Schott J W, Hoffmann D, Maetzig T, Müller F-J, Steinemann D, Zychlinski D, Cantz T, Baum C, Schambach A
1] Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany [2] Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.
Center for Integrated Psychiatry, Christian Albrechts University, Kiel School of Medicine, Kiel, Germany.
Gene Ther. 2014 Nov;21(11):938-49. doi: 10.1038/gt.2014.69. Epub 2014 Aug 7.
Retroviral vectors are versatile gene transfer vehicles widely used in basic research and gene therapy. Mutation of retroviral integrase converts these vectors into transient, integration-deficient gene delivery vehicles associated with a high degree of biosafety. We explored the option to use integration-deficient retroviral vectors to achieve transient ectopic expression of transcription factors, which is considered an important tool for induced cell fate conversion. Stepwise optimization of the retroviral episome transfer as exemplified for the transcription factor Oct4 enabled to improve both expression magnitude and endurance. Long terminal repeat-driven γ-retroviral vectors were identified as the most suitable vector architecture. Episomal expression was enhanced by epigenetic modifiers, and Oct4 activity was increased following fusion to a minimal transactivation motif of herpes simplex virus VP16. Based on kinetic analyses, we identified optimal time intervals for repeated vector administration and established prolonged expression windows of choice. Providing proof-of-concept, episomal transfer of Oct4 was potent to mediate conversion of human fibroblasts stably expressing Klf4, Sox2 and c-Myc into induced pluripotent stem cells, which were mainly free of residual Oct4 vector integration. This study provides evidence for suitability of retroviral episome transfer of transcription factors for cell fate conversion, allowing the generation of distinct patient- or disease-specific cell types.
逆转录病毒载体是广泛应用于基础研究和基因治疗的多功能基因转移工具。逆转录病毒整合酶的突变将这些载体转化为与高度生物安全性相关的瞬时、整合缺陷型基因递送工具。我们探索了使用整合缺陷型逆转录病毒载体来实现转录因子的瞬时异位表达的方法，这被认为是诱导细胞命运转变的重要工具。以转录因子Oct4为例，对逆转录病毒附加体转移进行逐步优化，能够提高表达量和持续时间。长末端重复序列驱动的γ-逆转录病毒载体被确定为最合适的载体结构。表观遗传修饰剂增强了附加体表达，并且在与单纯疱疹病毒VP16的最小反式激活基序融合后，Oct4活性增加。基于动力学分析，我们确定了重复载体给药的最佳时间间隔，并建立了选择的延长表达窗口。作为概念验证，Oct4的附加体转移能够有效地将稳定表达Klf4、Sox2和c-Myc的人成纤维细胞转化为诱导多能干细胞，这些细胞主要没有残留的Oct4载体整合。本研究为转录因子的逆转录病毒附加体转移适用于细胞命运转变提供了证据，从而能够产生不同的患者或疾病特异性细胞类型。
