An RNAi Therapeutic Targeting Antithrombin Increases Thrombin Generation in Nonhuman Primates

Abstract Abstract 3370 The hemostatic system balances the need to control blood loss with the need to prevent thrombosis. In hemophilia, the loss of certain procoagulant factors (Factor VIII (FVIII) and Factor IX (FIX), in the case of hemophilia A and B, respectively) results in an imbalance of the hemostatic system toward a bleeding phenotype. Interestingly, there have been reports suggesting that coinheritance of prothrombotic mutations (e.g. Factor V Leiden, protein C deficiency, protein S deficiency, antithrombin deficiency, prothrombin G20210A) may ameliorate the clinical phenotype in hemophilia. We are currently investigating the use of RNA interference (RNAi) to target the natural anticoagulant antithrombin (AT) as strategy to rebalance the hemostatic system and improve thrombin generation in hemophilia. Previously, a short interfering RNA (siRNA), ALN-AT3, employing a hepatocyte targeting ligand was developed against AT and demonstrated potent activity in both wild-type and hemophilia mice after single subcutaneous (SC) administration (ED50 ∼ 1 mg/kg). Further, treatment of hemophilia mice with ALN-AT3 has resulted in normalization of thrombin generation. In this work, we investigate the ability of ALN-AT3 to silence AT, and consequently, increase thrombin generation in nonhuman primates. A single SC administration of ALN-AT3 in nonhuman primates resulted in dose-dependent reductions of plasma AT of 50%, 70%, and 80% at 1, 3, and 10 mg/kg, respectively. Maximum AT reduction was achieved at approximately Day 15 post-administration. The duration of action was persistent, with >50% AT reduction maintained for over 40 days after a single SC dose of 10 mg/kg. All doses were well tolerated, with no injection site reactions, liver enzyme elevations, or changes in hematology parameters observed. To test the hypothesis that AT reduction results in an increase in thrombin generation, plasma samples were collected from animals at various time points and analyzed for thrombin generation using a Calibrated Automated Thrombinoscope (CAT). CAT assay results demonstrated that AT reduction was correlated with increased thrombin generation, with up to 4-fold increases in peak thrombin noted. Increases in thrombin generation capacity were persistent and decreased back to baseline levels as AT protein levels recovered to baseline levels. These data suggest that the use of a novel RNAi therapeutic targeting AT is a promising approach for the treatment of hemophilia, and potentially, other bleeding disorders. Further, the SC route of administration, long duration of action, and applicability to persons with hemophilia who have inhibitors, make this a particularly encouraging potential therapy. Disclosures: Akinc: Alnylam Pharmaceuticals: Employment. Sehgal:Alnylam Pharmaceuticals: Employment. Barros:Alnylam Pharmaceuticals: Employment. Hettinger:Alnylam Pharmaceuticals: Employment. Charisse:Alnylam Pharmaceuticals: Employment. Brodsky:Alnylam Pharmaceuticals: Employment. Qin:Alnylam Pharmaceuticals: Employment. Racie:Alnylam Pharmaceuticals: Employment.