Center of Biomedical Research, Faculty of Health Sciences, Universidad del Quindío, Armenia 630003, Colombia.
Molecular Biology and Virology Laboratory, Faculty of Medicine and Health Sciences, Corporación Universitaria Empresarial Alexander Von Humboldt, Armenia 630003, Colombia.
Viruses. 2022 Feb 7;14(2):339. doi: 10.3390/v14020339.
Dengue virus is a ssRNA+ flavivirus, which produces the dengue disease in humans. Currently, no specific treatment exists. siRNAs regulate gene expression and have been used systematically to silence viral genomes; however, they require controlled release. Liposomes show favorable results encapsulating siRNA for gene silencing. The objective herein was to design and evaluate in vitro siRNAs bound to liposomes that inhibit DENV replication. siRNAs were designed against DENV1-4 from conserved regions using siDirect2.0 and Web-BLOCK-iT™ RNAiDesigner; the initial in vitro evaluation was carried out through transfection into HepG2 cells. siRNA with silencing capacity was encapsulated in liposomes composed of D-Lin-MC3-DMA, DSPC, Chol. Cytotoxicity, hemolysis, pro-inflammatory cytokine release and antiviral activity were evaluated using plaque assay and RT-qPCR. A working concentration of siRNA was established at 40 nM. siRNA1, siRNA2, siRNA3.1, and siRNA4 were encapsulated in liposomes, and their siRNA delivery through liposomes led to a statistically significant decrease in viral titers, yielded no cytotoxicity or hemolysis and did not stimulate release of pro-inflammatory cytokines. Finally, liposomes were designed with siRNA against DENV, which proved to be safe in vitro.
登革热病毒是一种单股正链 RNA 黄病毒，可导致人类罹患登革热疾病。目前，尚无特效疗法。小干扰 RNA（siRNA）可调控基因表达，已被系统地用于沉默病毒基因组，但需要进行受控释放。脂质体在包封 siRNA 以实现基因沉默方面显示出良好的效果。本研究旨在设计并评估体外结合脂质体的 siRNAs，以抑制 DENV 的复制。使用 siDirect2.0 和 Web-BLOCK-iT™ RNAiDesigner 针对 DENV1-4 的保守区域设计 siRNA；通过转染 HepG2 细胞进行初步的体外评估。将具有沉默能力的 siRNA 包封于由 D-Lin-MC3-DMA、DSPC 和胆固醇组成的脂质体中。通过噬斑试验和 RT-qPCR 评估细胞毒性、溶血、促炎细胞因子释放和抗病毒活性。确定 40 nM 为工作浓度。将 siRNA1、siRNA2、siRNA3.1 和 siRNA4 包封于脂质体中，通过脂质体递送 siRNA 可显著降低病毒滴度，无细胞毒性或溶血作用，也不会刺激促炎细胞因子的释放。最后，设计了针对 DENV 的 siRNA 脂质体，体外实验证明其安全。
