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Enhancing rAAV vector expression in the lung
Author(s) -
VirellaLowell Isabel,
Zusman Benjamin,
Foust Kevin,
Loiler Scott,
Conlon Thomas,
Song Sihong,
Chesnut Kye A.,
Ferkol Thomas,
Flotte Terence R.
Publication year - 2005
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.759
Subject(s) - adeno associated virus , promoter , woodchuck hepatitis virus , reporter gene , biology , microbiology and biotechnology , capsid , genetic enhancement , expression cassette , enhancer , virology , luciferase , gene expression , gene , virus , recombinant dna , transfection , vector (molecular biology) , genetics , hepatitis b virus , hepadnaviridae
Despite favorable DNA transfer efficiency, gene expression from recombinant adeno‐associated virus (rAAV2) vectors in the lung has been variable in the context of cystic fibrosis (CF) gene therapy. This is due, in part, to the large size of the CF transmembrane regulator (CFTR)‐coding sequence which necessitates the use of compact endogenous promoter elements versus stronger exogenous promoters. We evaluated the possibility that gene expression from rAAV could be improved by using AAV capsid serotypes with greater tropism for the apical surface of airway cells (i.e. rAAV5 or rAAV1) and/or using strong promoters such as the cytomegalovirus (CMV) enhancer/chicken beta‐actin hybrid (Cβ) promoter. The relative activity of the CMV immediate‐early (CMVie) promoter, the Cβ promoter, and the Cβ promoter with a downstream woodchuck hepatitis virus post‐transcriptional regulatory element (wpre) were assessed in vitro and in vivo in C57\Bl6 mice using human alpha‐1 antitrypsin (hAAT) as a secreted reporter. In vivo , the Cβ‐AAT‐wpre group achieved maximum serum levels of 1.5 mg/ml of hAAT. AAV capsid serotypes were then compared in vivo utilizing the transcriptionally optimized CB‐wpre cassette in rAAV serotype 1, 2 or 5 capsids (rAAV1, rAAV2, and rAAV5), utilizing luciferase as a reporter to compare expression over a wide dynamic range. The pulmonary luciferase levels at 8 weeks were similar in rAAV5 and rAAV1 groups (2.9 × 10 6 relative light units (RLU)/g tissue and 2.7 × 10 6 RLU/g tissue, respectively), both of which were much higher than rAAV2. Although the advantage of rAAV5 over rAAV2 in the lung has already been described, the availability of another serotype (rAAV1) capable of efficient gene transfer in the lung could be useful. Copyright © 2005 John Wiley & Sons, Ltd.