Moving Forward Toward a Cure for Hemophilia B
Authors: Thierry VandenDriessche, Marinee K Chuah
Journal: Molecular Therapy (2015)

AAV 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.1Bainbridge JW Smith AJ Barker SS Robbie S Henderson R Balaggan K et al.Effect of gene therapy on visual function in Leber's congenital amaurosis.N Engl J Med. 2008; 358: 2231-2239Crossref PubMed Scopus (1610) Google Scholar,2Maguire AM High KA Auricchio A Wright JF Pierce EA Testa F et al.Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial.Lancet. 2009; 374 (erratum, Lancet 2010;375: 30): 1597-1605Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar,3Simonelli F Maguire AM Testa F Pierce EA Mingozzi F Bennicelli JL et al.Gene therapy for Leber's congenital amaurosis is safe and effective through 1.5 years after vector administration.Mol Ther. 2010; 18: 643-650Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar 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,4Carpentier AC Frisch F Labbé SM Gagnon R de Wal J Greentree S et al.Effect of alipogene tiparvovec (AAV1-LPL(S447X)) on postprandial chylomicron metabolism in lipoprotein lipase–deficient patients.J Clin Endocrinol Metab. 2012; 97: 1635-1644Crossref PubMed Scopus (128) Google Scholar,5Gaudet D Méthot J Déry S Brisson D Essiembre C Tremblay G et al.Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an open-label trial.Gene Ther. 2013; 20: 361-369Crossref PubMed Scopus (294) Google Scholar 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.6European Medicines Agency. Glybera (public assessment report). < http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/human/medicines/002145/human_med_001480.jsp&mid=WC0b01ac058001d124.>Google Scholar AAV has also been explored since the 1990s as a vector to treat hemophilia B via clotting FIX gene therapy.7Herzog 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: 5804-5809Crossref PubMed Scopus (406) Google Scholar Although FIX expression could be detected after liver-directed gene therapy, the levels were transient.8Manno 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: 342-347Crossref PubMed Scopus (1567) Google Scholar This transience was possibly due to the immune clearance of transduced hepatocytes by AAV-specific T cells,9Mingozzi F Maus MV Hui DJ Sabatino DE Murphy SL Rasko JE et al.CD8(+) T-cell responses to adeno-associated virus capsid in humans.Nat Med. 2007; 13: 419-422Crossref PubMed Scopus (529) Google Scholar consistent with the occurrence of transient liver toxicity as measured by elevated serum transaminases. In the muscle, however, FIX expression 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.10Kay MA Manno CS Ragni MV Larson PJ Couto LB McClelland A et al.Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.Nat Genet. 2000; 24: 257-261Crossref PubMed Scopus (878) Google Scholar,11Buchlis G Podsakoff GM Radu A Hawk SM Flake AW Mingozzi F et al.Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer.Blood. 2012; 119: 3038-3041Crossref PubMed Scopus (147) Google Scholar In a report published in the New England Journal of Medicine,12Nathwani AC Reiss UM Tuddenham EG Rosales C Chowdary P McIntosh J et al.Long-term safety and efficacy of factor IX gene therapy in hemophilia B.N Engl J Med. 2014; 371: 1994-2004Crossref PubMed Scopus (879) Google Scholar 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.13Nathwani AC Tuddenham EG Rangarajan S Rosales C McIntosh J Linch DC et al.Adenovirus-associated virus vector–mediated gene transfer in hemophilia B.N Engl J Med. 2011; 365: 2357-2365Crossref PubMed Scopus (1353) Google Scholar In this trial, patients suffering from severe hemophilia B (<1% FIX) were injected by peripheral vein administration with an AAV8 vector encoding a codon-optimized FIX. A single intravenous vector infusion in 10 hemophilia B patients resulted in a dose-dependent increase in circulating FIX levels between 1 and 6% of the normal value over a median period of 3.2 years. In the high-dose group (i.e., 2 × 1012 AAV8-FIX vg/kg), a consistent increase in FIX levels to 5.1 ± 1.7% was observed in all six patients. However, some short-term toxicity was apparent, in that most of the high-dose patients developed transaminitis between week 7 and week 10 after treatment, which could be resolved by short-term immune suppression with prednisolone. It is reassuring that no late toxic effects from the gene therapy were apparent. Most importantly, the levels that were attained resulted in a significant reduction in the bleeding frequency consistent with a diminished requirement for repeated FIX protein injections. This represents a significant improvement compared to conventional prophylactic protein substitution therapy based on plasma-derived or recombinant FIX proteins that required lifelong multiple injections per week. Obviously, the efficacy and safety of the gene therapy approach described by Nathwani and colleagues would need to be confirmed in larger cohorts in phase III clinical trials, prior to obtaining marketing authorization approval by the European Medicines Agency and the US Food and Drug Administration. Considering that patients received up to 2 × 1012 vg/kg, it is clear that advances in vector manufacturing are needed to move toward phase III trials and ultimately address the market needs. This could potentially be addressed by using baculovirus-based AAV production systems that are more readily amenable to upscaling than conventional production methods based on triple transfection. Using the same AAV-FIX vector design as the one used in the UCL–St. Jude trial, a clinical trial has been initiated based on the use of an AAV5-based serotype, manufactured using the baculovirus system. It is currently not yet clear whether AAV5 can achieve comparable gene transfer efficiencies compared to AAV8 in humans, although in nonhuman primates their performance was comparable.14Nathwani AC Gray JT McIntosh J Ng CY Zhou J Spence Y et al.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. 2007; 109: 1414-1421Crossref PubMed Scopus (217) Google Scholar Clearly, achieving sustained expression of therapeutic FIX levels for 3 years is an important milestone for hemophilia and for the field at large. It is also encouraging that the levels approximated the 5% threshold, which is sufficient to convert a severe to a mild phenotype (5–40%).15World Federation of Hemophilia. Severity of hemophilia <http://www.wfh.org/en/page.aspx?pid=643Google Scholar Nevertheless, the maximum FIX levels attained were insufficient to cure the bleeding diathesis. Indeed, the treated subjects were still at risk of bleeding and occasionally required FIX protein injections to treat their bleeding episodes. Moreover, the majority of patients enrolled in the UCL–St Jude trial receiving the highest vector dose (four out of six) experienced liver toxicity requiring short-term immune suppression with prednisolone. This treatment seemed to have been effective in diminishing inflammation and the ensuing liver toxicity. It was initially hypothesized that transient immune suppression may curtail the AAV capsid–specific T-cell response that accounts for the loss of AAV-transduced hepatocytes as measured by elevated transaminase levels.13Nathwani AC Tuddenham EG Rangarajan S Rosales C McIntosh J Linch DC et al.Adenovirus-associated virus vector–mediated gene transfer in hemophilia B.N Engl J Med. 2011; 365: 2357-2365Crossref PubMed Scopus (1353) Google Scholar This hypothesis is consistent with the results obtained in some of the earlier AAV-based clinical trials for hemophilia and LPL deficiency.8Manno 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: 342-347Crossref PubMed Scopus (1567) Google Scholar,16Mingozzi F Meulenberg JJ Hui DJ Basner-Tschakarjan E Hasbrouck NC Edmonson SA et al.AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells.Blood. 2009; 114: 2077-2086Crossref PubMed Scopus (219) Google Scholar However, some of the trial subjects in the UCL–St. Jude trial developed transaminitis without showing any detectable AAV-specific T cells, as measured in ELISPOT assays. Conversely, some trial subjects had detectable AAV-specific T cells but failed to develop transaminitis. Hence, other yet unknown factors or immune mechanisms may account for these apparent discrepancies. Ideally, FIX levels should be increased to normal levels (>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 "second-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 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.17Cantore A Nair N Della Valle P Di Matteo M Màtrai J Sanvito F et al.Hyperfunctional coagulation factor IX improves the efficacy of gene therapy in hemophilic mice.Blood. 