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Expression and maturation of Sendai virus vector-derived CFTR protein: functional and biochemical evidence using a GFP-CFTR fusion protein

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Abstract

Sendai virus (SeV) vector has been shown to efficiently transduce airway epithelial cells. As a precursor to the potential use of this vector for cystic fibrosis (CF) gene therapy, the correct maturation of the SeV vector-derived CF transmembrane conductance regulator (CFTR) protein was examined using biochemical and functional analyses. We constructed a recombinant SeV vector, based on the fusion (F) gene-deleted non-transmissible SeV vector, carrying the GFP-CFTR gene in which the N terminus of CFTR was fused to green fluorescence protein (GFP). This vector was recovered and propagated to high titers in the packaging cell line. Western blotting using an anti-GFP antibody detected both the fully glycosylated (mature) and the core-glycosylated (immature) proteins, indicating that SeV vector-derived GFP-CFTR was similar to endogenous CFTR. We also confirmed the functional channel activity of GFP-CFTR in an iodide efflux assay. The efficient expression of GFP-CFTR, and its apical surface localization, were observed in both MDCK cells in vitro, and in the nasal epithelium of mice in vivo. We concluded that recombinant SeV vector, a cytoplasmically maintained RNA vector, is able to direct production of a correctly localized, mature form of CFTR, suggesting the value of this vector for studies of CF gene therapy.

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Acknowledgements

We acknowledge B Moss for supplying vTF7-3; H Iba for supplying pCALNdLw; H Taira for supplying anti-F antibody; T Kanaya, K Washizawa, S Fujikawa and N Kurosawa for their excellent technical assistance. Members of the UK CF Gene Therapy Consortium are funded by the Cystic Fibrosis Trust.

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Authors and Affiliations

  1. DNAVEC Corporation, Tsukuba-shi, Ibaraki, Japan

    H Ban, M Inoue, A Iida & M Hasegawa

  2. Department of Gene Therapy, National Heart and Lung Institute, Imperial College, London, UK

    U Griesenbach, F Munkonge, M Chan & E W F W Alton

  3. UK Cystic Fibrosis Gene Therapy Consortium,

    U Griesenbach, F Munkonge, M Chan & E W F W Alton

Authors
  1. H Ban
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  2. M Inoue
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  3. U Griesenbach
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  4. F Munkonge
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  5. M Chan
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  6. A Iida
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  7. E W F W Alton
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  8. M Hasegawa
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Correspondence to M Inoue.

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Ban, H., Inoue, M., Griesenbach, U. et al. Expression and maturation of Sendai virus vector-derived CFTR protein: functional and biochemical evidence using a GFP-CFTR fusion protein. Gene Ther 14, 1688–1694 (2007). https://doi.org/10.1038/sj.gt.3303032

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  • DOI: https://doi.org/10.1038/sj.gt.3303032

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