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

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

Research output: Contribution to journalArticle

78 Citations (Scopus)

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 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
Volume7
Issue number22
StatePublished - 2000
Externally publishedYes

Fingerprint

Trans-Splicing
Spliceosomes
Cystic Fibrosis Transmembrane Conductance Regulator
RNA Precursors
Messenger RNA
Exons
Inborn Genetic Diseases
Introns
Phosphopeptides
HEK293 Cells
Feasibility Studies
Therapeutics
Regulator Genes
Cystic Fibrosis
Proteins
Western Blotting
Phosphorylation
RNA
Technology
Mutation

Keywords

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

ASJC Scopus subject areas

  • Genetics

Cite this

Mansfield, S. G., Kole, J., Puttaraju, M., Yang, C. C., Garcia-Blanco, M., Cohn, J. A., & Mitchell, L. G. (2000). Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing. Gene Therapy, 7(22), 1885-1895.

Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing. / Mansfield, S. G.; Kole, J.; Puttaraju, M.; Yang, C. C.; Garcia-Blanco, Mariano; Cohn, J. A.; Mitchell, L. G.

In: Gene Therapy, Vol. 7, No. 22, 2000, p. 1885-1895.

Research output: Contribution to journalArticle

Mansfield, SG, Kole, J, Puttaraju, M, Yang, CC, Garcia-Blanco, M, Cohn, JA & Mitchell, LG 2000, 'Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing', Gene Therapy, vol. 7, no. 22, pp. 1885-1895.
Mansfield SG, Kole J, Puttaraju M, Yang CC, Garcia-Blanco M, Cohn JA et al. Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing. Gene Therapy. 2000;7(22):1885-1895.
Mansfield, S. G. ; Kole, J. ; Puttaraju, M. ; Yang, C. C. ; Garcia-Blanco, Mariano ; Cohn, J. A. ; Mitchell, L. G. / Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing. In: Gene Therapy. 2000 ; Vol. 7, No. 22. pp. 1885-1895.
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