Ultrarapid, highly efficient viral gene transfer to the heart

Abstract

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...

In vivo myocardial gene transfer: optimization and evaluation of intracoronary gene delivery in vivo

“…When the duration of virus recirculation was increased to 20 min, the transfection efficiency increased to 40%. 10 These results are also in keeping with a more recent study by Logeart et al 18 Thus, it is likely that gene delivery in vivo would be less efficient due to the presence of blood, and that only one pass through the coronary circulation is unlikely to allow significant transfection.…”

“…9 by oxygenated perfluorocarbon emulsion as viral diluent Further understanding of the factors involved in gene transfer by intracoronary delivery of recombinant adenovirus has been gained through a series of experiments involving Langendorff perfusion. 10,11 These studies show that the coronary flow rate, absolute amount of virus, time spent in the coronary circulation and composition of perfusate were major factors affecting intracoronary gene transfer 10 and increasing capillary permeability leads to improvements in gene transfer efficiency. 11 A related model of intracoronary gene delivery has confirmed feasibility provided transcapillary pressure is increased by simultaneous occlusion of the aorta and pulmonary artery in the rat, 12 and more recently, the rabbit.…”

Lipid-mediated gene transfer of viral IL-10 prolongs vascularized cardiac allograft survival by inhibiting donor-specific cellular and humoral immune responses

“…[31][32][33][34] The higher efficiency of viral vectors at delivering genes in vivo than nonviral vectors is illustrated by a direct comparison in which luciferase transgene activity was 10-fold higher in rat cardiac grafts transduced with an adenoviral vector compared with a plasmid vector. 35 None the less, even with viral vectors, investigators have documented inefficient gene transfer by allograft perfusion, with most studies reporting less than 1% of the cells expressing the transgene.…”

A minimally invasive approach for efficient gene delivery to rodent hearts

“…19 Moreover, the replacement of the blood by a crystalloid solution may also have enhanced vascular permeability. 17 Using our protocol to block the aortic outflow and the right atrial inflow, we were able to mimic under closechest conditions the protocol previously developed by Hajjar et al 7 These authors used cross-clamping of the aorta and the pulmonary artery to perform adenoviral transfection under HIP. 4,7 We found that injection of adenoviral vectors within a short period (20 s) into the aortic root without cardiac arrest resulted in aortic root pressures above 300 mmHg but yielded only sparse and uneven gene transduction of the myocardium (3-5% of CMs).…”

“…Given the size of adenoviral particles (73 nm), which is too large to pass the normal endothelial barrier, disruption of the endothelial tight junction seemed to be a prerequisite to ensure access of the virus to the interstitium. 12 Therefore, factors capable of increasing endothelial permeability such as histamine, 3,13 bradykinin and serotonin 17 also improve gene transduction in the isolated hearts and in vivo. Surprisingly, pretreatment of coronary vessels with the nitric oxide (NO) donor SNAP or histamine, substances well known to elevate vascular permeability, did not increase the transfection rate in our study.…”