Department of Molecular and Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.
Hum Gene Ther. 2012 Nov;23(11):1200-8. doi: 10.1089/hum.2012.011. Epub 2012 Sep 18.
Combinational therapy with small RNA inhibitory agents against multiple viral targets allows efficient inhibition of viral production by controlling gene expression at critical time points. Here we explore combinations of different classes of therapeutic anti-HIV-1 RNAs expressed from within the context of an intronic MCM7 (minichromosome maintenance complex component-7) platform that naturally harbors 3 microRNAs (miRNAs). We replaced the endogenous miRNAs with anti-HIV small RNAs, including small interfering RNAs (siRNAs) targeting HIV-1 tat and rev messages that function to induce post-transcriptional gene silencing by the RNA interference pathway, a nucleolar-localizing RNA ribozyme that targets the conserved U5 region of HIV-1 transcripts for degradation, and finally nucleolar trans-activation response (TAR) and Rev-binding element (RBE) RNA decoys designed to sequester HIV-1 Tat and Rev proteins inside the nucleolus. We demonstrate the versatility of the MCM7 platform in expressing and efficiently processing the siRNAs as miRNA mimics along with nucleolar small RNAs. Furthermore, three of the combinatorial constructs tested potently suppressed viral replication during a 1-month HIV challenge, with greater than 5-log inhibition compared with untransduced, HIV-1-infected CEM T lymphocytes. One of the most effective constructs contains an anti-HIV siRNA combined with a nucleolar-localizing U5 ribozyme and TAR decoy. This represents the first efficacious example of combining Drosha-processed siRNAs with small nucleolar ribonucleoprotein (snoRNP)-processed nucleolar RNA chimeras from a single intron platform for effective inhibition of viral replication. Moreover, we demonstrated enrichment/selection for cells expressing levels of the antiviral RNAs that provide optimal inhibition under the selective pressure of HIV. The combinations of si/snoRNAs represent a new paradigm for combinatorial RNA-based gene therapy applications.
联合应用针对多种病毒靶点的小 RNA 抑制性药物通过在关键时间点控制基因表达来实现病毒生成的有效抑制。在此，我们探索了在天然含有 3 种 microRNA（miRNA）的内含子 MCM7（微小染色体维持复合物成分-7）平台内表达的不同类型的抗 HIV-1 RNA 药物联合应用。我们用抗 HIV 小 RNA 取代内源性 miRNA，包括靶向 HIV-1 tat 和 rev 信使的小干扰 RNA（siRNA），通过 RNA 干扰途径诱导转录后基因沉默，核仁定位的 RNA 核酶靶向 HIV-1 转录本的保守 U5 区域进行降解，最后是核仁转录激活反应（TAR）和 Rev 结合元件（RBE）RNA 诱饵，设计用于将 HIV-1 Tat 和 Rev 蛋白隔离在核仁内。我们证明了 MCM7 平台在表达和有效处理 siRNA 作为 miRNA 模拟物以及核仁小 RNA 方面的多功能性。此外，三种组合构建体在为期 1 个月的 HIV 挑战中强烈抑制病毒复制，与未经转导、HIV-1 感染的 CEM T 淋巴细胞相比，抑制率超过 5 个对数级。最有效的构建体之一包含抗 HIV siRNA 与核定位的 U5 核酶和 TAR 诱饵的组合。这代表了首次将 Drosha 处理的 siRNA 与来自单个内含子平台的小核仁核糖核蛋白（snoRNP）处理的核仁 RNA 嵌合体结合，用于有效抑制病毒复制的成功例子。此外，我们证明了在 HIV 的选择压力下，表达抗病毒 RNA 的细胞的富集/选择，为最佳抑制提供了所需的水平。si/snoRNA 的组合为基于 RNA 的组合基因治疗应用提供了一种新的范例。
