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INNOVATIVE METHODOLOGY

Adeno-associated virus mediated gene delivery into coronary microvessels of chronically instrumented dogs

Departments of¹Physiology and ²Medicine, New York Medical College, Valhalla, New York 10595;³Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520; and ⁴Department of Molecular Genetics and Microbiology, Centers for Gene Therapy and Mammalian Genetics, University of Florida College of Medicine, Gainesville, Florida 32610

Submitted 30 September 2002 ; accepted in final form 27 May 2003

The objective of this study was to assess the potential of adeno-associated virus (AAV)-mediated gene delivery into coronary microvessels in vivo in a large animal. Ten mongrel dogs were chronically instrumented and allowed to recover for 10 days. Dogs were reanesthetized, and the aorta was constricted by a hydraulic occluder, whereby left ventricular (LV) pressure increased by 30% and left circumflex coronary artery blood flow by 50%. Recombinant AAV (serotype 2, CMV enhancer/chicken -actin promoter) encoding for green fluorescent protein (GFP) was injected as a bolus into the left atrium during aortic constriction at total titers of 10¹⁰ or 10¹² infectious units. Dogs were followed for 2 (n = 4)or4wk(n = 6). Hemodynamics or body weight did not change. In LV tissue slices, a fluorescein-labeled antibody to GFP stained endothelial and smooth muscle cells but was absent in myocytes. To quantify transduction, slices were then stained with antibodies against -smooth muscle actin or von Willebrand factor. Approximately 4% of arterioles and 2% of microvessels stained positive for anti-GFP independent from viral titer or duration. By regression analyses, the percent of vessels transfected was proportional to the increase in LV systolic pressure during occlusion. AAV is a potential vector for gene transfer into the coronary microcirculation in large animals, including perhaps humans.

white cells; hemodynamics; green fluorescent protein; von Willebrand factor