Spanevello Francesca, Calistri Arianna, Del Vecchio Claudia, Mantelli Barbara, Frasson Chiara, Basso Giuseppe, Palù Giorgio, Cavazzana Marina, Parolin Cristina
Department of Molecular Medicine, University of Padova, Padova, Italy.
Oncohematology Laboratory, Department of Women's and Children's Health, University of Padova, Padova, Italy.
Mol Ther Nucleic Acids. 2016 Apr 19;5(4):e312. doi: 10.1038/mtna.2016.24.
Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1) were developed. The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combinatorial vectors in interfering with viral replication was evaluated in human primary CD4(+) T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application.
基因治疗在功能性治愈HIV-1感染方面具有巨大潜力，在这种背景下，基于RNA干扰（RNAi）的方法是强有力的策略。稳定表达靶向HIV基因或细胞辅助因子的小干扰RNA（siRNA）有可能使HIV-1易感细胞对感染产生抗性。为了抑制病毒生命周期的不同步骤，开发了在不同RNA聚合酶III启动子（U6、7SK、H1）控制下表达多种靶向CCR5细胞基因以及vif和tat/rev病毒转录本的siRNA的自失活慢病毒载体。还研究了使用单个RNA聚合酶III启动子驱动一个产生针对选定靶点的三种siRNA的序列（e-shRNA）的表达。采用荧光素酶测定法和抑制HIV-1在人Jurkat T细胞系中的复制来选择启动子/siRNA的最佳组合。在人原代CD4(+) T淋巴细胞中评估了所选开发的组合载体干扰病毒复制的效果。我们鉴定出两种有效的抗HIV组合载体，它们能提供针对R5和X4嗜性病毒的保护。总体而言，我们的结果表明抗病毒效果受不同因素影响，包括用于表达RNAi分子的启动子和所选的盒式组合。这些发现有助于进一步深入了解基于RNAi的针对HIV-1的基因治疗方法用于临床应用的设计。
