Development and Safety Evaluation of Chemo-Protective Gene Therapy Vectors

描述（由申请人提供）： 本项目的目的是评估利用甲基鸟嘌呤甲基转移酶（MGMT）的耐药基因治疗的安全性和有效性，结合体外和小鼠研究与临床相关的大动物模型，以开发更安全的基因治疗载体用于临床应用。我们已经证明了在使用γ逆转录病毒和慢病毒载体将MGMT递送至重建细胞后，在犬模型中实现高基因标记水平（>90%）和造血系统的化学保护的有效体内选择（参见初步数据）。这表明MGMT介导的体内选择将有益于广泛的遗传疾病，并且还在恶性疾病患者中引起骨髓化学保护的显著治疗益处。在我们的初步分析中，γ逆转录病毒整合体在非常接近转录起始位点处显示出最显著的增加，并且相对于慢病毒整合体，在原癌基因中和附近的频率更高，这表明γ逆转录病毒载体可能最容易发生不利的基因激活。因此，还必须制定战略，降低转化克隆的潜力，并建立一个平台，以测试正在进行的以安全性为目标的载体设计开发。因此，为了开发更安全的基因治疗载体，我们将在体内选择之前和之后对用含有强病毒启动子/增强子的慢病毒MGMT载体基因修饰的犬细胞的前病毒整合谱进行全面分析，作为当前慢病毒载体安全性的初步评估。我们将确定选择是否增加了原癌基因附近的逆转录病毒整合位点的百分比。作为降低启动子/增强子激活风险的策略，我们将表征和测试化疗诱导型启动子系统，并将最有前途的构建体纳入慢病毒自失活载体（SIN）中，在犬模型中测试最佳载体之前，在小鼠模型中检查其遗传毒性。该提案将结合联合收割机在体外和小鼠研究，以开发和表征更安全的基因治疗载体，并利用犬模型来评估MGMT介导的选择在临床相关的大型动物模型中的有效性和安全性。

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.