Development and Safety Evaluation of Chemo-Protective Gene Therapy Vectors

描述(由申请人提供)： 本项目的目的是评价利用甲基鸟嘌呤甲基转移酶(MGMT)进行耐药基因治疗的安全性和有效性，结合体外和小鼠的研究，结合临床相关的大动物模型，开发更安全的临床应用基因治疗载体。我们已经证明了高效的体内选择，实现了高基因标记水平(&gt；90%)，并在犬模型中对造血系统进行了化学保护，之后将MGMT转移到使用伽玛逆转录病毒和慢病毒载体的重新填充的细胞(见初步数据)。这表明，MGMT介导的体内选择将有益于广泛的遗传性疾病，并在恶性疾病患者的骨髓化学保护中获得显著的治疗益处。在我们的初步分析中，伽玛逆转录病毒整合子在非常接近转录起始点的地方表现出最显著的增加，与慢病毒整合子相比，原癌基因中和附近的频率更高，这表明伽玛逆转录病毒载体可能是最容易被不利的基因激活的。因此，还必须制定战略，减少转化克隆的潜力，并建立一个平台，以测试旨在安全的媒介设计的持续发展。因此，为了开发更安全的基因治疗载体，我们将全面分析含有强病毒启动子/增强子的慢病毒MGMT载体修饰的犬细胞在体内选择前后的前病毒整合情况，作为对当前慢病毒载体安全性的初步评估。我们将确定选择是否会增加原癌基因附近逆转录病毒整合位点的百分比。作为一种降低启动子/增强子激活风险的策略，我们将表征和测试化疗诱导的启动子系统，并将最有希望的构建物整合到慢病毒自失活载体(SIN)中，以在小鼠模型中检测它们的遗传毒性，然后在犬模型中测试最佳载体。这项建议将结合体外和小鼠研究，开发和表征更安全的基因治疗载体，并利用犬模型在临床相关的大型动物模型中评估MGMT介导的选择的有效性和安全性。

DESCRIPTION (provided by applicant): The objective of this project is to evaluate the safety and efficacy of drug resistance gene therapy utilizing methylguanine methyltransferase (MGMT) combining in vitro and murine studies with a clinically relevant large animal model to develop safer gene therapy vectors for clinical applications. We have demonstrated efficient in vivo selection achieving high gene marking levels (>90%) and chemo-protection of the hematopoietic system in the canine model after delivering MGMT to repopulating cells using gammaretroviral and lentiviral vectors (see preliminary data). This suggests that MGMT-mediated in vivo selection will benefit a broad range of genetic diseases and also elicit a significant therapeutic benefit of bone marrow chemo-protection in patients with malignant disease. In our initial analysis, gammaretroviral integrants showed the most significant increase very close to transcription start sites and a higher frequency in and near proto-oncogenes relative to lentiviral integrants, suggesting that gammaretroviral vectors may be the most prone to adverse gene activation. Thus it will also be important to develop strategies to reduce the potential of transformed clones and to have a platform in place to test ongoing developments in vector design aimed at safety. Therefore, to develop safer gene therapy vectors we will conduct a comprehensive analysis of the provirus integration profile of canine cells gene-modified with lentiviral MGMT vectors containing strong viral promoter/enhancers before and after in vivo selection as an initial assessment of current lentivirus vector safety. We will determine if selection increases the percentage of retroviral integration sites near proto-oncogenes. As a strategy to reduce the risk of promoter/enhancer activation, we will characterize and test chemotherapy-inducible promoter systems and incorporate the most promising constructs into lentivirus self-inactivating vectors (SIN) to examine their genotoxicity in the murine model before testing optimum vectors in the canine model. This proposal will combine in vitro and murine studies to develop and characterize safer gene therapy vectors and utilize the canine model to evaluate the efficacy and safety of MGMT-mediated selection in a clinically relevant large animal model.