We found that the use of a high-specific-activity transgene, factor IX–R338L, and codon-optimized expression cassette permitted the safe use of a low dose of vector (5×1011 vg per kilogram) and yielded high levels of sustained vector-derived factor IX coagulant activity and a low incidence of a capsid-directed immune response.7,10,11,20-22 These data support our hypothesis that a highly efficient vector can be administered at low doses to drive therapeutic factor IX expression while minimizing the risk of an AAV capsid-directed immune response.

We found that SPK-9001 was safe, a finding that is consistent with the results in previously published AAV gene-therapy trials.7,23 In addition, inhibitors to nonmutant FIX protein did not develop in any participants, and no marked ELISPOT response to factor IX–R338L was seen — findings that support the conclusions that expression of factor IX–R338L did not result in neoantigen formation and that sustained factor IX–R338L expression was safe over the time course that was examined in participants with either positive or negative results for cross-reacting material.

Furthermore, transgene-derived factor IX–specific activity was consistent with results from preclinical studies.13,24 The long-term safety of AAV gene transfer remains to be determined; however, to date, no genotoxic or gene-silencing events have been noted in human participants, including those who have been followed since the first AAV trials were reported in 1998.25 Transgene DNA is stabilized predominantly episomally. Thus, an advantage of AAV-mediated gene transfer is the reduced risk of insertional mutagenesis. Several features of the vector and the study design minimized the risks of insertional mutagenesis that have been defined in nonclinical studies, including the use of a vector dose that was lower by a factor of 4 to 120 times than doses used in other ongoing trials of gene therapy for hemophilia, the targeting of adult postmitotic hepatocytes, and the use of a liver-specific hAAT promoter.7,11,21,22,26 Although the long-term efficacy is unknown, stable expression after liver-directed AAV-mediated factor IX gene transfer has been reported for 4 years in a clinical trial and for 8 years in canine hemophilia B models.11,27 Continued follow-up of the participants will be necessary to determine long-term safety and durability of expression and to monitor vector shedding as it relates to any possible consequence to contacts of participants.

A major impediment to the advancement of in vivo AAV gene transfer is the lack of animal models that recapitulate the human cellular immune response to the AAV capsid; thus, the study of this phenomenon is limited to clinical investigation.7,28 Two participants were safely and successfully treated with glucocorticoids that were aimed to control an AAV capsid-directed cellular immune response; transgene expression and therapeutic efficacy were maintained in these participants. Our data suggest that the cellular immune response to this AAV vector is controllable with oral glucocorticoids. This favorable response may reflect properties of the vector, the dose, or both. The occurrence of the capsid-directed immune response highlights the need for sensitive immunomonitoring techniques, which we used during the at-risk window. Our data indicate that the combined evaluation of liver-function studies and assays to assess factor IX coagulant activity aid in the timely detection and appropriate management of a capsid-directed immune response and can be completed in any clinical laboratory. ELISPOT assay results provided meaningful data to confirm the cause of elevations in the aminotransferase levels but would be impractical for use in widespread adoption of this therapy.

Despite varied trial courses among the 10 participants, the mean steady-state factor IX coagulant activity was 33.7±18.5% of the normal value, and a therapeutic effect at this dose was observed in all the participants, even those who had preexisting (low titer) neutralizing antibodies or cellular immune responses that occurred after infusion. It is not clear what the basis was for the finding that the factor IX coagulant activity in Participant 9 was approximately 2 times as high as that in the other participants. Although the mechanism of transduction with AAV vectors has been extensively studied, many aspects, including binding to cell-surface receptors, entry by means of endosomal pathways, endosomal escape, uncoating, and entry into the nucleus, are poorly understood.29,30 The range of levels of factor IX coagulant activity that we observed may represent the inherent variability in transgene-derived protein levels with this new class of therapeutic agents. The two participants who had a capsid-directed immune response also had the most rapid initial rise in factor IX coagulant activity. More extensive experience will be required in order to determine whether these events occurred by chance or represent a useful predictor of immune response. It is also unclear whether the immune response somehow facilitated vector expression. In addition, the plateau in the factor IX coagulant activity in Participant 6, which was lower than that in the other participants, is consistent with an expected reduction in hepatocyte transduction in the presence of the 1:1 titer for neutralizing antibody (anticipated to neutralize 50% of vector) to AAV (Table 1); this finding supports the hypothesis that our AAV neutralizing antibody assay is sensitive. A current limitation of in vivo AAV gene transfer is the existence of AAV neutralizing antibodies in approximately 30 to 40% of the population, who would not be eligible for this therapy as it is currently formulated.31 A sensitive AAV neutralizing antibody assay is essential for the identification of patients with a negative titer and those with low titers, who may still benefit from this therapy but who would be predicted to have a transgene expression level that is lower than the level in patients with a negative titer, given the current vector formulation.32

We found consistent clinical outcomes despite the baseline heterogeneity of the participants, including the extent of hemophilic arthropathy, age, and coexisting conditions, such as previous HCV infection, which affects more than 90% of men with severe hemophilia B who are older than 40 years of age.33 The long-term outcomes regarding sustained, stable factor IX coagulant activity on joint health and quality of life are not yet known. Recent preclinical work has shown that factor IX products with an extended half-life improved synovial and osteochondral healing after hemarthrosis in mice with hemophilia B, whereas short-term factor IX replacement for hemostasis did not prevent arthropathy.34 This information suggests that sustained factor IX coagulant activity, such as the results we observed, may function to prevent acute hemarthrosis and to minimize the long-term sequelae of joint disease. Finally, even in a small cohort with limited follow-up, the reduction in the use of factor IX concentrate represented a marked cost savings.

In conclusion, we observed that a one-time intravenous infusion of SPK-9001 safely resulted in a sustained level of factor IX coagulant activity of approximately 30% that consistently permitted the termination of prophylaxis, prevented bleeding, and nearly completely eliminated the need for exogenous factor in 10 men with hemophilia B. This early success requires confirmation in a larger cohort and long-term monitoring of safety and efficacy.