Recombinant Adeno-Associated Viral Vectors Expressing Human Coagulation FIX-E456H Variant in Hemophilia B Mice

Abstract

Gene therapy using recombinant adeno-associated virus (AAV) has induced sustained long-term coagulation human factor IX (hFIX) levels in hemophilia B (HB) patients. However, asymptomatic transient liver toxicity was observed at high vector doses, highlighting the need to improve the potency of these vectors. We report the generation of an AAV transgene cassette containing the hyperfunctional hFIX-E456H variant showing improved binding to platelets, with a comparison to wild-type hFIX (hFIX-WT) and hFIX-R384L variant (Padua) transgenes, containing F9 truncated-intron 1 (I1). In vitro specific activity was increased by 3.2- and 4.2-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using chromogenic assay, and by 7-and 8.6-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using one-stage assay. The transgenes were packaged into single-stranded AAV2/8 vectors that were tail vein injected at 5 × 10⁹, 2 × 10¹⁰, and 5 × 10¹⁰ vg per mouse in HB mice. Plasma FIX activity level, assessed by chromogenic assay, was up to fourfold higher for hFIX-E456H compared with hFIX-WT and was not different compared with hFIX-R384L, among the three dose cohorts. Overall, the in vivo specific activity was increased by threefold for hFIX-E456H and 4.9-fold for hFIX-R384L compared with hFIX-WT. At the lower dose of 5 × 10⁹ vg, the blood loss was significantly lower for hFIX-E456H compared with hFIX-WT, but did not differ compared with hFIX-R384L. The results found for the hFIX-E456H variant indicate that it might be a suitable alternative for gene therapy of HB.

Keywords

gene therapy - hemophilia B - factor IX - platelets - factor IX Padua

Authors' Contributions

S.L.Q., A.P.D., C.N., A.C.N., and N.E. contributed to the design of the study. S.L.Q., A.P.D., E.B., Y.D., J.C.B., T.M., and M.G.B. performed experiments and technical studies relative to the work. S.L.Q. analyzed the data and wrote the manuscript. A.P.D., F.M., C.N., A.C.N., and N.E. critically revised the manuscript.

Note: This work was carried out at all the institutions cited above.

