Characteristics of the Study Participants

Table 1. Table 1. Characteristics of the Six Study Participants at Baseline and after Gene Transfer, According to Dose of Vector.

Six men with severe hemophilia B (FIX activity, <1% of normal values) were enrolled under a protocol approved by the relevant ethics boards and regulatory agencies, after providing written informed consent. All except one participant received regular prophylaxis with FIX concentrates (two or three times per week) before gene transfer (Table 1). Participant 4 was receiving targeted prophylaxis (about once weekly), which was tailored to his sports activities and the associated risk of traumatic injury. Participant 2 had a null mutation in the FIX gene, and Participant 6 had a promoter mutation; consequently, neither had FIX protein expression. The other four participants had missense mutations, which resulted in normal plasma levels of FIX antigen but less than 1% clotting activity. All participants had modest levels (>5 relative units) of anti-AAV2 IgG antibodies before gene transfer.

Safety and Efficacy of Peripheral-Vein Infusion of Vector

No immediate changes in vital signs were noted during or after vector infusion in any participant, despite the persistence of the vector in bodily fluids (including plasma) for up to 15 days after gene transfer (Fig. 1 in the Supplementary Appendix). There were no clinically significant changes in serum chemical values (including liver enzyme levels) in any participant for the first 6 weeks after vector infusion (Fig. 2 in the Supplementary Appendix). Three adverse events were reported: anemia developed in Participants 1 and 2 at 5 and 7 weeks, respectively, after gene transfer, and Participant 3 had a transient period of bradycardia at 16 weeks after gene transfer, when he was being prepared for surgery on his left knee (see the Supplementary Appendix for details).

Low-Dose Group

Figure 1. Figure 1. Factor IX (FIX) Activity after Peripheral-Vein Infusion of the Adenovirus-Associated Virus (AAV) Vector in the Six Study Participants. A one-stage clotting assay was used to determine FIX coagulation activity at prespecified time points (arrows) after the administration of the AAV vector (scAAV2/8-LP1-hFIXco). Panels A and B show FIX levels in Participants 1 and 2, respectively, who received the low dose of vector (2×1011 vector genomes [vg] per kilogram of body weight). Panels C and D show FIX levels in Participants 3 and 4, respectively, who received the intermediate dose of vector (6×1011 vg per kilogram). Panels E and F show both FIX levels and alanine aminotransferase (ALT) levels in Participants 5 and 6, respectively, who received the high dose of vector (2×1012 vg per kilogram). The duration of treatment with prednisolone (Pred), which was initiated at a dose of 60 mg per day and then gradually tapered, is also shown.

In Participant 1, after the initial clearance of exogenously administered FIX protein, plasma FIX activity stabilized at 2% of normal levels on day 14 after the infusion of 2×1011 vg of scAAV2/8-LP1-hFIXco per kilogram. FIX activity remained relatively stable at this level for more than 16 months after gene transfer, despite the discontinuation of twice-weekly FIX prophylaxis (Figure 1A). This level was substantially higher than his baseline FIX activity level (<1% of normal levels) and is consistent with endogenous synthesis of FIX. He had no spontaneous hemorrhages during this period, but FIX prophylaxis was required on five occasions to provide protection in the event of accidental injuries and during elective surgery.

In Participant 2, the plasma FIX level was 1% of normal levels 18 days after gene transfer, which was 3 weeks after his last injection of FIX concentrate. Although this level was consistent with endogenous synthesis of transgenic FIX, he continued to have spontaneous hemorrhages and resumed regular FIX prophylaxis. Over time, he gradually increased the interval between prophylactic FIX injections to 2 weeks, with no spontaneous hemorrhages. At 23 weeks after gene transfer and 3 weeks after the last FIX concentrate injection, his plasma FIX level was 2% of normal values, which suggested ongoing endogenous FIX synthesis (Figure 1B).

Intermediate-Dose Group

In Participant 3, the level of FIX activity in plasma consistently drifted downward to below 1% of normal values in the absence of FIX concentrate during the first 5 weeks after the infusion of 6×1011 vg of scAAV2/8-LP1-hFIXco per kilogram. This change raised the possibility that the transduction of hepatocytes was blocked because the level of anti-AAV8 antibody before gene transfer was higher in this participant than in the other participants (Table 1). However, over time, Participant 3 extended the period between prophylactic FIX injections to 2 weeks. Between weeks 20 and 33, he did not receive FIX prophylaxis and remained free of spontaneous bleeding. During this period, his plasma FIX level was between 1 and 3% of normal values, which is consistent with endogenous synthesis of transgenic protein (Figure 1C).

Plasma FIX levels in Participant 4 increased from a baseline value of less than 1% of normal values to a peak of 4% 4 weeks after gene transfer. FIX expression remained at this level for almost 3 months before declining to 2 to 3% of normal values (Figure 1D). The reason for this decline is unclear, since the results of liver-function tests remained in the normal range, and assays for neutralizing anti-FIX antibodies were consistently negative. Despite this drop in FIX activity, he had no bleeding episodes, even though he engaged in physical activities that had previously provoked such episodes (e.g., playing soccer and cricket without having received targeted prophylaxis). Approximately 9 weeks after gene transfer, however, he did receive a bolus of FIX concentrate to prevent severe bleeding after a fall.

