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Adeno‐associated Virus 9 Vector in the Gene Therapy of Occlusive Vascular Diseases
Author(s) -
Agrawal Devendra Kumar,
Pankajakshan Divya,
Del Core Michael,
Pipinos Iraklis,
Hatzoudis George
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.116.6
Subject(s) - green fluorescent protein , microbiology and biotechnology , expression vector , transfection , reporter gene , gene expression , vector (molecular biology) , lac operon , coronary arteries , staining , biology , viral vector , gene , pathology , artery , medicine , biochemistry , recombinant dna
We analyzed the efficiency and duration of transfection of AAV9 vector containing SM22α promoter with either eGFP or LacZ as reporter genes. The vectors (1 × 10 12 pfu) were incorporated into coronary and carotid arteries of swine using microporous balloon catheter‐based gene delivery approach. Segments from all vessels were excised at various time points and immediately frozen for mRNA/protein expression or were processed for frozen sections for X‐Gal, GFP or H&E staining. GFP was visualized distinctly in the medial layer. Frozen sections were stained and β‐galactosidase gene transfer was assessed by blue‐stained cell nuclei. The endothelial and adventitial layers were carefully scraped off from the harvested vessel and total RNA was isolated from the remaining medial layer for qPCR. In the medial layer of coronary and femoral arteries LacZ mRNA expression was visualized 7 days after vector administration. The GFP mRNA expression was detected at 7 days and lasted for at least 2 months showing the prolonged expression of the AAV9‐vector. The GFP expression was confirmed by Western blot. Control arteries did not show any expression of GFP or LacZ. These findings support the use of AAV9 as a vector to effectively transduce a gene in coronary arteries. (Supported by LB692 Nebraska Tobacco settlement Funds and NHLBI‐sponsored Penn Vector Core, University of Pennsylvania)