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Transgene expression levels and kinetics determine risk of humoral immune response modeled in factor IX knockout and missense mutant mice

Gene Therapy volume 14, pages 429–440 (2007)Cite this article

Abstract

Immune responses leading to antibody-mediated elimination of the transgenic protein are a concern in gene replacement for congenital protein deficiencies, for which hemophilia is an important model. Although most hemophilia B patients have circulating non-functional but immunologically crossreactive factor IX (FIX) protein (CRM+ phenotype), inciting factors for FIX neutralizing antibody (inhibitor) development have been studied in crossreactive material-negative (CRM−) animal models. For this study, determinants of FIX inhibitor development were compared in hemophilia B mice, in which circulating FIX protein is absent (CRM− factor IX knockout (FIXKO) model) or present (CRM+ missense R333Q-hFIX model) modeling multiple potential therapies. The investigations compare for the first time different serotypes of adeno-associated virus (AAV) vectors (AAV2 and AAV1), each at multiple doses, in the setting of two different FIX mutations. The comparisons demonstrate in the FIXKO background (CRM− phenotype) that neither vector serotype nor vector particle number independently determine the inhibitor trigger, which is influenced primarily by the level and kinetics of transgene expression. In the CRM+ missense background, inhibitor development was never stimulated by AAV gene therapy or protein therapy, despite the persistence of lymphocytes capable of responding to FIX with non-inhibitory antibodies. This genotype/phenotype is strongly protective against antibody formation in response to FIX therapy.

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References

  1. Giannelli F, Green PM, Sommer SS, Poon M, Ludwig M, Schwaab R et al. Haemophilia B: database of point mutations and short additions and deletions – eighth edition. Nucleic Acids Res 1998; 26: 265–268.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Herzog RW, Hagstrom JN, Kung SH, Tai SJ, Wilson JM, Fisher KJ et al. 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: 5804–5809.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Monahan PE, Samulski RJ, Tazelaar J, Xiao X, Nichols TC, Bellinger DA et al. Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia. Gene Therapy 1998; 5: 40–49.

    Article  CAS  PubMed  Google Scholar 

  4. Snyder RO, Miao CH, Patijn GA, Spratt SK, Danos O, Nagy D et al. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors. Nat Genet 1997; 16: 270–276.

    Article  CAS  PubMed  Google Scholar 

  5. Chao H, Samulski R, Bellinger D, Monahan P, Nichols T, Walsh C . Persistent expression of canine factor IX in hemophilia B canines. Gene Therapy 1999; 6: 1695–1704.

    Article  CAS  PubMed  Google Scholar 

  6. Wang L, Nichols TC, Read MS, Bellinger DA, Verma IM . Sustained expression of therapeutic level of factor IX in hemophilia B dogs by AAV-mediated gene therapy in liver. Mol Ther 2000; 1: 154–158.

    Article  CAS  PubMed  Google Scholar 

  7. 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: 1258–1265.

    Article  CAS  PubMed  Google Scholar 

  8. Mount JD, Herzog RW, Tillson DM, Goodman SA, Robinson N, McCleland ML et al. Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy. Blood 2002; 99: 2670–2676.

    Article  CAS  PubMed  Google Scholar 

  9. Arruda VR, Schuettrumpf J, Herzog RW, Nichols TC, Robinson N, Lotfi Y et al. Safety and efficacy of factor IX gene transfer to skeletal muscle in murine and canine hemophilia B models by adeno-associated viral vector serotype 1. Blood 2004; 103: 85–92.

    Article  CAS  PubMed  Google Scholar 

  10. Wang L, Dobrzynski E, Schlachterman A, Cao O, Herzog RW . Systemic protein delivery by muscle-gene transfer is limited by a local immune response. Blood 2005; 105: 4226–4234.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Sarukhan A, Soudais C, Danos O, Jooss K . Factors influencing cross-presentation of non-self antigens expressed from recombinant adeno-associated virus vectors. J Gene Med 2001; 3: 260–270.

