ENHANCEMENT OF hFVIII ACTIVITY THROUGH LC MODIFICATIONS FOR GENE THERAPY OF HEMOPHILIA A

Abstract

Gene therapy for Hemophilia A (HA) using the recombinant Adeno-associated virus (rAAV) offers an alternative to classic treatment, which consists of FVIII protein infusions. However, due to limitations associated with rAAV and the FVIII protein itself, the end result is a transgene expression below therapeutic limits. One approach to improving the therapeutic value of rAAV gene therapy for HA is to engineer a more active FVIII protein through genetic modifications. Preliminary testing revealed that canine FVIII Light Chain (kLC) enhances coagulation activity, and that it would be possible to improve FVIII activity through modifications of the light chain. Through the process of engineering, evaluation, and negative selection of kLC, a final construct was engineered. The hLC-K12 is a human Light Chain (hLC) construct containing 12 amino acid changes that work together to enhance coagulation activity. A comparison of the FVIII clotting activity to the amount of protein produced determined that hLC-K12 produced a 3.28 fold increase in specific activity over hLC in vitro. Similar in vitro results were observed when hLC-k12 was tested with the X5 heavy chain (X5HC), a heavy chain that has been genetically modified to enhance production. CD4KO/HA mice were injected with a rAAV vector carrying the hLC-K12 gene in conjugation with a rAAV vector carrying the X5HC gene. Replacing the hLC vector with the hLC-K12 vector produced an average 7.43 fold increase in FVIII clotting activity. An ELISA assay revealed no significant difference between productions of the heavy or light chains at any time point. By comparing the clotting activity to the amount of protein produced, it was determined that the increase in coagulation activity was due to an increase in specific activity. In fact, replacing the hLC vector with the hLC-K12 vector resulted in an average 5.8 fold increase in FVIII specific activity. The K12 modifications were evaluated using a single chain FVIII conformation. In vitro, the addition of the K12 mutations to the human heavy chain, hHCK12BDD, resulted in a 4.3 fold increase in clotting activity, but no increase in protein production. There was however, a 3.3 fold increase in specific activity of the protein. Adding the K12 mutations to the X5 heavy chain, X5K12BDD, in vitro, resulted in a 2.7 fold increase in clotting activity and a 1.42 fold increase in specific activity of the protein. Single chain rAAV vectors were packaged and delivered to CD4KO/HA mice. Compared to mice injected with hFVIIIBDD, the hHCK12BDD produced an average 4.6 fold increase in clotting activity. An ELISA revealed no significant difference in production between these two groups. However, mice injected with hHCK12BDD produced FVIII with an average of 4.13 fold increase in specific activity. Similarly, when compared to mice injected with X5FVIIIBDD, the X5K12BDD produced an average 2.14 fold increase in clotting activity. An ELISA assay demonstrated no significant increase in protein production between these two groups. However, when compared to X5BDD, mice injected with the X5K12BDD vector produced FVIII with an average 1.98 fold increase in specific activity. Results demonstrate that the K12 light chain modifications are able to enhance clotting activity of hFVIII both in vitro and in vivo, using either a dual chain or single chain delivery method. In order to determine the mechanism of enhancement, hFVIIIBDD and hHCK12BDD protein was partially purified and tested for activity. Results demonstrated that the hHCK12BDD protein produced a specific activity of 39,153.69 Units/mg, which is a 6.28 fold increase over hFVIIIBDD specific activity, which was 6,237.92 Units/mg. Measurement of conversion from FX to FXa revealed that the hHCK12BDD protein generated a higher amount of FXa at a quicker rate. In conclusion, these results provide evidence that the K12 modifications enhance specific activity through an increase in FXa generation.