Reversal of Cystic Fibrosis Phenotype in a Cultured Delta508 Cystic Fibrosis Transmembrane Conductance Regulator Cell Line by Oligonucleotide Insertion

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2004 May 19

PMID 15148387

Citations 15

Authors

Paul C Zamecnik

Malay K Raychowdhury

David R Tabatadze

Horacio F Cantiello

Affiliations

Soon will be listed here.

Abstract

Cystic fibrosis (CF) is a lethal genetic disorder that is due to mutations in the gene encoding the cAMP-activated anion CF transmembrane conductance regulator (CFTR) channel. A three-nucleotide base deletion (TTT), encoding phenylalanine in position 508 of the translatable CFTR sequence (accompanied by a C to T replacement immediately 5' to the deletion), accounts for approximately 75% of cases of the disease. In the present study, an oligonucleotide complex (CF4-CF6, 2'-0-methyl RNA-unmodified RNA oligonucleotide duplex, respectively) was used to restore CFTR function by insertion of missing bases in Delta508 CFTR mRNA from a cultured (Delta508) cell line. cAMP-activated whole-cell currents and Cl- transport were detected in CF4-CF6-treated, but not control Delta508, cells by patch-clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence (SPQ) quenching analyses, respectively. Further, the nucleotide addition in the deleted region of Delta508 CFTR was determined after amplification by RT-PCR. Insertion of UGU and replacement of U by C immediately 5' to the deletion site in Delta508 mRNA appear to have taken place, with phenotypic but not genotypic reversion in tissue culture of treated cells. The mechanism of insertion of nucleotides has yet to be determined.

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References

Skerra A . Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity. Nucleic Acids Res. 1992; 20(14):3551-4. PMC: 334000. DOI: 10.1093/nar/20.14.3551. View

Kandimalla E, Zhu F, Bhagat L, Yu D, Agrawal S . Toll-like receptor 9: modulation of recognition and cytokine induction by novel synthetic CpG DNAs. Biochem Soc Trans. 2003; 31(Pt 3):654-8. DOI: 10.1042/bst0310654. View

Denning G, Anderson M, Amara J, Marshall J, Smith A, Welsh M . Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature. 1992; 358(6389):761-4. DOI: 10.1038/358761a0. View

Stephenson M, Zamecnik P . Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc Natl Acad Sci U S A. 1978; 75(1):285-8. PMC: 411231. DOI: 10.1073/pnas.75.1.285. View

Liu X, Jiang Q, Mansfield S, Puttaraju M, Zhang Y, Zhou W . Partial correction of endogenous DeltaF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing. Nat Biotechnol. 2001; 20(1):47-52. DOI: 10.1038/nbt0102-47. View

Verkman A . Development and biological applications of chloride-sensitive fluorescent indicators. Am J Physiol. 1990; 259(3 Pt 1):C375-88. DOI: 10.1152/ajpcell.1990.259.3.C375. View

Pasyk E, Foskett J . Mutant (delta F508) cystic fibrosis transmembrane conductance regulator Cl- channel is functional when retained in endoplasmic reticulum of mammalian cells. J Biol Chem. 1995; 270(21):12347-50. DOI: 10.1074/jbc.270.21.12347. View

LETSINGER R, Elghanian R, Viswanadham G, Mirkin C . Use of a steroid cyclic disulfide anchor in constructing gold nanoparticle-oligonucleotide conjugates. Bioconjug Chem. 2000; 11(2):289-91. DOI: 10.1021/bc990152n. View

Brown C, Biwersi J, Verkman A, Welch W . Chemical chaperones correct the mutant phenotype of the delta F508 cystic fibrosis transmembrane conductance regulator protein. Cell Stress Chaperones. 1996; 1(2):117-25. PMC: 248464. DOI: 10.1379/1466-1268(1996)001<0117:ccctmp>2.3.co;2. View

10.

Riordan J, Rommens J, Kerem B, Alon N, Rozmahel R, Grzelczak Z . Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989; 245(4922):1066-73. DOI: 10.1126/science.2475911. View

11.

Li C, Ramjeesingh M, Reyes E, Jensen T, Chang X, Rommens J . The cystic fibrosis mutation (delta F508) does not influence the chloride channel activity of CFTR. Nat Genet. 1993; 3(4):311-6. DOI: 10.1038/ng0493-311. View

12.

Taubes G . Gene therapy. The strange case of chimeraplasty. Science. 2002; 298(5601):2116-20. DOI: 10.1126/science.298.5601.2116. View

13.

Zamecnik P, Stephenson M . Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci U S A. 1978; 75(1):280-4. PMC: 411230. DOI: 10.1073/pnas.75.1.280. View

14.

Bedwell D, Kaenjak A, Benos D, Bebok Z, Bubien J, Hong J . Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line. Nat Med. 1997; 3(11):1280-4. DOI: 10.1038/nm1197-1280. View

15.

Dechecchi M, Tamanini A, Berton G, Cabrini G . Protein kinase C activates chloride conductance in C127 cells stably expressing the cystic fibrosis gene. J Biol Chem. 1993; 268(15):11321-5. View

16.

Wang H, Yang Y, Schofield M, Du C, Fridman Y, Lee S . DNA bending and unbending by MutS govern mismatch recognition and specificity. Proc Natl Acad Sci U S A. 2003; 100(25):14822-7. PMC: 299810. DOI: 10.1073/pnas.2433654100. View

17.

Cantiello H, Prat A, Reisin I, Ercole L, Abraham E, Amara J . External ATP and its analogs activate the cystic fibrosis transmembrane conductance regulator by a cyclic AMP-independent mechanism. J Biol Chem. 1994; 269(15):11224-32. View

18.

Padmapriya A, Tang J, Agrawal S . Large-scale synthesis, purification, and analysis of oligodeoxynucleotide phosphorothioates. Antisense Res Dev. 1994; 4(3):185-99. DOI: 10.1089/ard.1994.4.185. View

19.

Yang X, Otero F, Skene R, McRee D, Schimmel P, Ribas de Pouplana L . Crystal structures that suggest late development of genetic code components for differentiating aromatic side chains. Proc Natl Acad Sci U S A. 2003; 100(26):15376-80. PMC: 307575. DOI: 10.1073/pnas.2136794100. View

20.

Taton T, Mirkin C, LETSINGER R . Scanometric DNA array detection with nanoparticle probes. Science. 2000; 289(5485):1757-60. DOI: 10.1126/science.289.5485.1757. View