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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Akopian AN et al. Trans-splicing of a voltage-gated sodium channel is regulated by nerve growth factor FEBS Lett 1999 445: 177–182
Kawasaki T et al. RNA maturation of the rice SPK gene may involve trans-splicing Plant J 1999 18: 625–632
Li BL et al. Human acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) gene organization and evidence that the 4.3-kilobase ACAT-1 mRNA is produced from two different chromosomes J Biol Chem 1999 274: 11060–11071
Zaphiropoulos PG . RNA molecules containing exons originating from different members of the cytochrome P450 2C gene subfamily (CYP2C) in human epidermis and liver Nucleic Acids Res 1999 27: 2585–2590
Caudevilla C et al. Natural trans-splicing in carnitine octanoyltransferase pre-mRNAs in rat liver Proc Natl Acad Sci USA 1998 95: 12185–12190
Malek O, Brennicke A, Knoop V . Evolution of trans-splicing plant mitochondrial introns in pre-Permian times Proc Natl Acad Sci USA 1997 94: 553–558
Davis RE et al. RNA trans-splicing in flatworms J Biol Chem 1995 270: 21813–21819
Eul J, Graessmann M, Graessmann A . Experimental evidence for RNA trans-splicing in mammalian cells EMBO J 1995 14: 3226–3235
Rajkovic A, Davis RE, Simonsen JN, Rottman FM . A spliced leader is present on a subset of mRNAs from the human parasite Schistosoma mansoni Proc Natl Acad Sci USA 1990 87: 8879–8883
Puttaraju M et al. Spliceosome-mediated RNA trans-splicing as a tool for gene therapy Nature Biotech 1999 17: 246–252
Davis PB, Drumm M, Konstan MW . Cystic fibrosis Am J Respir Crit Care Med 1996 154: 1229–1256
Welsh MJ, Smith AE . Molecular mechanism of CFTR channel dysfunction in cystic fibrosis Cell 1993 73: 1251–1254
Zielenski J, Tsui L-C . Cystic fibrosis: genotypic and phenotypic variations Annu Rev Genet 1995 29: 777–807
Riordan JR et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA Science 1989 245: 1066–1072
Kartner N et al. Mislocalization of delta F508 CFTR in cystic fibrosis sweat gland Nature Genet 1992 1: 321–327
Olsen JC et al. Correction of the apical membrane chloride permeability defect in polarized cystic fibrosis airway epithelia following retroviral-mediated gene transfer Hum Gene Ther 1992 3: 253–266
Johnson LG et al. Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis Nat Genet 1992 2: 21–25
Rich DP et al. Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells Nature 1990 347: 358–363
Engelhardt JF, Wilson JM . Gene therapy of cystic fibrosis lung disease J Pharm Pharmacol 1992 1: 165–167
Engelhardt JF, Yankaskas JR, Wilson JM . In vivo retroviral gene transfer into human bronchial epithelia of xenografts J Clin Invest 1992 90: 2598–2607
Hyde SC . Correction of the ion transport defect in cystic fibrosis transgenic mice by gene therapy Nature 1993 362: 250–255
Grubb BR . Inefficient gene transfer by adenovirus vector to cystic fibrosis airway epithelia of mice and humans Nature 1994 371: 802–806
Harvey B-G et al. Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus J Clin Invest 1999 104: 1245–1255
Zabner J et al. Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis Cell 1993 75: 207–216
Wagner JA et al. Efficient and persistent gene transfer of AAV-CFTR in maxillary sinus Lancet 1998 351: 1702–1703
Cohn J et al. Characterization of the cystic fibrosis transmembrane conductance regulator in a colonocyte cell line Proc Natl Acad Sci USA 1992 89: 2340–2344
Yang Y et al. Molecular basis of defective anion transport in L cells expressing recombinant forms of CFTR Hum Mol Genet 1993 2: 1253–1261
Zarrinkar PP, Sullenger BA . Optimizing the substrate specificity of a group I intron ribozyme Biochemistry 1999 38: 3426–3432
Kren BT, Metz R, Kumar R, Steer CJ . Gene repair using chimeric RNA/DNA oligonucleotides Semin Liver Dis 1999 19: 93–104
Friedman KJ et al. Correction of aberrant splicing of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by antisense oligonucleotides J Biol Chem 1999 274: 36193–36199
Goncz KK, Kunzelmann K, Xu Z, Gruenert DC . Targeted replacement of normal and mutant CFTR sequences in human airway epithelial cells using DNA fragments Hum Mol Genet 1998 7: 1913–1919
Flotte TR, Carter BJ . Adeno-associated virus vectors for gene therapy of cystic fibrosis Meth Enzymol 1998 292: 717–732
Zhang L et al. Efficient expression of CFTR function with adeno-associated virus vectors that carry shortened CFTR genes Proc Natl Acad Sci USA 1998 95: 10158–10163
Wang D et al. Efficient CFTR expression from AAV vectors packaged with promoters – the second generation Gene Therapy 1999 6: 667–675
Chow YH et al. Development of an epithelium-specific expression cassette with human DNA regulatory elements for transgene expression in lung airways Proc Natl Acad Sci USA 1997 94: 14695–14700
Schiavi SC et al. Biosynthetic and growth abnormalities are associated with high-level expression of CFTR in heterologous cells Am J Physiol 1996 270: C341–C351
Mohammad-Panah R et al. Hyperexpression of recombinant CFTR in heterologous cells alters its physiological properties Am J Physiol 1998 274: C310–C318
Mathews DH, Sabina J, Zuker M, Turner DH . Expanded sequence dependence of thermodynamic parameters provides robust prediction of RNA secondary structure J Mol Biol 1999 288: 911–940
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.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.gt.3301307
Keywords
This article is cited by
-
Construction and validation of an RNA trans-splicing molecule suitable to repair a large number of COL7A1 mutations
Gene Therapy (2016)
-
A Gene Gun-mediated Nonviral RNA trans-splicing Strategy for Col7a1 Repair
Molecular Therapy - Nucleic Acids (2016)
-
Repair of Rhodopsin mRNA by Spliceosome-Mediated RNA Trans -Splicing: A New Approach for Autosomal Dominant Retinitis Pigmentosa
Molecular Therapy (2015)
-
Trans-splicing repair of mutant p53 suppresses the growth of hepatocellular carcinoma cells in vitro and in vivo
Scientific Reports (2015)
-
Replacement of huntingtin exon 1 by trans-splicing
Cellular and Molecular Life Sciences (2012)