ENGINEERED THYMIDINE KINASES FOR CANCER GENE THERAPY

到目前为止，疱疹病毒胸苷激酶(HSV-TK)和更昔洛韦(GCV)的异源表达已经在不同肿瘤的基因治疗中显示出巨大的临床前景。最初的临床试验结果总体上很有希望，在HSV-TK/GCV治疗后，一小部分患者的肿瘤完全抑制。然而，大多数患者对该疗法只有部分或最小的反应。本文提出的工作目标是通过使用合理设计的HSV-TK突变组合来产生GCV激酶(GCVK)来提高这一有希望的治疗方法的疗效。与目前使用的HSV-TKS相比，这种GCVK将具有更高的GCV磷酸化活性和最低的胸苷激酶活性。我们的假设是，在抗癌基因治疗中，这些GCVK将导致更多的肿瘤细胞杀伤，潜在的体内免疫反应，并可能更安全，因为需要较低的GCV剂量。为了实现这一目标，几种原型GCVK已经被表征为具有增强的GCVK活性或降低的TK活性，或者两者兼而有之，通过酶分析和细胞培养表达来评估。对于这项提议，我们将继续设计和表征这些原型GCVK的新版本和混合版本。最有希望的具有所需酶特性的GCVK的治疗效果将在结肠肿瘤细胞系中进行评估，以了解它们对GCV代谢、细胞杀伤和旁观者效应的影响。对新的GCVK的任何增加的治疗益处的进一步分析将在多灶性大鼠肿瘤癌症模型中进行评估。拟议系统中的治疗益处的成功展示将为这些用于治疗转移性肝病的新的GCVK的临床测试以及目前采用HSV-TK/GCV系统的任何遗传疗法提供基础。

To date, heterologous expression of herpesvirus thymidine kinase (HSV- TK) in combination with ganciclovir (GCV) has shown great clinical promise for a genetic therapy of different cancers. Initial clinical trial results have been overall quite promising, with a small percentage of patients having complete tumor repressions after HSV-TK/GCV treatments. However, the majority of patients have had only partial or minimal responses to the therapy. The goal of the work proposed herein is to increase the efficacy of this promising therapy by using a combination of rationally-designed mutations of HSV-TK to generate a GCV kinase (GCVK). Relative to currently used HSV-TKs, this GCVK will have improved phosphorylation activities for GCV and minimal thymidine kinase activities. Our hypothesis is that in anti-cancer gene therapies, these GCVKs will result in increased tumor cell killing, potential the in vivo immune response and potentially be safer due to lower required GCV doses. Towards this goal, several prototype GCVKs have already been characterized that possess either increased GCVK activity, reduced TK activity, or both, as evaluated by enzyme assays and cell culture expression. For this proposal, we will continue to design and characterize new and hybrid versions of these prototype GCVKs. The therapeutic efficacy of the most promising GCVKs with the desired enzymatic properties will be evaluated in colon tumor cell lines for their effect on GCV metabolism, cell killing and bystander effect. Further analysis of any increased therapeutic benefits of the new GCVKs will be evaluated in a multi-focal rat-tumor model of cancer. Successful demonstration of a therapeutic benefit in the proposed systems should provide the basis for clinical testing of these new GCVKs for treatment of metastatic liver disease and any genetic therapy currently employing the HSV-TK/GCV system.