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AAV-based neonatal gene therapy for hemophilia A: long-term correction and avoidance of immune responses in mice

Gene Therapy volume 19, pages 1166–1176 (2012)Cite this article

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Abstract

Hemophilia A gene therapy has been hampered by immune responses to vector-associated antigens and by neutralizing antibodies or inhibitors against the factor VIII (FVIII) protein; these ‘inhibitors’ more commonly affect hemophilia A patients than those with hemophilia B. A gene replacement strategy beginning in the neonatal period may avoid the development of these immune responses and lead to prolonged expression with correction of phenotype, thereby avoiding long-term consequences. A serotype rh10 adeno-associated virus (AAV) was developed splitting the FVIII coding sequence into heavy and light chains with the chicken β-actin promoter/CMV enhancer for dual recombinant adeno-associated viral vector delivery. Virions of each FVIII chain were co-injected intravenously into mice on the second day of life. Mice express sustained levels of FVIII antigen ⩾5% up to 22 months of life without development of antibodies against FVIII. Phenotypic correction was manifest in all AAV-FVIII-treated mice as demonstrated by functional assay and reduction in bleeding time. This study demonstrates the use of AAV in a gene replacement strategy in neonatal mice that establishes both long-term phenotypic correction of hemophilia A and lack of antibody development against FVIII in this disease model where AAV is administered shortly after birth. These studies support the consideration of gene replacement therapy for diseases that are diagnosed in utero or in the early neonatal period.

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References

  1. Takahashi M, Ilan Y, Chowdhury NR, Guida J, Horwitz M, Chowdhury JR . Long term correction of bilirubin-UDP-glucuronosyltransferase deficiency in Gunn rats by administration of a recombinant adenovirus during the neonatal period. J Biol Chem 1996; 271: 26536–26542.

    Article  CAS  PubMed  Google Scholar 

  2. Xu L, Haskins ME, Melniczek JR, Gao C, Weil MA, O’Malley TM et al. Transduction of hepatocytes after neonatal delivery of a Moloney murine leukemia virus based retroviral vector results in long-term expression of beta-glucuronidase in mucopolysaccharidosis VII dogs. Mol Ther 2002; 5: 141–153.

    Article  CAS  PubMed  Google Scholar 

  3. Xu L, Mango RL, Sands MS, Haskins ME, Ellinwood NM, Ponder KP . Evaluation of pathological manifestations of disease in mucopolysaccharidosis VII mice after neonatal hepatic gene therapy. Mol Ther 2002; 6: 745–758.

    Article  CAS  PubMed  Google Scholar 

  4. Ponder KP, Melniczek JR, Xu L, Weil MA, O’Malley TM, O′Donnell PA et al. Therapeutic neonatal hepatic gene therapy in mucopolysaccharidosis VII dogs. Proc Natl Acad Sci USA 2002; 99: 13102–13107.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Mah C, Cresawn KO, Fraites Jr TJ, Pacak CA, Lewis MA, Zolotukhin I et al. Sustained correction of glycogen storage disease type II using adeno-associated virus serotype 1 vectors. Gene Therapy 2005; 12: 1405–1409.

    Article  CAS  PubMed  Google Scholar 

  6. Chandler RJ, Venditti CP . Long-term rescue of a lethal murine model of methylmalonic acidemia using adeno-associated viral gene therapy. Mol Ther 2010; 18: 11–16.

    Article  CAS  PubMed  Google Scholar 

  7. Carrillo-Carrasco N, Chandler RJ, Chandrasekaran S, Venditti CP . Liver-directed recombinant adeno-associated viral gene delivery rescues a lethal mouse model of methylmalonic acidemia and provides long-term phenotypic correction. Hum Gene Ther 2010; 21: 1147–1154.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Sabatino DE, Mackenzie TC, Peranteau W, Edmonson S, Campagnoli C, Liu YL et al. Persistent expression of hF.IX After tolerance induction by in utero or neonatal administration of AAV-1-F.IX in hemophilia B mice. Mol Ther 2007; 15: 1677–1685.

    Article  CAS  PubMed  Google Scholar 

  9. Zhang J, Xu L, Haskins ME, Parker Ponder K . Neonatal gene transfer with a retroviral vector results in tolerance to human factor IX in mice and dogs. Blood 2004; 103: 143–151.

    Article  CAS  PubMed  Google Scholar 

  10. Foust KD, Wang X, McGovern VL, Braun L, Bevan AK, Haidet AM et al. Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN. Nat Biotechnol 2010; 28: 271–274.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kaufman RJ . Advances toward gene therapy for hemophilia at the millennium. Hum Gene Ther 1999; 10: 2091–2107.

