Creation of AlkB Mutants for Protection of Bone Marrow

描述（由申请人提供）：甲基化剂是治疗癌症的主要药物，是少数几种可用于治疗脑肿瘤的药物之一。 负责保持血细胞供应的细胞，“血液祖细胞”，对这些药物高度敏感，这会导致严重的副作用，包括出血和感染。 血液祖细胞可以通过抗性基因保护免受甲基化的毒性作用。最有效的一种是AGT，它直接消除由细胞遗传物质（DNA）甲基化引起的毒性修饰。然而，AGT不识别某些有毒的DNA修饰。在这里，我建议使用一种不同的基因（AlkB），它靶向这些替代病变中的一些，以提高对甲基化毒性作用的保护。这个基因，这是最近才被表征，因此应该增强AGT的保护。此外，与AGT不同，AlkB在介导修复反应后保持活性，这意味着AlkB修复应该更易于调节。 在这里，我提出了一个强大的方法来识别一个人类同源的AlkB（ABH2）的变体，具有改善的修复性能。具体来说，我想增强酶识别临床使用的药物所产生的病变的能力。将在培养的人祖细胞和小鼠动物模型中测试选定的变体，以保护其免受高剂量这些药物的治疗。最终，在这项工作中确定的突变体应该改善甲基化剂副作用的管理，并且还可以使用更高的剂量进行更有效的治疗。

DESCRIPTION (provided by applicant): Methylating agents are mainstay drugs for the treatment of cancer and are amongst the few agents available against brain tumors. Cells responsible for keeping the blood cell supply, "blood progenitor cells", are highly sensitive to these agents, which results in severe side-effects, including bleeding and infection. Blood progenitor cells can be protected from the toxic effects of methylation by resistance genes. The most effective one is AGT, which directly removes toxic modifications caused by methylation of the cell's genetic material (DNA). However, AGT does not recognize certain toxic DNA modifications. Here I propose the use of a different gene (AlkB), which targets some of these alternative lesions, to improve protection against the toxic effects of methylation. This gene, which has only recently been characterized, should therefore enhance AGT protection. Moreover, unlike AGT, AlkB remains active after mediating a repair reaction, which implies that AlkB repair should be more amenable to modulation. Here I propose a powerful approach to identify variants of one human homologue of AlkB (ABH2) with improved repair performance. Specifically, I want to enhance the enzyme's ability to recognize lesions generated by agents used in the clinic. Selected variants will be tested in human progenitor cells in culture and in a mouse animal model for protection against treatment with high doses of these agents. Ultimately, the mutants identified in this work should improve the management of side effects of methylating agents and would also allow the use of higher doses for more effective treatment.