RESUMO
Suicide gene therapy using gene-directed enzyme prodrug therapy (GDEPT) is based on delivering a gene-encoded enzyme to cells that converts a nontoxic prodrug into its toxic metabolite. The bystander effect is thought to compensate for inefficiencies in delivery and expression because the produced toxic metabolite can spread to adjacent non-expressing cells.The purpose of this study was to assess the significance of bystander effect in GDEPT over the long term in vivo.We performed experiments using mixtures of yeast cytosine deaminase (yCD) expressing and empty vector (EV) containing cells. First, the bystander effect was assessed in various ratios of colon cancer cell lines RKO with yCD/EV in 2D and 3D culture. Next, tumors raised from RKO with yCD/EV in mice were treated with the prodrug 5-fluorocytosine (5-FC) for 42 days to assess bystander effect in vivo. Cell types constituting relapsed tumors were determined by 5-FC treatment and PCR.We were able to demonstrate bystander effect in both 2D and 3D. In mice, tumors initially regressed, but they all eventually recurred including those produced from 80% yCD expressing cells. Cells explanted from the recurrent tumors demonstrated that suicide gene expressing cells had been selected against during in vivo treatment with 5-FC.We conclude that gene therapy of malignant tumors in patients using the yCD/5-FC system will require targeting well over 80% of the malignant cells, and therefore will likely require improved bystander effect or repeated treatment.
