Avoidance of Immune Response Prolongs Expression of Genes Delivered to the Adult Rat Myocardium by Replication-Defective Adenovirus

Abstract

Background Gene delivery is a rapidly expanding field with potential applications to every human organ system. Recently, adenoviruses have been used as efficient vectors for in vivo gene transfer into the myocardium. These methods, however, have shown a sharp decline of gene expression after 1 week. To test the hypothesis that an immune-effector mechanism is involved in this decline, we compared the results after injection of adenovirus-5 carrying the β-galactosidase gene (Adβ-gal) into the left ventricular myocardium of athymic nude rats (NDRs) versus immunocompetent Sprague-Dawley rats (SDRs). Methods and Results Adβ-gal (5.0×10 9 PFU/mL) was injected into the left ventricle of NDRs (n=16) and SDRs (n=22). Hearts were harvested, embedded in paraffin, and sectioned and stained for β-gal activity, hematoxylin and eosin and picrosirius red at 4, 21, 35, 85, and 120 days. Representative samples were immunostained with antibodies directed at inflammatory markers. β-gal activity was quantified by digital planimetry and expressed as area of staining (%±SEM). Peak β-gal activity was highest at 4 days, with NDRs displaying significantly greater staining (83±3.0% versus 54±8.0%; P =.03). SDRs sustained a rapid drop in activity, such that at 35 (1±0.19%) and 85 (1±0.4%) days, only occasional cells stained positive and by 120 days (0.3±0.0%), activity had been extinguished. NDRs continued to show transgene expression at all time periods (35 and 85 days, 25±7.1% and 7.4±2.7%, respectively) and was still readily detected at 120 days. An inflammatory response was limited in NDRs compared with SDRs, in which there was intense mononuclear cell infiltration, with collagen deposition and scar formation. Immunostaining identified the majority of these inflammatory cells as not being of lymphocyte lineage, although small numbers of lymphocytes and phagocytic and activated plasma cells were identified. Conclusions Our data suggest that immune-effector mechanisms can severely affect the expression of genes delivered by adenovirus. The present model provides efficient gene expression for at least 120 days without significant inflammatory reaction.