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 Crystal

Research output: Contribution to journalArticle

33 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
Externally publishedYes

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Trans-Splicing
Cystic Fibrosis Transmembrane Conductance Regulator
Cystic Fibrosis
Epithelial Cells
Complementary DNA
Plasmids
Messenger RNA
RNA Precursors
Microinjections
Product Packaging

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

Cite this

Functional cystic fibrosis transmembrane conductance regulator expression in cystic fibrosis airway epithelial cells by AAV6.2-mediated segmental trans-splicing. / Song, Yuhu; Lou, Howard H.; Boyer, Julie L.; Limberis, Maria P.; Vandenberghe, Luk H.; Hackett, Neil R.; Leopold, Philip L.; Wilson, James M.; Crystal, Ronald.

In: Human Gene Therapy, Vol. 20, No. 3, 01.03.2009, p. 267-281.

Research output: Contribution to journalArticle

Song, Yuhu ; Lou, Howard H. ; Boyer, Julie L. ; Limberis, Maria P. ; Vandenberghe, Luk H. ; Hackett, Neil R. ; Leopold, Philip L. ; Wilson, James M. ; Crystal, Ronald. / Functional cystic fibrosis transmembrane conductance regulator expression in cystic fibrosis airway epithelial cells by AAV6.2-mediated segmental trans-splicing. In: Human Gene Therapy. 2009 ; Vol. 20, No. 3. pp. 267-281.
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