Long-term correction of hemorrhagic diathesis in hemophilia A mice by an AAV-delivered hybrid FVIII composed of the human heavy chain and the rat light chain

Abstract

Conventional therapies for hemophilia A (HA) are prophylactic or on-demand intravenous FVIII infusions. However, they are expensive and inconvenient to perform. Thus, better strategies for HA treatment must be developed. In this study, a recombinant FVIII cDNA encoding a human/rat hybrid FVIII with an enhanced procoagulant potential for adeno-associated virus (AAV)-delivered gene therapy was developed. Plasmids containing human FVIII heavy chain (hHC), human light chain (hLC), and rat light chain (rLC) were transfected into cells and hydrodynamically injected into HA mice. Purified AAV viruses were intravenously injected into HA mice at two doses. Results showed that the hHC+ rLC protein had a higher activity than the hHC+ hLC protein at comparable expression levels. The specific activity of hHC+ rLC was about 4- to 8-fold higher than that of their counterparts. Hydrodynamic injection experiments obtained consistent results. Notably, the HA mice undergoing the AAV-delivered hHC+ rLC treatment exhibited a visibly higher activity than those treated with hHC+ hLC, and the therapeutic effects lasted for up to 40 weeks. In conclusion, the application of the hybrid FVIII (hHC+ rLC) via an AAV-delivered gene therapy substantially improved the hemorrhagic diathesis of the HA mice. These data might be of help to the development of optimized FVIII expression cassette for HA gene therapy.

Keywords

hemophilia A / adeno-associated virus (AAV) / human/rat hybrid factor VIII / gene therapy / dual chain strategy

References

[1] Makris M, Oldenburg J, Mauser-Bunschoten EP, Peerlinck K, Castaman G, Fijnvandraat K; subcommittee on Factor VIII, Factor IX and Rare Bleeding Disorders.The definition, diagnosis and management of mild hemophilia A: communication from the SSC of the ISTH. J Thromb Haemost 2018; 16(12): 2530–2533

[2] Bolton-Maggs PHB, Pasi KJ. Haemophilias A and B. Lancet 2003; 361(9371): 1801–1809

[3] Lai J, Hough C, Tarrant J, Lillicrap D. Biological considerations of plasma-derived and recombinant factor VIII immunogenicity. Blood 2017; 129(24): 3147–3154

[4] Morfini M, Rapisarda CAP. Safety of recombinant coagulation factors in treating hemophilia. Expert Opin Drug Saf 2019; 18(2): 75–85

[5] Perrin GQ, Herzog RW, Markusic DM. Update on clinical gene therapy for hemophilia. Blood 2019; 133(5): 407–414

[6] Borsotti C, Follenzi A. New technologies in gene therapy for inducing immune tolerance in hemophilia A. Expert Rev Clin Immunol 2018; 14(12): 1013–1019

[7] Asokan A, Schaffer DV, Samulski RJ. The AAV vector toolkit: poised at the clinical crossroads. Mol Ther 2012; 20(4): 699–708

[8] Zaiss AK, Liu Q, Bowen GP, Wong NC, Bartlett JS, Muruve DA. Differential activation of innate immune responses by adenovirus and adeno-associated virus vectors. J Virol 2002; 76(9): 4580–4590

[9] Nathwani AC, Tuddenham EGD, Rangarajan S, Rosales C, McIntosh J, Linch DC, Chowdary P, Riddell A, Pie AJ, Harrington C, O’Beirne J, Smith K, Pasi J, Glader B, Rustagi P, Ng CYC, Kay MA, Zhou J, Spence Y, Morton CL, Allay J, Coleman J, Sleep S, Cunningham JM, Srivastava D, Basner-Tschakarjan E, Mingozzi F, High KA, Gray JT, Reiss UM, Nienhuis AW, Davidoff AM. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 2011; 365(25): 2357–2365

[10] Rangarajan S, Walsh L, Lester W, Perry D, Madan B, Laffan M, Yu H, Vettermann C, Pierce GF, Wong WY, Pasi KJ. AAV5-Factor VIII gene transfer in severe hemophilia A. N Engl J Med 2017; 377(26): 2519–2530

[11] Nathwani AC, Davidoff AM, Tuddenham EGD. Gene therapy for hemophilia. Hematol Oncol Clin North Am 2017; 31(5): 853–868

[12] Nathwani AC, Nienhuis AW, Davidoff AM. Current status of gene therapy for hemophilia. Curr Hematol Rep 2003; 2(4): 319–327

[13] Nambiar B, Sookdeo C C, Berthelette P, Jackson R, Piraino S, Burnham B, Nass S, Souza D, O’Riordan CR, Vincent KA, Cheng SH, Armentano D, Kyostio-Moore S. Characteristics of minimally oversized adeno-associated virus vectors encoding human factor VIII generated using producer cell lines and triple transfection. Hum Gene Ther Methods 2017; 28(1): 23–38

