Nwokeoha Sandra, Carlisle Robert, Cleveland Robin O
Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford, United Kingdom.
Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford, United Kingdom.
Ultrasound Med Biol. 2016 Oct;42(10):2478-92. doi: 10.1016/j.ultrasmedbio.2016.06.001. Epub 2016 Jul 18.
The delivery of genes into cells through the transfer of ribonucleic acids (RNAs) has been found to cause a change in the level of target protein expression. RNA-based transfection is conceptually more efficient than commonly delivered plasmid DNA because it does not require division or damage of the nuclear envelope, thereby increasing the chances of the cell remaining viable. Shock waves (SWs) have been found to induce cellular uptake by transiently altering the permeability of the plasma membrane, thereby overcoming a critical step in gene therapy. However, accompanying SW bio-effects include dose-dependent irreversible cell injury and cytotoxicity. Here, the effect of SWs generated by a clinical lithotripter on the viability and permeabilisation of three different cell lines in vitro was investigated. Comparison of RNA stability before and after SW exposure revealed no statistically significant difference. Optimal SW exposure parameters were identified to minimise cell death and maximise permeabilisation, and applied to enhanced green fluorescent protein (eGFP) messenger RNA (mRNA) or anti-eGFP small interfering RNA delivery. As a result, eGFP mRNA expression levels increased up to 52-fold in CT26 cells, whereas a 2-fold decrease in GFP expression was achieved after anti-eGFP small interfering RNA delivery to MCF-7/GFP cells. These results indicate that SW parameters can be employed to achieve effective nucleotide delivery, laying the foundation for non-invasive and high-tolerability RNA-based gene therapy.
通过核糖核酸（RNA）转移将基因导入细胞已被发现会导致靶蛋白表达水平发生变化。基于RNA的转染在概念上比常用的质粒DNA转染更有效，因为它不需要核膜分裂或损伤，从而增加了细胞保持存活的机会。已发现冲击波（SWs）可通过瞬时改变质膜通透性诱导细胞摄取，从而克服基因治疗中的关键步骤。然而，伴随的SW生物效应包括剂量依赖性不可逆细胞损伤和细胞毒性。在此，研究了临床碎石机产生的SWs对三种不同细胞系体外活力和通透性的影响。SW暴露前后RNA稳定性的比较显示无统计学显著差异。确定了最佳SW暴露参数以最小化细胞死亡并最大化通透性，并将其应用于增强型绿色荧光蛋白（eGFP）信使RNA（mRNA）或抗eGFP小干扰RNA递送。结果，在CT26细胞中eGFP mRNA表达水平提高了52倍，而在将抗eGFP小干扰RNA递送至MCF-7/GFP细胞后，GFP表达降低了2倍。这些结果表明，可以采用SW参数实现有效的核苷酸递送，为基于RNA的非侵入性和高耐受性基因治疗奠定基础。
