Platelet-Derived Codon-Optimized Hyperfunctional FIX Gene Therapy of Hemophilia B Mice

Our previous studies demonstrate that targeting factor IX (FIX) expression to platelets under control of the platelet-specific αIIb promoter (2bF9) can restore hemostasis and induce immune tolerance in hemophilia B (HB) (FIX^null) mice (Chen, et al. Mol Ther 2014). However, functional platelet-FIX activity levels in transduced mice were only around 3% in whole blood even when a lethal 11Gy total body irradiation (TBI) was employed. To reduce potential toxicities associated with this gene therapy protocol from preconditioning and insertion site-mediated mutagenesis, it is desirable to optimize our vector for better clinical efficacy and safety. Recent studies have demonstrated that the combined effect of codon optimization and hyperfunctional FIX Padua can significantly enhance the efficacy of hepatocyte-targeted FIX gene therapy in HB. Thus, we engineered a novel lentiviral vector, 2bCoF9R338L, in which codon-optimized FIX Padua was used to replace the normal FIX expression cassette in our 2bF9 construct. FIX^null mice that we used in this study were originally developed by Lin, et al. (Blood 1997). Platelet-FIX expression was introduced by 2bCoF9R338L lentivirus transduction and syngeneic transplantation under a clinically relevant non-myeloablative preconditioning regimen 6.6Gy TBI. The levels of FIX expression were determined by ELISA for FIX antigen (FIX:Ag) and chromogenic assay for functional FIX activity (FIX:C). Both antigen and activity levels of FIX in platelets from 2bCoF9R338L-transduced recipients were significantly higher than those from normal 2bF9LV-transduced animals. There are approximately a 5.8-fold higher antigen (10.9±3.9 vs. 1.9±1.3 mU/10⁸ platelets) and 28-fold activity (29.1±9.8 vs. 1.1±0.3 mU/10⁸ platelets) levels, respectively, in the 2bCoF9R338L group compared to the 2bF9 group. Flow cytometry analysis showed that 17.7±5.8% of platelets expressed hFIX, which was not significantly different from the 2bF9 group (14.8±10.7%), demonstrating that lentivirus harboring 2bCoF9R338L has similar transduction efficiency as the 2bF9 lentivirus.

To assess whether the bleeding phenotype was rescued in FIX^null mice after receiving 2bCoF9R338L-transduced HSCs, we used a 6-hour tail bleeding test. All 2bCoF9R338L-transduced recipients' tail bleeding clotted within 6 hours with a clotting time of 2.5±0.6 hours and the remaining hemoglobin level of 69.3±8.8%, which were not significantly different from those of the wild type controls (1.9±0.3 hours and 67.2±4.2%). In contrast, none of the FIX^null control mice clotted within 6 hours and the remaining hemoglobin level (40.5±1.9%) was significantly lower than in the 2bCoF9R338L group. To investigate whether anti-FIX immune tolerance was induced in 2bCoF9R338L-transduced recipients, 6 months after HSCT, animals were immunized with recombinant human FIX (rhF9) at a dose of 200 U/kg via intraperitoneal injection two times with a 3-week interval, and the anti-FIX inhibitory antibodies (inhibitors) were determined by Bethesda assay. We found that none of the FIX^null mice that received 2bCoF9R338L-transduced HSCs developed anti-FIX inhibitors even after extensive rhF9 immunization in the presence of adjuvant. In contrast, all FIX^null control mice developed anti-FIX inhibitors when the same immunization protocol was employed. Of note, anaphylaxis can occur in these FIX^null mice with rhF9 infusion if the immune system was primed by FIX. To confirm that platelet-FIX expression is sustained in 2bCoF9R338L-transduced recipients, sequential transplantation was carried out using bone marrow from primary recipients that received 2bCoF9R338L-transduced HSCs. Platelet lysate FIX assays showed that hyperfunctional platelet-FIX was sustained in the secondary recipients resulting in phenotypic correction and immune tolerance in the secondary transplantation FIX^null recipients. Together, our data strongly suggest that immune tolerance is induced in FIX^null mice after 2bCoF9R338L gene therapy.

In summary, we have demonstrated that we are able to significantly augment platelet-FIX expression utilizing codon-optimized FIX Padua for platelet-specific gene therapy of HB, resulting in phenotypic correction and immune tolerance induction in FIX^null mice. Our data suggest that platelet-targeted codon-optimized gain-of-function FIX gene therapy is a promising approach for gene therapy of HB.