Schambach Axel, Baum Christopher
Department of Experimental Hematology, Hannover Medical School, Hannover, Germany.
DNA Repair (Amst). 2007 Aug 1;6(8):1187-96. doi: 10.1016/j.dnarep.2007.03.017. Epub 2007 May 7.
Enhancing DNA repair activity of hematopoietic cells by stably integrating gene vectors that express O(6)-methylguanine-DNA-methyltransferase (MGMT) is of major interest for innovative approaches in tumor chemotherapy and for the control of hematopoietic chimerism in the treatment of multiple other acquired or inherited disorders. Crucial determinants of this selection principle are the stringency of treatment with O(6)-alkylating agents and the level of transgenic MGMT expression. Attempts to generate clinically useful MGMT vectors focus on the design of potent expression cassettes, an important component of which is formed by enhancer sequences that are active in primitive as well as more differentiated hematopoietic cells. However, recent studies have revealed that vectors harboring strong enhancer sequences are more likely to induce adverse events related to insertional mutagenesis. Safety-improved vectors that maintain high levels of MGMT expression may be constructed based on the following principles: choice of enhancer-promoter sequences with relatively mild long-distance effects despite a high transcription rate, improved RNA processing (export, stability and translation), and protein design. The need for optimizing MGMT protein design is supported by recent observations suggesting that the P140K mutant of MGMT, developed to be resistant to inhibitors such as O(6)-benzylguanine, may confer a selective disadvantage when expressed at high levels. Here, we provide a review of the literature exploring MGMT expression vectors for bone marrow chemoprotection, and describe experimental evidence suggesting that high expression of MGMT P140K induces a selective disadvantage in the absence of alkylating agents. We conclude that the appropriate design of expression vectors and MGMT protein features will be crucial for the long-term prospects of this promising selection principle.
通过稳定整合表达O(6)-甲基鸟嘌呤-DNA甲基转移酶（MGMT）的基因载体来增强造血细胞的DNA修复活性，对于肿瘤化疗的创新方法以及在治疗多种其他获得性或遗传性疾病中控制造血嵌合体具有重要意义。这种选择原则的关键决定因素是O(6)-烷基化剂治疗的严格程度和转基因MGMT的表达水平。生成临床有用的MGMT载体的尝试集中在高效表达盒的设计上，其中一个重要组成部分是由在原始以及更分化的造血细胞中具有活性的增强子序列形成的。然而，最近的研究表明，携带强增强子序列的载体更有可能引发与插入诱变相关的不良事件。可以基于以下原则构建保持高水平MGMT表达的安全性改进载体：选择具有相对温和的长距离效应但转录率高的增强子-启动子序列，改进RNA加工（输出、稳定性和翻译）以及蛋白质设计。最近的观察结果支持了优化MGMT蛋白质设计的必要性，这些观察结果表明，为对诸如O(6)-苄基鸟嘌呤等抑制剂具有抗性而开发的MGMT的P140K突变体，在高水平表达时可能会带来选择性劣势。在这里，我们综述了探索用于骨髓化学保护的MGMT表达载体的文献，并描述了实验证据，表明在没有烷基化剂的情况下，MGMT P140K的高表达会导致选择性劣势。我们得出结论，表达载体和MGMT蛋白质特征的适当设计对于这一有前景的选择原则的长期前景至关重要。
