Mostoslavsky Gustavo, Kotton Darrell N, Fabian Attila J, Gray John T, Lee Jeng-Shin, Mulligan Richard C
Department of Genetics, Harvard Medical School and Molecular Medicine, Children's Hospital, Boston, MA 02115, USA.
Mol Ther. 2005 Jun;11(6):932-40. doi: 10.1016/j.ymthe.2005.01.005.
The development of leukemias in several children with severe combined immunodeficiency disease who were transplanted with retroviral vector-transduced bone marrow cells has renewed concerns about the risks associated with the random integration of proviral sequences into chromosomal DNA. One theoretical way to reduce the risks of insertional mutagenesis would be to employ transduction/transplantation protocols that minimize the total number of genetically modified cells and associated proviral integration "events" introduced into recipients. Toward this end, we have developed a transduction protocol that involves the short-term incubation of highly purified murine stem cells with high-titer recombinant lentivirus vectors in the presence of serum-free medium and the cytokines SCF and TPO. Competitive repopulation studies showed that stem cells transduced in this way possessed the same reconstitutive ability as fresh, unmanipulated cells. Animals transplanted with only 200-2000 transduced cells were efficiently reconstituted with the genetically modified cells, and most hematopoietic cells in the recipients expressed the transgene. In contrast, the use of high-titer oncoretroviral vectors in conjunction with the same transduction/transplantation protocol resulted in only low levels of gene marking in vivo. The use of a similar transduction/transplantation strategy in future clinical studies may offer distinct advantages over current protocols.
几名严重联合免疫缺陷病儿童在接受逆转录病毒载体转导的骨髓细胞移植后发生白血病，这再次引发了人们对原病毒序列随机整合到染色体DNA中所带来风险的担忧。降低插入诱变风险的一种理论方法是采用转导/移植方案，尽量减少引入受体的基因改造细胞总数和相关的原病毒整合“事件”。为此，我们开发了一种转导方案，该方案包括在无血清培养基以及细胞因子SCF和TPO存在的情况下，将高度纯化的小鼠干细胞与高滴度重组慢病毒载体进行短期孵育。竞争性再增殖研究表明，以这种方式转导的干细胞具有与新鲜未处理细胞相同的重建能力。仅移植200 - 2000个转导细胞的动物能够有效地被基因改造细胞重建，并且受体中的大多数造血细胞都表达了转基因。相比之下，使用高滴度的γ逆转录病毒载体结合相同的转导/移植方案，在体内仅产生低水平的基因标记。在未来的临床研究中使用类似的转导/移植策略可能比当前方案具有明显优势。
