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
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