Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center, Im Neuenheimer Feld 350, Heidelberg, Germany.
Gene Ther. 2011 Feb;18(2):210-2. doi: 10.1038/gt.2010.126. Epub 2010 Nov 11.
More than 10 years ago, we developed an efficient protocol for serum-free retroviral transduction of human hematopoietic stem cells derived from mobilized peripheral blood. After upscaling of the methodology, serum-free retroviral gibbon-ape leukemia virus (GALV) pseudotype PG13/LN vector supernatant produced under strict good manufacturing practice (GMP) conditions was used in the first clinical gene-marking trial in Germany. In this study, we analyzed the titer and transduction efficiency of this serum-free clinical-grade retroviral supernatant 10 years after production to evaluate the long-term stability. Long-term storage and transport on dry ice resulted in modestly decreased titers and levels of transduction efficiency in CD34+ cells ranging from 38.4 to 49.1%. We conclude that the stability of retroviral vectors in serum-free medium allows extended storage and distribution of approved clinical-grade retroviral vector stocks to distant sites in multicenter clinical trials.
10 多年前，我们开发了一种高效的无血清逆转录病毒转导方案，用于从动员的外周血中分离的人造血干细胞。该方法经过放大后，在严格的良好生产规范 (GMP) 条件下生产的无血清 gibbon-ape leukemia virus (GALV) 假型 PG13/LN 载体上清液被用于德国首例临床基因标记试验。在这项研究中，我们分析了生产 10 年后这种无血清临床级逆转录病毒上清液的滴度和转导效率，以评估其长期稳定性。长期在干冰上储存和运输导致滴度适度下降，CD34+细胞的转导效率从 38.4%降至 49.1%。我们的结论是，无血清培养基中逆转录病毒载体的稳定性允许批准的临床级逆转录病毒载体库存的扩展储存和分配到多中心临床试验的偏远地点。
