Improved retroviral episome transfer of transcription factors enables sustained cell fate modification
Improved retroviral episome transfer of transcription factors enables sustained cell fate modification
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DOI：
10.1038/gt.2014.69
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发表时间：
2014-11-01
期刊：
影响因子：
5.1
通讯作者：
Schambach, A.
中科院分区：
文献类型：
--
作者：
Schott, J. W.;Hoffmann, D.;Schambach, A.
逆转录病毒载体是一种多功能的基因转移载体，广泛应用于基础研究和基因治疗。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相关的瞬时、整合缺陷的基因输送载体。我们探索了使用整合缺陷的逆转录病毒载体来实现转录因子的瞬时异位表达的选择，这被认为是诱导细胞命运转换的重要工具。逆转录病毒Episome转移的逐步优化，如转录因子Oct4，能够提高表达幅度和耐受性。长末端重复序列驱动的伽马逆转录病毒载体被认为是最合适的载体结构。表观遗传修饰物增强了表观基因的表达，融合到单纯疱疹病毒VP16的最小反式激活基序后，Oct4的活性增加。在动力学分析的基础上，我们确定了重复给药的最佳时间间隔，并建立了延长的表达窗口。OCT4的异体转移提供了概念验证，可以有效地介导稳定表达KLF4、SOX2和c-Myc的人成纤维细胞向诱导的多能干细胞转化，诱导的多能干细胞主要没有残留的Oct4载体整合。这项研究提供了逆转录病毒Episome转移转录因子用于细胞命运转换的适宜性证据，允许产生不同的患者或疾病特定细胞类型。
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 gamma-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.