LAUSR

Our previous studies have demonstrated that platelet‐targeted factor IX (FIX) gene therapy can introduce sustained platelet‐FIX expression in hemophilia B (FIXnull) mice. In this study, we aimed to enhance platelet‐FIX expression in FIXnull mice with O6‐methylguanine‐DNA‐methyltransferase (MGMT)‐mediated in vivo drug selection of transduced cells under nonmyeloablative preconditioning. We constructed a novel lentiviral vector (2bF9/MGMT lentivirus vector), which harbors dual genes, the FIX gene driven by the αIIb promoter (2bF9) and the MGMT P140K gene under the murine stem cell virus promoter. Platelet‐FIX expression in FIXnull mice was introduced by 2bF9/MGMT‐mediated hematopoietic stem cell transduction and transplantation. The 2bF9/MGMT‐transduced cells were effectively enriched after drug selection by O6‐benzylguanine/1,3‐bis‐2‐chloroethyl‐1‐nitrosourea. There were a 2.9‐fold higher FIX antigen and a 3.7‐fold higher FIX activity in platelets, respectively, posttreatment compared with pretreatment. When a 6‐hr tail bleeding test was used to grade the bleeding phenotype, the clotting time in treated animals was 2.6 ± 0.5 hr. In contrast, none of the FIXnull control mice were able to clot within 6 hr. Notably, none of the recipients developed anti‐FIX antibodies after gene therapy. One of four recipients developed a low titer of inhibitors when challenged with rhF9 together with adjuvant. In contrast, all FIXnull controls developed inhibitors after the same challenge. Anti‐FIX immunoglobulin G were barely detectable in recipients (1.08 ± 0.54 µg/ml), an 875‐fold lower level than in the FIXnull controls. Our data demonstrate that using the MGMT‐mediated drug selection system in 2bF9 gene therapy can significantly enhance therapeutic platelet‐FIX expression, resulting in sustained phenotypic correction and immune tolerance in FIXnull mice.