Van Beusechem V W, Bakx T A, Kaptein L C, Bart-Baumeister J A, Kukler A, Braakman E, Valerio D
Department of Gene Therapy, TNO Institute of Applied Radiobiology and Immunology, Rijswijk, The Netherlands.
Hum Gene Ther. 1993 Jun;4(3):239-47. doi: 10.1089/hum.1993.4.3-239.
We have generated a cell line, designated POAM-P1, shedding amphotropic recombinant retroviruses carrying the human adenosine deaminase (hADA) gene. It exhibits a 1 log increased retrovirus titer on NIH-3T3 cells and a five-fold more efficient transduction of human ADA-deficient T lymphocytes, as compared to the previously generated cell line POC-1 which produces the same recombinant hADA retrovirus. To study whether the titer of retrovirus-producing cell lines influences the transduction efficiency of hematopoietic stem cells in a co-culture setting, we compared the POAM-P1 and POC-1 cell lines with respect to their gene transfer efficiency on rhesus monkey bone marrow. Following co-cultivation of rhesus monkey bone marrow with POAM-P1 cells, successful transduction could be demonstrated in approximately 10% of myeloid progenitor colonies (CFU-C) and 0.1% of peripheral blood mononuclear cells (PBMC) and granulocytes in vivo until > 1 year after autologous transplantation. In addition, the presence of functional hADA enzyme was detected in red blood cells, PBMC, and granulocytes. Monkeys receiving POC-1 co-cultured bone marrow carried transduced blood cells for > 2 years after transplantation. Despite the higher retrovirus titer of POAM-P1 cells as compared to POC-1 cells, no difference was observed in gene transfer efficiency into CFU-C and long-term repopulating stem cells. This shows that in our co-cultivation procedure the retrovirus titer was not limiting the transduction efficiency of primate hematopoietic stem cells.
我们已经构建了一个细胞系，命名为POAM-P1，它能分泌携带人腺苷脱氨酶（hADA）基因的双嗜性重组逆转录病毒。与之前构建的产生相同重组hADA逆转录病毒的细胞系POC-1相比，它在NIH-3T3细胞上的逆转录病毒滴度提高了1个对数，对人ADA缺陷型T淋巴细胞的转导效率提高了5倍。为了研究逆转录病毒产生细胞系的滴度在共培养环境中是否会影响造血干细胞的转导效率，我们比较了POAM-P1和POC-1细胞系对恒河猴骨髓的基因转移效率。将恒河猴骨髓与POAM-P1细胞共培养后，在自体移植后超过1年的时间里，在体内约10%的髓系祖细胞集落（CFU-C）、0.1%的外周血单个核细胞（PBMC）和粒细胞中可证明成功转导。此外，在红细胞、PBMC和粒细胞中检测到了功能性hADA酶。接受POC-1共培养骨髓的猴子在移植后2年以上仍携带转导的血细胞。尽管POAM-P1细胞的逆转录病毒滴度高于POC-1细胞，但在CFU-C和长期重建造血干细胞的基因转移效率方面未观察到差异。这表明在我们的共培养过程中，逆转录病毒滴度并未限制灵长类造血干细胞的转导效率。
