Characteristics of the Participants

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

Nine men with severe hemophilia A were enrolled into one of the three dose cohorts and were followed through the week 52 visit (Table 1). Eight participants had been receiving regular factor VIII prophylaxis before the study. Participant 4 used on-demand factor VIII for bleeding episodes.

Safety of AAV5-hFVIII-SQ Infusion

The adverse events that were reported in at least three of the nine participants were an increased alanine aminotransferase level (in seven), arthralgia (in six), back pain (in four), an increased aspartate aminotransferase level (in three), fatigue (in three), and productive cough (in three); all these events were of mild severity except for one moderate event of arthralgia and one moderately increased level of aspartate aminotransferase. The only serious adverse event was progression of chronic arthropathy in Participant 6, which occurred at an anatomical site that had often been a bleeding site before treatment and for which he subsequently had surgical knee replacement.

Figure 1. Figure 1. Individual Participant Profiles, Including Factor VIII Activity Levels, Bleeding Episodes, Levels of Alanine Aminotransferase, and Glucocorticoid Use. Participant 1 was in the low-dose cohort (dose, 6×1012 vector genomes [vg] per kilogram of body weight), Participant 2 in the intermediate-dose cohort (dose, 2×1013 vg per kilogram), and Participants 3 through 9 in the high-dose cohort (dose, 6×1013 vg per kilogram). The dose (dashed vertical line) was administered on day 0. Factor VIII values are from the one-stage assay performed by a central laboratory (Esoterix); the normal range is 50 to 150 IU per deciliter. The normal range for the alanine aminotransferase (ALT) level is 6 to 43 U per liter; the upper limit of the normal range is indicated by the horizontal dashed line. Arrows indicate bleeding episodes that were reported by the participant. The dose of glucocorticoids (values associated with green bars) in Participants 3 through 9 is shown in milligrams per day; Participants 1 and 2 did not use glucocorticoids.

Eight participants (one in the low-dose cohort and seven in the high-dose cohort) had increased laboratory values regarding alanine aminotransferase, with the first observed value above the upper limit of the normal range at week 3; peak values ranged from 59 to 128 U per liter (normal range, 6 to 43) (Figure 1, and Fig. S2 in the Supplementary Appendix). All these events were asymptomatic and resolved without sequelae. In two participants (Participants 1 and 5), the rise in the alanine aminotransferase level was concurrent with alcohol consumption, hepatotoxic medication (celecoxib), or vigorous exercise (or a combination of these events). No participant had abnormal elevations in the bilirubin or alkaline phosphatase level.

All the participants in the high-dose cohort received glucocorticoids (Figure 1 and Table 1), and all had the glucocorticoids successfully tapered off. Participants 1 and 2 (in the two lower-dose cohorts) did not receive glucocorticoids during the study. There was no clear association between the resolution of the elevated alanine aminotransferase level and prednisolone use. The increased alanine aminotransferase level was accompanied by a decline in the factor VIII activity level in only one participant (Participant 4), in whom the level declined from 227 IU per deciliter to 52 IU per deciliter in conjunction with an increase in the alanine aminotransferase level from week 28 to week 35 (peak value, 95 U per liter) (Figure 1). A decline in the alanine aminotransferase level was noted before the initiation of therapeutic prednisolone. The factor VIII activity level subsequently increased, and no bleeding was reported.

Efficacy Results

Low-Dose Cohort

In the low-dose cohort (AAV5-hFVIII-SQ at a dose of 6×1012 vg per kilogram), the factor VIII activity level remained under 1 IU per deciliter through week 54 in Participant 1 (Figure 1). The consumption of factor VIII was 95 infusions per year (3792 IU per kilogram per year) before the study and 123 infusions per year (3461 IU per kilogram per year) after gene transfer. The participant resumed factor VIII prophylaxis after week 16.

