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Partial correction of endogenous ΔF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing

Abstract

Spliceosome-mediated RNA trans-splicing (SMaRT) was investigated as a means for functionally correcting endogenous ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) transcripts using in vitro human cystic fibrosis (CF) polarized airway epithelia and in vivo human CF bronchial xenografts. Recombinant adenovirus (Ad.CFTR-PTM) encoding a pre-therapeutic molecule (PTM) targeted to CFTR intron 9 corrected transepithelial cyclic AMP (cAMP)-sensitive short-circuit current (Isc) in ΔF508 homozygous epithelia to a level 16% of that observed in normal human bronchial epithelia. Molecular analyses using RT-PCR and western blotting confirmed SMaRT-mediated partial correction of endogenous ΔF508 messenger RNA (mRNA) transcripts and protein. In an in vivo model of ΔF508 CF airway epithelia, human CF bronchial xenografts infected with Ad.CFTR-PTM also demonstrated partial correction of CFTR-mediated Cl permeability at a level 22% of that seen in non-CF xenografts. These results provide functional evidence for SMaRT-mediated repair of mutant endogenous CFTR mRNA in intact polarized CF airway epithelial models.

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Figure 1: Schematic diagrams of CFTR PTMs and pre-mRNA target.
Figure 2: Transepithelial short-circuit currents in a ΔF508-homozygous CF model of polarized airway epithelia.
Figure 3: Evaluation of RNA trans-splicing repair by allele-specific RT-PCR and allele-specific oligonucleotide hybridization.
Figure 4: Immunodetection of trans-spliced CFTR protein.
Figure 5: In vivo complementation of CFTR Cl permeability defects in ΔF508/ΔF508 CF bronchial xenografts.

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Acknowledgements

This work was supported by SBIR grant R44 DK56526 from the National Institutes of Health (L.G.M.) and a grant from the Cystic Fibrosis Foundation (S.G.M.) to Intronn, and by the Iowa Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases co-funded by the National Institutes of Health and the Cystic Fibrosis Foundation (P30 DK54759; J.F.E.). The corresponding author has no financial interests in Intronn Inc. and does not consult for this company.

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Correspondence to John F. Engelhardt.

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Liu, X., Jiang, Q., Mansfield, S. et al. Partial correction of endogenous ΔF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing. Nat Biotechnol 20, 47–52 (2002). https://doi.org/10.1038/nbt0102-47

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