High-Dose Group

Fourteen days after peripheral-vein infusion of 2×1012 vg of scAAV2/8-LP1-hFIXco per kilogram (17 days after the last infusion of FIX concentrate), the plasma FIX level in Participant 5 was 7% of normal values, and this level was maintained until week 7 without the administration of concentrate. On day 49 after the vector infusion, aspartate aminotransferase and alanine aminotransferase levels were elevated, reaching peaks of 143 and 202 IU per liter, respectively, at 58 days (upper limit of the normal range, 37 and 41 IU per liter, respectively). He was asymptomatic, with normal bilirubin levels and prothrombin time, but the plasma FIX level dropped to 3% of normal values (Figure 1E). After ruling out possible causes of this reduction, such as autoimmune responses, toxic factors (alcohol, drugs, or toxins), or infectious viral causes (hepatitis A, B, C, or E virus, herpes simplex virus, Epstein–Barr virus, cytomegalovirus, human immunodeficiency virus, or human T-lymphotrophic virus type I or II), we considered an immunologic response to scAAV2/8-LP1-hFIX as a potential explanation for the liver injury. This was reported as a grade 3 adverse event related to the study agent. Treatment with prednisolone was begun on day 58 at a dose of 60 mg per day, with subsequent tapering of the dose, rapidly reducing aminotransferase levels. Nine weeks after glucocorticoid therapy was discontinued, transgene expression persisted at the 3% level, and the results of liver-function tests remained in the normal range. The patient was free of spontaneous hemorrhage for more than 6 months after the gene transfer but required three boluses of FIX concentrate to prevent bleeding due to traumatic injuries sustained during a recent geologic field trip.

In Participant 6, plasma FIX expression remained between 8 and 12% of normal levels during an 8-week period after vector administration. He was able to stop FIX prophylaxis even while he was training for a marathon. Clinical and laboratory measures remained within the normal range until week 9 (day 62 after gene transfer), when aspartate aminotransferase and alanine aminotransferase values roughly doubled, as compared with baseline levels (with both reaching 36 IU per liter). This change followed a weekend of intensive physical activity, which included participation in a half-marathon, and was associated with a rise in the level of lactate dehydrogenase (to 523 IU per liter; normal range, 240 to 480). When FIX activity declined to 5% of normal levels, Participant 6 also began a short (4-week) course of prednisolone for suspected immune-mediated hepatocyte clearance after other possible causes had been ruled out. Aminotransferase levels subsequently returned to baseline values, and FIX expression remained at a level that was between 8 and 12% of normal values (Figure 1F). However, even at their peak, liver enzyme levels in Participant 6 remained below the upper limit of the normal range and therefore did not meet the criteria for an adverse event.

Immune Responses to Vector and Transgene

Figure 2. Figure 2. Humoral Immune Response in Participant 1. The profile of the humoral immune response in Participant 1 is representative of the responses seen in the other participants. Plasma samples obtained after peripheral-vein infusion of the scAAV2/8-LP1-hFIXco vector were analyzed with the use of an enzyme-linked immunosorbent assay for the presence of AAV8-specific IgM antibodies, total IgG antibodies, and IgG isotypes. Data for the IgG1 isotype are shown. IgG2 and IgG4 levels did not increase above baseline (data not shown).

Neutralizing antibodies to FIX were not detected at any time point in any participant. The kinetic characteristics of the humoral immune response to the AAV8 capsid were similar in all six participants and were consistent with a primary immune response to AAV8 (Figure 2 and Table 1, and Results in the Supplementary Appendix).

Figure 3. Figure 3. Cellular Immune Response in the Six Study Participants. Results of the interferon-γ enzyme-linked immunosorbent spot assay for capsid-specific T-cell responses are shown as the maximum number of spot-forming units (SFU) per 1 million peripheral-blood mononuclear cells (PBMCs) in response to AAV8-capsid peptide pools. The horizontal lines in each panel represent the threshold for positivity, defined as three times the T-cell response for the negative control (medium only) and as at least 50 SFU per 1 million PBMCs. Liver-enzyme levels were elevated in Participant 5 at weeks 8 and 9 and in Participant 6 at week 9. In Participant 6, poor cell recovery and viability were noted at weeks 4 through 8.

T-cell–mediated immune responses to the FIX transgene or putative products of translation from alternative open reading frames in the codon-optimized FIX complementary DNA17 were not detectable in any of the participants (data not shown), as determined with the use of an interferon-γ enzyme-linked immunosorbent spot (ELISPOT) assay. The same assay showed that participants in the low-dose cohort did not have significant AAV8-capsid–specific T-cell responses after gene transfer. At the intermediate-dose level, a significant increase in the frequency of AAV8-capsid–specific T cells was observed. In particular, Participant 3 had T-cell reactivity to the AAV capsid until week 33 after the gene transfer (Figure 3), and Participant 4 had a very strong capsid-specific T-cell response at week 2 (about 1700 spot-forming units [SFU] per 1 million peripheral-blood mononuclear cells [PBMCs]). At the highest dose level, an increase in circulating AAV8-capsid–specific T cells was observed in Participant 5 starting at week 5 after transduction and reaching a peak of more than 500 SFU per 1 million PBMCs by week 8, which was concomitant with the increase in liver enzyme levels. In this participant, broad reactivity was observed across the six capsid peptide pools assessed (Fig. 3 in the Supplementary Appendix). By week 10, capsid-specific T cells were once again undetectable. No T-cell reactivity to the AAV capsid was detectable in PBMCs from Participant 6 until week 8 after the gene transfer, although this may have been due in part to reduced cell recovery and viability. At weeks 9 and 10, capsid-specific T cells became detectable, with levels of up to approximately 200 SFU per 1 million PBMCs, a finding that was concomitant with the minor increase in liver enzyme levels.