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  • Research Article
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Self-inactivating retroviral vectors with improved RNA processing

Abstract

Three RNA features have been identified that elevate retroviral transgene expression: an intron in the 5′ untranslated region (5′UTR), the absence of aberrant translational start codons and the presence of the post-transcriptional regulatory element (PRE) of the woodchuck hepatitis virus in the 3′UTR. To include such elements into self-inactivating (SIN) vectors with potentially improved safety, we excised the strong retroviral promoter from the U3 region of the 3′ long terminal repeat (LTR) and inserted it either downstream or upstream of the retroviral RNA packaging signal (Ψ). The latter concept is new and allows the use of an intron in the 5′UTR, taking advantage of retroviral splice sites surrounding Ψ. Three LTR and four SIN vectors were compared to address the impact of RNA elements on titer, splice regulation and transgene expression. Although titers of SIN vectors were about 20-fold lower than those of their LTR counterparts, inclusion of the PRE allowed production of more than 106 infectious units per ml without further vector optimizations. In comparison with state-of-the-art LTR vectors, the intron-containing SIN vectors showed greatly improved splicing. With regard to transgene expression, the intron-containing SIN vectors largely matched or even exceeded the LTR counterparts in all cell types investigated (embryonic carcinoma cells, fibroblasts, primary T cells and hematopoietic progenitor cells).

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Acknowledgements

This work was supported by grants from European Community (QLK3-2001-01265, QLRT-2001-00427) and the DFG (BA 1837/4-1). We thank Andreas Rimek for flow-cytometry sorting of the EGFP+ mass cultures of transduced cells and Cordula Grüttner for her technical assistance.

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Kraunus, J., Schaumann, D., Meyer, J. et al. Self-inactivating retroviral vectors with improved RNA processing. Gene Ther 11, 1568–1578 (2004). https://doi.org/10.1038/sj.gt.3302309

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