Highly efficient ex vivo gene transfer to the transplanted heart by means of hypothermic perfusion with a low dose of adenoviral vector.

Abstract

Hypothermic conditions required for donor heart preservation may reduce gene-transfer efficiency. Experiments were designed to determine whether a perfusion technique could improve the efficiency of gene transfer to donor hearts. An adenoviral vector encoding beta-galactosidase (3.5 x 10(8) plaque-forming units) was infused into explanted rat hearts under 4 conditions (each n = 6): (1) the virus was diluted in 350 microL of University of Wisconsin solution and infused as a high-pressure bolus into the coronary arteries of donor hearts through the aortic root; (2) the virus was diluted in 5 mL of University of Wisconsin solution and circulated by means of a peristaltic pump (flow, 0.75 mL/min) through the vasculature of the donor heart for 30 minutes; (3) 5 mL of viral solution was circulated as for group 2 for 15 minutes; and (4) 5 mL of viral solution was circulated for 5 minutes at a flow rate of 2.4 mL/min. Transduced hearts were transplanted into the abdomen of syngeneic rats, and transgene expression was assessed by means of immunoassay 4 days later. The median beta-galactosidase content was (1) 45.0 ng/mg protein (25th-75th percentile, 33-73 ng/mg), (2) 640 ng/mg protein (25th-75th percentile, 614-878 ng/mg), (3) 493.8 ng/mg protein (25th-75th percentile, 456-527 ng/mg), and (4) 503.3 ng/mg protein (25th-75th percentile, 475-562 ng/mg; P <.01 for group 2 vs group 1, and P <.05 for groups 3 and 4 vs group 1). Transgene expression was predominantly in myocytes and favored the subepicardial region of the right ventricle. Hypothermic perfusion of the donor heart with an adenoviral vector resulted in efficient transgene expression compared with that induced by a single bolus injection.