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Article: Episomal expression of wild-type CFTR corrects cAMP-dependent chloride transport in respiratory epithelial cells

TitleEpisomal expression of wild-type CFTR corrects cAMP-dependent chloride transport in respiratory epithelial cells
Authors
KeywordsCystic fibrosis
EBV expression vectors
Gene therapy
Issue Date1996
PublisherNature Publishing Group. The Journal's web site is located at http://www.nature.com/gt
Citation
Gene Therapy, 1996, v. 3 n. 5, p. 427-436 How to Cite?
AbstractThe isolation of the gene responsible for the Cl- ion transport defect in cystic fibrosis (CF) has provided important information about the relationship between the disease pathology and the underlying genetic and biochemical mechanisms. In addition, new areas of investigation and therapy are now possible. Most notably, the isolation of the CF gene, the cystic fibrosis transmembrane conductance regulator (CFTR) has led to the development of different gene therapy strategies. To circumvent possible complications due to insertional mutagenesis and virally induced immune responses, we have employed Epstein-Barr virus (EBV)-based expression vectors for correction of the cAMP-dependent Cl- transport defect associated with CF. A CFTR-containing expression construct under the regulation of the Rous sarcoma virus (RSV) long terminal repeat (LTR) (pREP5/CFTR) was introduced into transformed human airway epithelial cells defective in cAMP-dependent Cl- transport. Transfected cells were assayed for Cl- transport. Transfected cells were assayed for Cl- ion transport by 36Cl- efflux, SPQ, and patch clamp and showed restoration of intact cAMP-dependent transport. CFTR transcription from pREP5/CFTR was detected by Northern hybridization. The level of response to agonists appeared to be dependent on the level of CFTR expression. When cells were tested for functional expression of CFTR after removal of selection pressure, they showed a continuous decrease in responsiveness to forskolin as a function of time after removal of selection. This decreased correlated with a loss of CFTR mRNA and in the loss of CFTR mRNA and in the loss of the pREP5/CFTR. After 12 to 15 weeks growth without selection both cAMP-dependent Cl- transport and plasmid-derived CFTR mRNA were not detectable. However, it was still possible to rescue cAMP-dependent Cl- transport in these transfected cells by reselection suggesting the presence of the CFTR containing plasmid in a portion of the cells. Analysis of DNA indicated that the pREP5/CFTR vector copy number was reduced from 30 copies per cell with continuous selection, to approximately 0.3 copies per cell after 20 weeks without hygromycin B.
Persistent Identifierhttp://hdl.handle.net/10722/44300
ISSN
2015 Impact Factor: 3.242
2015 SCImago Journal Rankings: 1.519
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorLei, DCen_HK
dc.contributor.authorKunzelmann, Ken_HK
dc.contributor.authorKoslowsky, Ten_HK
dc.contributor.authorYezzi, MJen_HK
dc.contributor.authorEscobar, LCen_HK
dc.contributor.authorXu, Zen_HK
dc.contributor.authorEllison, ARen_HK
dc.contributor.authorRommens, JMen_HK
dc.contributor.authorTsui, L-Cen_HK
dc.contributor.authorTykocinski, Men_HK
dc.contributor.authorGruenert, DCen_HK
dc.date.accessioned2007-09-12T03:50:57Z-
dc.date.available2007-09-12T03:50:57Z-
dc.date.issued1996en_HK
dc.identifier.citationGene Therapy, 1996, v. 3 n. 5, p. 427-436en_HK
dc.identifier.issn0969-7128en_HK
dc.identifier.urihttp://hdl.handle.net/10722/44300-
