QUANTITATIVE-DETERMINATION OF ADENOVIRUS-MEDIATED GENE DELIVERY TO RAT CARDIAC MYOCYTES IN-VITRO AND IN-VIVO

To optimize the use of modified adenoviruses as vectors for gene deliv ery to the myocardium, we have characterized infection of cultured fet al and adult rat cardiac myocytes in vitro and of adult cardiac myocyt es in vivo by using a replication-defective adenovirus carrying the ch loramphenicol acetyltransferase (CAT) reporter gene driven by the cyto megalovirus promoter (AdCMVCATgD). In vitro, virtually all fetal or ad ult cardiocytes express the CAT gene when infected with 1 plaque-formi ng unit of virus per cell. CAT enzymatic activity can be detected in t hese cells as early as 4 hr after infection, reaching near-maximal lev els at 48 hr. In fetal cells, CAT expression was maintained without a loss in activity for at least 1 week. Using in vitro studies as a guid e, we introduced the AdCMVCATgD virus directly into adult rat myocardi um and compared the expression results obtained from virus injection w ith those obtained by direct injection of pAdCMVCATgD plasmid DNA. The amount of CAT activity resulting from adenovirus infection of the myo cardium was orders of magnitude higher than that seen from DNA injecti on and was proportional to the amount of input virus. Immunostaining f or CAT protein in cardiac tissue sections following adenovirus injecti on demonstrated large numbers of positive cells, reaching nearly 100% of the myocytes in many regions of the heart. Expression of genes intr oduced by adenovirus peaked at 5 days but was still detectable 55 days following infection. Adenoviruses are therefore a very useful tool fo r high-efficiency gene transfer into the cardiovascular system.