[14] Mah C, Sarkar R, Zolotukhin I, Schleissing M, Xiao X, Kazazian HH, Byrne BJ. Dual vectors expressing murine factor VIII result in sustained correction of hemophilia A mice. Hum Gene Ther 2003; 14(2): 143–152

[15] Wang Q, Dong B, Firrman J, Roberts S, Moore AR, Cao W, Diao Y, Kapranov P, Xu R, Xiao W. Efficient production of dual recombinant adeno-associated viral vectors for factor VIII delivery. Hum Gene Ther Methods 2014; 25(4): 261–268

[16] Doshi BS, Arruda VR. Gene therapy for hemophilia: what does the future hold? Ther Adv Hematol 2018; 9(9): 273–293

[17] Shestopal SA, Hao JJ, Karnaukhova E, Liang Y, Ovanesov MV, Lin M, Kurasawa JH, Lee TK, Mcvey JH, Sarafanov AG. Expression and characterization of a codon-optimized blood coagulation factor VIII. J Thromb Haemost 2017; 15(4): 709–720

[18] McIntosh J, Lenting PJ, Rosales C, Lee D, Rabbanian S, Raj D, Patel N, Tuddenham EGD, Christophe OD, McVey JH, Waddington S, Nienhuis AW, Gray JT, Fagone P, Mingozzi F, Zhou SZ, High KA, Cancio M, Ng CYC, Zhou J, Morton CL, Davidoff AM, Nathwani AC. 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

[19] Nguyen GN, George LA, Siner JI, Davidson RJ, Zander CB, Zheng XL, Arruda VR, Camire RM, Sabatino DE. Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A. J Thromb Haemost 2017; 15(1): 110–121

[20] Kosloski MP, Shetty KA, Wakabayashi H, Fay PJ, Balu-Iyer SV. Effects of replacement of factor VIII amino acids Asp519 and Glu665 with Val on plasma survival and efficacy in vivo. AAPS J 2014; 16(5): 1038–1045

[21] Doering CB, Healey JF, Parker ET, Barrow RT, Lollar P. High level expression of recombinant porcine coagulation factor VIII. J Biol Chem 2002; 277(41): 38345–38349

[22] Doering CB, Healey JF, Parker ET, Barrow RT, Lollar P. Identification of porcine coagulation factor VIII domains responsible for high level expression via enhanced secretion. J Biol Chem 2004; 279(8): 6546–6552

[23] Wang Q, Dong B, Firrman J, Wu W, Roberts S, Moore AR, Liu LS, Chin MPS, Diao Y, Kost J, Xiao W. Evaluation of the biological differences of canine and human factor VIII in gene delivery: implications in human hemophilia treatment. Gene Ther 2016; 23(7): 597–605

[24] Sabatino DE, Freguia CF, Toso R, Santos A, Merricks EP, Kazazian HH, Nichols TC, Camire RM, Arruda VR. Recombinant canine B-domain-deleted FVIII exhibits high specific activity and is safe in the canine hemophilia A model. Blood 2009; 114(20): 4562–4565

[25] Lollar P, Parker ET, Fay PJ. Coagulant properties of hybrid human/porcine factor VIII molecules. J Biol Chem 1992; 267(33): 23652–23657

[26] Zhang W, Mao J, Shen Y, Zhang G, Shao Y, Ruan Z, Wang Y, Wu W, Wang X, Zhu J, Chen S, Xiao W, Xi X. Evaluation of the activity levels of rat FVIII and human FVIII delivered by adeno-associated viral vectors both in vitro and in vivo. Blood Cells Mol Dis 2018; 73: 47–54

[27] Scallan CD, Liu T, Parker AE, Patarroyo-White SL, Chen H, Jiang H, Vargas J, Nagy D, Powell SK, Wright JF, Sarkar R, Kazazian HH, McClelland A, Couto LB. Phenotypic correction of a mouse model of hemophilia A using AAV2 vectors encoding the heavy and light chains of FVIII. Blood 2003; 102(12): 3919–3926

[28] Mueller C, Ratner D, Zhong L, Esteves-Sena M, Gao G. Production and discovery of novel recombinant adeno-associated viral vectors. Curr Protoc Microbiol 2012; Chapter 14: s26

[29] Wang J, Xie J, Lu H, Chen L, Hauck B, Samulski RJ, Xiao W. Existence of transient functional double-stranded DNA intermediates during recombinant AAV transduction. Proc Natl Acad Sci USA 2007; 104(32): 13104–13109

