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

Abstract

Canine bone marrow stromal cells (BMSCs), transduced ex vivo with retroviral vectors, expressed and secreted biologically active human and canine coagulation factor IX (hFIX and cFIX) in vitro, and on autologous reinfusion expressed hFIX into the circulation of normal (nonhemophiliac) dogs. Human FIX, when expressed in vitro by BMSCs of two dogs at 1.22 and 1.39 μg/10⁶ cells/24 hr in medium supplemented with vitamin K, respectively, exhibited 28.1 and 27.3% normal biological activity as determined on the basis of a one-stage clotting assay. BMSCs of two additional dogs expressed 1.54 and 4.81 μg of cFIX/10⁶ cells/24 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 × 10⁸ transduced BMSCs, expressing 1.22 and 2.61 μg of hFIX/10⁶ cells/24 hr or 3.24 and 7.82 μg of cFIX/10⁶ cells/24 hr were reintroduced 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 antibodies, which were detected as early as 2–4 days after reinfusion of BMSCs expressing hFIX, may have masked potentially longer duration expression in vivo. Peak plasma levels of hFIX represented 2.1 and 5.8% normal human hFIX levels. When adjusted for percent normal one-stage clotting activity determined in vitro, these levels represented 0.6 and 1.6% normal human hFIX activity levels. Thus, we have demonstrated that retroviral vector-modified BMSCs can deliver human therapeutic levels of hFIX to the circulation of dogs.

Overview summary

Bone marrow stromal cells (BMSCs) represent a promising cell type for delivery of secreted proteins to the circulation in ex vivo gene therapy. We used BMSCs to deliver coagulation factor IX (FIX) to the circulation of dogs. Canine BMSCs secreted high levels of biologically active human or canine FIX (hFIX or cFIX) in vitro after ex vivo transduction with retroviral vectors. Autologous reinfusion of modified BMSCs into normal dogs resulted in peak plasma levels of hFIX that were clinically relevant. Human FIX was detectable in the plasma for 7 to 12 days. Development of canine anti-hFIX antibodies may have masked longer duration and higher level expression in vivo. Thus, we have demonstrated that BMSCs have great potential as a cell type for the delivery of FIX to the circulation in the treatment of hemophilia B.