• References

• 1 Bolton-Maggs PH, Pasi KJ. Haemophilias A and B. Lancet 2003; 361 (9371): 1801-1809
• 2 Nazeef M, Sheehan JP. New developments in the management of moderate-to-severe hemophilia B. J Blood Med 2016; 7: 27-38
• 3 Nathwani AC, Tuddenham EG, Rangarajan S. , et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 2011; 365 (25) 2357-2365
• 4 Nathwani AC, Reiss UM, Tuddenham EG. , et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med 2014; 371 (21) 1994-2004
• 5 George LA, Sullivan SK, Giermasz A. , et al. Hemophilia B gene therapy with a high-specific-activity factor IX variant. N Engl J Med 2017; 377 (23) 2215-2227
• 6 Evens H, Chuah MK, VandenDriessche T. Haemophilia gene therapy: from trailblazer to gamechanger. Haemophilia 2018; 24 (Suppl. 06) 50-59
• 7 Perot E, Enjolras N, Le Quellec S. , et al. Expression and characterization of a novel human recombinant factor IX molecule with enhanced in vitro and in vivo clotting activity. Thromb Res 2015; 135 (05) 1017-1024
• 8 Harthé C, Claustrat B. A sensitive and practical competitive radioassay for plasma biotin. Ann Clin Biochem 2003; 40 (Pt 3): 259-263
• 9 Mustard JF, Perry DW, Ardlie NG, Packham MA. Preparation of suspensions of washed platelets from humans. Br J Haematol 1972; 22 (02) 193-204
• 10 Enjolras N, Dargaud Y, Pérot E, Guillaume F, Becchi M, Négrier C. Human hepatoma cell line HuH-7 is an effective cellular system to produce recombinant factor IX with improved post-translational modifications. Thromb Res 2012; 130 (05) e266-e273
• 11 Nathwani AC, Davidoff A, Hanawa H, Zhou JF, Vanin EF, Nienhuis AW. Factors influencing in vivo transduction by recombinant adeno-associated viral vectors expressing the human factor IX cDNA. Blood 2001; 97 (05) 1258-1265
• 12 Nathwani AC, Hanawa H, Vandergriff J, Kelly P, Vanin EF, Nienhuis AW. Efficient gene transfer into human cord blood CD34+ cells and the CD34+CD38- subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV. Gene Ther 2000; 7 (03) 183-195
• 13 Allay JA, Sleep S, Long S. , et al. Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial. Hum Gene Ther 2011; 22 (05) 595-604
• 14 Hermens WT, ter Brake O, Dijkhuizen PA. , et al. Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system. Hum Gene Ther 1999; 10 (11) 1885-1891
• 15 Strobel B, Miller FD, Rist W, Lamla T. Comparative analysis of cesium chloride- and iodixanol-based purification of recombinant adeno-associated viral vectors for preclinical applications. Hum Gene Ther Methods 2015; 26 (04) 147-157
• 16 Wang L, Zoppè M, Hackeng TM, Griffin JH, Lee KF, Verma IM. A factor IX-deficient mouse model for hemophilia B gene therapy. Proc Natl Acad Sci U S A 1997; 94 (21) 11563-11566
• 17 McIntosh J, Lenting PJ, Rosales C. , et al. Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant. Blood 2013; 121 (17) 3335-3344
• 18 Enjolras N, Plantier JL, Rodriguez MH. , et al. Two novel mutations in EGF-like domains of human factor IX dramatically impair intracellular processing and secretion. J Thromb Haemost 2004; 2 (07) 1143-1154
• 19 Enjolras N, Rodriguez MH, Plantier JL, Maurice M, Attali O, Négrier C. The three in-frame ATG, clustered in the translation initiation sequence of human factor IX gene, are required for an optimal protein production. Thromb Haemost 1999; 82 (04) 1264-1269
• 20 Le Quellec S, Enjolras N, Perot E, Girard J, Negrier C, Dargaud Y. Fusion of factor IX to factor XIII-B sub-unit improves the pharmacokinetic profile of factor IX. Thromb Haemost 2018; 118 (12) 2053-2063
• 21 Dargaud Y, Spronk HM, Leenders P, Hemker HC, Ten Cate H. Monitoring platelet dependent thrombin generation in mice. Thromb Res 2010; 126 (05) 436-441
• 22 Monahan PE, Sun J, Gui T. , et al. Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial. Hum Gene Ther 2015; 26 (02) 69-81
• 23 Schuettrumpf J, Herzog RW, Schlachterman A, Kaufhold A, Stafford DW, Arruda VR. Factor IX variants improve gene therapy efficacy for hemophilia B. Blood 2005; 105 (06) 2316-2323
• 24 Ahmad SS, Rawala-Sheikh R, Cheung WF, Jameson BA, Stafford DW, Walsh PN. High-affinity, specific factor IXa binding to platelets is mediated in part by residues 3-11. Biochemistry 1994; 33 (40) 12048-12055
• 25 Wilkinson FH, London FS, Walsh PN. Residues 88-109 of factor IXa are important for assembly of the factor X activating complex. J Biol Chem 2002; 277 (08) 5725-5733
• 26 Simioni P, Tormene D, Tognin G. , et al. X-linked thrombophilia with a mutant factor IX (factor IX Padua). N Engl J Med 2009; 361 (17) 1671-1675
• 27 Hart PD, Young MR. Ammonium chloride, an inhibitor of phagosome-lysosome fusion in macrophages, concurrently induces phagosome-endosome fusion, and opens a novel pathway: studies of a pathogenic mycobacterium and a nonpathogenic yeast. J Exp Med 1991; 174 (04) 881-889
• 28 Lacave R, Ouar Z, Paulais M. , et al. Lysosomotropic agents increase vinblastine efflux from mouse MDR proximal kidney cells exhibiting vectorial drug transport. J Cell Physiol 1999; 178 (02) 247-257
• 29 Branchini A, Campioni M, Mazzucconi MG. , et al. Replacement of the Y450 (c234) phenyl ring in the carboxyl-terminal region of coagulation factor IX causes pleiotropic effects on secretion and enzyme activity. FEBS Lett 2013; 587 (19) 3249-3253
• 30 Bottema CD, Bottema MJ, Ketterling RP. , et al. Why does the human factor IX gene have a G + C content of 40%?. Am J Hum Genet 1991; 49 (04) 839-850
• 31 Drost JB, Scaringe WA, Jaloma-Cruz AR. , et al. Novel hotspot detector software reveals a non-CpG hotspot of germline mutation in the factor IX gene (F9) in Latin Americans. Hum Mutat 2000; 16 (03) 203-210
• 32 Tartary M, Vidaud D, Piao Y. , et al. Detection of a molecular defect in 40 of 44 patients with haemophilia B by PCR and denaturing gradient gel electrophoresis. Br J Haematol 1993; 84 (04) 662-669
• 33 Samelson-Jones BJ, Arruda VR. Protein-engineered coagulation factors for hemophilia gene therapy. Mol Ther Methods Clin Dev 2018; 12: 184-201
• 34 Cantore A, Nair N, Della Valle P. , et al. Hyperfunctional coagulation factor IX improves the efficacy of gene therapy in hemophilic mice. Blood 2012; 120 (23) 4517-4520
• 35 Nathwani AC, Gray JT, Ng CY. , et al. Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver. Blood 2006; 107 (07) 2653-2661
• 36 Nair N, Rincon MY, Evens H. , et al. Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy. Blood 2014; 123 (20) 3195-3199
• 37 Tchaikovski SN, VAN Vlijmen BJ, Rosing J, Tans G. Development of a calibrated automated thrombography based thrombin generation test in mouse plasma. J Thromb Haemost 2007; 5 (10) 2079-2086
• 38 Crudele JM, Finn JD, Siner JI. , et al. AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice. Blood 2015; 125 (10) 1553-1561