Functional cystic fibrosis transmembrane conductance regulator expression in cystic fibrosis airway epithelial cells by AAV6.2-mediated segmental trans-splicing.

Publication Type	Academic Article
Authors	Song Y, Lou H, Boyer J, Limberis M, Vandenberghe L, Hackett N, Leopold P, Wilson J, Crystal R
Journal	Hum Gene Ther
Volume	20
Issue	3
Pagination	267-81
Date Published	03/01/2009
ISSN	1557-7422
Keywords	Adenoviridae, Cystic Fibrosis Transmembrane Conductance Regulator, Epithelial Cells, 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.
DOI	10.1089/hum.2008.173
PubMed ID	19257851
PubMed Central ID	PMC2855253