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

Yuhu Song, Howard H. Lou, Julie L. Boyer, Maria P. Limberis, Luk H. Vandenberghe, Neil R. Hackett, Philip L. Leopold, James M. Wilson, Ronald G. Crystal

Research output: Contribution to journalArticle

36 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)267-281
Number of pages15
JournalHuman gene therapy
Volume20
Issue number3
DOIs
Publication statusPublished - 1 Mar 2009

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

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