Cytotoxicity and Bystander Killing for HSV TK Substrates

描述(由申请人提供)：最常用的 涉及自杀基因转移/前药物治疗的方法是疱疹 单纯病毒胸苷激酶(HSV-TK)/更昔洛韦(GCV)系统。虽然这件事 酶/前药疗法在几种动物中产生了肿瘤消退 在模型中，患者的结果不那么令人鼓舞。临床上的一个主要障碍 这种和其他基因治疗模式的有效性在于基因的低程度 体内转移，通常转移到肿瘤内不到10%的细胞。 为了改进这种方法的治疗，必须改善对旁观者的杀戮。在……里面 在前面的应用中，我们演示了HSV-TK/GCV是唯一能够 在多种人类肿瘤细胞系中诱导多对数细胞杀伤，由于一种 低水平的GCV三磷酸具有高度细胞毒性的新机制。 这些研究还表明，旁观者杀人是有效的，因为转移 即使是低水平的GCV磷酸对非HSV-TK表达的细胞也会导致 高细胞毒性。进一步的研究表明，略微减少 通过添加核苷酸还原酶抑制剂来竞争dGTP池， 羟基脲，增强对HSVtk细胞的杀伤作用，而它产生 协同增加旁观者细胞的杀伤力。初步研究表明 这种效应可以在活体动物模型中实现。在当前 应用，这些研究将通过确定更有效的 核糖核苷酸还原酶抑制剂，或更特异的dGTP抑制剂 合成，可以提高这种旁观者细胞在体外和体内的杀伤作用。在……里面 此外，我们还注意到细胞内GCV磷酸转移的一种新机制 这不涉及GJIC，我们已经设计了一组等基因细胞系 在GJIC的水平上不同，以比较新机制和 GJIC参与了旁观者细胞的杀戮。生化调节器的能力 增强对精通或缺乏GJIC意志细胞的旁观者杀伤 被评估。GJIC增加与生化调节治疗的疗效比较 在动物模型中根除肿瘤，在动物模型中只有一部分肿瘤 HSV-TK的表达将被确定。这些研究的结果应该是 证明有助于理解GJIC在以下情况下的旁观者杀戮中的作用 临床相关的情况，并可能有助于设计临床方案 HSV-TK/前药联合生化调节剂治疗。

DESCRIPTION (provided by the applicant): One of the most commonly used approaches involving suicide gene transfer/prodrug therapy is the herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) system. While this enzyme/prodrug approach has produced tumor regressions in several animal models, results in patients are less encouraging. A major obstacle to clinical efficacy for this and other modes of gene therapy is the low extent of gene transfer in vivo, typically to fewer than 10 percent of cells within a tumor. To improve therapy with this approach, bystander killing must be improved. In the previous application, we demonstrated that HSV-TK/GCV is uniquely able to induce multi-log cell killing in a variety of human tumor cell lines, due to a novel mechanism in which low levels of GCV triphosphate are highly cytotoxic. These studies also showed that bystander killing was effective because transfer of even low levels of GCV phosphates to non-HSV-TK-expressing cells resulted in high cytotoxicity. Further studies demonstrated that a small reduction in the competing dGTP pool by the addition of a ribonucleotide reductase inhibitor, hydroxyurea, enhanced killing of HSVTK cells additively whereas it produced a synergistic increase in bystander cell killing. Preliminary studies suggest that this effect can be achieved in animal models in vivo. In the current application, these studies will be extended by determining whether more potent inhibitors of ribonucleotide reductase, or more specific inhibitors of dGTP synthesis, can improve this bystander cell killing in vitro and in vivo. In addition, we have noted a novel mechanism of GCV phosphate transfer in cells that does not involve GJIC, and we have devised a panel of isogenic cell lines that vary in the level of GJIC to compare the roles of the novel mechanism vs. GJIC in bystander cell killing. The ability of the biochemical modulators to enhance bystander killing in cells either proficient or deficient in GJIC will be evaluated. The efficacy of increased GJIC vs. biochemical modulation to eradicate tumors in animal models in which only a portion of the tumor expresses HSV-TK will be determined. The results from these studies should prove useful in understanding the role of GJIC in bystander killing under clinically relevant conditions, and may help in designing clinical protocols combining HSV-TK/prodrug therapy with a biochemical modulator.