RETROVIRAL VECTOR-MODIFIED BONE-MARROW STROMAL CELLS SECRETE BIOLOGICALLY-ACTIVE FACTOR-IX IN-VITRO AND TRANSIENTLY DELIVER THERAPEUTIC LEVELS OF HUMAN FACTOR-IX TO THE PLASMA OF DOGS AFTER REINFUSION

Canine bone marrow stromal cells (BMSCs), transduced ex vivo with retr oviral vectors, expressed and secreted biologically active human and c anine coagulation factor IX (hFIX and cFIX) in vitro, and on autologou s reinfusion expressed hFIX into the circulation of normal (nonhemophi liac) dogs. Human FIX, when expressed in vitro by BMSCs of two dogs at 1.22 and 1.39 mu g/10(6) cells/24 hr in medium supplemented with vita min K, respectively, exhibited 28.1 and 27.3% normal biological activi ty as determined on the basis of a one-stage clotting assay. BMSCs of two additional dogs expressed 1.54 and 4.81 mu g of cFIX/10(6) cells/2 4 hr in vitamin K-supplemented medium and the expressed cFIX possessed 58.4 and 32.9% normal activity, respectively. Between 2.33 and 3.35 x 10(8) transduced BMSCs, expressing 1.22 and 2.61 mu g of hFIX/10(6) c ells/24 hr or 3.24 and 7.82 mu g of cFIX/10(6) cells/24 hr were reintr oduced into the four donor dogs by intravenous infusion. Human FIX was detected in plasma for 7 or 12 days after BMSC reinfusion, with peak levels of 85.8 and 233.0 ng/ml observed at 2 days. Canine anti-hFIX an tibodies, which were detected as early as 2-4 days after reinfusion of BMSCs expressing hFIX, may have masked potentially longer duration ex pression in vivo. Peak plasma levels of hFIX represented 2.1 and 5.8% normal human hFIX levels. When adjusted for percent normal one-stage c lotting activity determined in vitro, these levels represented 0.6 and 1.6% normal human hFIX activity levels. Thus, we have demonstrated th at retroviral vector-modified BMSCs can deliver human therapeutic leve ls of hFIX to the circulation of dogs.