    Article  CAS  PubMed  Google Scholar 

  12. Chen J, Wu Q, Yang P, Hsu HC, Mountz JD . Determination of specific CD4 and CD8T cell epitopes after AAV2- and AAV8-hF.IX gene therapy. Mol Ther 2006; 13: 260–269.

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

    CAS  PubMed  Google Scholar 

  15. Chao H, Monahan PE, Liu Y, Samulski RJ, Walsh CE . Sustained and complete phenotype correction of hemophilia B mice following intramuscular injection of AAV1 serotype vectors. Mol Ther 2001; 4: 217–222.

    Article  CAS  PubMed  Google Scholar 

  16. Fields PA, Arruda VR, Armstrong E, Chu K, Mingozzi F, Hagstrom JN et al. Risk and prevention of anti-factor IX formation in AAV-mediated gene transfer in the context of a large deletion of F9. Mol Ther 2001; 4: 201–210.

    Article  CAS  PubMed  Google Scholar 

  17. Liu XL, Clark KR, Johnson PR . Production of recombinant adeno-associated virus vectors using a packaging cell line and a hybrid recombinant adenovirus. Gene Therapy 1999; 6: 293–299.

    Article  CAS  PubMed  Google Scholar 

  18. Jin DY, Zhang TP, Gui T, Stafford DW, Monahan PE . Creation of a mouse expressing defective human factor IX. Blood 2004; 104: 1733–1739.

    Article  CAS  PubMed  Google Scholar 

  19. Fields PA, Kowalczyk DW, Arruda VR, Armstrong E, McCleland ML, Hagstrom JN et al. Role of vector in activation of T cell subsets in immune responses against the secreted transgene product factor IX. Mol Ther 2000; 1: 225–235.

    Article  CAS  PubMed  Google Scholar 

  20. Cao O, Armstrong E, Schlachterman A, Wang L, Okita DK, Conti-Fine B et al. Immune deviation by mucosal antigen administration suppresses gene-transfer-induced inhibitor formation to factor IX. Blood 2006; 108: 480–486.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Orstavik KH, Miller CH . IgG subclass identification of inhibitors to factor IX in haemophilia B patients. Br J Haematol 1988; 68: 451–454.

    Article  CAS  PubMed  Google Scholar 

  22. Sawamoto Y, Shima M, Yamamoto M, Kamisue S, Nakai H, Tanaka I et al. Measurement of anti-factor IX IgG subclasses in haemophilia B patients who developed inhibitors with episodes of allergic reactions to factor IX concentrates. Thromb Res 1996; 83: 279–286.

    Article  CAS  PubMed  Google Scholar 

  23. Herzog RW, Fields PA, Arruda VR, Brubaker JO, Armstrong E, McClintock D et al. Influence of vector dose on factor IX-specific T and B cell responses in muscle-directed gene therapy. Hum Gene Ther 2002; 13: 1281–1291.

    Article  CAS  PubMed  Google Scholar 

  24. Ponder K . A more clinically relevant mouse model of hemophilia B. Blood 2004; 104: 1595.

    Article  CAS  Google Scholar 

  25. Smith KJ, Thompson AR, McMullen BA, Frazier D, Lin SW, Stafford D et al. Carrier testing in hemophilia B with an immunoassay that distinguishes a prevalent factor IX dimorphism. Blood 1987; 70: 1006–1013.

    CAS  PubMed  Google Scholar 

  26. Frazier D, Smith KJ, Cheung WF, Ware J, Lin SW, Thompson AR et al. Mapping of monoclonal antibodies to human factor IX. Blood 1989; 74: 971–977.

    CAS  PubMed  Google Scholar 

  27. Qian J, Collins M, Sharpe AH, Hoyer LW . Prevention and treatment of factor VIII inhibitors in murine hemophilia A. Blood 2000; 95: 1324–1329.

