© The American Society of Gene & Cell Therapy
commentary
Moving Forward Toward a Cure for Hemophilia B Thierry VandenDriessche1,2 and Marinee K Chuah1,2 doi:10.1038/mt.2015.56
A
AV vectors are among the most promising to treat hereditary diseases by gene therapy. Long-term expression of therapeutic genes has been demonstrated in preclinical models and in clinical trials after AAV delivery in various tissues. In particular, AAV gene transfer to the retina resulted in long-term correction of RPE65 deficiency, a rare form of congenital blindness.1–3 Similarly, AAV-based gene therapy to the skeletal muscle resulted in a sustained reduction of postprandial chylomicron levels in patients afflicted by lipoprotein lipase (LPL) deficiency,4,5 possibly reducing the risk of pancreatitis, one of the hallmarks of this genetic disease. This latest clinical advance constitutes the basis of the first Marketing Authorization Approval—albeit under specific restrictive conditions—of a gene therapy product (i.e., Glybera, developed by uniQure) by the European Medicines Agency.6 AAV has also been explored since the 1990s as a vector to treat hemophilia B via clotting FIX gene therapy.7 Although FIX expression could be detected after liver-directed gene therapy, the levels were transient.8 This transience was possibly due to the immune clearance of transduced hepatocytes by AAV-specific T cells,9 consistent with the occurrence of transient liver toxicity as measured by elevated serum transaminases. In the muscle, however, FIX expression 1 Department of Gene Therapy and Regenerative Medicine, Free University of Brussels (VUB), Brussels, Belgium; 2Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, University of Leuven, Leuven, Belgium Correspondence: Thierry VandenDriessche and Marinee Chuah, Department of Gene Therapy and Regenerative Medicine, Faculty of Medicine and Pharmacy, Building D, Room D306, Free University of Brussels (VUB), Laarbeeklaan 103 B-1090, Brussels, Belgium. E-mail: [email protected] and [email protected]
Molecular Therapy vol. 23 no. 5 may 2015
was sustained for 10 years and no transient toxicity was apparent, but the FIX production by the myofibers was insufficient to yield detectable FIX levels in the blood.10,11 In a report published in the New England Journal of Medicine,12 Nathwani and colleagues (University College London (UCL) and St. Jude Children’s Research Hospital) provided an update on an AAV serotype 8 (AAV8) vector-based gene therapy trial for hemophilia B (ClinicalTrials.gov NCT00979238), extending the findings of a previous report.13 In this trial, patients suffering from severe hemophilia B (50%) and the risk of transaminitis should be further reduced, obviating the need for immune suppression. Consequently, there is still a need to further improve the efficacy and safety of gene therapy for hemophilia. This justifies the development of improved “secondgeneration” AAV vectors that allow for higher FIX expression levels at lower and thus potentially safer vector doses. This could possibly be achieved by using a hyperactive FIX-R338L (Padua) transgene. We had demonstrated that incorporation of a gain-of-function R338L mutation in the FIX protein resulted in a 5- to 10-fold increase in clotting activity using both lentiviral and AAV vectors.17,18 This hyperactivating mutation was previously identified in thrombophilic patients who express FIX protein with more than eight times the normal specific activity, presumably with more efficient generation of thrombin.19 When incorporated into a gene therapy vector, this gain-of-function R338L mutation translated into a significant dose advantage, as substantially lower vector doses could be used to achieve a comparable therapeutic effect. These encouraging studies prompted preclinical studies in canine hemophilia B models20–22 that confirmed our initial results in hemophilic mice regarding the enhanced functionality of FIX-R338L Padua.17,18 These preclinical studies justified the use of the hyperactive FIX-R338L for clinical applications in patients suffering from severe hemophilia B. It is noteworthy that a clinical trial is ongoing in patients suffering from severe hemophilia B, based on AAV8-mediated liver-directed gene therapy of the hyperactive FIX-R338L Padua (ClinicalTrials.gov NCT 01687608). It is particularly encouraging that FIX activity levels at 10% levels or higher had been reported.23 Another strategy that could be used to boost FIX expression levels is based on the design of more robust liver-specific promoters. We have recently identified hepatocyte-specific transcriptional cis-regulatory modules (CRMs)
810
