Gene therapy for hemophilia B mice with scAAV8-LP1-hFIX

Abstract

Hemophilia B is a hemorrhagic disease caused by the deficiency of clotting factor IX (FIX). Gene therapy might be the ultimate strategy for the disease. However, two main problems that should be solved in gene therapy for hemophilia B are immunity and safety. Self-complementary adeno-associated virus serotype 8 (scAAV8), a non-human primate AAV featuring low immunogenicity and high transfection efficiency in liver cells, might be a potential vector for hemophilia B gene therapy. A strong liver-specific promoter-1 (LP1) was inserted and mutant human FIX Arg338Ala was introduced into plasmid scAAV8-LP1 to develop an optimized AAV8 vector that expresses human clotting factor FIX (hFIX). The efficiency of scAAV8-LP1-hFIX administered through normal systemic injection or hydrodynamic injection was compared. A high expression was achieved using hydrodynamic injection, and the peak hFIX expression levels in the 5×10¹¹ and 1×10¹¹ virus genome (vg) cohorts were 31.94% and 25.02% of normal level, respectively, at 60 days post-injection. From the perspective of long-term (200 days) expression, both injection methods presented promising results with the concentration value maintained above 4% of normal plasma. The results were further verified by enzyme-linked immunosorbent assay and activated partial thromboplastin time. Our study provides a potential gene therapy method for hemophilia B.

Keywords

hemophilia B / AAV8 / hFIX / gene therapy

References

[1] Nathwani AC, Tuddenham EG. Epidemiology of coagulation disorders. Baillieres Clin Haematol 1992; 5(2): 383–439

[2] Löfqvist T, Nilsson IM, Berntorp E, Pettersson H. Haemophilia prophylaxis in young patients—a long-term follow-up. J Intern Med 1997; 241(5): 395–400

[3] Ljung RC. Can haemophilic arthropathy be prevented? Br J Haematol 1998; 101(2): 215–219

[4] Miller AD. Human gene therapy comes of age. Nature 1992; 357(6378): 455–460

[5] Crystal RG, McElvaney NG, Rosenfeld MA, Chu CS, Mastrangeli A, Hay JG, Brody SL, Jaffe HA, Eissa NT, Danel C. Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet 1994; 8(1): 42–51

[6] Flotte T, Carter B, Conrad C, Guggino W, Reynolds T, Rosenstein B, Taylor G, Walden S, Wetzel R. A phase I study of an adeno-associated virus-CFTR gene vector in adult CF patients with mild lung disease. Hum Gene Ther 1996; 7(9): 1145–1159

[7] Levine BL, Humeau LM, Boyer J, MacGregor RR, Rebello T, Lu X, Binder GK, Slepushkin V, Lemiale F, Mascola JR, Bushman FD, Dropulic B, June CH. Gene transfer in humans using a conditionally replicating lentiviral vector. Proc Natl Acad Sci USA 2006; 103(46): 17372–17377

[8] Qiu X, Lu D, Zhou J, Wang J, Yang J, Meng P, Hsueh JL. Implantation of autologous skin fibroblast genetically modified to secrete clotting factor IX partially corrects the hemorrhagic tendencies in two hemophilia B patients. Chin Med J (Engl) 1996; 109(11): 832–839

[9] Tsui LV, Kelly M, Zayek N, Rojas V, Ho K, Ge Y, Moskalenko M, Mondesire J, Davis J, Roey MV, Dull T, McArthur JG. Production of human clotting factor IX without toxicity in mice after vascular delivery of a lentiviral vector. Nat Biotechnol 2002; 20(1): 53–57

[10] Brown BD, Cantore A, Annoni A, Sergi LS, Lombardo A, Della Valle P, D’Angelo A, Naldini L. A microRNA-regulated lentiviral vector mediates stable correction of hemophilia B mice. Blood 2007; 110(13): 4144–4152

[11] Ehrhardt A, Kay MA. A new adenoviral helper-dependent vector results in long-term therapeutic levels of human coagulation factor IX at low doses in vivo. Blood 2002; 99(11): 3923–3930

