Gene therapy for common myocardial diseases will require effective and homogeneous gene delivery throughout the intact heart. We created two experimental models to identify and optimize parameters important for adenovirus-mediated cardiac gene transfer. In cultured rabbit ventricular myocytes, the percentage of infected cells increased with higher absolute numbers of virus particles, longer durations of virus exposure, physiological temperatures, and specific culture media compositions. Simulating the in vitro conditions, we delivered adenovirus to intact rabbit hearts by intracoronary perfusion. The percentage of infected cells increased with higher coronary f low rates, longer virus exposure times, and higher virus concentrations. Under optimal conditions, nearly 100% of myocytes expressed the reporter gene ␤-galactosidase after ex vivo infection. This novel delivery method, the first to demonstrate virtually complete transduction of any intact organ, could be adapted to achieve widespread gene transfer in vivo.Diseases such as congestive heart failure, familial hypertrophic cardiomyopathy, and the long QT syndrome result from alterations of myocardial function on a cellular or subcellular level, and effecting a cure will necessitate modification of a majority of the diseased cells. Adenovirus-mediated gene transfer has been used to introduce recombinant genes to cardiac myocytes, offering the potential to treat both rare and common cardiac disorders. Previous attempts utilized intramyocardial injection of viral vectors and achieved locally intense gene delivery, limited to an area within 1-2 mm of the needle track (1, 2). More diffuse but less effective gene transfer was demonstrated with coronary arterial delivery, either by percutaneous in vivo delivery or in explanted hearts prior to cardiac transplantation (3)(4)(5). Despite all of these attempts, no strategy yet devised has achieved high levels of homogeneous gene expression throughout the intact heart. These observations motivated us to evaluate infection conditions in a more controlled environment to identify parameters that would increase infection efficiency.Our work identifies several key variables that influence recombinant adenoviral gene transfer in cultured cardiac myocytes and intact hearts. We first evaluated infection conditions in primary cultures of adult rabbit ventricular myocytes using recombinant adenoviruses encoding the reporter gene for either ␤-galactosidase (␤-gal) (Ad␤gal) or luciferase (AdLuc). Infection of cultured cells is most effective at 37ЊC in crystalloid solutions with elevated virus concentrations and virus-to-cell ratios. Using such conditions in intact hearts, we achieve reporter gene expression in 96% of cardiac myocytes infected by coronary perfusion. If appropriately modified for in vivo applications, this delivery strategy would be a viable method for gene therapy in the heart and other solid organs. MATERIALS AND METHODSAdenovirus Vectors. Ad␤gal contained the Escherichia coli lac Z gene driven by the huma...

Previous attempts at adenoviral gene transfer to the intact heart have been limited by the requirement for prolonged exposure to high virus concentrations. In an ex vivo coronary perfusion model of intact adult rabbit hearts, we previously reported gene transfer to 96% of cardiac myocytes after a 60 min exposure to 1.6 x 10(9) p.f.u./ml Ad beta gal, a recombinant adenovirus encoding beta-galactosidase. Here we sought to decrease the virus exposure time by enhancing microvascular permeability to increase the efficiency of adenoviral gene transfer. Baseline perfusion with 1.0 x 10(8) p.f.u./ml Ad beta gal in normal Krebs solution (1 mM calcium) caused infection of 22% of myocytes at 30 min and 40% at 60 and 120 min. Increasing the virus concentration, decreasing perfusate calcium concentration, or pretreating with serotonin or bradykinin in Krebs solution or L-NAME in heparinized rabbit blood significantly decreased the necessary exposure time. Under optimal conditions of serotonin pretreatment, 50 mumol/l perfusate calcium, and a virus concentration of 1.6 x 10(9) p.f.u./ml, 2 min of coronary perfusion sufficed to produce near-total infection. This profound enhancement of infection parameters has important implications for in vivo myocardial gene transfer, where a similar strategy could facilitate gene therapy for common myocardial disorders.

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