dc.description.abstractThe isolation of the gene responsible for the Cl- ion transport defect in cystic fibrosis (CF) has provided important information about the relationship between the disease pathology and the underlying genetic and biochemical mechanisms. In addition, new areas of investigation and therapy are now possible. Most notably, the isolation of the CF gene, the cystic fibrosis transmembrane conductance regulator (CFTR) has led to the development of different gene therapy strategies. To circumvent possible complications due to insertional mutagenesis and virally induced immune responses, we have employed Epstein-Barr virus (EBV)-based expression vectors for correction of the cAMP-dependent Cl- transport defect associated with CF. A CFTR-containing expression construct under the regulation of the Rous sarcoma virus (RSV) long terminal repeat (LTR) (pREP5/CFTR) was introduced into transformed human airway epithelial cells defective in cAMP-dependent Cl- transport. Transfected cells were assayed for Cl- transport. Transfected cells were assayed for Cl- ion transport by 36Cl- efflux, SPQ, and patch clamp and showed restoration of intact cAMP-dependent transport. CFTR transcription from pREP5/CFTR was detected by Northern hybridization. The level of response to agonists appeared to be dependent on the level of CFTR expression. When cells were tested for functional expression of CFTR after removal of selection pressure, they showed a continuous decrease in responsiveness to forskolin as a function of time after removal of selection. This decreased correlated with a loss of CFTR mRNA and in the loss of CFTR mRNA and in the loss of the pREP5/CFTR. After 12 to 15 weeks growth without selection both cAMP-dependent Cl- transport and plasmid-derived CFTR mRNA were not detectable. However, it was still possible to rescue cAMP-dependent Cl- transport in these transfected cells by reselection suggesting the presence of the CFTR containing plasmid in a portion of the cells. Analysis of DNA indicated that the pREP5/CFTR vector copy number was reduced from 30 copies per cell with continuous selection, to approximately 0.3 copies per cell after 20 weeks without hygromycin B.en_HK
dc.languageengen_HK
dc.publisherNature Publishing Group. The Journal's web site is located at http://www.nature.com/gten_HK
dc.relation.ispartofGene Therapyen_HK
dc.subjectCystic fibrosisen_HK
dc.subjectEBV expression vectorsen_HK
dc.subjectGene therapyen_HK
dc.subject.meshChlorides - metabolismen_HK
dc.subject.meshCyclic amp - metabolismen_HK
dc.subject.meshCystic fibrosis - genetics - therapyen_HK
dc.subject.meshCystic fibrosis transmembrane conductance regulator - genetics - metabolismen_HK
dc.subject.meshGene therapyen_HK
dc.titleEpisomal expression of wild-type CFTR corrects cAMP-dependent chloride transport in respiratory epithelial cellsen_HK
dc.typeArticleen_HK
dc.identifier.emailTsui, LC: tsuilc@hkucc.hku.hken_HK
dc.identifier.authorityTsui, LC=rp00058en_HK
dc.description.naturelink_to_subscribed_fulltexten_HK
dc.identifier.pmid9156804-
dc.identifier.scopuseid_2-s2.0-9344221644en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-9344221644&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume3en_HK
dc.identifier.issue5en_HK
dc.identifier.spage427en_HK
dc.identifier.epage436en_HK
dc.identifier.isiWOS:A1996UK58900008-
dc.publisher.placeUnited Kingdomen_HK
dc.identifier.scopusauthoridLei, DC=7006221963en_HK
dc.identifier.scopusauthoridKunzelmann, K=7005750781en_HK
dc.identifier.scopusauthoridKoslowsky, T=16162359400en_HK
dc.identifier.scopusauthoridYezzi, MJ=6602327840en_HK
dc.identifier.scopusauthoridEscobar, LC=7007110199en_HK
dc.identifier.scopusauthoridXu, Z=7405423384en_HK
dc.identifier.scopusauthoridEllison, AR=7103219845en_HK
dc.identifier.scopusauthoridRommens, JM=7006884140en_HK
dc.identifier.scopusauthoridTsui, LC=7102754167en_HK
dc.identifier.scopusauthoridTykocinski, M=7005612284en_HK
dc.identifier.scopusauthoridGruenert, DC=7005195617en_HK

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