[12] Ehrhardt A, Xu H, Dillow AM, Bellinger DA, Nichols TC, Kay MA. A gene-deleted adenoviral vector results in phenotypic correction of canine hemophilia B without liver toxicity or thrombocytopenia. Blood 2003; 102(7): 2403–2411

[13] Cooper M, Nayak S, Hoffman BE, Terhorst C, Cao O, Herzog RW. Improved induction of immune tolerance to factor IX by hepatic AAV-8 gene transfer. Hum Gene Ther 2009; 20(7): 767–776

[14] Allay JA, Sleep S, Long S, Tillman DM, Clark R, Carney G, Fagone P, McIntosh JH, Nienhuis AW, Davidoff AM, Nathwani AC, Gray JT. Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial. Hum Gene Ther2011; 22(5):595-604

[15] Nathwani AC, Rosales C, McIntosh J, Rastegarlari G, Nathwani D, Raj D, Nawathe S, Waddington SN, Bronson R, Jackson S, Donahue RE, High KA, Mingozzi F, Ng CY, Zhou J, Spence Y, McCarville MB, Valentine M, Allay J, Coleman J, Sleep S, Gray JT, Nienhuis AW, Davidoff AM. Long-term safety and efficacy following systemic administration of a self-complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins. Mol Ther 2011; 19(5): 876–885

[16] Nathwani AC, Tuddenham EG, 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 CY, 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

[17] Nathwani AC, Reiss UM, Tuddenham EG, Rosales C, Chowdary P, McIntosh J, Della Peruta M, Lheriteau E, Patel N, Raj D, Riddell A, Pie J, Rangarajan S, Bevan D, Recht M, Shen YM, Halka KG, Basner-Tschakarjan E, Mingozzi F, High KA, Allay J, Kay MA, Ng CY, Zhou J, Cancio M, Morton CL, Gray JT, Srivastava D, Nienhuis AW, Davidoff AM. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med 2014; 371(21): 1994–2004

[18] Chang J, Jin J, Lollar P, Bode W, Brandstetter H, Hamaguchi N, Straight DL, Stafford DW. Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity. J Biol Chem 1998; 273(20): 12089–12094

[19] Haberman RA, Kroner-Lux G, McCown TJ, Samulski RJ. Current protocols in neuroscience. New York, N.Y.: John Wiley and Sons, 1999

[20] Lin HF, Maeda N, Smithies O, Straight DL, Stafford DW. A coagulation factor IX-deficient mouse model for human hemophilia B. Blood 1997; 90(10): 3962–3966

[21] Zhang G, Budker V, Wolff JA. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum Gene Ther 1999; 10(10): 1735–1737

[22] Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther 1999; 6(7): 1258–1266

[23] Kim HS, Kim JC, Lee YK, Kim JS, Park YS. Hepatic control elements promote long-term expression of human coagulation factor IX gene in hydrodynamically transfected mice. J Gene Med 2011; 13(7-8): 365–372

[24] Herzog RW, Hagstrom JN, Kung SH, Tai SJ, Wilson JM, Fisher KJ, High KA. Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus. Proc Natl Acad Sci USA 1997; 94(11): 5804–5809

[25] Miao CH, Thompson AR, Loeb K, Ye X. Long-term and therapeutic-level hepatic gene expression of human factor IX after naked plasmid transfer in vivo. Mol Ther 2001; 3(6): 947–957

[26] Xu ZX, Chen JZ, Yue YB, Zhang JQ, Li ZH, Feng DM, Ruan ZC, Tian L, Xue JL, Wang QJ, Jia W. A 16-bp RBE element mediated Rep-dependent site-specific integration in AAVS1 transgenic mice for expression of hFIX. Gene Ther 2009; 16(5): 589–595

[27] Xu Z, Ye J, Zhang A, Xie L, Shen Q, Xue J, Chen J. Gene therapy for hemophilia B with liver-specific element mediated by Rep-RBE site-specific integration system. J Cardiovasc Pharmacol 2015; 65(2): 153–159

[28] Graham T, McIntosh J, Work LM, Nathwani A, Baker AH. Performance of AAV8 vectors expressing human factor IX from a hepatic-selective promoter following intravenous injection into rats. Genet Vaccines Ther 2008; 6(1): 9

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