214 Several recent clinical trials have demonstrated the feasibility of using plasmid 1 and adenoviral ap- proaches 2 to deliver therapeutic genes to the myocardium. Despite these advances, myocardial gene delivery continues to be limited by factors such as blood flow, endothelial barrier, and the terminally differentiated state of cardiomyocytes. 3 Modalities that enhance penetration of vectors across the endothelium offer one strategy to enhance transfec- tion rates. Such improvements in transfection effi- ciency of plasmid-based systems are particularly attractive because of the relative safety of these agents compared with adenoviruses. Preliminary data of ultrasound-mediated destruction of micro- bubbles, either conjugated with plasmid 4 or ade- noviral delivery systems, have shown promise. 5 Insonification of microbubbles in vivo causes microvascular rupture, which may enhance the abil- ity of these acoustically active particles to deliver material across the endothelium, 6,7 in addition to having incremental effects on gene expression. 8 To the best of our knowledge,there have been no reports of in vivo transfection by disruption of gas- filled cationic liposomes linked to plasmid, using diagnostic ultrasound frequencies in large animals. The aim of this study was to evaluate the efficiency of transgene expression in dogs receiving low doses of cationic microbubble-linked plasmid administered intravenously and subjected to exposure to ultra- sound insonation. METHODS The study was approved by the University Committee for the Use and Care of Animals (UCUCA) at the University of Michigan (Ann Arbor) and conformed to the American Heart Association (AHA) guidelines for the use of animals in research. Animal Preparation Sixteen closed-chest mongrel dogs of either sex, weighing 15 to 20 kg, served as the study animals.After establishing venous access in the cephalic vein, they were subcuta- neously sedated with 0.075 mL/kg butorphanol, acepro- mazine, and glycopyrrolate, intubated, and ventilated with an oxygen/isoflurane mixture at an isoflurane rate of 1.5% to 2%. Oxygen saturation and electrocardiogram (ECG) were monitored. From the Department of Internal Medicine, Division of Cardi- ology, University of Michigan Health System, Ann Arbor; ImaRx Therapeutics Inc, Tucson, Ariz (T.M.); National Institute of Health, Bethesda, Md (E.N.); and the Department of Radiology, University of Arizona, Tucson (E.U.). Reprint requests: Mani A. Vannan, MBBS, Hahnemann University Hospital, 245 N 15th St, MS-470, Philadelphia, PA 19102 (E-mail: mav25@drexel.edu). Copyright 2002 by the American Society of Echocardiography. 0894-7317/2002/$35.00 + 0 27/1/119913 doi:10.1067/mje.2002.119913 We tested the hypothesis that targeted disruption of cationic microbubble-linked plasmid DNA, using diagnostic ultrasound, may aid transfection of large animal myocardium. Plasmid DNA encoding for CAT (pCAT, chloramphenicol acetyltransferase) was bound to a novel cationic microbubble containing MRX-225 for intravenous administration, and 16 dogs in 4 groups variously received this conjugate or plasmid only, or were exposed to ultrasound. Histochemical staining and enzyme-linked immuno- sorbent assay analysis showed CAT activity in the myocardium of only those animals that received microbubble-linked DNA and were exposed to ultrasound. Thus, disruption of cationic-linked, low- dose plasmid systems by diagnostic ultrasound may facilitate transfection of large animal hearts. (J Am Soc Echocardiogr 2002;15:214-8.) Ultrasound-Mediated Transfection of Canine Myocardium by Intravenous Administration of Cationic Microbubble-Linked Plasmid DNA Mani Vannan, MBBS, Thomas McCreery, MSc, Peng Li, MD, Zhenguo Han, MD, Evan Unger, MD, Bettina Kuersten, MD, Elizabeth Nabel, MD, and Sanjay Rajagopalan, MD, Ann Arbor, Michigan; Tucson, Arizona; and Bethesda, Maryland