Suitability of oligonucleotide-mediated cystic fibrosis gene repair in airway epithelial cells

David de Semir, Marga Nadal, Juan R. González, Sara Larriba, Anna Avinyó, Virginia Nunes, Teresa Casals, Xavier P. Estivill, Josep M. Aran

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Background: Non-viral vector-mediated targeted gene repair could become a useful alternative to classical gene addition strategies. The methodology guarantees a physiologically regulated and persistent expression of the repaired gene, with reported gene conversion and phenotypic correction efficiencies approaching 40-50% in some in vitro and in vivo models of disease. This is particularly important for cystic fibrosis (CF) because of its complex pathophysiology and the cellular heterogeneity of the cystic fibrosis transmembrane conductance regulator (CFTR) gene expression and function in the lung. Methods: A cell-free biochemical assay was applied to assess the ability of CF airway epithelial cells to support chimeraplast-mediated repair. In addition, a methodology allowing the relative quantification of the percentage of W1282X mutation repair in a heterozygous background using the PCR/oligonucleotide ligation assay (PCR/OLA) was developed. The performance of different chimeraplast and short single-stranded oligonucleotide structures delivered by non-viral vectors and electroporation was evaluated. Results: Chimeraplast-mediated repair competency was corroborated in GF airway epithelial cells. However, their repair activity was about 5-fold lower than that found in liver cells. Moreover, regardless of the corrector oligonucleotide structure applied to our GF bronchial epithelial cells, of compound heterozygous genotype (F508del/W1282X), the percentage of their resulting wild-type allele in the W1282X (exon 20) locus of the CFTR gene was not significantly different from that of the control untreated cells by our PCR/OLA assay (confidence interval at 95% ±4 allele wild-type). Conclusions: Oligonucleotide-mediated CFTR gene repair is an inefficient process in CF airway epithelial cells. Further improvements in oligonucleotide structure, nuclear delivery and/or the capability for mismatch repair stimulation will be necessary to achieve therapeutic levels of mutation correction in these cells.

Original languageEnglish
Pages (from-to)625-639
Number of pages15
JournalJournal of Gene Medicine
Volume5
Issue number7
DOIs
Publication statusPublished - 2003
Externally publishedYes

Fingerprint

Cystic Fibrosis
Oligonucleotides
Epithelial Cells
Cystic Fibrosis Transmembrane Conductance Regulator
Regulator Genes
Genes
Targeted Gene Repair
Ligation
Alleles
Gene Expression
Gene Conversion
Polymerase Chain Reaction
Mutation
DNA Mismatch Repair
Electroporation
Exons
Genotype
Confidence Intervals
Lung
Liver

Keywords

  • Chimeraplasts
  • Non-viral vectors
  • PCR/OLA
  • W1282X mutation correction

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Drug Discovery
  • Genetics(clinical)

Cite this

de Semir, D., Nadal, M., González, J. R., Larriba, S., Avinyó, A., Nunes, V., ... Aran, J. M. (2003). Suitability of oligonucleotide-mediated cystic fibrosis gene repair in airway epithelial cells. Journal of Gene Medicine, 5(7), 625-639. https://doi.org/10.1002/jgm.374

Suitability of oligonucleotide-mediated cystic fibrosis gene repair in airway epithelial cells. / de Semir, David; Nadal, Marga; González, Juan R.; Larriba, Sara; Avinyó, Anna; Nunes, Virginia; Casals, Teresa; Estivill, Xavier P.; Aran, Josep M.

In: Journal of Gene Medicine, Vol. 5, No. 7, 2003, p. 625-639.

Research output: Contribution to journalArticle

de Semir, D, Nadal, M, González, JR, Larriba, S, Avinyó, A, Nunes, V, Casals, T, Estivill, XP & Aran, JM 2003, 'Suitability of oligonucleotide-mediated cystic fibrosis gene repair in airway epithelial cells', Journal of Gene Medicine, vol. 5, no. 7, pp. 625-639. https://doi.org/10.1002/jgm.374
de Semir, David ; Nadal, Marga ; González, Juan R. ; Larriba, Sara ; Avinyó, Anna ; Nunes, Virginia ; Casals, Teresa ; Estivill, Xavier P. ; Aran, Josep M. / Suitability of oligonucleotide-mediated cystic fibrosis gene repair in airway epithelial cells. In: Journal of Gene Medicine. 2003 ; Vol. 5, No. 7. pp. 625-639.
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AU - Nunes, Virginia

AU - Casals, Teresa

AU - Estivill, Xavier P.

AU - Aran, Josep M.

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AB - Background: Non-viral vector-mediated targeted gene repair could become a useful alternative to classical gene addition strategies. The methodology guarantees a physiologically regulated and persistent expression of the repaired gene, with reported gene conversion and phenotypic correction efficiencies approaching 40-50% in some in vitro and in vivo models of disease. This is particularly important for cystic fibrosis (CF) because of its complex pathophysiology and the cellular heterogeneity of the cystic fibrosis transmembrane conductance regulator (CFTR) gene expression and function in the lung. Methods: A cell-free biochemical assay was applied to assess the ability of CF airway epithelial cells to support chimeraplast-mediated repair. In addition, a methodology allowing the relative quantification of the percentage of W1282X mutation repair in a heterozygous background using the PCR/oligonucleotide ligation assay (PCR/OLA) was developed. The performance of different chimeraplast and short single-stranded oligonucleotide structures delivered by non-viral vectors and electroporation was evaluated. Results: Chimeraplast-mediated repair competency was corroborated in GF airway epithelial cells. However, their repair activity was about 5-fold lower than that found in liver cells. Moreover, regardless of the corrector oligonucleotide structure applied to our GF bronchial epithelial cells, of compound heterozygous genotype (F508del/W1282X), the percentage of their resulting wild-type allele in the W1282X (exon 20) locus of the CFTR gene was not significantly different from that of the control untreated cells by our PCR/OLA assay (confidence interval at 95% ±4 allele wild-type). Conclusions: Oligonucleotide-mediated CFTR gene repair is an inefficient process in CF airway epithelial cells. Further improvements in oligonucleotide structure, nuclear delivery and/or the capability for mismatch repair stimulation will be necessary to achieve therapeutic levels of mutation correction in these cells.

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