title: Functional cystic fibrosis transmembrane conductance regulator expression in cystic fibrosis airway epithelial cells by AAV6.2-mediated segmental trans-splicing.
abstract: Cystic fibrosis is characterized by deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) transporter. The packaging constraints of adeno-associated viral (AAV) vectors preclude delivery of both an active promoter and CFTR cDNA to target cells. We hypothesized that segmental trans-splicing, in which two AAV vectors deliver the 5' and 3' halves of the CFTR cDNA, could mediate splicing of two pre-mRNAs into a full-length, functional CFTR mRNA. Using a segmental trans-splicing 5' donor-3' acceptor pair that split the CFTR cDNA between exons 14a and 14b, cotransfection of donor and acceptor plasmids into CFTR(-) cells resulted in full-length CFTR message and protein. Microinjection of plasmids into CFTR(-) cells produced cAMP-activated Cl(-) conductance. Vectors created with an engineered human serotype, AAV6.2, were used to deliver CFTR donor and acceptor constructs, resulting in full-length CFTR mRNA and protein as well as cAMP-activated Cl(-) conductance in CFTR(-) cells, including human CF airway epithelial IB3-1 cells. Thus, segmental trans-splicing can be used with AAV vectors to mediate expression of CFTR, a strategy potentially applicable to individuals with CF.
authors.affiliation: Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10065, USA.
authors.forename: Yuhu
authors.initials: Y
authors.lastname: Song
authors.forename: Howard H
authors.initials: HH
authors.lastname: Lou
authors.forename: Julie L
authors.initials: JL
authors.lastname: Boyer
authors.forename: Maria P
authors.initials: MP
authors.lastname: Limberis
authors.forename: Luk H
authors.initials: LH
authors.lastname: Vandenberghe
authors.forename: Neil R
authors.initials: NR
authors.lastname: Hackett
authors.forename: Philip L
authors.initials: PL
authors.lastname: Leopold
authors.forename: James M
authors.initials: JM
authors.lastname: Wilson
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Crystal
mesh_terms: D000256:Adenoviridae; D000818:Animals; D002460:Cell Line; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000235:genetics*; D004847:Epithelial Cells / Q000378:metabolism*; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D012322:RNA Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020040:Trans-Splicing
chemical_list: D012322:RNA Precursors; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator