The primary gene therapy targets for hemophilia B to date have been liver and muscle and these lead to increased levels of plasma factor IX (FIX). In the presence of FIX inhibitory antibodies, plasma FIX is undetectable. Thus, these strategies may not be successful in patients with FIX inhibitors. We have previously developed a FVIII gene therapy strategy for murine hemophilia A in which FVIII is ectopically expressed in platelets. This approach was found to be effective even in the presence of high-titer FVIII inhibitory antibodies. This prompted our current study to explore a similar strategy for gene therapy of hemophilia B to express FIX in platelets by using a platelet specific promoter. Our hypothesis is that expressing FIX in platelets might enable storage of FIX in platelet α-granules where it might be protected from inhibitory or anaphylactic antibodies and be released locally at the site of vascular injury. We constructed a FIX expression cassette in which FIX cDNA was placed under the control of the platelet αIIb promoter (2bF9), a promoter that has been previously demonstrated to be platelet specific, and as a non-specific control, a second plasmid with FIX under the control of the CMV promoter (CMVF9). Using Dami (a megakaryocytic cell line), HepG2, and AtT20 cell lines, FIX antigen was determined by FIX ELISA and the activity of the expressed FIX determined by one-stage clotting and/or chromogenic assay. The 2bF9 expressed well in Dami cells but not in HepG2 or AtT20 cells, while the CMVF9 expressed in all three cell lines. The specific activity of produced FIX suggests effective γ-carboxylation. We next made a 2bF9 expressing lentiviral vector from which we developed 2bF9 transgenic mice by lentiviral-mediated transgenesis. Normally, FIX is not produced in hematopoietic cells and is not present in platelet lysate. In several lines of mice containing the platelet-specific FIX transgene, the platelet human FIX antigen could be measured with levels ranging up to 0.82 mU per108 platelets. Comparable levels of FIX were measured by ELISA, one-stage clotting and chromogenic assays suggesting that FIX can undergo γ-carboxylation in platelets. Human FIX was also detected in the plasma, indicating that unlike our experiments with 2bF8, all of the FIX is not retained in platelets and is therefore found in plasma. However, nearly 85% of total human FIX in blood is stored in platelets and only 15% in plasma. When platelets were stimulated with agonist more than 80% of the FIX in platelets can be released by agonist stimulation - a result that is similar to our previous experience with releasable platelet-FVIII following platelet-directed expression. These results demonstrate that targeting FIX expression in platelets can form a FIX storage pool in platelets, and the stored FIX is functional and can be release by agonist-induced stimulation. Thus, targeting FIX expression in platelets could be a new strategy in gene therapy for hemophilia B in which the stored FIX might be protected from FIX inhibitory antibodies. Release of FIX at the site of vascular injury would increase the local concentration of FIX and achieve effective hemostasis in hemophilia B even in patients with FIX inhibitory antibodies.

Author notes

Disclosure:Research Funding: Postdoctoral fellowship from ZLB-Behring Foundation.