    Article  CAS  PubMed  Google Scholar 

  12. Kay MA, High K . Gene therapy for the hemophilias. Proc Natl Acad Sci USA 1999; 96: 9973–9975.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ et al. Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response. Nat Med 2006; 12: 342–347.

    Article  CAS  PubMed  Google Scholar 

  14. Ponder KP . Hemophilia gene therapy: a Holy Grail found. Mol Ther 2011; 19: 427–428.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Nathwani AC, Tuddenham E, Rosales C, McIntosh J, Riddel A, Rustagi PK et al. Early clinical trial results following administration of a low dose of a novel self complementary adeno-associated viral vector encoding human factor IX in two subjects with severe hemophilia B. Blood 2010; 116: 114.

    Google Scholar 

  16. Mah C, Sarkar R, Zolotukhin I, Schleissing M, Xiao X, Kazazian HH et al. Dual vectors expressing murine factor VIII result in sustained correction of hemophilia A mice. Hum Gene Ther 2003; 14: 143–152.

    Article  CAS  PubMed  Google Scholar 

  17. VandenDriessche T, Vanslembrouck V, Goovaerts I, Zwinnen H, Vanderhaeghen ML, Collen D et al. Long-term expression of human coagulation factor VIII and correction of hemophilia A after in vivo retroviral gene transfer in factor VIII-deficient mice. Proc Natl Acad Sci USA 1999; 96: 10379–10384.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Xiao X, Li J, Samulski RJ . Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J Virol 1996; 70: 8098–8108.

    CAS  PubMed Central  PubMed  Google Scholar 

  19. Matsushita T, Elliger S, Elliger C, Podsakoff G, Villarreal L, Kurtzman GJ et al. Adeno-associated virus vectors can be efficiently produced without helper virus. Gene Therapy 1998; 5: 938–945.

    Article  CAS  PubMed  Google Scholar 

  20. Hu C, Busuttil RW, Lipshutz GS . RH10 provides superior transgene expression in mice when compared with natural AAV serotypes for neonatal gene therapy. J Gene Med 2010; 12: 766–778.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gao GP, Alvira MR, Wang L, Calcedo R, Johnston J, Wilson JM . Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci USA 2002; 99: 11854–11859.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sanlioglu S, Monick MM, Luleci G, Hunninghake GW, Engelhardt JF . Rate limiting steps of AAV transduction and implications for human gene therapy. Curr Gene Ther 2001; 1: 137–147.

    Article  CAS  PubMed  Google Scholar 

  23. Thomas CE, Storm TA, Huang Z, Kay MA . Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotyped adeno-associated virus vectors. J Virol 2004; 78: 3110–3122.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Wang J, Xie J, Lu H, Chen L, Hauck B, Samulski RJ et al. Existence of transient functional double-stranded DNA intermediates during recombinant AAV transduction. Proc Natl Acad Sci USA 2007; 104: 13104–13109.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wang L, Wang H, Bell P, McCarter RJ, He J, Calcedo R et al. Systematic evaluation of AAV vectors for liver directed gene transfer in murine models. Mol Ther 2010; 18: 118–125.

    Article  CAS  PubMed  Google Scholar 

  26. Qian J, Borovok M, Bi L, Kazazian Jr HH, Hoyer LW . Inhibitor antibody development and T cell response to human factor VIII in murine hemophilia A. Thromb Haemost 1999; 81: 240–244.

    Article  CAS  PubMed  Google Scholar 

  27. Scallan CD, Lillicrap D, Jiang H, Qian X, Patarroyo-White SL, Parker AE et al. Sustained phenotypic correction of canine hemophilia A using an adeno-associated viral vector. Blood 2003; 102: 2031–2037.

    Article  CAS  PubMed  Google Scholar 

  28. Jiang H, Lillicrap D, Patarroyo-White S, Liu T, Qian X, Scallan CD et al. Multiyear therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor VIII to hemophilia A mice and dogs. Blood 2006; 108: 107–115.

    Article  CAS  PubMed  Google Scholar 

  29. Sarkar R, Mucci M, Addya S, Tetreault R, Bellinger DA, Nichols TC et al. Long-term efficacy of adeno-associated virus serotypes 8 and 9 in hemophilia a dogs and mice. Hum Gene Ther 2006; 17: 427–439.

    Article  CAS  PubMed  Google Scholar 

  30. Sabatino DE, Lange AM, Altynova ES, Sarkar R, Zhou S, Merricks EP et al. Efficacy and safety of long-term prophylaxis in severe hemophilia A dogs following liver gene therapy using AAV vectors. Mol Ther 2011; 19: 442–449.

