State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200438, China.
Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Viruses. 2019 Oct 8;11(10):920. doi: 10.3390/v11100920.
Bicistronic transgene expression mediated by internal ribosome entry site (IRES) elements has been widely used. It co-expresses heterologous transgene products from a message RNA driven by a single promoter. Hematologic gene delivery is a promising treatment for both inherited and acquired diseases. A combined strategy was recently documented for potential genome editing in hematopoietic cells. A transduction efficiency exceeding ~90% can be achieved by capsid-optimized recombinant adeno-associated virus serotype 6 (rAAV6) vectors. In this study, to deliver an encephalomyocarditis virus (EMCV) IRES-containing rAAV6 genome into hematopoietic cells, we observed that EMCV IRES almost completely shut down the transgene expression during the process of mRNA-protein transition. In addition, position-dependent behavior was observed, in which only the EMCV IRES element located between a promoter and the transgenes had an inhibitory effect. Although further studies are warranted to evaluate the involvement of cellular translation machinery, our results propose the use of specific IRES elements or an alternative strategy, such as the 2A system, to achieve bicistronic transgene expression in hematopoietic cells.
双顺反子转基因表达由内部核糖体进入位点（IRES）元件介导，已被广泛应用。它可从单个启动子驱动的信使 RNA 共表达异源转基因产物。血液基因传递是治疗遗传性和获得性疾病的一种很有前途的方法。最近的一项联合策略记录了用于造血细胞的潜在基因组编辑。通过衣壳优化的重组腺相关病毒血清型 6(rAAV6)载体，可实现超过~90%的转导效率。在这项研究中，为了将含有脑炎心肌炎病毒（EMCV）IRES 的 rAAV6 基因组递送到造血细胞中，我们观察到 EMCV IRES 在 mRNA-蛋白转换过程中几乎完全关闭了转基因的表达。此外，还观察到位置依赖性行为，其中只有位于启动子和转基因之间的 EMCV IRES 元件具有抑制作用。虽然需要进一步的研究来评估细胞翻译机制的参与，但我们的结果表明，在造血细胞中实现双顺反子转基因表达时，应使用特定的 IRES 元件或替代策略，如 2A 系统。