using a computational strategy that increased FIX levels more than 10-fold.18,24,25 Vector efficacy could be enhanced further, by combining these novel hepatocyte-specific cis-regulatory modules with a codonoptimized hyperfunctional FIX-R338L Padua transgene, yielding one of the most robust vector designs for hemophilia B gene therapy to date. Finally, the use of novel hepatotropic AAV vectors that selectively increase gene transfer efficiency into human hepatocytes compared to AAV8 could potentially result in higher FIX expression levels.26 Because the clinical trial data revealed a vector dose–response, administering higher vector doses could, theoretically at least, result in increased circulating FIX levels. However, it is likely that increasing the AAV vector dose would also inadvertently increase the risk of liver inflammation and toxicity. Though this undesirable side effect could potentially be mitigated by transient immune suppression, it is not clear whether there would be a threshold vector dose, above which transient immune suppression would become less effective. In a worst-case scenario, the high vector doses could provoke an uncontrolled inflammatory reaction that could potentially be dangerous. This could be compounded by a more potent innate immune reaction at higher AAV vector doses.27 From a safety perspective, this would again justify the need to generate more robust gene therapy vectors that express higher levels of functional FIX proteins. At first glance, it seems reassuring that vector doses exceeding the highest dose used in the trial (i.e., 2 × 1012 vg/kg) had not been associated with any dose-limiting toxicity in preclinical animal models. However, it has been particularly challenging to model this AAV-specific immune response and ensuing liver toxicity in mice, dogs, or even nonhuman primates. Recently, Herzog and colleagues established an alternative murine model that overcomes this limitation and that is well suited to assess this potential risk.28 In this model, ex vivo– expanded AAV capsid-specific CD8+ T cells are adoptively transferred in mice that were subjected to AAV2- or AAV8-based gene therapy. Consequently, this provoked the loss of AAV2- or AAV8-transduced hepatocytes consistent with transaminitis. Would it be possible to diminish the risk of liver toxicity by modifying the vector rather than by subjecting the patient to
immune suppression, which is in itself not a risk-free intervention? It is unlikely that changing AAV serotypes would suffice, because they share common T-cell e pitopes that are recognized by the peripheral blood mononuclear cells obtained from the treated patients.9 Instead, it would make more sense to modify the intracellular processing of the AAV capsids to the extent that the presentation of antigenic peptides on major histocompatibility complex class I is reduced. For instance, this could be accomplished by using capsid mutants that are less susceptible to proteasomal degradation and concomitantly resulted in reduced hepatotoxicity.28 We have recently generated an alternative “immune stealth” AAV capsid that inhibits the proteasome directly and efficiently prevents major histocompatibility complex class I presentation of AAV capsid–derived peptides, without interfering with the transduction efficiency per se (unpublished observations). The recent clinical findings by Nathwani and colleagues12 constitute an important step in the right direction. Nevertheless, gene therapy for hemophilia B is not quite ready for ‘prime-time’. A bona fide cure of hemophilia B has not yet been established and some short-term safety issues remain to be addressed. Consequently, these clinical data justify the development of improved ‘next-generation’ AAV vectors that allow for higher FIX expression levels at lower and thus potentially safer vector doses. This could possibly be achieved by using hyperactive FIX (Padua), more robust cis-regulatory modules to drive the therapeutic transgene and/or improved hepatotropic AAV capsid variants. It is therefore important to continue to move forward cautiously towards the development of a safe and effective cure for hemophilia in order to ultimately maximize the benefits for those patients and their families that are afflicted by this severe bleeding disorder. ACKNOWLEDGMENTS Some of the research described in this Commentary was supported by grants from European Union Framework Program 7 PERSIST, Association Française Contre les Myopathies (AFM), the Fund for Scientific Research (FWO), Free University of Brussels Geconcerteerde Onderzoeksacties, Strategic Research Program (“Grower”), and Industrieel Onderzoeksfonds (Genefix) and Willy Gepts Fund.
