ABSTRACT

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.

摘要

使用基因定向酶前药疗法 (GDEPT) 的自杀基因疗法是基于将基因编码的酶传递给细胞，将无毒前药转化为其有毒代谢物。旁观者效应被认为可以弥补传递和表达的低效率，因为产生的有毒代谢物可以扩散到相邻的非表达细胞。

本研究的目的是评估长期体内GDEPT 中旁观者效应的重要性。

我们使用表达酵母胞嘧啶脱氨酶( yCD ) 和含有空载体 (EV) 的细胞的混合物进行了实验。首先，在 2D 和 3D 培养中评估了不同比例的结肠癌细胞系 RKO 与 yCD/EV 的旁观者效应。接下来，用前药 5-氟胞嘧啶 (5-FC) 治疗小鼠 yCD/EV 从 RKO 产生的肿瘤 42 天，以评估体内旁观者效应。通过5-FC处理和PCR确定构成复发肿瘤的细胞类型。

我们能够在 2D 和 3D 中展示旁观者效应。在小鼠中，肿瘤最初消退，但它们最终都会复发，包括由 80% yCD表达细胞产生的肿瘤。从复发性肿瘤中取出的细胞表明，在用 5-FC进行体内治疗期间，已经选择了表达自杀基因的细胞。

我们得出的结论是，使用 yCD/5-FC 系统对患者的恶性肿瘤进行基因治疗将需要靶向超过 80% 的恶性细胞，因此可能需要改善旁观者效应或重复治疗。