    CAS  PubMed  Google Scholar 

  28. Xiao W, Chirmule N, Berta SC, McCullough B, Gao G, Wilson JM . Gene therapy vectors based on adeno-associated virus type 1. J Virol 1999; 73: 3994–4003.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Chao H, Liu Y, Rabinowitz J, Li C, Samulski RJ, Walsh CE . Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors. Mol Ther 2000; 2: 619–623.

    Article  CAS  PubMed  Google Scholar 

  30. Chao H, Monahan P, Elia J, Camp A, Nichols T, Bellinger D et al. Intramuscular delivery of AAV1 vectors resulted in sustained expression of therapeutic levels of FIX in hemophilia B dogs. Blood 2002; 100: 19a.

    Google Scholar 

  31. Liu YL, Mingozzi F, Rodriguez-Colon SM, Joseph S, Dobrzynski E, Suzuki T et al. Therapeutic levels of factor IX expression using a muscle-specific promoter and adeno-associated virus serotype 1 vector. Hum Gene Ther 2004; 15: 783–792.

    Article  CAS  PubMed  Google Scholar 

  32. Gao G, Lebherz C, Weiner DJ, Grant R, Calcedo R, McCullough B et al. Erythropoietin gene therapy leads to autoimmune anemia in macaques. Blood 2004; 103: 3300–3302.

    Article  CAS  PubMed  Google Scholar 

  33. Wang L, Cao O, Swalm B, Dobrzynski E, Mingozzi F, Herzog RW . Major role of local immune responses in antibody formation to factor IX in AAV gene transfer. Gene Therapy 2005; 12: 1453–1464.

    Article  CAS  PubMed  Google Scholar 

  34. Herzog RW, Dobrzynski E . Immune implications of gene therapy for hemophilia. Semin Thromb Hemost 2004; 30: 215–226.

    Article  CAS  PubMed  Google Scholar 

  35. Chuah MK, Collen D, Vandendriessche T . Preclinical and clinical gene therapy for haemophilia. Haemophilia 2004; 10 (Suppl 4): 119–125.

    Article  PubMed  Google Scholar 

  36. Chao H, Walsh CE . AAV vectors for hemophilia B gene therapy. Mt Sinai J Med 2004; 71: 305–313.

    PubMed  Google Scholar 

  37. Wang L, Herzog RW . AAV-mediated gene transfer for treatment of hemophilia. Curr Gene Ther 2005; 5: 349–360.

    Article  CAS  PubMed  Google Scholar 

  38. Sabatino DE, Armstrong E, Edmonson S, Liu YL, Pleimes M, Schuettrumpf J et al. Novel hemophilia B mouse models exhibiting a range of mutations in the Factor IX gene. Blood 2004; 104: 2767–2774.

    Article  CAS  PubMed  Google Scholar 

  39. Bril WS, van Helden PM, Hausl C, Zuurveld MG, Ahmad RU, Hollestelle MJ et al. Tolerance to factor VIII in a transgenic mouse expressing human factor VIII cDNA carrying an Arg(593) to Cys substitution. Thromb Haemost 2006; 95: 341–347.

    Article  CAS  PubMed  Google Scholar 

  40. Greenwood R, Wang B, Midkiff K, White II GC, Lin HF, Frelinger JA . Identification of T-cell epitopes in clotting factor IX and lack of tolerance in inbred mice. J Thromb Haemost 2003; 1: 95–102.

    Article  CAS  PubMed  Google Scholar 

  41. Lozier JN, Tayebi N, Zhang P . Mapping of genes that control the antibody response to human factor IX in mice. Blood 2005; 105: 1029–1035.

    Article  CAS  PubMed  Google Scholar 

  42. Zhang HG, High KA, Wu Q, Yang P, Schlachterman A, Yu S et al. Genetic analysis of the antibody response to AAV2 and factor IX. Mol Ther 2005; 11: 866–874.