www.moleculartherapy.org vol. 23 no. 5 may 2015
© The American Society of Gene & Cell Therapy
references
1. Bainbridge, JW, Smith, AJ, Barker, SS, Robbie, S, Henderson, R, Balaggan, K et al. (2008). Effect of gene therapy on visual function in Leber’s congenital amaurosis. N Engl J Med 358: 2231–2239. 2. Maguire, AM, High, KA, Auricchio, A, Wright, JF, Pierce, EA, Testa, F et al. (2009). Age-dependent effects of RPE65 gene therapy for Leber’s congenital amaurosis: a phase 1 dose-escalation trial. Lancet 374: 1597–1605; erratum, Lancet 2010;375: 30. 3. Simonelli, F, Maguire, AM, Testa, F, Pierce, EA, Mingozzi, F, Bennicelli, JL et al. (2010). Gene therapy for Leber’s congenital amaurosis is safe and effective through 1.5 years after vector administration. Mol Ther 18: 643–650. 4. Carpentier, AC, Frisch, F, Labbé, SM, Gagnon, R, de Wal, J, Greentree, S et al. (2012). Effect of alipogene tiparvovec (AAV1-LPL(S447X)) on postprandial chylomicron metabolism in lipoprotein lipase–deficient patients. J Clin Endocrinol Metab 97: 1635–1644. 5. Gaudet, D, Méthot, J, Déry, S, Brisson, D, Essiembre, C, Tremblay, G et al. (2013). Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an openlabel trial. Gene Ther 20: 361–369. 6. European Medicines Agency. Glybera (public assessment report). 7. Herzog, RW, Hagstrom, JN, Kung, SH, Tai, SJ, Wilson, JM, Fisher, KJ et al. (1997). Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus. Proc Natl Acad Sci USA 94: 5804–5809. 8. Manno, CS, Pierce, GF, Arruda, VR, Glader, B, Ragni, M, Rasko, JJ et al. (2006). Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response. Nat Med 12: 342–347. 9. Mingozzi, F, Maus, MV, Hui, DJ, Sabatino, DE, Murphy, SL, Rasko, JE et al. (2007). CD8(+) T-cell responses to adeno-associated virus capsid in humans. Nat Med 13: 419–422.
Molecular Therapy vol. 23 no. 5 may 2015
commentary 10. Kay, MA, Manno, CS, Ragni, MV, Larson, PJ, Couto, LB, McClelland, A et al. (2000). Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet 24: 257–261. 11. Buchlis, G, Podsakoff, GM, Radu, A, Hawk, SM, Flake, AW, Mingozzi, F et al. (2012). Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer. Blood 119: 3038–3041. 12. Nathwani, AC, Reiss, UM, Tuddenham, EG, Rosales, C, Chowdary, P, McIntosh, J et al. (2014). Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med 371: 1994–2004. 13. Nathwani, AC, Tuddenham, EG, Rangarajan, S, Rosales, C, McIntosh, J, Linch, DC et al. (2011). Adenovirus-associated virus vector–mediated gene transfer in hemophilia B. N Engl J Med 365: 2357–2365. 14. Nathwani, AC, Gray, JT, McIntosh, J, Ng, CY, Zhou, J, Spence, Y et al. (2007). Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates. Blood 109: 1414–1421. 15. World Federation of Hemophilia. Severity of hemophilia . 16. Mingozzi, F, Meulenberg, JJ, Hui, DJ, Basner-Tschakarjan, E, Hasbrouck, NC, Edmonson, SA et al. (2009). AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells. Blood 114: 2077–2086. 17. Cantore, A, Nair, N, Della Valle, P, Di Matteo, M, Màtrai, J, Sanvito, F et al. (2012). Hyperfunctional coagulation factor IX improves the efficacy of gene therapy in hemophilic mice. Blood 120: 4517–4520. 18. Nair, N, Rincon, MY, Evens, H, Sarcar, S, Dastidar, S, Samara-Kuko, E et al. (2014). Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy. Blood 123: 3195–3199. 19. Simioni, P, Tormene, D, Tognin, G, Gavasso, S, Bulato, C, Iacobelli, NP et al. (2009). X-linked thrombophilia with a mutant factor IX (factor IX Padua). N Engl J Med 361: 1671–1675.
