Acceleration of widespread adenoviral gene transfer to intact rabbit hearts by coronary perfusion with low calcium and serotonin

Abstract

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 × 10⁹ p.f.u./ml Adβgal, a recombinant adenovirus encoding β-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 × 10⁸ p.f.u./ml Adβgal 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 μmol/l perfusate calcium, and a virus concentration of 1.6 × 10⁹ 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.