Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Department of Pediatrics, Medical University of Vienna, Austria.
Antiviral Res. 2013 Jan;97(1):10-23. doi: 10.1016/j.antiviral.2012.10.008. Epub 2012 Nov 2.
Human adenoviruses are rarely associated with life-threatening infections in healthy individuals. However, immunocompromised patients, and particularly allogeneic hematopoietic stem cell transplant recipients, are at high risk of developing disseminated and potentially fatal disease. The efficacy of commonly used drugs to treat adenovirus infections (i.e., cidofovir in most cases) is limited, and alternative treatment options are needed. Artificial microRNAs (amiRNAs) are a class of synthetic RNAs resembling cellular miRNAs, and, similar to their natural relatives, can mediate the knockdown of endogenous gene expression. This process, termed RNA interference, can be harnessed to target and potentially silence both cellular and viral genes. In this study, we designed amiRNAs directed against adenoviral E1A, DNA polymerase, and preterminal protein (pTP) mRNAs in order to inhibit adenoviral replication in vitro. For the expression of amiRNA-encoding sequences, we utilized replication-deficient adenoviral vectors. In cells transduced with the recombinant vectors and infected with the wild-type (wt) adenovirus, one particular amiRNA that was directed against the pTP mRNA was capable of decreasing the output of infectious wt virus progeny by 2.6 orders of magnitude. This inhibition rate could be achieved by concatemerizing amiRNA-encoding sequences to allow for high intracellular amiRNA concentrations. Because superinfecting wt virus induces the replication and amplification of the recombinant adenoviral vector, amiRNA concentrations were increased in cells infected with wt adenovirus. Furthermore, a combination of amiRNA expression and treatment of infected cells with cidofovir resulted in additive effects that manifested as a total reduction of infectious virus progeny by greater than 3 orders of magnitude.
人类腺病毒很少与健康个体的危及生命的感染有关。然而，免疫功能低下的患者，特别是异基因造血干细胞移植受者，有发生弥散性和潜在致命疾病的高风险。目前常用的治疗腺病毒感染的药物（即在大多数情况下使用西多福韦）疗效有限，因此需要替代治疗方案。人工 microRNA（amiRNA）是一类类似于细胞 microRNA 的合成 RNA，与天然 microRNA 类似，可介导内源性基因表达的下调。这种过程称为 RNA 干扰，可以用来靶向并可能沉默细胞和病毒基因。在这项研究中，我们设计了针对腺病毒 E1A、DNA 聚合酶和末端前蛋白（pTP）mRNA 的 amiRNA，以抑制腺病毒在体外的复制。为了表达 amiRNA 编码序列，我们利用了复制缺陷型腺病毒载体。在转导了重组载体并感染野生型（wt）腺病毒的细胞中，一种针对 pTP mRNA 的特定 amiRNA 能够使感染性 wt 病毒的产量降低 2.6 个数量级。通过串联 amiRNA 编码序列以允许细胞内高浓度的 amiRNA 来实现这种抑制率。由于超感染 wt 病毒会诱导重组腺病毒载体的复制和扩增，因此在感染 wt 腺病毒的细胞中，amiRNA 浓度增加。此外，amiRNA 表达与用西多福韦处理感染细胞相结合，产生了相加的效果，导致感染性病毒的产量完全减少了 3 个以上数量级。
