 mg/m 2 /day. PNP expression was evaluated by enzymic conversion of its substrate using reverse phase HPLC. OAdV623 showed excellent in vitro transcriptional specificity for CaP cells. In vivo, expression of PNP persisted for 46 days after OAdV623 injection and a single treatment provided 100% increase in tumor doubling time and 450% inhibition of tumor growth for both LNCaP-LN3 and PC3 lines, with increased tumor necrosis and apoptosis and decreased tumor cell proliferation. OAdV623 significantly suppressed the growth of AS and AI human CaP xenografts in mice.

Inhibition of prostate cancer by suicide gene targeting the FCY1 and HSV-TK genes

“…LNCaP cells were chosen in this study due to its dependence on androgen at earlier stages (9,19,26). More efficient tumor formation was observed in castrated nude mice when high-passage LNCaP cells were injected.…”

“…However, when both compounds were administrated, growth inhibition reached ~70%. Since PSMA promoter activity is less dependent on androgen, we explored the inhibition ratio of the lessdependent androgen cells, PC-3 (26). Growth inhibition in PC-3 cells by 5-FC was ~50%, while GCV inhibited only 40%, similar to that of untransfected PC-3 cells (data not shown).…”

“…Driven by the PSMA promoter, the FCY1 and HSV-TK suicide genes can be expressed in the androgen-dependent LNCaP cells (19,26). We further investigated whether the recombinant plasmids can inhibit the growth of prostate cancer cells mediated by pro-drugs in vitro.…”

“…More than 80 passages of these cells can decrease androgen dependence in vivo (26). LNCaP cells that underwent >80 passages were employed to observe tumor formation efficiency in the castrated nude mice.…”

Development of gene therapy in association with clinically used cytotoxic deoxynucleoside analogues

“…48 Both prodrug-activating systems induce apoptosis in Transduction of PNP gene followed by treatment with nucleoside analogues has been studied in vivo on bladder tumors, pancreatic adenocarcinomas, gliomas, ovarian or prostate tumors and showed different levels of growth inhibition. 40,44,[49][50][51][52][53][54][55][56] To increase the effect obtained with this gene-therapy approach by using new genes and new drugs, a study based on crystallographic and computer modeling of E. coli PNP, was performed. The mutated protein (M64V) had a more than 100-fold increased enzymatic efficiency (kcat/Km) toward the nontoxic 9-(6-deoxy-a-L-talofuranosyl)-6-methylpurine as compared to the wt protein.…”