Sorg Ursula R, Kleff Veronika, Fanaei Sepideh, Schumann Alexandra, Moellmann Michael, Opalka Bertram, Thomale Jürgen, Moritz Thomas
Department of Internal Medicine (Cancer Research), West German Cancer Center, University of Duisburg-Essen Medical School, Hufelandstr. 55, 45122 Essen, Germany.
DNA Repair (Amst). 2007 Aug 1;6(8):1197-209. doi: 10.1016/j.dnarep.2007.03.021. Epub 2007 May 11.
As haematopoietic stem cell gene therapy utilizing O(6)-methylguanine-DNA-methyltransferase has reached the clinical stage, safety-related questions become increasingly important. These issues concern insertional mutagenesis of viral vectors, the acute toxicity of pre-transplant conditioning protocols and in vivo selection regimens as well as potential genotoxic side effects of the alkylating drugs administered in this context. To address these questions, we have investigated toxicity-reduced conditioning regimens combining low-dose alkylator application with sublethal irradiation and have analysed their influence on engraftment and subsequent selectability of transduced haematopoietic stem cells. In addition, a strategy to monitor the acute and long-term genotoxic effects of drugs with high guanine-O(6) alkylating potential, such as chloroethylnitrosoureas or temozolomide is introduced. For this purpose, assays were implemented which allow an assessment of the generation and fate of primary drug-induced adducts as well as their long-term effect on chromosomal integrity at the single cell level.
随着利用O(6)-甲基鸟嘌呤-DNA-甲基转移酶的造血干细胞基因疗法进入临床阶段，与安全性相关的问题变得越来越重要。这些问题涉及病毒载体的插入诱变、移植前预处理方案和体内选择方案的急性毒性以及在此背景下施用的烷化剂的潜在基因毒性副作用。为了解决这些问题，我们研究了将低剂量烷化剂应用与亚致死性照射相结合的毒性降低的预处理方案，并分析了它们对转导造血干细胞植入和随后可选择性的影响。此外，还介绍了一种监测具有高鸟嘌呤-O(6)烷基化潜力的药物（如氯乙基亚硝脲或替莫唑胺）急性和长期基因毒性作用的策略。为此，实施了一些检测方法，这些方法可以在单细胞水平上评估原发性药物诱导加合物的产生和命运以及它们对染色体完整性的长期影响。
