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An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia

Nature Medicine volume 21, pages 492–497 (2015)Cite this article

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Abstract

Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia1,2,3,4,5,6,7,8,9, we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia. When administered subcutaneously, ALN-AT3 showed potent, dose-dependent, and durable reduction of AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates (NHPs). In NHPs, a 50% reduction in AT levels was achieved with weekly dosing at approximately 0.125 mg/kg, and a near-complete reduction in AT levels was achieved with weekly dosing at 1.5 mg/kg. Treatment with ALN-AT3 promoted hemostasis in mouse models of hemophilia and led to improved thrombin generation in an NHP model of hemophilia A with anti-factor VIII inhibitors. This investigational compound is currently in phase 1 clinical testing in subjects with hemophilia A or B.

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Figure 1: Therapeutic approach.
Figure 2: ALN-AT3 pharmacokinetics and pharmacodynamics in mice.
Figure 3: ALN-AT3 pharmacology in mice.
Figure 4: ALN-AT3 pharmacology in NHPs.

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References

  1. Escuriola Ettingshausen, C. et al. Symptomatic onset of severe hemophilia A in childhood is dependent on the presence of prothrombotic risk factors. Thromb. Haemost. 85, 218–220 (2001).

    Article  CAS  Google Scholar 

  2. Franchini, M. & Lippi, G. Factor V Leiden and hemophilia. Thromb. Res. 125, 119–123 (2010).

    Article  CAS  Google Scholar 

  3. Franchini, M. et al. Interpatient phenotypic inconsistency in severe congenital hemophilia: a systematic review of the role of inherited thrombophilia. Semin. Thromb. Hemost. 35, 307–312 (2009).

    Article  Google Scholar 

  4. Ghosh, K., Shetty, S. & Mohanty, D. Milder clinical presentation of haemophilia A with severe deficiency of factor VIII as measured by one-stage assay. Haemophilia 7, 9–12 (2001).

    Article  CAS  Google Scholar 

  5. Kurnik, K. et al. Effects of the factor V G1691A mutation and the factor II G20210A variant on the clinical expression of severe hemophilia A in children–results of a multicenter study. Haematologica 92, 982–985 (2007).

    Article  Google Scholar 

  6. Lee, D.H. et al. Effect of the factor V Leiden mutation on the clinical expression of severe hemophilia A. Thromb. Haemost. 83, 387–391 (2000).

    Article  CAS  Google Scholar 

  7. Negrier, C., Berruyer, M., Durin, A., Philippe, N. & Dechavanne, M. Increased thrombin generation in a child with a combined factor IX and protein C deficiency. Blood 81, 690–695 (1993).

    Article  CAS  Google Scholar 

  8. Shetty, S. et al. Contribution of natural anticoagulant and fibrinolytic factors in modulating the clinical severity of haemophilia patients. Br. J. Haematol. 138, 541–544 (2007).

    Article  CAS  Google Scholar 

  9. Tizzano, E.F., Cornet, M., Domenech, M. & Baiget, M. Modifier genes in haemophilia: Their expansion in the human genome. Haemophilia 8, 250–254 (2002).

    Article  CAS  Google Scholar 

  10. Bertina, R.M. et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 369, 64–67 (1994).

    Article  CAS  Google Scholar 

  11. Langlois, N.J. & Wells, P.S. Risk of venous thromboembolism in relatives of symptomatic probands with thrombophilia: a systematic review. Thromb. Haemost. 90, 17–26 (2003).

    Article  CAS  Google Scholar 

  12. Rees, D.C., Cox, M. & Clegg, J.B. World distribution of factor V Leiden. Lancet 346, 1133–1134 (1995).

    Article  CAS  Google Scholar 

  13. Bucciarelli, P. et al. Risk of venous thromboembolism and clinical manifestations in carriers of antithrombin, protein C, protein S deficiency, or activated protein C resistance: a multicenter collaborative family study. Arterioscler. Thromb. Vasc. Biol. 19, 1026–1033 (1999).