Intermediate-Dose Cohort

In the intermediate-dose cohort (AAV5-hFVIII-SQ at a dose of 2×1013 vg per kilogram), Participant 2 had a stable but low factor VIII activity level (1 to 3 IU per deciliter) through week 54 (Figure 1). He discontinued factor VIII prophylaxis. The consumption of factor VIII fell from 104 infusions per year before the study to 14 infusions per year after gene transfer (reduction in consumption, from 3029 to 366 IU per kilogram per year), but the annualized bleeding rate increased from 3 to 11 events. The participant elected to continue with on-demand therapy.

High-Dose Cohort

Figure 2. Figure 2. Factor VIII Activity Levels in the High-Dose Cohort. Factor VIII values are from the one-stage assay performed by a central laboratory (Esoterix), with normal values of 50 to 150 IU per deciliter (shaded area). Data were available for all seven participants in the high-dose cohort, except that data were not available for Participant 7 at week 32. The plot is based on the median values of factor VIII activity within 4-week windows for each participant. The horizontal line within each box indicates the median value among the participants. The lower and upper boundaries of the box represent the 25th and 75th percentiles, respectively. The ends of the whisker lines represent the minimum and maximum values within 1.5 times the interquartile range from the lower and upper box boundaries. Mean values are indicated by diamonds. Factor VIII activity levels below the limit of quantitation were imputed as being 0.5 IU per deciliter. Factor VIII activity levels within a 72-hour period since the last consumption of factor VIII were excluded. Data points beyond the range (i.e., 1.5 times the interquartile range from the lower and upper box boundaries) were considered to be outliers and are marked with an ×.

After the infusion in the high-dose cohort (AAV5-hFVIII-SQ at a dose of 6×1013 vg per kilogram), the factor VIII activity level gradually increased and then appeared to plateau at or above physiologic levels in weeks 20 through 24 (Figure 1 and Figure 2). At week 16 (the prespecified time point for the efficacy assessment), all seven participants had a factor VIII activity level that was more than 5 IU per deciliter (5 IU per deciliter is the cutoff for moderate vs. mild hemophilia); this level occurred in four participants by week 2 (Table 1). After week 20, the factor VIII activity level was consistently more than 50 IU per deciliter in six of seven participants, and in the remaining participant the level typically ranged from 12 to 32 IU per deciliter. At week 52, the median factor VIII activity level was 77 IU per deciliter (range, 19 to 164; mean [±SD] level, 93±48). The factor VIII activity levels reported here are from the one-stage clotting assay and were consistently approximately 1.65 times as high as the factor VIII levels from the chromogenic assay (see the Comparison of One-Stage and Chromogenic Assays for Factor VIII Activity section and Fig. S3 in the Supplementary Appendix).

Figure 3. Figure 3. Annualized Bleeding Rate and Rate of Factor VIII Use among Six Participants in the High-Dose Cohort Who Were Receiving Factor VIII Prophylaxis before the Study. Panel A shows the mean annualized bleeding rate (±SD) among the six participants in the high-dose cohort who were receiving exogenous factor VIII prophylaxis before study enrollment. The annualized bleeding rate was calculated as follows: (number of bleeding episodes÷total number of days during the calculation period)×365.25. Median values with interquartile ranges (IQR) are also shown. Panel B shows the mean annualized use of factor VIII in these six participants. The annualized use of factor VIII was calculated as follows: (number of infusions of exogenous factor VIII replacement therapy÷total number of days during the calculation period)×365.25.

In the six participants who had received factor VIII prophylaxis before the study, the median annualized bleeding rate dropped from 16 events per year before the study to 1 event per year after gene transfer (mean reduction in rate, from 16 to 2 events) (Figure 3A). The median annualized use of factor VIII fell from 138 infusions per year before the study to 2 infusions per year after gene transfer (mean reduction in rate, from 137 to 5 infusions per year) and was 0 after week 2 (i.e., when the endogenous factor VIII activity level reached 1 to 5 IU per deciliter) (Figure 3B). The median consumption of factor VIII decreased from 5286 to 65 IU per kilogram per year. One participant did not use factor VIII at all after gene transfer, and four ceased factor VIII use after week 2. Only Participant 6 used factor VIII for self-reported bleeding after week 2 — at week 21 for a knee that had undergone multiple radiosynovectomy procedures previously; at this time, his circulating factor VIII level was 12 IU per deciliter. He subsequently underwent total knee replacement, with perioperative use of factor VIII, at week 54.

