Highly efficient adenovirus-mediated gene transfer to cardiac myocytes after single-pass coronary delivery

Efficient and homogeneous gene transfer to cardiac myocytes is a major targ et in myocardial gene therapy. The aim of this study was to determine the c onditions permitting efficient, homogeneous, adenovirus-mediated gene trans fer to cardiac myocytes, with a view to application during coronary artery catheterization. Gene transfer to adult rat ventricular myocytes was conduc ted using type 5 adenoviruses carrying the lacZ reporter gene, Adenovirus d elivery via coronary arteries was performed on isolated perfused rat hearts , and gene transfer efficiency was analyzed on whole ventricles, freshly is olated myocytes, and cultured myocytes. Single-pass delivery of 1 X 10(9) P FU associated with 1 min of no-flow yielded only I +/- 0.5% of positive myo cytes. Pretreatment by histamine perfusion (10(-5) M final concentration) i ncreased this value to 30 +/- 9% (p < 0.001), and pretreatment by Ca2+-free buffer perfusion increased it to 67 +/- 8% (p < 0.001). Combination of the two pretreatments had no additional effect. Increasing the viral dose to 3 X 10(9) PFU increased transfection efficiency only in permeabilized vessel s. The 1-min no-flow period after adenovirus delivery was crucial for effic ient gene transfer: despite histamine pretreatment, only 2 +/- 1% positive myocytes were observed without flow interruption (p < 0.05 versus 1 min of no-flow). Gene transfer was shown to occur in situ during cardiac perfusion , rather than during heart digestion or myocyte isolation. This study shows that highly efficient adenovirus-mediated gene transfer to cardiac myocyte s in situ can be achieved by single-pass intracoronary vector delivery, pro vided that vascular permeability is first increased and coronary flow is br iefly interrupted.