    Article  CAS  Google Scholar 

  14. Patnaik, M.M. & Moll, S. Inherited antithrombin deficiency: a review. Haemophilia 14, 1229–1239 (2008).

    Article  CAS  Google Scholar 

  15. Bolliger, D., Szlam, F., Suzuki, N., Matsushita, T. & Tanaka, K.A. Heterozygous antithrombin deficiency improves in vivo haemostasis in factor VIII-deficient mice. Thromb. Haemost. 103, 1233–1238 (2010).

    Article  CAS  Google Scholar 

  16. Di Micco, B. et al. Inhibition of antithrombin by protein SV-IV normalizes the coagulation of hemophilic blood. Eur. J. Pharmacol. 391, 1–9 (2000).

    Article  CAS  Google Scholar 

  17. Beltrán-Miranda, C.P., Khan, A., Jaloma-Cruz, A.R. & Laffan, M.A. Thrombin generation and phenotypic correlation in haemophilia A. Haemophilia 11, 326–334 (2005).

    Article  Google Scholar 

  18. Dargaud, Y. et al. Evaluation of thrombin generating capacity in plasma from patients with haemophilia A and B. Thromb. Haemost. 93, 475–480 (2005).

    Article  CAS  Google Scholar 

  19. Santagostino, E. et al. Severe hemophilia with mild bleeding phenotype: molecular characterization and global coagulation profile. J. Thromb. Haemost. 8, 737–743 (2010).

    Article  CAS  Google Scholar 

  20. van Boven, H.H., Vandenbroucke, J.P., Briët, E. & Rosendaal, F.R. Gene-gene and gene-environment interactions determine risk of thrombosis in families with inherited antithrombin deficiency. Blood 94, 2590–2594 (1999).

    Article  CAS  Google Scholar 

  21. de Fougerolles, A., Vornlocher, H.P., Maraganore, J. & Lieberman, J. Interfering with disease: a progress report on siRNA-based therapeutics. Nat. Rev. Drug Discov. 6, 443–453 (2007).

    Article  CAS  Google Scholar 

  22. Novina, C.D. & Sharp, P.A. The RNAi revolution. Nature 430, 161–164 (2004).

    Article  CAS  Google Scholar 

  23. Coelho, T. et al. Safety and efficacy of RNAi therapy for transthyretin amyloidosis. N. Engl. J. Med. 369, 819–829 (2013).

    Article  CAS  Google Scholar 

  24. Fitzgerald, K. et al. Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled, phase 1 trial. Lancet 383, 60–68 (2014).

    Article  CAS  Google Scholar 

  25. Tabernero, J. et al. First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement. Cancer Discov. 3, 406–417 (2013).

    Article  CAS  Google Scholar 

  26. Nair, J.K. et al. Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J. Am. Chem. Soc. 136, 16958–16961 (2014).

    Article  CAS  Google Scholar 

  27. Bi, L. et al. Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A. Nat. Genet. 10, 119–121 (1995).

    Article  CAS  Google Scholar 

  28. Falati, S., Gross, P., Merrill-Skoloff, G., Furie, B.C. & Furie, B. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse. Nat. Med. 8, 1175–1181 (2002).

    Article  CAS  Google Scholar 

  29. Ivanciu, L. et al. A zymogen-like factor Xa variant corrects the coagulation defect in hemophilia. Nat. Biotechnol. 29, 1028–1033 (2011).

    Article  CAS  Google Scholar 

  30. Schlachterman, A. et al. Factor V Leiden improves in vivo hemostasis in murine hemophilia models. J. Thromb. Haemost. 3, 2730–2737 (2005).

    Article  CAS  Google Scholar 

  31. Peng, A., Straubinger, R.M. & Balu-Iyer, S.V. Phosphatidylinositol containing lipidic particles reduces immunogenicity and catabolism of factor VIII in hemophilia a mice. AAPS J. 12, 473–481 (2010).

    Article  CAS  Google Scholar 

  32. Pastoft, A.E. et al. A sensitive venous bleeding model in haemophilia A mice: effects of two recombinant FVIII products (N8 and Advate((R))). Haemophilia 18, 782–788 (2012).