20. Finn, JD, Nichols, TC, Svoronos, N, Merricks, EP, Bellenger, DA, Zhou, S et al. (2012). The efficacy and the risk of immunogenicity of FIX Padua (R338L) in hemophilia B dogs treated by AAV muscle gene therapy. Blood 120: 4521–4523. 21. Crudele, JM, Finn, JD, Siner, JI, Martin, NB, Niemeyer, GP, Zhou, S et al. (2015). AAV liver expression of FIXPadua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice. Blood 125: 1553–1561. 22. Cantore, A, Ranzani, M, Bartholomae, CC, Volpin M, Valle, PD, Sanvito, F, et al. Liver-directed lentiviral gene therapy in a dog model of hemophilia B.Sci Transl Med. 4;7:277ra28. 23. Baxter Healthcare Corporation. Baxter provides progress update on gene therapy program, including phase I/II clinical trial of BAX 335, investigational gene therapy for hemophilia B Press release, 12 February 2015 24. Chuah, MK, Petrus, I, De Bleser, P, Le Guiner, C, Gernoux, G, Adjali, O et al. (2014). Liver-specific transcriptional modules identified by genome-wide in silico analysis enable efficient gene therapy in mice and nonhuman primates. Mol Ther 22: 1605–1613. 25. Di Matteo, M, Samara-Kuko, E, Ward, NJ, Waddington, SN, McVey, JH, Chuah, MK et al. (2014). Hyperactive piggyBac transposons for sustained and robust liver-targeted gene therapy. Mol Ther 22: 1614–1624. 26. Lisowski, L, Dane, AP, Chu, K, Zhang, Y, Cunningham, SC, Wilson, EM et al. (2014). Selection and evaluation of clinically relevant AAV variants in a xenograft liver model. Nature 506: 382–386. 27. Martino, AT, Suzuki, M, Markusic, DM, Zolotukhin, I, Ryals, RC, Moghimi, B et al. (2011). The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9–dependent innate immune responses in the liver. Blood 117: 6459–6468. 28. Martino, AT, Basner-Tschakarjan, E, Markusic, DM, Finn, JD, Hinderer, C, Zhou, S et al. (2013). Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells. Blood 121: 2224–2233.
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commentary
Moving Forward Toward a Cure for Hemophilia B Thierry VandenDriessche1,2 and Marinee K Chuah1,2 doi:10.1038/mt.2015.56
A
AV vectors are among the most promising to treat hereditary diseases by gene therapy. Long-term expression of therapeutic genes has been demonstrated in preclinical models and in clinical trials after AAV delivery in various tissues. In particular, AAV gene transfer to the retina resulted in long-term correction of RPE65 deficiency, a rare form of congenital blindness.1–3 Similarly, AAV-based gene therapy to the skeletal muscle resulted in a sustained reduction of postprandial chylomicron levels in patients afflicted by lipoprotein lipase (LPL) deficiency,4,5 possibly reducing the risk of pancreatitis, one of the hallmarks of this genetic disease. This latest clinical advance constitutes the basis of the first Marketing Authorization Approval—albeit under specific restrictive conditions—of a gene therapy product (i.e., Glybera, developed by uniQure) by the European Medicines Agency.6 AAV has also been explored since the 1990s as a vector to treat hemophilia B via clotting FIX gene therapy.7 Although FIX expression could be detected after liver-directed gene therapy, the levels were transient.8 This transience was possibly due to the immune clearance of transduced hepatocytes by AAV-specific T cells,9 consistent with the occurrence of transient liver toxicity as measured by elevated serum transaminases. In the muscle, however, FIX expression 1 Department of Gene Therapy and Regenerative Medicine, Free University of Brussels (VUB), Brussels, Belgium; 2Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, University of Leuven, Leuven, Belgium Correspondence: Thierry VandenDriessche and Marinee Chuah, Department of Gene Therapy and Regenerative Medicine, Faculty of Medicine and Pharmacy, Building D, Room D306, Free University of Brussels (VUB), Laarbeeklaan 103 B-1090, Brussels, Belgium. E-mail: [email protected] and [email protected]
Molecular Therapy vol. 23 no. 5 may 2015
