Kaleko M, Garcia J V, Osborne W R, Miller A D
Program in Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
Blood. 1990 Apr 15;75(8):1733-41.
A high titer retroviral vector was used to transfer a human adenosine deaminase (h-ADA) cDNA into murine bone marrow cells in vitro. The h-ADA cDNA was linked to the retroviral promoter, and the vector also contained a neomycin phosphotransferase gene as a selectable marker. Infected marrow was transplanted into syngeneic W/Wv recipients, and h-ADA expression was monitored for 5.5 months. Several weeks after transplantation, h-ADA was detected in the erythrocytes of all nine recipients, eight of which expressed levels equal to the endogenous enzyme. This level of expression persisted in two of six surviving mice, while expression in three others stabilized at lower, but readily detectable, levels. Only one mouse had no detectable h-ADA after 5.5 months. Vector DNA sequences with common integration sites were found in hematopoietic and lymphoid tissues of the mice at 5.5 months, providing evidence that hematopoietic stem cells had been infected. Furthermore, all mice transplanted with marrow that had been selected in G418 before infusion had multiple vector copies per genome. While this category included the two highest h-ADA expressors, it also included the negative mouse. Thus, multiple copies of the vector were not sufficient to guarantee long-term h-ADA expression. Mice were monitored for "helper virus" infections with an assay designed to detect a wide range of replication-competent retroviruses, including those endogenous to the mouse genome. No helper virus was detected in the two highest h-ADA expressors, ruling out helper-assisted vector spread as a cause of the high h-ADA expression. These results help provide a foundation for the development of somatic gene therapy techniques to be used in the treatment of human disease.
使用高滴度逆转录病毒载体在体外将人腺苷脱氨酶（h-ADA）cDNA 导入小鼠骨髓细胞。h-ADA cDNA 与逆转录病毒启动子相连，该载体还包含新霉素磷酸转移酶基因作为选择标记。将感染的骨髓移植到同基因 W/Wv 受体中，并监测 h-ADA 表达 5.5 个月。移植后几周，在所有九只受体的红细胞中检测到 h-ADA，其中八只表达水平与内源性酶相当。这种表达水平在六只存活小鼠中的两只中持续存在，而另外三只中的表达稳定在较低但仍可检测到的水平。5.5 个月后只有一只小鼠未检测到 h-ADA。在 5.5 个月时，在小鼠的造血和淋巴组织中发现了具有共同整合位点的载体 DNA 序列，这证明造血干细胞已被感染。此外，所有移植了输注前在 G418 中筛选过的骨髓的小鼠，每个基因组都有多个载体拷贝。虽然这一组包括两个 h-ADA 表达最高的小鼠，但也包括那只呈阴性的小鼠。因此，载体的多个拷贝不足以保证 h-ADA 的长期表达。用一种旨在检测多种具有复制能力的逆转录病毒（包括小鼠基因组内源性逆转录病毒）的检测方法对小鼠进行“辅助病毒”感染监测。在两个 h-ADA 表达最高的小鼠中未检测到辅助病毒，排除了辅助病毒介导的载体传播是 h-ADA 高表达原因的可能性。这些结果为开发用于治疗人类疾病的体细胞基因治疗技术奠定了基础。
