ADENOVIRALLY MEDIATED GENE-TRANSFER OF FUNCTIONAL HUMAN TISSUE-TYPE PLASMINOGEN-ACTIVATOR TO MURINE LUNGS

As several forms of lung injury are associated with alveolar fibrin de position, and fibrin has been pathogenically implicated in the lung fi brotic response, we sought to develop an in vivo gene transfer model o f fibrinolytic protease overexpression. To this end, human tissue-type plasminogen activator (t-PA) possesses a high degree of specificity f or proteolytic activation of fibrin-bound plasminogen to its active fo rm, plasmin. To construct an effective vector, the cDNA for human t-PA was inserted downstream of a cytomegalovirus early enhancer-promoter into the Fl position of a replication-deficient adenovirus. The adenov irally expressed t-PA was found to be of the expected size and appropr iate functional activity both in vitro and in vivo. A single intratrac heal instillation of the adenoviral-t-PA construct resulted in a dose- dependent, tissue-specific expression of increased levels of t-PA anti gen (100-fold) and t-PA protease activity (4-fold) for at least 2 wk i n whole lung lysates. The expressed protein localized to the bronchiol ar epithelium and peribronchiolar alveolar cells and did not result in increases in total lung protein or alveolar cell counts at 3 d after instillation. In conclusion, a single intratracheal instillation of ad enoviral-cytomegalovirus-t-PA construct will generate dramatic broncho alveolar compartment overexpression of functional recombinant human t- PA for at least 2 wk. This vector can now be utilized for the determin ation of the therapeutic potential of t-PA in a number of in vivo mode l systems.