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CLINICAL RESEARCH: CARDIAC ULTRASOUND

Optimization of ultrasound parameters for cardiac gene delivery of adenoviral or plasmid deoxyribonucleic acid by Ultrasound-Targeted microbubble destruction

Shuyuan Chen, MD^*, Ralph V. Shohet, MD, FACC^*, Raffi Bekeredjian, MD^*, Peter Frenkel, MD, FACC^*, and Paul A. Grayburn, MD, FACC^{*, ,*}

^* Division of Cardiology, Department of Internal Medicine, University of Texas Southwestern Medical School, Dallas, Texas, USA
Department of Veterans Affairs Medical Center, Dallas, Texas, USA
Baylor University Medical Center, Dallas, Texas, USA

Manuscript received November 25, 2002; revised manuscript received April 10, 2003, accepted April 14, 2003.

^* Reprint requests and correspondence: Dr. Paul A. Grayburn, Director of Cardiology Research, Baylor Heart and Vascular Institute, 3500 Gaston Avenue, Dallas, Texas 75216, USA.
paulgr{at}baylorhealth.edu

OBJECTIVES: This study was undertaken to optimize echocardiographic parameters for successful gene delivery to the heart and to extend the method from adenoviral to plasmid deoxyribonucleic acid (DNA).

BACKGROUND: We have previously shown that ultrasound-targeted microbubble destruction can direct tissue expression of adenoviral transgenes to the heart. The optimal echocardiographic parameters for this technique have not been reported.

METHODS: Adenoviral or plasmid DNA encoding the luciferase reporter gene was incorporated into liposome microbubbles and infused intravenously into anesthetized rats. We systematically evaluated the effects of ultrasound parameters known to influence microbubble destruction, including electrocardiogram (ECG) triggering, ultrasound frequency, mode of ultrasound, and mechanical index, on gene expression in rat myocardium four days after treatment. In addition, gene expression in heart, liver, and skeletal muscle were compared between adenoviral and plasmid DNA.

RESULTS: Optimal ultrasound parameters for this technique include low-transmission frequency (1.3 MHz), maximal mechanical index, and ECG triggering to allow complete filling of the myocardial capillary bed by microbubbles. No difference was seen between ultraharmonics and power Doppler mode. Using adenoviral DNA, optimal ultrasound parameters yielded myocardial luciferase activity on the order of 10⁴ relative light units/mg protein/min but with even higher liver activity. Plasmid DNA was expressed in rat myocardium at similar levels but without detectable liver expression.

CONCLUSIONS: Ultrasound-targeted microbubble destruction can be used to deliver adenoviral or plasmid DNA to the myocardium. This technique holds great promise in applying the rapidly expanding repertoire of gene therapies being developed for cardiac disease.

Abbreviations and Acronyms   AdCMV-luc   adenovirus encoding the luciferase gene under a cytomegalovirus promoter   ANOVA   analysis of variance   DNA   deoxyribonucleic acid   ECG   electrocardiogram/electrocardiograph/ electrocardiographic   LV   left ventricle/ventricular   UTMD   ultrasound-targeted microbubble destruction

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