Functional cystic fibrosis transmembrane conductance regulator expression in cystic fibrosis airway epithelial cells by AAV6.2-mediated segmental trans-splicing.
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.
Song
Yuhu
Y
Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10065, USA.
Lou
Howard H
HH
Boyer
Julie L
JL
Limberis
Maria P
MP
Vandenberghe
Luk H
LH
Hackett
Neil R
NR
Leopold
Philip L
PL
Wilson
James M
JM
Crystal
Ronald G
RG
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
D012322:RNA Precursors; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator