FIX It in One Go: Enhanced Factor IX Gene Therapy for Hemophilia B

A phase 1/2 clinical trial of AAV-mediated gene therapy in patients with hemophilia B using an enhanced specific activity factor IX (FIX) transgene reports sustained levels of FIX levels, leading to the near elimination of bleeding for more than a year and without serious adverse side effects. These results are the best outcome to date for hemophilia gene therapy. A phase 1/2 clinical trial of AAV-mediated gene therapy in patients with hemophilia B using an enhanced specific activity factor IX (FIX) transgene reports sustained levels of FIX levels, leading to the near elimination of bleeding for more than a year and without serious adverse side effects. These results are the best outcome to date for hemophilia gene therapy. Patients with hemophilia A or B suffer from recurrent bleeds due to a deficiency in the clotting factors factor VIII and factor IX (FIX), respectively. With an incidence of 1/5,000 for hemophilia A and 1/25,000 live male births for hemophilia B, they are among the most common hereditary bleeding disorders. Current hemophilia management requires frequent (at least once per week) intravenous protein replacement therapy of the deficient clotting factor. This treatment is inconvenient and costly, and in some patients (∼30% for FVIII and ∼4% for FIX), it results in a neutralizing antibody response to the therapy. For all of these reasons, the prospect of single-administration gene therapy being able to deliver long-term therapeutic clotting factor levels, and possibly disease cure, is a significant advance. Now, a phase 1/2 clinical trial, sponsored by Spark Therapeutics and published in the New England Journal of Medicine, reports persistent expression of FIX activity levels in hemophilia B patients following a single-administration of adeno-associated virus (AAV) gene transfer of a gain-of-function FIX variant (George et al., 2017George L.A. Sullivan S.K. Giermasz A. Rasko J.E.J. Samelson-Jones B.J. Ducore J. Cuker A. Sullivan L.M. Majumadar S. Teitel J. et al.Hemophilia B gene therapy with a high-specific-activity factor IX variant.N. Engl. J. Med. 2017; 377: 2215-2227Crossref PubMed Scopus (442) Google Scholar) (Figure 1). Normal FIX levels range from 50% to 150%, but the values of >30% documented in the ten patients in this trial are quite sufficient to prevent most bleeding episodes. Progress with the clinical application of hemophilia gene therapy has been significant since the first report of persistent FIX expression at therapeutically relevant levels in 2011 (Nathwani et al., 2011Nathwani A.C. Tuddenham E.G.D. Rangarajan S. Rosales C. McIntosh J. Linch D.C. Chowdary P. Riddell A. Pie A.J. Harrington C. et al.Adenovirus-associated virus vector-mediated gene transfer in hemophilia B.N. Engl. J. Med. 2011; 365: 2357-2365Crossref PubMed Scopus (1359) Google Scholar). Since then, several phase 1/2 trials of FIX gene transfer have reported persistence of expression for many months, and in the past year, the first successful clinical study of factor VIII gene transfer has been reported (Pasi et al., 2016Pasi K. Wong W. Rangarajan S. Wilde J. Perry D. Interim results of an open-label phase 1/2 study of BMN 270 an AAV5-FVIII gene transfer in severe hemophilia A.Haemophilia. 2016; 22: 151Google Scholar). While such clinical advances of hemophilia gene therapy have shown much promise, there are still several challenges to its widespread introduction. At the outset, depending upon the type of AAV vector being used, 30%–50% of candidate patients will have pre-existing immunity to AAV (due to prior exposure to the wild-type AAV virus in the environment) that will neutralize attempts at transgene delivery. Following successful transgene delivery, two limitations have been documented: (1) the development of transient liver cell damage in ∼25% of recipients and (2) the consistent attainment of therapeutically useful levels of clotting factor. The study of George et al. is significant for several important safety and efficacy outcomes. The first of these positive aspects of the trial relates to the use of a gain-of-function FIX transgene, the Padua variant that results in a 7-fold increase in specific coagulant activity of the protein (Simioni et al., 2009Simioni P. Tormene D. Tognin G. Gavasso S. Bulato C. Iacobelli N.P. Finn J.D. Spiezia L. Radu C. Arruda V.R. X-linked thrombophilia with a mutant factor IX (factor IX Padua).N. Engl. J. Med. 2009; 361: 1671-1675Crossref PubMed Scopus (228) Google Scholar). This variant of FIX, a single point mutation, R338L, was initially discovered in a thrombophilic patient and is postulated to possess increased procoagulant activity due to an enhanced incorporation into the intrinsic tenase complex in the clotting factor cascade. Prior pre-clinical studies of FIX Padua in gene therapy confirmed its benefit in this context. The structure/function benefit of this variant FIX enables low-vector doses to be used to attain substantive and persistent plasma FIX levels (mean FIX 33.7%). This level of FIX not only is sufficient to eliminate the risk of spontaneous bleeding in these patients (11.1 bleeds pre versus 0.4 bleeds post) but should also be enough to minimize the risk of bleeding from interventions and trauma. There was also a highly significant reduction in the requirement for additional clotting factor replacement therapy (2,908 units FIX/kg pre versus 49 units/kg post). The results in the George et al. study have demonstrated persistence of FIX expression out to beyond one year. This finding is consistent with prior reports in large animal models of hemophilia, and a previous human study in which persistence of the transgene expression to produce therapeutic levels of clotting protein has continued for >7 years (Nathwani et al., 2014Nathwani A.C. Reiss U.M. Tuddenham E.G.D. Rosales C. Chowdary P. McIntosh J. Della Peruta M. Lheriteau E. Patel N. Raj D. 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 (885) Google Scholar). These observations pose the question of whether a single administration of AAV-mediated gene therapy could be curative for hemophilia, and if this is the case, how is this achieved with a non-integrating vector? All current hemophilia gene therapy trials are using AAV vectors of different serotypes, and while there is variability in the prevalence of pre-existing immunity with the different AAV serotypes, as noted above, this limitation will still eliminate between 30% and 50% of patients who would otherwise be eligible for these trials (Louis Jeune et al., 2013Louis Jeune V. Joergensen J.A. Hajjar R.J. Weber T. Pre-existing anti-adeno-associated virus antibodies as a challenge in AAV gene therapy.Hum. Gene Ther. Methods. 2013; 24: 59-67Crossref PubMed Scopus (184) Google Scholar). This obstacle requires further work on the discovery or generation of AAV capsids that evade pre-existing neutralizing antibodies or the further development of alternative vector systems for the ineligible patient population. The study of George et al. has been conducted with a novel AAV vector that has bioengineered changes to the vector capsid. This revision may increase the likelihood of finding patients without pre-existing immunity to AAV that would neutralize the ability of the vector to deliver the FIX transgene. The other complication that has been documented in ∼25% of patients receiving systemically delivered, liver-directed AAV gene transfer is the development of elevated liver enzyme levels, indicative of liver cell damage. In the study of George et al., the AAV vector is targeted to hepatocytes, but only two of the patients experienced the hepatotoxicity that has been described in all systemic AAV trials to date. These episodes of “transaminitis” usually occur between 2 and 12 weeks following vector delivery and are not associated with any clinical symptoms. However, with persistence of the transaminitis, there is usually a fall in transgene protein expression as vector copies are lost as a consequence of liver cell injury. In some instances, this complication has been associated with the documentation of a cytotoxic T cell response to vector capsid proteins (Mingozzi and High, 2011Mingozzi F. High K.A. Immune responses to AAV in clinical trials.Curr. Gene Ther. 2011; 11: 321-330Crossref PubMed Scopus (134) Google Scholar), but this finding is not consistent, and the pathogenesis of the liver cell damage remains under investigation. To date, fortunately, these episodes of transaminitis have responded well to transient corticosteroid administration that can be stopped after 2–3 months. Lastly, while AAV is a non-integrating vector, the fact that trillions of vector particles are delivered to these patients inevitably results in a low level of vector integration into the host cell genome. Despite this, there is very little evidence of genotoxic outcomes from long-term pre-clinical studies, and examination of integration sites in non-human primates and humans that have received AAV vectors demonstrates a low-level, random integration of AAV sequences with no obvious involvement of oncogenic loci (Gil-Farina et al., 2016Gil-Farina I. Fronza R. Kaeppel C. Lopez-Franco E. Ferreira V. D’Avola D. Benito A. Prieto J. Petry H. Gonzalez-Aseguinolaza G. Schmidt M. Recombinant AAV Integration Is Not Associated With Hepatic Genotoxicity in Nonhuman Primates and Patients.Mol. Ther. 2016; 24: 1100-1105Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Collectively, AAV-mediated gene transfer for hemophilia has advanced significantly in the past 5 years, and the study of George et al. represents the best result to date in terms of persistent levels of clotting factor activity and clinical outcome efficacy following a single intravenous infusion. The initiation and outcome of phase 3 hemophilia gene therapy studies are now keenly awaited.