2012; 120: 4517-4520Crossref PubMed Scopus (77) Google Scholar,18Nair N Rincon MY Evens H Sarcar S Dastidar S Samara-Kuko E et al.Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy.Blood. 2014; 123: 3195-3199Crossref PubMed Scopus (63) Google Scholar 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.19Simioni P Tormene D Tognin G Gavasso S Bulato C Iacobelli NP et al.X-linked thrombophilia with a mutant factor IX (factor IX Padua).N Engl J Med. 2009; 361: 1671-1675Crossref PubMed Scopus (225) Google Scholar 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 models20Finn JD Nichols TC Svoronos N Merricks EP Bellenger DA Zhou S et al.The efficacy and the risk of immunogenicity of FIX Padua (R338L) in hemophilia B dogs treated by AAV muscle gene therapy.Blood. 2012; 120: 4521-4523Crossref PubMed Scopus (82) Google Scholar,21Crudele JM Finn JD Siner JI Martin NB Niemeyer GP Zhou S et al.AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice.Blood. 2015; 125: 1553-1561Crossref PubMed Scopus (111) Google Scholar,22Cantore 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. 2015; 4;7: 277ra28Crossref Scopus (97) Google Scholar that confirmed our initial results in hemophilic mice regarding the enhanced functionality of FIX-R338L Padua.17Cantore A Nair N Della Valle P Di Matteo M Màtrai J Sanvito F et al.Hyperfunctional coagulation factor IX improves the efficacy of gene therapy in hemophilic mice.Blood. 2012; 120: 4517-4520Crossref PubMed Scopus (77) Google Scholar,18Nair N Rincon MY Evens H Sarcar S Dastidar S Samara-Kuko E et al.Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy.Blood. 2014; 123: 3195-3199Crossref PubMed Scopus (63) Google Scholar 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.23Baxter 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.http://www.baxter.com/press_room/press_releases/2015/02_12_15_bax355.htmlGoogle Scholar 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) using a computational strategy that increased FIX levels more than 10-fold.18Nair N Rincon MY Evens H Sarcar S Dastidar S Samara-Kuko E et al.Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy.Blood. 2014; 123: 3195-3199Crossref PubMed Scopus (63) Google Scholar,24Chuah MK Petrus I De Bleser P Le Guiner C Gernoux G Adjali O et al.Liver-specific transcriptional modules identified by genome-wide in silico analysis enable efficient gene therapy in mice and nonhuman primates.Mol Ther. 2014; 22: 1605-1613Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar,25Di Matteo M Samara-Kuko E Ward NJ Waddington SN McVey JH Chuah MK et al.Hyperactive piggyBac transposons for sustained and robust liver-targeted gene therapy.Mol Ther. 2014; 22: 1614-1624Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Vector efficacy could be enhanced further, by combining these novel hepatocyte-specific cis-regulatory modules with a codon-optimized 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.26Lisowski L Dane AP Chu K Zhang Y Cunningham SC Wilson EM et al.Selection and evaluation of clinically relevant AAV variants in a xenograft liver model.Nature. 2014; 506: 382-386Crossref PubMed Scopus (297) Google Scholar 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.27Martino AT Suzuki M Markusic DM Zolotukhin I Ryals RC Moghimi B et al.The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9–dependent innate immune responses in the liver.Blood. 2011; 117: 6459-6468Crossref PubMed Scopus (145) Google Scholar 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.28Martino AT Basner-Tschakarjan E Markusic DM Finn JD Hinderer C Zhou S et al.Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells.Blood. 2013; 121: 2224-2233Crossref PubMed Scopus (117) Google Scholar 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 epitopes 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.28Martino AT Basner-Tschakarjan E Markusic DM Finn JD Hinderer C Zhou S et al.Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells.Blood. 2013; 121: 2224-2233Crossref PubMed Scopus (117) Google Scholar 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 colleagues12Nathwani AC Reiss UM Tuddenham EG Rosales C Chowdary P McIntosh J et al.Long-term safety and efficacy of factor IX gene therapy in hemophilia B.N Engl J Med. 2014; 371: 1994-2004Crossref PubMed Scopus (879) Google Scholar 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. 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.