[30] Kuang Y, Wang J, Lu X, Lu S, Zhang L, Shen C, Fei J, Wang Z. Generation of factor VIII gene knockout mouse by tetraploid embryo complementation technology. Chin J Med Genet (Zhonghua Yi Xue Yi Chuan Xue Za Zhi) 2010; 27(1): 1–6 (in Chinese)

[31] Wang D, Zhang G, Gu J, Shao X, Dai Y, Li J, Pan X, Yao S, Xu A, Jin Y, Huang J, Shi Q, Zhu J, Xi X, Chen Z, Chen S. In vivo generated hematopoietic stem cells from genome edited induced pluripotent stem cells are functional in platelet-targeted gene therapy of murine hemophilia A. Haematologica 2020; 105(4): e175–e179

[32] Tiede A. Half-life extended factor VIII for the treatment of hemophilia A. J Thromb Haemost 2015; 13(Suppl 1): S176–S179

[33] Persson E, Foscolo T, Hansen M. Reagent-specific underestimation of turoctocog alfa pegol (N8-GP) clotting activity owing to decelerated activation by thrombin. Res Pract Thromb Haemost 2019; 3(1): 114–120

[34] Shapiro AD, Ragni MV, Kulkarni R, Oldenberg J, Srivastava A, Quon DV, Pasi KJ, Hanabusa H, Pabinger I, Mahlangu J, Fogarty P, Lillicrap D, Kulke S, Potts J, Neelakantan S, Nestorov I, Li S, Dumont JA, Jiang H, Brennan A, Pierce GF. Recombinant factor VIII Fc fusion protein: extended-interval dosing maintains low bleeding rates and correlates with von Willebrand factor levels. J Thromb Haemost 2014; 12(11): 1788–1800

[35] Lippi G, Favaloro EJ. Emicizumab (ACE910): clinical background and laboratory assessment of hemophilia A. Adv Clin Chem 2019; 88: 151–167

[36] High KA, Anguela XM. Adeno-associated viral vectors for the treatment of hemophilia. Hum Mol Genet 2016; 25(R1): R36–R41

[37] Lisowski L, Dane AP, Chu K, Zhang Y, Cunningham SC, Wilson EM, Nygaard S, Grompe M, Alexander IE, Kay MA. Selection and evaluation of clinically relevant AAV variants in a xenograft liver model. Nature 2014; 506(7488): 382–386

[38] George LA, Sullivan SK, Giermasz A, Rasko JEJ, Samelson-Jones BJ, Ducore J, Cuker A, Sullivan LM, Majumdar S, Teitel J, McGuinn CE, Ragni MV, Luk AY, Hui D, Wright JF, Chen Y, Liu Y, Wachtel K, Winters A, Tiefenbacher S, Arruda VR, van der Loo JCM, Zelenaia O, Takefman D, Carr ME, Couto LB, Anguela XM, High KA. Hemophilia B gene therapy with a high-specific-activity factor IX variant. N Engl J Med 2017; 377(23): 2215–2227

[39] Okuyama T, Huber RM, Bowling W, Pearline R, Kennedy SC, Flye MW, Ponder KP. Liver-directed gene therapy: a retroviral vector with a complete LTR and the ApoE enhancer-α₁-antitrypsin promoter dramatically increases expression of human α₁-antitrypsin in vivo. Hum Gene Ther 1996; 7(5): 637–645

[40] Simioni P, Tormene D, Tognin G, Gavasso S, Bulato C, Iacobelli NP, Finn JD, Spiezia L, Radu C, Arruda VR. X-linked thrombophilia with a mutant factor IX (factor IX Padua). N Engl J Med 2009; 361(17): 1671–1675

[41] Takeyama M, Wakabayashi H, Fay PJ. Contribution of factor VIII light-chain residues 2007–2016 to an activated protein C-interactive site. Thromb Haemost 2013; 109(2): 187–198

[42] O’Brien LM, Mastri M, Fay PJ. Regulation of factor VIIIa by human activated protein C and protein S: inactivation of cofactor in the intrinsic factor Xase. Blood 2000; 95(5): 1714–1720

[43] Pratt KP, Shen BW, Takeshima K, Davie EW, Fujikawa K, Stoddard BL. Structure of the C2 domain of human factor VIII at 1.5 A resolution. Nature 1999; 402(6760): 439–442

[44] Novakovic VA, Cullinan DB, Wakabayashi H, Fay PJ, Baleja JD, Gilbert GE. Membrane-binding properties of the Factor VIII C2 domain. Biochem J 2011; 435(1): 187–196

[45] Watzka M, Geisen C, Seifried E, Oldenburg J. Sequence of the rat factor VIII cDNA. Thromb Haemost 2004; 91(1): 38–42

RIGHTS & PERMISSIONS

Higher Education Press