Hemophilia B (HB) is an X chromosome-linked bleeding disorder resulting from a deficiency of factor FIX (FIX). Our previous studies have demonstrated that platelet-targeted FIX gene therapy can provide sustained therapeutic levels of FIX expression in platelets without inhibitory antibody (inhibitor) development, indicating platelet gene therapy could be a promising strategy for the treatment of HB patients. In this study, we aimed to enhance platelet-FIX expression in HB mice with MGMT P140K-mediated in vivo selection of hematopoietic stem cells (HSCs) under a nonmyeloablative preconditioning. We constructed a novel lentiviral vector (2bF9/MGMT LV), which harbors dual genes, the FIX gene driven by the αIIb promoter (2bF9) and the drug-resistance MGMT P140K gene. Platelet-FIX expression was introduced in nonmyeloablative preconditioned HB mice after 2bF9/MGMT LV-mediated HSC transduction and transplantation. After 8 weeks of bone marrow reconstitution, recipients were administered with O6-benzylguanine/1, 3-bis-2-chloroethyl-1- nitrosourea (BG/BCNU) once every 4 weeks for a total of 3 treatments. Animals were analyzed starting at 4 weeks after transplantation by PCR, quantitative real-time PCR (qPCR), FIX assays, inhibitor assays, and tail bleeding test.

PCR results showed that 2bF9 proviral DNA was detected in all recipients that received 2bF9/MGMT LV-transduced HSCs. Quantitative PCR results confirmed that 2bF9/MGMT LV-transduced cells were effectively enriched after each round of BG/BCNU selective treatment in vivo. The average copy number of the 2bF9 cassette per cell after three BG/BCNU treatments was 0.50 ± 0.04, which was significantly higher than pre-BG/BCNU treatment (0.13 ± 0.01). There was an approximately 2.9-fold higher FIX antigen (FIX:Ag) (4.11 ± 0.76 vs. 1.43 ± 0.06 mU/108 platelets) and a 3.7-fold FIX activity (FIX:C) (2.56 ± 0.50 vs. 0.69 ± 0.04 mU/108 platelets) level, respectively, post-treatment compared to pre-treatment. There was a small amount of FIX:Ag detected in the 2bF9/MGMT LV-transduced recipient plasma. When we normalized FIX:Ag levels to total whole blood FIX content, the results demonstrated that 96.32 ± 0.32% of whole blood FIX in the BG/BCNU treated animals was stored in platelets. To assess whether the bleeding phenotype was rescued in HB mice after receiving 2bF9/MGMT LV-transduced HSCs, we used a 6-hour tail bleeding test. All 2bF9/MGMT LV-transduced recipients stopped bleeding within 6 hours with a clotting time of 2.6 ± 0.5 hours and a remaining hemoglobin level of 65.1 ± 4.9%, which were not significantly different from those of the wild-type control group (1.7 ± 0.9 hours and 70.6 ± 13.9%). In contrast, none of the FIXnull mice stopped bleeding within 6 hours with a remaining hemoglobin level of 38.8 ± 6.7%.

Notably, none of the recipients developed anti-FIX inhibitory antibodies as measured by a modified Bethesda assay. To investigate whether immune tolerance was induced in 2bF9/MGMT LV-transduced recipients, all the recipients were challenged with 200U/kg recombinant human FIX (rhFIX) in the presence of Incomplete Freund's adjuvant (IFA) at 9 months after transplantation. Only one out of four recipients developed a low titer of inhibitors (1.3 BU/ml). In contrast, all of the HB controls developed inhibitors ranging from 17-120 BU/ml after the same challenge (n = 4). When we used the ELISA assay for the quantification of total anti-FIX IgG antibodies, low titer antibodies were found in the recipients after they were challenged with rhFIX plus IFA. However, the antibody levels in the HB control mice under the same conditioning were 213-fold higher than those developed in 2bF9 MGMT LV-transduced recipients.

In conclusion, our data demonstrated 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 suppression in HB mice. Our studies suggest that platelet-targeted FIX gene therapy together with in vivo enrichment of engineered cells may provide a promising strategy for the treatment of HB patients.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.