    Article  CAS  PubMed  Google Scholar 

  31. Lofqvist T, Nilsson IM, Berntorp E, Pettersson H . Haemophilia prophylaxis in young patients—a long-term follow-up. J Intern Med 1997; 241: 395–400.

    Article  CAS  PubMed  Google Scholar 

  32. Finn JD, Ozelo MC, Sabatino DE, Franck HW, Merricks EP, Crudele JM et al. Eradication of neutralizing antibodies to factor VIII in canine hemophilia A after liver gene therapy. Blood 2010; 116: 5842–5848.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Fausto N, Webber EM (eds). The Liver: Biology and Pathobiology. Raven: New York, 1994.

    Google Scholar 

  34. Fausto N, Campbell JS . The role of hepatocytes and oval cells in liver regeneration and repopulation. Mech Dev 2003; 120: 117–130.

    Article  CAS  PubMed  Google Scholar 

  35. Magami Y, Azuma T, Inokuchi H, Kokuno S, Moriyasu F, Kawai K et al. Cell proliferation and renewal of normal hepatocytes and bile duct cells in adult mouse liver. Liver 2002; 22: 419–425.

    Article  PubMed  Google Scholar 

  36. Nakai H, Yant SR, Storm TA, Fuess S, Meuse L, Kay MA . Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo. J Virol 2001; 75: 6969–6976.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Miao CH, Snyder RO, Schowalter DB, Patijn GA, Donahue B, Winther B et al. The kinetics of rAAV integration in the liver. Nat Genet 1998; 19: 13–15.

    Article  CAS  PubMed  Google Scholar 

  38. Cunningham SC, Dane AP, Spinoulas A, Logan GJ, Alexander IE . Gene delivery to the juvenile mouse liver using AAV2/8 vectors. Mol Ther 2008; 16: 1081–1088.

    Article  CAS  PubMed  Google Scholar 

  39. Wang L, Bell P, Lin J, Calcedo R, Tarantal AF, Wilson JM . AAV8-mediated hepatic gene transfer in infant rhesus monkeys (Macaca mulatta). Mol Ther 2011; 19: 2012–2020.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Malik P, McQuiston SA, Yu XJ, Pepper KA, Krall WJ, Podsakoff GM et al. Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line. J Virol 1997; 71: 1776–1783.

    CAS  PubMed Central  PubMed  Google Scholar 

  41. Flotte TR . Adeno-associated virus-based gene therapy for inherited disorders. Pediatr Res 2005; 58: 1143–1147.

    Article  CAS  PubMed  Google Scholar 

  42. Grimm D, Pandey K, Nakai H, Storm TA, Kay MA . Liver transduction with recombinant adeno-associated virus is primarily restricted by capsid serotype not vector genotype. J Virol 2006; 80: 426–439.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Conlon TJ, Cossette T, Erger K, Choi YK, Clarke T, Scott-Jorgensen M et al. Efficient hepatic delivery and expression from a recombinant adeno-associated virus 8 pseudotyped alpha1-antitrypsin vector. Mol Ther 2005; 12: 867–875.

    Article  CAS  PubMed  Google Scholar 

  44. Solomon JB . Foetal and neonatal immunology. In: Neuberger A, Tatum EL (eds). Frontiers of Biology, vol. 20. North-Holland Publishing Company: Amsterdam/London, 1971, pp 353–354.

    Google Scholar 

  45. Lechardeur D, Sohn KJ, Haardt M, Joshi PB, Monck M, Graham RW et al. Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Therapy 1999; 6: 482–497.

    Article  CAS  PubMed  Google Scholar 

  46. Hacein-Bey-Abina S, Garrigue A, Wang GP, Soulier J, Lim A, Morillon E et al. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Invest 2008; 118: 3132–3142.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003; 302: 415–419.

    Article  CAS  PubMed  Google Scholar 

  48. Scallan CD, Liu T, Parker AE, Patarroyo-White SL, Chen H, Jiang H et al. Phenotypic correction of a mouse model of hemophilia A using AAV2 vectors encoding the heavy and light chains of FVIII. Blood 2003; 102: 3919–3926.

    Article  CAS  PubMed  Google Scholar 

  49. Sabatino DE, Lange AM, Altynova ES, Sarkar R, Zhou Ss, Merricks EP et al. Efficacy and safety of long-term prophylaxis in severe hemophilia A dogs following liver gene therapy using AAV vectors. Mol Ther 2011; 19: 442–449.

