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 |