Department of Biochemistry and Physiology, School of Pharmacy and Food Sciences, Nanoscience and Nanotechnology Institute, IN2UB, University of Barcelona, 08028 Barcelona, Spain.
ICREA, Institute of Neurosciences at UAB, 08193 Bellaterra, Spain.
Int J Mol Sci. 2021 Sep 16;22(18):10025. doi: 10.3390/ijms221810025.
PolyPurine Reverse Hoogsteen Hairpins (PPRHs) are gene-silencing DNA-oligonucleotides developed in our laboratory that are formed by two antiparallel polypurine mirror repeat domains bound intramolecularly by Hoogsteen bonds. The aim of this work was to explore the feasibility of using viral vectors to deliver PPRHs as a gene therapy tool. After treatment with synthetic RNA, plasmid transfection, or viral infection targeting the gene, viability was determined by the MTT assay, mRNA was determined by RT-qPCR, and protein levels were determined by Western blot. We showed that the RNA-PPRH induced a decrease in cell viability in a dose-dependent manner and an increase in apoptosis in PC-3 and HeLa cells. Both synthetic RNA-PPRH and RNA-PPRH intracellularly generated upon the transfection of a plasmid vector were able to reduce survivin mRNA and protein levels in PC-3 cells. An adenovirus type-5 vector encoding the PPRH against was also able to decrease survivin mRNA and protein levels, leading to a reduction in HeLa cell viability. In this work, we demonstrated that PPRHs can also work as RNA species, either chemically synthesized, transcribed from a plasmid construct, or transcribed from viral vectors. Therefore, all these results are the proof of principle that viral vectors could be considered as a delivery system for PPRHs.
多聚嘌呤反向 Hoogsteen 发夹 (PPRHs) 是我们实验室开发的基因沉默 DNA 寡核苷酸，由两个反平行的多聚嘌呤镜像重复结构域通过 Hoogsteen 键在分子内结合形成。本研究旨在探索利用病毒载体将 PPRHs 作为基因治疗工具进行递送的可行性。在经过合成 RNA、质粒转染或针对 基因的病毒感染处理后，通过 MTT 测定法确定细胞活力，通过 RT-qPCR 测定 mRNA，通过 Western blot 测定蛋白水平。结果表明，RNA-PPRH 以剂量依赖的方式诱导细胞活力降低，并在 PC-3 和 HeLa 细胞中诱导凋亡增加。在 PC-3 细胞中，合成的 RNA-PPRH 和质粒载体转染后在细胞内产生的 RNA-PPRH 均能降低 survivin mRNA 和蛋白水平。携带针对 基因的 PPRH 的 5 型腺病毒载体也能降低 survivin mRNA 和蛋白水平，导致 HeLa 细胞活力降低。在这项工作中，我们证明了 PPRHs 也可以作为 RNA 发挥作用，无论是化学合成的、从质粒构建物转录的还是从病毒载体转录的。因此，所有这些结果都证明了病毒载体可以被认为是 PPRHs 的递送系统。
