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

Publication ,  Journal Article
Mansfield, SG; Kole, J; Puttaraju, M; Yang, CC; Garcia-Blanco, MA; Cohn, JA; Mitchell, LG
Published in: Gene Ther
November 2000

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 (deltaF508) 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.

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Published In

Gene Ther

DOI

ISSN

0969-7128

Publication Date

November 2000

Volume

7

Issue

22

Start / End Page

1885 / 1895

Location

England

Related Subject Headings

  • Transfection
  • Spliceosomes
  • Reverse Transcriptase Polymerase Chain Reaction
  • RNA Splice Sites
  • RNA Precursors
  • Lipids
  • Kidney
  • Humans
  • Genetic Therapy
  • Genetic Engineering
 

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Mansfield, S. G., Kole, J., Puttaraju, M., Yang, C. C., Garcia-Blanco, M. A., Cohn, J. A., & Mitchell, L. G. (2000). Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing. Gene Ther, 7(22), 1885–1895. https://doi.org/10.1038/sj.gt.3301307
Mansfield, S. G., J. Kole, M. Puttaraju, C. C. Yang, M. A. Garcia-Blanco, J. A. Cohn, and L. G. Mitchell. “Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing.Gene Ther 7, no. 22 (November 2000): 1885–95. https://doi.org/10.1038/sj.gt.3301307.
Mansfield SG, Kole J, Puttaraju M, Yang CC, Garcia-Blanco MA, Cohn JA, et al. Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing. Gene Ther. 2000 Nov;7(22):1885–95.
Mansfield, S. G., et al. “Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing.Gene Ther, vol. 7, no. 22, Nov. 2000, pp. 1885–95. Pubmed, doi:10.1038/sj.gt.3301307.
Mansfield SG, Kole J, Puttaraju M, Yang CC, Garcia-Blanco MA, Cohn JA, Mitchell LG. Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing. Gene Ther. 2000 Nov;7(22):1885–1895.

Published In

Gene Ther

DOI

ISSN

0969-7128

Publication Date

November 2000

Volume

7

Issue

22

Start / End Page

1885 / 1895

Location

England

Related Subject Headings

  • Transfection
  • Spliceosomes
  • Reverse Transcriptase Polymerase Chain Reaction
  • RNA Splice Sites
  • RNA Precursors
  • Lipids
  • Kidney
  • Humans
  • Genetic Therapy
  • Genetic Engineering