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  • Inherited Disease
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Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing

Gene Therapy volume 7, pages 1885–1895 (2000)Cite this article

Abstract

Most messenger RNA precursors (pre-mRNA) undergo cis-splicing in which introns are excised and the adjoining exons from a single pre-mRNA are ligated together to form mature messenger RNA. This reaction is driven by a complex known as the spliceosome. Spliceosomes can also combine sequences from two independently transcribed pre-mRNAs in a process known as trans-splicing. Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology in which RNA pre-therapeutic molecules (PTMs) are designed to recode a specific pre-mRNA by suppressing cis-splicing while enhancing trans-splicing between the PTM and its pre-mRNA target. This study examined the feasibility of SMaRT as a potential therapy for genetic diseases to correct mutations using cystic fibrosis (CF) as an example. We used several versions of a cystic fibrosis transmembrane conductance regulator (CFTR) mini-gene expressing mutant (ΔF508) pre-mRNA targets and tested this against a number of PTMs capable of binding to the CFTR target intron 9 and trans-splicing in the normal coding sequences for exons 10–24 (containing F508). When 293T cells were cotransfected with both constructs, they produced a trans-spliced mRNA in which normal exon 10–24 replaced mutant exon 10. To test whether SMaRT produced mature CFTR protein, proteins were immunoprecipitated from lysates of co- transfected cells and detected by Western blotting and PKA-phosphorylation. Tryptic phosphopeptide mapping confirmed the identity of CFTR. This proof-of-concept study demonstrates that exon replacement by SMaRT can repair an abnormal pre-mRNA associated with a genetic disease.

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Acknowledgements

MAG-B is also Associate Professor of Genetics, Microbiology and Medicine at Duke University Medical Center. This work was funded by Proteome Sciences, plc, in the United Kingdom, and by an SBIR NIH grant (No. 1 R43 DK56526-01). Additional support was provided by a NIH R01 grant (1R01-DK54023). We wish to thank Richard C Boucher, Larry G Johnson, and R Jude Samulski (Centers of Cystic Fibrosis and Pulmonary Research, and of Gene Therapy, UNC-Chapel Hill, Chapel Hill, NC, USA) for helpful suggestions. We also wish to thank Russell Nugent for proofreading the manuscript.

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Authors and Affiliations

  1. Intronn, LLC, Durham, NC, USA

    S G Mansfield, M Puttaraju, C C Yang, M A Garcia-Blanco & L G Mitchell

  2. Department of Medicine, Duke University and VA Medical Centers, Durham, NC, USA

    J Kole & J A Cohn

  3. Department of Genetics, Duke University and VA Medical Centers, Durham, NC, USA

    M A Garcia-Blanco

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  1. S G Mansfield
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  2. J Kole
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  3. M Puttaraju
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  4. C C Yang
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  6. J A Cohn
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  7. L G Mitchell
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Mansfield, S., Kole, J., Puttaraju, M. et al. Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing. Gene Ther 7, 1885–1895 (2000). https://doi.org/10.1038/sj.gt.3301307

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