Abstract
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.
Highlights
Bicistronic transgene expression is currently essential in gene therapy and biomedical research.The application of internal ribosome entry site (IRES) elements can co-express dual heterologous transgene products from a message RNA driven by a single promoter [1,2]
We further found that recombinant adeno-associated virus serotype 6 (rAAV6) vectors led to a ~10% transduction efficiency in the primary CD34+ hematopoietic stem cells (HSCs) and CD4+ T cells at an MOI of 10,000 vgs/cell (Figure 1C)
We investigated transgene expression when encephalomyocarditis virus (EMCV) IRES-gfp was integrated in the host genome by using a lentiviral system
Summary
The application of internal ribosome entry site (IRES) elements can co-express dual heterologous transgene products from a message RNA driven by a single promoter [1,2]. IRES elements that mimic the 5’ cap structure allow for translation in an RNA cap-independent manner [7,8]. Trans-acting factors vary with distinct IRES elements, resulting in different translational efficiencies based on cell types and cellular conditions [12]. One type of IRES element that is derived from encephalomyocarditis virus (EMCV) has been widely used for pharmaceutical and biomedical applications. It initiates a higher translation efficiency than other viral and non-viral IRES
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