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Escherichia coli-cloned CFTR loci relevant for human artificial chromosome therapy

2010-09Journal article Restricted access
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dc.contributor.authorRocchi, Lucia
dc.contributor.authorBraz, Carla
dc.contributor.authorCattani, Sonja
dc.contributor.authorRamalho, Anabela
dc.contributor.authorChristan, Sulith
dc.contributor.authorEdlinger, Marlene
dc.contributor.authorAscenzioni, Fiorentina
dc.contributor.authorLaner, Andreas
dc.contributor.authorKraner, Simone
dc.contributor.authorAmaral, Margarida
dc.contributor.authorSchindelhauer, Dirk
dc.date.accessioned2011-09-20T11:45:29Z
dc.date.available2011-09-20T11:45:29Z
dc.date.issued2010-09
dc.description.abstractClassical gene therapy for cystic fibrosis has had limited success because of immune response against viral vectors and short-term expression of cDNA-based transgenes. These limitations could be overcome by delivering the complete genomic CFTR gene on nonintegrating human artificial chromosomes (HACs). Here, we report reconstruction of the genomic CFTR locus and analyze incorporation into HACs of three P1 phage-based and F factor bacteria-based artificial chromosomes (PACs/BACs) of various sizes: (1) 5A, a large, nonselectable BAC containing the entire wild-type CFTR locus extending into both adjacent genes (296.8-kb insert, from kb -58.4 to +51.4) containing all regulators; (2) CGT21, a small, selectable, telomerized PAC (134.7 kb, from kb -60.7 to + 2) containing a synthetic last exon joining exon 10, EGFP, exon 24, and the 3' untranslated region; and (3) CF225, a midsized, nonselectable PAC (225.3 kb, from kb -60.7 to +9.8) ligated from two PACs with optimized codons and a silent XmaI restriction variant to discriminate transgene from endogenous expression. Cotransfection with telomerized, blasticidin-S-selectable, centromere-proficient α-satellite constructs into HT1080 cells revealed a workable HAC formation rate of 1 per ∼25 lines when using CGT21 or 5A. CF225 was not incorporated into a de novo HAC in 122 lines analyzed, but integrants were expressed. Stability analyses suggest the feasibility of prefabricating a large, tagged CFTR transgene that stably replicates in the proximity of a functional centromere. Although definite conclusions about HAC-proficient construct configurations cannot be drawn at this stage, important transfer resources were generated and characterized, demonstrating the promise of de novo HACs as potentially ideal gene therapy vector systems.por
dc.identifier.citationHum Gene Ther. 2010 Sep;21(9):1077-92por
dc.identifier.issn1043-0342
dc.identifier.otherdoi:10.1089/hum.2009.225
dc.identifier.urihttp://hdl.handle.net/10400.18/219
dc.language.isoengpor
dc.peerreviewedyespor
dc.publisherMary Ann Liebertpor
dc.relation.publisherversionhttp://www.liebertonline.com/doi/abs/10.1089/hum.2009.225por
dc.subjectDoenças Genéticaspor
dc.titleEscherichia coli-cloned CFTR loci relevant for human artificial chromosome therapypor
dc.typejournal article
dspace.entity.typePublication
oaire.citation.endPage1092por
oaire.citation.startPage1077por
oaire.citation.titleHuman Gene Therapypor
rcaap.rightsrestrictedAccesspor
rcaap.typearticlepor

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