    Article  CAS  PubMed  Google Scholar 

  43. Rawle FE, Shi CX, Brown B, McKinven A, Tinlin S, Graham FL et al. Heterogeneity of the immune response to adenovirus-mediated factor VIII gene therapy in different inbred hemophilic mouse strains. J Gene Med 2004; 6: 1358–1368.

    Article  CAS  PubMed  Google Scholar 

  44. DiMichele D, Kroner B . The maintenance of tolerance after successful immune tolerance induction in hemophilia A and B: the North American Registry. Factor VIII/IX Subcommittee of the International Society for Thrombosis and Hemostasis. Haematologica 2000; 85 (Suppl 10): 40–42; discussion 2–4.

    CAS  PubMed  Google Scholar 

  45. Gilles JG, Saint-Remy JM . Healthy subjects produce both anti-factor VIII and specific anti-idiotypic antibodies. J Clin Invest 1994; 94: 1496–1505.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Hausl C, Ahmad RU, Sasgary M, Doering CB, Lollar P, Richter G et al. High-dose factor VIII inhibits factor VIII-specific memory B cells in hemophilia A with factor VIII inhibitors. Blood 2005; 106: 3415–3422.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Reding MT, Wu H, Krampf M, Okita DK, Diethelm-Okita BM, Key NS et al. CD4+ T cell response to factor VIII in hemophilia A, acquired hemophilia, and healthy subjects. Thromb Haemost 1999; 82: 509–515.

    Article  CAS  PubMed  Google Scholar 

  48. Chenuaud P, Larcher T, Rabinowitz JE, Provost N, Cherel Y, Casadevall N et al. Autoimmune anemia in macaques following erythropoietin gene therapy. Blood 2004; 103: 3303–3304.

    Article  CAS  PubMed  Google Scholar 

  49. Zinkernagel RM . Localization dose and time of antigens determine immune reactivity. Semin Immunol 2000; 12: 163–171; discussion 257–344.

    Article  CAS  PubMed  Google Scholar 

  50. Weiner HL . Induction and mechanism of action of transforming growth factor-beta-secreting Th3 regulatory cells. Immunol Rev 2001; 182: 207–214.

    Article  CAS  PubMed  Google Scholar 

  51. Gallucci S, Matzinger P . Danger signals: SOS to the immune system. Curr Opin Immunol 2001; 13: 114–119.

    Article  CAS  PubMed  Google Scholar 

  52. Brown BD, Lillicrap D . Dangerous liaisons: the role of ‘danger’ signals in the immune response to gene therapy. Blood 2002; 100: 1133–1140.

    Article  CAS  PubMed  Google Scholar 

  53. Haberman RP, Samulski RJ, McCown TJ . Attenuation of seizures and neuronal death by adeno-associated virus vector galanin expression and secretion. Nat Med 2003; 9: 1076–1080.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by NIH Grant: PO1 HL66973 (DWS and PEM) and Career Development Awards from the National Hemophilia Foundation (PEM) and Hemophilia of Georgia (PEM). We thank Joseph Elia and Nathan Laborde for valuable assistance with assays and graphics and R Jude Samulski and Nigel Key for critical review.

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Authors and Affiliations

  1. The Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    T-P Zhang & P E Monahan

  2. Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    D-Y Jin, T Gui & D W Stafford

  3. Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    R M Wardrop III

  4. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    R Maile & J A Frelinger

  5. Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    R Maile

  6. Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    P E Monahan

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  1. T-P Zhang
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Correspondence to P E Monahan.

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Zhang, TP., Jin, DY., Wardrop, R. et al. Transgene expression levels and kinetics determine risk of humoral immune response modeled in factor IX knockout and missense mutant mice. Gene Ther 14, 429–440 (2007). https://doi.org/10.1038/sj.gt.3302881

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