was sustained for 10 years and no transient toxicity was apparent, but the FIX production by the myofibers was insufficient to yield detectable FIX levels in the blood.10,11 In a report published in the New England Journal of Medicine,12 Nathwani and colleagues (University College London (UCL) and St. Jude Children’s Research Hospital) provided an update on an AAV serotype 8 (AAV8) vector-based gene therapy trial for hemophilia B (ClinicalTrials.gov NCT00979238), extending the findings of a previous report.13 In this trial, patients suffering from severe hemophilia B (50%) and the risk of transaminitis should be further reduced, obviating the need for immune suppression. Consequently, there is still a need to further improve the efficacy and safety of gene therapy for hemophilia. This justifies the development of improved “secondgeneration” AAV vectors that allow for higher FIX expression levels at lower and thus potentially safer vector doses. This could possibly be achieved by using a hyperactive FIX-R338L (Padua) transgene. We had demonstrated that incorporation of a gain-of-function R338L mutation in the FIX protein resulted in a 5- to 10-fold increase in clotting activity using both lentiviral and AAV vectors.17,18 This hyperactivating mutation was previously identified in thrombophilic patients who express FIX protein with more than eight times the normal specific activity, presumably with more efficient generation of thrombin.19 When incorporated into a gene therapy vector, this gain-of-function R338L mutation translated into a significant dose advantage, as substantially lower vector doses could be used to achieve a comparable therapeutic effect. These encouraging studies prompted preclinical studies in canine hemophilia B models20–22 that confirmed our initial results in hemophilic mice regarding the enhanced functionality of FIX-R338L Padua.17,18 These preclinical studies justified the use of the hyperactive FIX-R338L for clinical applications in patients suffering from severe hemophilia B. It is noteworthy that a clinical trial is ongoing in patients suffering from severe hemophilia B, based on AAV8-mediated liver-directed gene therapy of the hyperactive FIX-R338L Padua (ClinicalTrials.gov NCT 01687608). It is particularly encouraging that FIX activity levels at 10% levels or higher had been reported.23 Another strategy that could be used to boost FIX expression levels is based on the design of more robust liver-specific promoters. We have recently identified hepatocyte-specific transcriptional cis-regulatory modules (CRMs)
810
using a computational strategy that increased FIX levels more than 10-fold.18,24,25 Vector efficacy could be enhanced further, by combining these novel hepatocyte-specific cis-regulatory modules with a codonoptimized hyperfunctional FIX-R338L Padua transgene, yielding one of the most robust vector designs for hemophilia B gene therapy to date. Finally, the use of novel hepatotropic AAV vectors that selectively increase gene transfer efficiency into human hepatocytes compared to AAV8 could potentially result in higher FIX expression levels.26 Because the clinical trial data revealed a vector dose–response, administering higher vector doses could, theoretically at least, result in increased circulating FIX levels. However, it is likely that increasing the AAV vector dose would also inadvertently increase the risk of liver inflammation and toxicity. Though this undesirable side effect could potentially be mitigated by transient immune suppression, it is not clear whether there would be a threshold vector dose, above which transient immune suppression would become less effective. In a worst-case scenario, the high vector doses could provoke an uncontrolled inflammatory reaction that could potentially be dangerous. This could be compounded by a more potent innate immune reaction at higher AAV vector doses.27 From a safety perspective, this would again justify the need to generate more robust gene therapy vectors that express higher levels of functional FIX proteins. At first glance, it seems reassuring that vector doses exceeding the highest dose used in the trial (i.e., 2 × 1012 vg/kg) had not been associated with any dose-limiting toxicity in preclinical animal models. However, it has been particularly challenging to model this AAV-specific immune response and ensuing liver toxicity in mice, dogs, or even nonhuman primates. Recently, Herzog and colleagues established an alternative murine model that overcomes this limitation and that is well suited to assess this potential risk.28 In this model, ex vivo– expanded AAV capsid-specific CD8+ T cells are adoptively transferred in mice that were subjected to AAV2- or AAV8-based gene therapy. Consequently, this provoked the loss of AAV2- or AAV8-transduced hepatocytes consistent with transaminitis. Would it be possible to diminish the risk of liver toxicity by modifying the vector rather than by subjecting the patient to
