title: Escherichia coli-cloned CFTR loci relevant for human artificial chromosome therapy.
abstract: Classical 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.
authors.affiliation: Life Sciences Center Weihenstephan, Technical University Munich, Freising, Germany.
authors.forename: Lucia
authors.initials: L
authors.lastname: Rocchi
authors.forename: Carla
authors.initials: C
authors.lastname: Braz
authors.forename: Sonja
authors.initials: S
authors.lastname: Cattani
authors.forename: Anabela
authors.initials: A
authors.lastname: Ramalho
authors.forename: Sulith
authors.initials: S
authors.lastname: Christan
authors.forename: Marlene
authors.initials: M
authors.lastname: Edlinger
authors.forename: Fiorentina
authors.initials: F
authors.lastname: Ascenzioni
authors.forename: Andreas
authors.initials: A
authors.lastname: Laner
authors.forename: Simone
authors.initials: S
authors.lastname: Kraner
authors.forename: Margarida
authors.initials: M
authors.lastname: Amaral
authors.forename: Dirk
authors.initials: D
authors.lastname: Schindelhauer
mesh_terms: D045744:Cell Line, Tumor; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D022222:Chromosomes, Artificial, Human / Q000235:genetics*; D003001:Cloning, Molecular; D003550:Cystic Fibrosis / Q000235:genetics / Q000628:therapy*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D056426:Genetic Loci / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D017422:Sequence Analysis, DNA; D014162:Transfection
chemical_list: D019005:Cystic Fibrosis Transmembrane Conductance Regulator