Dubé I D, Kruth S, Abrams-Ogg A, Kamel-Reid S, Lutzko C, Nanji S, Ruedy C, Singaraja R, Wild A, Krygsman P, Chu P, Messner H, Reddy V, McGarrity G, Stewart A K
Department of Laboratory Medicine, Sunnybrook Health Science Centre, University of Toronto, Ontario, Canada.
Hum Gene Ther. 1996 Nov 10;7(17):2089-100. doi: 10.1089/hum.1996.7.17-2089.
Long-term marrow cultures (LTMCs) were established from 27 human marrows. Hematopoietic cells were subjected to multiple rounds of exposure to retroviral vectors during 3 weeks of culture. Seven different retroviral vectors were evaluated. LTMCs were assessed for viability, replication-competent retrovirus, progenitors capable of proliferating in immune-deficient mice, and gene transfer. The average number of adherent cells and committed granulocyte-macrophage progenitors (CFU-GM) recovered from LTMCs was 28% and 11% of the input totals, respectively. There was no evidence by marker rescue assay or polymerase chain reaction (PCR) of replication-competent virus production during LTMC. No toxicity to cellular proliferation due to the transduction procedure was observed. The adherent layers of LTMCs exposed to retroviral vectors were positive for proviral DNA by PCR and by Southern blot analysis. Fifty-three percent of 1,427 individual CFU-GM from transduced LTMC adherent layers were positive for vector-derived DNA. For neocontaining vectors, the average G418 resistance was 28% of 1,393 LTMC-derived CFU-GM. Forty percent of 187 tissues from 30 immune-deficient mice injected with human LTMC cells were positive for human DNA 4-5 weeks after adoptive transfer. These studies indicate that multiple exposures of human LTMCs to retroviral vectors result in consistent and reproducible LTMC viability and gene transfer into committed progenitors. Our results further support the use of transduced LTMC cells in clinical trials of hematopoietic stem cell gene transfer.
从27份人类骨髓中建立了长期骨髓培养物（LTMC）。在3周的培养过程中，造血细胞接受了多轮逆转录病毒载体的暴露。评估了七种不同的逆转录病毒载体。对LTMC进行了活力、复制能力强的逆转录病毒、能够在免疫缺陷小鼠中增殖的祖细胞以及基因转移方面的评估。从LTMC中回收的贴壁细胞和定向粒细胞-巨噬细胞祖细胞（CFU-GM）的平均数量分别为输入总数的28%和11%。在LTMC培养期间，通过标记挽救试验或聚合酶链反应（PCR）未发现有复制能力的病毒产生的证据。未观察到转导过程对细胞增殖的毒性作用。通过PCR和Southern印迹分析，暴露于逆转录病毒载体的LTMC的贴壁层中前病毒DNA呈阳性。来自转导的LTMC贴壁层的1427个单个CFU-GM中，53%的细胞载体衍生DNA呈阳性。对于含新霉素的载体，平均G418抗性为1393个LTMC衍生的CFU-GM的28%。在过继转移4-5周后，向30只免疫缺陷小鼠注射人类LTMC细胞，187个组织中有40%的组织人类DNA呈阳性。这些研究表明，人类LTMC多次暴露于逆转录病毒载体可导致一致且可重复的LTMC活力以及基因转移到定向祖细胞中。我们的结果进一步支持在造血干细胞基因转移的临床试验中使用转导的LTMC细胞。