immune suppression, which is in itself not a risk-free intervention? It is unlikely that changing AAV serotypes would suffice, because they share common T-cell e pitopes that are recognized by the peripheral blood mononuclear cells obtained from the treated patients.9 Instead, it would make more sense to modify the intracellular processing of the AAV capsids to the extent that the presentation of antigenic peptides on major histocompatibility complex class I is reduced. For instance, this could be accomplished by using capsid mutants that are less susceptible to proteasomal degradation and concomitantly resulted in reduced hepatotoxicity.28 We have recently generated an alternative “immune stealth” AAV capsid that inhibits the proteasome directly and efficiently prevents major histocompatibility complex class I presentation of AAV capsid–derived peptides, without interfering with the transduction efficiency per se (unpublished observations). The recent clinical findings by Nathwani and colleagues12 constitute an important step in the right direction. Nevertheless, gene therapy for hemophilia B is not quite ready for ‘prime-time’. A bona fide cure of hemophilia B has not yet been established and some short-term safety issues remain to be addressed. Consequently, these clinical data justify the development of improved ‘next-generation’ AAV vectors that allow for higher FIX expression levels at lower and thus potentially safer vector doses. This could possibly be achieved by using hyperactive FIX (Padua), more robust cis-regulatory modules to drive the therapeutic transgene and/or improved hepatotropic AAV capsid variants. It is therefore important to continue to move forward cautiously towards the development of a safe and effective cure for hemophilia in order to ultimately maximize the benefits for those patients and their families that are afflicted by this severe bleeding disorder. ACKNOWLEDGMENTS Some of the research described in this Commentary was supported by grants from European Union Framework Program 7 PERSIST, Association Française Contre les Myopathies (AFM), the Fund for Scientific Research (FWO), Free University of Brussels Geconcerteerde Onderzoeksacties, Strategic Research Program (“Grower”), and Industrieel Onderzoeksfonds (Genefix) and Willy Gepts Fund.
www.moleculartherapy.org vol. 23 no. 5 may 2015
© The American Society of Gene & Cell Therapy
references
1. Bainbridge, JW, Smith, AJ, Barker, SS, Robbie, S, Henderson, R, Balaggan, K et al. (2008). Effect of gene therapy on visual function in Leber’s congenital amaurosis. N Engl J Med 358: 2231–2239. 2. Maguire, AM, High, KA, Auricchio, A, Wright, JF, Pierce, EA, Testa, F et al. (2009). Age-dependent effects of RPE65 gene therapy for Leber’s congenital amaurosis: a phase 1 dose-escalation trial. Lancet 374: 1597–1605; erratum, Lancet 2010;375: 30. 3. Simonelli, F, Maguire, AM, Testa, F, Pierce, EA, Mingozzi, F, Bennicelli, JL et al. (2010). Gene therapy for Leber’s congenital amaurosis is safe and effective through 1.5 years after vector administration. Mol Ther 18: 643–650. 4. Carpentier, AC, Frisch, F, Labbé, SM, Gagnon, R, de Wal, J, Greentree, S et al. (2012). Effect of alipogene tiparvovec (AAV1-LPL(S447X)) on postprandial chylomicron metabolism in lipoprotein lipase–deficient patients. J Clin Endocrinol Metab 97: 1635–1644. 5. Gaudet, D, Méthot, J, Déry, S, Brisson, D, Essiembre, C, Tremblay, G et al. (2013). Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an openlabel trial. Gene Ther 20: 361–369. 6. European Medicines Agency. Glybera (public assessment report). 7. Herzog, RW, Hagstrom, JN, Kung, SH, Tai, SJ, Wilson, JM, Fisher, KJ et al. (1997). Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus. Proc Natl Acad Sci USA 94: 5804–5809. 8. Manno, CS, Pierce, GF, Arruda, VR, Glader, B, Ragni, M, Rasko, JJ et al. (2006). Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response. Nat Med 12: 342–347. 9. Mingozzi, F, Maus, MV, Hui, DJ, Sabatino, DE, Murphy, SL, Rasko, JE et al. (2007). CD8(+) T-cell responses to adeno-associated virus capsid in humans. Nat Med 13: 419–422.
