Boehme Philip, Zhang Wenli, Solanki Manish, Ehrke-Schulz Eric, Ehrhardt Anja
Department of Human Medicine, Faculty of Health, Institute of Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), University of Witten/Herdecke, Witten, Germany.
Mol Ther Nucleic Acids. 2016 Jul 19;5(7):e337. doi: 10.1038/mtna.2016.44.
For efficient delivery of required genetic elements we utilized high-capacity adenoviral vectors in the past allowing high transgene capacities of up to 36 kb. Previously we explored the hyperactive Sleeping Beauty (SB) transposase (HSB5) for somatic integration from the high-capacity adenoviral vectors genome. To further improve this hybrid vector system we hypothesized that the previously described hyperactive SB transposase SB100X will result in significantly improved efficacies after transduction of target cells. Plasmid based delivery of the SB100X system revealed significantly increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5. After optimizing experimental setups for high-capacity adenoviral vectors-based delivery of the SB100X system we observed up to eightfold and 100-fold increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5 and the inactive transposase mSB, respectively. Furthermore, transposon copy numbers per cell were doubled with SB100X compared with HSB5 when using the identical multiplicity of infection. We believe that this improved hybrid vector system represents a valuable tool for achieving stabilized transgene expression in cycling cells and for treatment of numerous genetic disorders. Especially for in vivo approaches this improved adenoviral hybrid vector system will be advantageous because it may potentially allow reduction of the applied viral dose.
为了高效递送所需的遗传元件，我们过去使用了高容量腺病毒载体，其允许高达36 kb的高转基因容量。此前，我们探索了用于从高容量腺病毒载体基因组进行体细胞整合的超活性睡美人（SB）转座酶（HSB5）。为了进一步改进这种杂交载体系统，我们推测先前描述的超活性SB转座酶SB100X在转导靶细胞后将显著提高效率。基于质粒的SB100X系统递送显示，与先前发表的超活性SB转座酶HSB5相比，整合效率显著提高。在优化了基于高容量腺病毒载体的SB100X系统递送的实验设置后，我们观察到与先前发表的超活性SB转座酶HSB5和无活性转座酶mSB相比，整合效率分别提高了高达8倍和100倍。此外，当使用相同的感染复数时，与HSB5相比，SB100X使每个细胞的转座子拷贝数增加了一倍。我们相信，这种改进的杂交载体系统是在循环细胞中实现稳定转基因表达以及治疗多种遗传疾病的有价值工具。特别是对于体内方法，这种改进的腺病毒杂交载体系统将具有优势，因为它可能潜在地允许减少所应用的病毒剂量。
