Chemoresistance and Stem Cell Selection

描述（由申请人提供）：治疗严重联合免疫缺陷（SCID）的基因治疗试验的成功提供了体内选择转导造血细胞群的力量和效用的证据。在过去的十年里，我们和其他人已经证明了结合基因转移和药物方法在体内选择利用化疗耐药基因的造血干细胞的有效性。特别是，O6甲基鸟嘌呤DNA甲基转移酶（MGMT）突变体与该蛋白抑制剂（如6-苄基鸟嘌呤（6-BG））联合转移，并使用烷基化剂处理，已被证明对造血细胞，特别是造血干细胞的体内选择非常有效。MGMT的基因转移具有广泛的应用，不仅在干细胞选择方面，而且在产生耐化疗造血方面也有广泛的应用，这可能使癌症治疗中的剂量增强成为可能。虽然这种方法是有效的，但对于功能性干细胞区室的潜在损伤，以及在这种选择后存在于长寿细胞中的残留DNA损伤的长期影响，都知之甚少。此外，尽管整合逆转录病毒引起的插入突变风险最近受到了广泛关注，并且是基因转移应用于人类疾病所面临的一个重要问题，但我们认为这些风险不仅与整合事件有关，而且可能还取决于特定的转基因表达和涉及被操纵细胞的其他因素，如增殖应激和增殖史。在这个互动的、多研究者的资助申请中，我们建议研究体内干细胞选择的有效性，这种选择对长期干细胞功能的影响，体内选择后存在的残留DNA损伤的数量和质量，最后，除了载体整合之外，这些因素的组合与体内造血细胞白血病转化发生率的病理生理相关性。拟议的工作包括许多在造血、病毒载体设计、DNA损伤检测和表征以及载体整合鉴定/生物信息学方面具有专业知识的有成就和富有成效的研究人员的合作努力。该研究计划直接模拟和评估体内选择和DNA损伤对关键、敏感和治疗相关的生物室（造血干细胞）的影响。这些研究的结果将为病毒载体在遗传疾病和癌症中的应用以及保护造血干细胞免受DNA损伤的详细后果和益处提供强有力的见解。

DESCRIPTION (provided by applicant): The success of the gene therapy trial in treatment of severe combined immunodeficiency (SCID) provides evidence of the power and utility of in vivo selection of transduced hematopoietic cell populations. Over the past decade, we and others have demonstrated the effectiveness of a combined gene transfer and pharmacologic approach for in vivo selection of hematopoietic stem cells utilizing chemotherapy-resistance genes. In particular, transfer of mutants of O6 methylguanine DNA methyltransferase (MGMT) in combination with inhibitors of this protein, such as 6-benzylguanine (6-BG), and treatment with alkylating agents have been demonstrated to be highly effective for in vivo selection of hematopoietic cells, specifically hematopoietic stem cells. Gene transfer of MGMT has wide application, not only in stem cell selection, but also in generating chemo-resistant hematopoiesis that may allow dose intensification in cancer therapies. While this approach is effective, little is known neither about the potential damage to the functional stem cell compartment nor the long term effects of residual DNA damage present in long-lived cells after such selection. In addition, although the risk of insertional mutagenesis resulting from integrating retroviruses has received wide-spread recent attention and is an important issue facing application of gene transfer to human diseases, we believe that these risks are related not simply to the integration event, but are likely also dependent upon the specific transgene expressed and other factors involving the manipulated cells, such as proliferative stress and proliferative history. In this interactive, multi-investigator grant application, we propose to examine the effectiveness of in vivo stem cell selection, the effects of such selection on long-term stem cell function, the quantity and quality of residual DNA damage that is present after in vivo selection and finally the pathophysiological relevance of the combination of these factors in addition to vector integration on the incidence of leukemia transformation of hematopoietic cells in vivo. The proposed work includes collaborative efforts of a number of accomplished and productive investigators with expertise in hematopoiesis, virus vector design, detection and characterization of DNA damage and vector integration identification/bioinformatics. The research plan directly models and evaluates the effects of in vivo selection and DNA damage on a critical, sensitive, and therapeutically relevant biological compartment (hematopoietic stem cells). The results from these studies will provide powerful insight into the application of virus vectors to genetic diseases and cancer as well as detailed consequences and benefits of protecting hematopoietic stem cells from DNA damage.