In the one participant (Participant 4) who had previously used on-demand factor VIII therapy, factor VIII consumption fell from 833 IU per kilogram per year before the study to 81 IU per kilogram per year after gene transfer. After week 2, the participant used factor VIII only once for self-diagnosed bleeding at week 7; the factor VIII activity level at the time was 34 to 66 IU per deciliter.

Host Immune Response to Factor VIII and AAV5

AAV5 capsid-specific antibodies developed after gene transfer in all the participants by week 8 (the first time point assessed). However, cellular immune responses that were specific for AAV5 capsid peptides were not detected at any time point, according to interferon-γ enzyme-linked immunospot (ELISPOT) assay. In Participants 6 and 9, at one time point each, the results on the interferon-γ ELISPOT assay above the established threshold for positivity (50 spot-forming units [SFU] per 106 peripheral-blood mononuclear cells) were detected against FVIII-SQ peptides. These responses, with a maximal value of 120 SFU per 106 peripheral-blood mononuclear cells, were not consistently associated with increasing levels of alanine aminotransferase or declines in measures of factor VIII and returned to negative 4 weeks later. No participant tested positive for factor VIII inhibitors at any time point according to the Nijmegen–Bethesda assay.

Vector Shedding

Vector DNA was detected by the quantitative polymerase-chain-reaction (PCR) assay in blood, semen, saliva, urine, and feces within 72 hours after infusion in all the participants, with peak levels in weeks 1 through 4. Samples were reported as negative only if no signal on the quantitative PCR assay above the threshold of amplification was observed; otherwise, the results were reported as positive. Positive samples at or above the limit of quantitation were reported with numerical results, and positive samples below the limit of quantitation were reported as being below the limit of quantitation. Clearance for each matrix was defined as having had negative results at three consecutive visits.

Overall, all the samples of biologic fluids and feces showed decreasing quantities of residual vector DNA over the study period. For the two participants in the two lower-dose cohorts, the fastest clearing biologic fluids were urine (5 weeks and 11 weeks) and semen (11 weeks and 13 weeks). Each of these participants had two consecutive negative results in saliva at week 52. The samples from Participant 1 (in the low-dose cohort) indicated that feces was cleared and the blood level was below the limit of quantitation at week 52. The samples from Participant 2 (in the intermediate-dose cohort) indicated one negative result in the feces compartment and a blood level above the limit of quantitation at week 52.

Figure 4. Figure 4. Median Levels of Vector DNA in Biologic Fluids in the High-Dose Cohort. The plot is based on the median level of vector DNA at each visit. When multiple test results were available for a participant in a visit window, the maximum result was picked for the participant for the median calculation of that visit. Negative values were carried over to the following visits without additional testing, after three consecutive negative test results were observed (i.e., after clearance was observed). The data-cutoff date was based on the 52-week observation window. Values below the limit of quantitation (LOQ) were imputed as one half the validated LOQ of 50 vg per quantitative polymerase chain reaction and were then back-calculated to the theoretically corresponding vector genomes per standard unit of biologic specimen.

In the high-dose cohort, residual levels of vector DNA were present in all seven participants at week 52 in blood, with all values above the limit of quantitation. The fastest clearing biologic fluid was urine, with all participants having urine cleared at or before 28 weeks (range, 6 to 28) (Figure 4). Four of the seven participants had semen cleared at or before 36 weeks (range, 16 to 36); of the remaining three participants, one had two consecutive negative results (this participant had semen cleared at week 56 when three consecutive negative results were obtained), and two had levels below the limit of quantitation in semen at week 52. On further investigation, we found that vector DNA was not present in purified sperm cells that were obtained from these two participants, which ruled out the risk of inadvertent germline modification. All the participants in the high-dose cohort had samples showing that saliva was cleared at or before 52 weeks (range, 40 to 52). No participant in the high-dose cohort had a sample showing that feces was cleared at week 52, but all the levels were below the limit of quantitation. The household contacts of the participants were not examined.