ONCOGENIC ACTIVITY OF RETROVIRAL VECTORS USED IN HUMAN GENE THERAPY

联邦政府批准的第一项逆转录病毒载体基因临床试验 人类遗传病的治疗已经开始。有几个问题令人担忧 在将逆转录病毒介导的基因转移应用于 人类体细胞遗传病。一个令人担忧的问题是，向量，或 其成分，可能具有致癌作用。我们正在调查是否 使用逆转录病毒载体进行基因治疗可能会产生不利的影响 结果通过检查这些向量对变换的影响 体外培养的细胞。该项目的初始阶段涉及感染C3H10T 带有逆转录病毒载体骨架PE501/G1Na(Ltr-neo-Ltr)的1/2细胞， 由安德森博士(美国国立卫生研究院)生态包装和供应。这个向量是 已发现可有效感染C3H10T 1/2细胞并增强对 抗生素G418，转移到转基因细胞。进行了转化实验 在三种不同的方案下进行：一次接触病媒 在不同的滴度，重复暴露和暴露，然后重复 这些细胞。Southern杂交显示，该载体整合到了 被感染的细胞和整合载体拷贝的数量 随着载体滴度的增加而增加。测量的每个实验 三种作用：细胞毒性、G418抗性和转化。3- 甲基胆蒽(3-MC)，一种已知的致癌物质，它正在转化为 相对无毒，作为阳性对照。在任何情况下都不会暴露 与载体PE501/G1Na的结合，会导致存活率的降低或 C3H10T 1/2细胞转化与未处理(阴性)的比较 控制。

The first federally approved clinical trial of retroviral vector based gene therapy for human genetic disease has begun. There are several concerns that must be addressed when applying retroviral-mediated gene transfer to human somatic cell genetic diseases. One concern is that the vector, or components of it, may have oncogenic effects. We are investigating whether the use of retroviral vectors for gene therapy may have adverse consequences by examining the effects of such vectors on transformation of cells in vitro. The initial phase of the project involved infecting C3H10T 1/2 cells with the retroviral vector backbone PE501/G1Na (LTR-neo-LTR), ecotropically packaged and supplied by Dr. Anderson (NIH). This vector was found to efficiently infect C3H10T 1/2 cells and to confer resistance to the antibiotic G418, to transfected cells. Transformation experiments were performed under three different regimens: a single exposure to the vector at varying titer, repeated exposures, and exposure followed by replating of the cells. Southern blots show that the vector integrated into the DNA of the infected cells and that the number of integrated vector copies increased with increasing titer of the vector. Each experiment measured three effects: cytotoxicity, G418 resistance, and transformation. 3- methylcholanthrene (3-MC), a known carcinogen which is transforming but relatively nontoxic, served as a positive control. In no case did exposure to the vector, PE501/G1Na, cause any decrease in survival or increase in transformation of C3H10T 1/2 cells compared to untreated (negative) controls.