Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing

S. G. Mansfield, J. Kole, M. Puttaraju, C. C. Yang, M. A. Garcia-Blanco, J. A. Cohn, L. G. Mitchell

Research output: Contribution to journalArticlepeer-review

86 Scopus citations


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 cotransfected 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.

Original languageEnglish (US)
Pages (from-to)1885-1895
Number of pages11
JournalGene Therapy
Issue number22
StatePublished - 2000
Externally publishedYes


  • CFTR protein
  • Gene repair
  • Gene therapy
  • Sequence deletion
  • Therapeutics
  • Trans-splicing

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics


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