Molecular Therapy vol. 23 no. 5 may 2015
commentary 10. Kay, MA, Manno, CS, Ragni, MV, Larson, PJ, Couto, LB, McClelland, A et al. (2000). Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet 24: 257–261. 11. Buchlis, G, Podsakoff, GM, Radu, A, Hawk, SM, Flake, AW, Mingozzi, F et al. (2012). Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer. Blood 119: 3038–3041. 12. Nathwani, AC, Reiss, UM, Tuddenham, EG, Rosales, C, Chowdary, P, McIntosh, J et al. (2014). Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med 371: 1994–2004. 13. Nathwani, AC, Tuddenham, EG, Rangarajan, S, Rosales, C, McIntosh, J, Linch, DC et al. (2011). Adenovirus-associated virus vector–mediated gene transfer in hemophilia B. N Engl J Med 365: 2357–2365. 14. Nathwani, AC, Gray, JT, McIntosh, J, Ng, CY, Zhou, J, Spence, Y et al. (2007). Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates. Blood 109: 1414–1421. 15. World Federation of Hemophilia. Severity of hemophilia . 16. Mingozzi, F, Meulenberg, JJ, Hui, DJ, Basner-Tschakarjan, E, Hasbrouck, NC, Edmonson, SA et al. (2009). AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells. Blood 114: 2077–2086. 17. Cantore, A, Nair, N, Della Valle, P, Di Matteo, M, Màtrai, J, Sanvito, F et al. (2012). Hyperfunctional coagulation factor IX improves the efficacy of gene therapy in hemophilic mice. Blood 120: 4517–4520. 18. Nair, N, Rincon, MY, Evens, H, Sarcar, S, Dastidar, S, Samara-Kuko, E et al. (2014). Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy. Blood 123: 3195–3199. 19. Simioni, P, Tormene, D, Tognin, G, Gavasso, S, Bulato, C, Iacobelli, NP et al. (2009). X-linked thrombophilia with a mutant factor IX (factor IX Padua). N Engl J Med 361: 1671–1675.
20. Finn, JD, Nichols, TC, Svoronos, N, Merricks, EP, Bellenger, DA, Zhou, S et al. (2012). The efficacy and the risk of immunogenicity of FIX Padua (R338L) in hemophilia B dogs treated by AAV muscle gene therapy. Blood 120: 4521–4523. 21. Crudele, JM, Finn, JD, Siner, JI, Martin, NB, Niemeyer, GP, Zhou, S et al. (2015). AAV liver expression of FIXPadua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice. Blood 125: 1553–1561. 22. Cantore, A, Ranzani, M, Bartholomae, CC, Volpin M, Valle, PD, Sanvito, F, et al. Liver-directed lentiviral gene therapy in a dog model of hemophilia B.Sci Transl Med. 4;7:277ra28. 23. Baxter Healthcare Corporation. Baxter provides progress update on gene therapy program, including phase I/II clinical trial of BAX 335, investigational gene therapy for hemophilia B Press release, 12 February 2015 24. Chuah, MK, Petrus, I, De Bleser, P, Le Guiner, C, Gernoux, G, Adjali, O et al. (2014). Liver-specific transcriptional modules identified by genome-wide in silico analysis enable efficient gene therapy in mice and nonhuman primates. Mol Ther 22: 1605–1613. 25. Di Matteo, M, Samara-Kuko, E, Ward, NJ, Waddington, SN, McVey, JH, Chuah, MK et al. (2014). Hyperactive piggyBac transposons for sustained and robust liver-targeted gene therapy. Mol Ther 22: 1614–1624. 26. Lisowski, L, Dane, AP, Chu, K, Zhang, Y, Cunningham, SC, Wilson, EM et al. (2014). Selection and evaluation of clinically relevant AAV variants in a xenograft liver model. Nature 506: 382–386. 27. Martino, AT, Suzuki, M, Markusic, DM, Zolotukhin, I, Ryals, RC, Moghimi, B et al. (2011). The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9–dependent innate immune responses in the liver. Blood 117: 6459–6468. 28. Martino, AT, Basner-Tschakarjan, E, Markusic, DM, Finn, JD, Hinderer, C, Zhou, S et al. (2013). Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells. Blood 121: 2224–2233.
811
