Prevention of interferon-stimulated gene expression using microRNA-designed hairpins
Prevention of interferon-stimulated gene expression using microRNA-designed hairpins
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DOI：
10.1038/gt.2008.123
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发表时间：
2009-01-01
期刊：
影响因子：
5.1
通讯作者：
Ueffing, M.
中科院分区：
文献类型：
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作者：
Bauer, M.;Kinkl, N.;Ueffing, M.
RNA干扰允许选择性基因沉默，并广泛用于脊椎动物细胞中个体基因的功能分析，并且代表了治疗中枢神经系统疾病的有吸引力的治疗选择。然而，越来越多的证据表明，短发夹 RNA (shRNA) 的表达可以触发细胞免疫反应，导致非特异性细胞表型和严重的副作用。我们发现，慢病毒载体（LV）介导的原代皮质培养物中shRNA的表达导致干扰素刺激的基因寡聚腺苷酸合成酶1（Oas1）的强烈表达，同时伴随着细胞凋亡加速和大量的净神经元损失。通过实现天然存在的 microRNA30 (miR-30) 前体的特征来修饰 shRNA 构建体，避免了转导原代培养物中的 Oas1 诱导，从而过客链的修饰似乎是规避干扰素刺激的基因表达的关键特征。这项工作代表了第一个实验研究，表明基于 miR-30 的 shRNA 构建体可以防止 Oas1 通路相关的脱靶效应，我们认为这是 shRNA 在未来基因治疗方法中使用的重要先决条件。
RNA interference allows selective gene silencing, and is widely used for functional analysis of individual genes in vertebrate cells and represents an attractive therapeutic option for treating central nervous system diseases. However, growing evidence exists that the expression of short hairpin RNAs (shRNAs) can trigger cellular immune response resulting in unspecific cellular phenotypes and severe side effects. We found that lentiviral vector (LV)mediated expression of shRNAs in primary cortical cultures resulted in strong expression of the interferon-stimulated gene oligoadenylate synthetase 1 (Oas1), which was accompanied by accelerated apoptosis and substantial net neuron loss. Modification of the shRNA construct by implementing features of the naturally occurring microRNA30 (miR-30) precursor avoided Oas1 induction in transduced primary cultures, whereby modification of the passenger strand seems to be a crucial feature to circumvent interferon-stimulated gene expression. This work represents the first experimental study showing that an miR-30-based shRNA construct prevents Oas1 pathway associated off-target effects, which we consider as an essential prerequisite for shRNA use in future gene therapeutic approaches.