    Article  CAS  PubMed  Google Scholar 

  50. Hu C, Cela RG, Suzuki M, Lee B, Lipshutz GS . Neonatal helper-dependent adenoviral vector gene therapy mediates correction of hemophilia A and tolerance to human factor VIII. Proc Natl Acad Sci USA 2011; 108: 2082–2087.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Mingozzi F, Liu YL, Dobrzynski E, Kaufhold A, Liu JH, Wang Y et al. Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer. J Clin Invest 2003; 111: 1347–1356.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Jamieson BD, Butler LD, Ahmed R . Effective clearance of a persistent viral infection requires cooperation between virus-specific Lyt2+ T cells and nonspecific bone marrow-derived cells. J Virol 1987; 61: 3930–3937.

    CAS  PubMed Central  PubMed  Google Scholar 

  53. Jamieson BD, Ahmed R . T-cell tolerance: exposure to virus in utero does not cause a permanent deletion of specific T cells. Proc Natl Acad Sci USA 1988; 85: 2265–2268.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Calcedo R, Vandenberghe LH, Gao G, Lin J, Wilson JM . Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J Infect Dis 2009; 199: 381–390.

    Article  PubMed  Google Scholar 

  55. Boutin S, Monteilhet V, Veron P, Leborgne C, Benveniste O, Montus MF et al. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum Gene Ther 2010; 21: 704–712.

    Article  CAS  PubMed  Google Scholar 

  56. Lollar P . Pathogenic antibodies to coagulation factors. Part one: factor VIII and factor IX. J Thromb Haemost 2004; 2: 1082–1095.

    Article  CAS  PubMed  Google Scholar 

  57. Fadel S, Sarzotti M . Cellular immune responses in neonates. Int Rev Immunol 2000; 19: 173–193.

    Article  CAS  PubMed  Google Scholar 

  58. Evans MM, Williamson WG, Irvine WJ . The appearance of immunological competence at an early age in New Zealand black mice. Clin Exp Immunol 1968; 3: 375–383.

    CAS  PubMed Central  PubMed  Google Scholar 

  59. Fan X, Ang A, Pollock-Barziv SM, Dipchand AI, Ruiz P, Wilson G et al. Donor-specific B-cell tolerance after ABO-incompatible infant heart transplantation. Nat Med 2004; 10: 1227–1233.

    Article  CAS  PubMed  Google Scholar 

  60. Kay HE, Doe J, Hockley A . Response of human foetal thymocytes to phytohaemagglutinin (PHA). Immunology 1970; 18: 393–396.

    CAS  PubMed Central  PubMed  Google Scholar 

  61. Yonemura H, Sugawara K, Nakashima K, Nakahara Y, Hamamoto T, Mimaki I et al. Efficient production of recombinant human factor VIII by co-expression of the heavy and light chains. Protein Eng 1993; 6: 669–674.

    Article  CAS  PubMed  Google Scholar 

  62. Nguyen AT, Dow AC, Kupiec-Weglinski J, Busuttil RW, Lipshutz GS . Evaluation of gene promoters for liver expression by hydrodynamic gene transfer. J Surg Res 2008; 148: 60–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Bi L, Sarkar R, Naas T, Lawler AM, Pain J, Shumaker SL et al. Further characterization of factor VIII-deficient mice created by gene targeting: RNA and protein studies. Blood 1996; 88: 3446–3450.

    CAS  PubMed  Google Scholar 

  64. Sabatino DE, Freguia CF, Toso R, Santos A, Merricks EP, Kazazian Jr HH et al. Recombinant canine B-domain-deleted FVIII exhibits high specific activity and is safe in the canine hemophilia A model. Blood 2009; 114: 4562–4565.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Dejana E, Callioni A, Quintana A, de Gaetano G . Bleeding time in laboratory animals. II—a comparison of different assay conditions in rats. Thromb Res 1979; 15: 191–197.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by grants from National Institutes of Health (5K08HD057555-03,4, 3KO8HD057555-03S1 and 1R01NS071076-01A1) and from the Stein–Oppenheimer Foundation to GSL. We thank Tamara Horwich, MD, MS (UCLA), for assistance with statistical analysis; Denise Sabatino, PhD (University of Pennsylvania), for discussion of FVIII antigen, activity, and light and heavy chain determination, and for providing antibodies that are no longer commercially available; and Robin Rosenblatt, Fides Lay, Eun K Lee and Luciano Castaneda for technical assistance.

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  1. Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

    C Hu & G S Lipshutz

  2. Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

    G S Lipshutz

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Hu, C., Lipshutz, G. AAV-based neonatal gene therapy for hemophilia A: long-term correction and avoidance of immune responses in mice. Gene Ther 19, 1166–1176 (2012). https://doi.org/10.1038/gt.2011.200

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