Hemophila B is an X-linked inherited disorder caused by the lack of a functional Factor IX (FIX) and characterized by a bleeding diathesis of variable severity. Currently, treatment of hemophilia B is performed by intravenous infusion of plasma-derived or recombinant FIX. However, the high cost associated with the protein concentrates poses a financial burden to protein replacement therapy. Other treatments, such as gene therapy and tissue implant techniques, are also under study but not yet developed for clinical use. An alternative approach is to develop a FIX molecule with higher specific activity that will reduce the amount of the clotting factor consumed in treatment. For this purpose, we used alanine-scanning mutagenesis and generated 7 alanine-replaced FIX variants that were expressed in HEK-293 cells and purified. These variants have either single (n=3), double (n=3) or triple (n=1) amino acid changes and each was characterized by ELISA and aPTT assay. We observed that the FIX triple mutation variant (R86A/E277A/R338A, FIX-Triple) exhibited 10-12 times higher clotting activity than wild-type FIX (FIX-WT). The affinity of IX-Triple to human Factor VIIIa increased 10-fold (Kd = 0.19 nM vs. 2.4 nM, FIX-Triple vs. FIX-WT) in a Factor Xa-generation assay, consistent with the fact that the amino acid substitutions were in two domains important for factor VIII interaction. Protein infusion into hemophilia B (HB) mice showed that recombinant FIX-Triple was also more effective than FIX-WT in shortening the clotting time in these HB mice. To further evaluate the medium and long-term efficacy of FIX-Triple expression, we used two methods of gene delivery in (HB) mice. For medium-term expression, we performed tail vein hydrodynamic plasmid injections of either FIX-WT or FIX-Triple, expressed from a liver-specific promoter. We assayed specific activity (clotting activity/antigen, U/mg) 24h post-delivery and found that FIX-Triple had a 3.5-fold higher specific activity than FIX-WT. For long-term expression, we performed tail vein administration of a serotype 8 recombinant Adeno-associated vector (AAV8) expressing either FIX-WT or FIX-Triple from a liver-specific promoter at vector doses of 4x1012 (high dose) and 8x1010 (low dose) vector genomes (v.g.)/kg. These mice were sacrificed 8 weeks post vector administration and blood samples were analyzed for FIX antigen (ELISA) and clotting activity (aPTT). Corroborating our previous observations, we found that the FIX-Triple variant had 7 times higher specific activity vs. FIX-WT, for either vector dose used. More importantly, mice that received AAV8-FIX-Triple exhibited activity of 23% of normal pooled human plasma (12-38%, n=6), in contrast to mice that received FIX-WT that only reached 4% (1-8%, n = 4), following 8x1010 v.g./kg vector administration. Activity and antigen level for both transgenes was dose-dependent. Lastly, we generated knock-in mice for FIX-WT and FIX-Triple (FIX-KI-WT and FIX-KI-Triple) by exchanging the mouse sequence for the human, keeping the endogenous promoter intact. Confirming our observations with hydrodynamic injection and AAV administration, mice with FIX-KI-Triple exhibited 7 fold higher specific activity than those with FIX-KI-WT (2040 ± 253 vs 279 ± 33 U/mg). Collectively, our results indicate that FIX-Triple variant exhibits significantly enhanced clotting activity relative to FIX-WT due to tighter binding to Factor VIIIa, as demonstrated both in vitro and in vivo. Therefore, Factor IX-Triple is a good candidate for further evaluation in protein replacement therapy as well as other, gene-based therapeutic strategies.

Disclosures: No relevant conflicts of interest to declare.

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