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 酶/前药方法在几种动物中产生了肿瘤消退 然而，在患者身上的结果却不那么令人鼓舞。临床应用的主要障碍 这种和其他基因治疗模式的疗效是基因治疗的低程度， 在体内转移，通常转移到肿瘤内少于10%的细胞。 为了改善这种方法的治疗，必须改善旁观者杀伤。在 在先前的应用中，我们证明了HSV-TK/GCV是唯一能够 在多种人肿瘤细胞系中诱导多对数细胞杀伤， 新的机制，其中低水平的GCV三磷酸是高度细胞毒性的。 这些研究还表明，旁观者杀死是有效的，因为转移 即使是低水平的GCV磷酸盐对非HSV-TK表达细胞的作用， 高细胞毒性。进一步的研究表明， 通过加入核糖核苷酸还原酶抑制剂竞争dGTP库， 羟基脲，增加了对HSVTK细胞的杀伤作用，而它产生了一种抑制HSVTK细胞增殖的作用。 协同增加旁观者细胞杀伤。初步研究表明 这种效果可以在体内动物模型中实现。在当前 应用，这些研究将通过确定是否更有效 核糖核苷酸还原酶抑制剂，或更特异的dGTP抑制剂 合成，可以在体外和体内提高这种旁观者细胞杀伤。在 此外，我们还发现了GCV磷酸转移的一种新机制 不涉及GJIC，我们设计了一组同基因细胞系， GJIC水平不同，以比较新机制与 GJIC在旁观者细胞杀伤中的作用生物化学调节剂的能力， 增强GJIC熟练或缺乏的细胞中的旁观者杀伤将 被评价。增加的GJIC与生化调节相比， 在动物模型中根除肿瘤， 将测定表达HSV-TK的细胞。这些研究的结果应该 证明有助于理解GJIC在旁观者杀伤中的作用， 临床相关的条件，并可能有助于设计临床方案 将HSV-TK/前药疗法与生化调节剂组合。

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.