    Article  CAS  Google Scholar 

  33. Pastoft, A.E., Ezban, M., Tranholm, M., Lykkesfeldt, J. & Lauritzen, B. Prolonged effect of a new O-glycoPEGylated FVIII (N8-GP) in a murine saphenous vein bleeding model. Haemophilia 19, 913–919 (2013).

    Article  CAS  Google Scholar 

  34. Buyue, Y., Whinna, H.C. & Sheehan, J.P. The heparin-binding exosite of factor IXa is a critical regulator of plasma thrombin generation and venous thrombosis. Blood 112, 3234–3241 (2008).

    Article  CAS  Google Scholar 

  35. Feng, D., Whinna, H., Monroe, D. & Stafford, D.W. FVIIa as used pharmacologically is not TF dependent in hemophilia B mice. Blood 123, 1764–1766 (2014).

    Article  CAS  Google Scholar 

  36. Landesman, Y. et al. In vivo quantification of formulated and chemically modified small interfering RNA by heating-in-Triton quantitative reverse transcription polymerase chain reaction (HIT qRT-PCR). Silence 1, 16 (2010).

    Article  Google Scholar 

  37. Hui, K.Y., Haber, E. & Matsueda, G.R. Monoclonal antibodies to a synthetic fibrin-like peptide bind to human fibrin but not fibrinogen. Science 222, 1129–1132 (1983).

    Article  CAS  Google Scholar 

  38. Weiler-Guettler, H. et al. A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state. J. Clin. Invest. 101, 1983–1991 (1998).

    Article  CAS  Google Scholar 

  39. Hemker, H.C. et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol. Haemost. Thromb. 33, 4–15 (2003).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank B. Bettencourt for assistance with statistical analysis.

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Authors and Affiliations

  1. Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA

    Alfica Sehgal, Scott Barros, June Qin, Tim Racie, Julia Hettinger, Mary Carioto, Yongfeng Jiang, Josh Brodsky, Harsha Prabhala, Xuemei Zhang, Husain Attarwala, Renta Hutabarat, Don Foster, Stuart Milstein, Klaus Charisse, Satya Kuchimanchi, Martin A Maier, Lubo Nechev, Pachamuthu Kandasamy, Alexander V Kel'in, Jayaprakash K Nair, Kallanthottathil G Rajeev, Muthiah Manoharan, Rachel Meyers, Benny Sorensen, Amy R Simon & Akin Akinc

  2. Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA

    Lacramioara Ivanciu & Rodney M Camire

  3. Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, North Carolina, USA

    Brian Cooley

  4. Unité d'Hémostase Clinique, Hôpital Edouard Herriot, Université Lyon 1, Lyon, France

    Yesim Dargaud & Claude Negrier

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Contributions

A.S. directed the pharmacology studies in rodents and NHPs, led research collaborations, and contributed to manuscript preparation. S.B., J.H., M.C. and Y.J. planned and conducted the toxicology studies. J.Q., T.R., J.B., H.P. conducted the rodent pharmacology studies and generation measurements. L.I. and R.M.C. planned and conducted the microvessel laser injury model studies. B.C. planned and conducted the saphenous vein bleed model studies. X.Z., H.A. and R.H. planned and conducted the pharmacokinetics studies. D.F., S.M., K.C., S.K., M.A.M., L.N., P.K., A.V.K., J.K.N., K.G.R. and M.M. contributed to the design, synthesis, screening and scale-up of ALN-AT3. R.M., B.S., A.R.S., Y.D. and C.N. provided program guidance and medical expertise in the area of hemophilia. A.A. directed the program and wrote the manuscript.

Corresponding author

Correspondence to Akin Akinc.

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Competing interests

A.S., S.B., J.Q., T.R., J.H., M.C., Y.J., J.B., H.P., X.Z., H.A., R.H., D.F., S.M., K.C., S.K., M.A.M., L.N., B.S., A.R.S., and A.A. are all employees of Alnylam Pharmaceuticals. L.I., B.C., Y.D., and R.M.C. received research funding from Alnylam Pharmaceuticals. C.N. received research funding and a consultancy from Alnylam Pharmaceuticals.

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Sehgal, A., Barros, S., Ivanciu, L. et al. An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia. Nat Med 21, 492–497 (2015). https://doi.org/10.1038/nm.3847

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