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Sustained Clinical Benefit of AAV Gene Therapy in Severe Hemophilia B. BACKGROUND: Adeno-associated virus (AAV)-mediated gene therapy has emerged as a promising treatment for hemophilia B. Data on safety and durability from 13 years of follow-up in a cohort of patients who had been successfully treated with scAAV2/8-LP1-hFIXco gene therapy are now available. METHODS: Ten men with severe hemophilia B received a single intravenous infusion of the scAAV2/8-LP1-hFIXco vector in one of three dose groups (low-dose: 2×1011 vector genomes [vg] per kilogram of body weight [in two participants]; intermediate-dose: 6×1011 vg per kilogram [in two]; or high-dose: 2×1012 vg per kilogram [in six]). Efficacy outcomes included factor IX activity, the annualized bleeding rate, and factor IX concentrate use. Safety assessments included clinical events, liver function, and imaging. RESULTS: Participants were followed for a median of 13.0 years (range, 11.1 to 13.8). Factor IX activity remained stable across the dose cohorts, with mean factor IX levels of 1.7 IU per deciliter in the low-dose group, 2.3 IU per deciliter in the intermediate-dose group, and 4.8 IU per deciliter in the high-dose group. Seven of the 10 participants did not receive prophylaxis. The median annualized bleeding rate decreased from 14.0 episodes (interquartile range, 12.0 to 21.5) to 1.5 episodes (interquartile range, 0.7 to 2.2), which represented a reduction by a factor of 9.7. Use of factor IX concentrate decreased by a factor of 12.4 (interquartile range, 2.2 to 27.1). A total of 15 vector-related adverse events occurred, primarily transient elevations in aminotransferase levels. Factor IX inhibitor, thrombosis, or chronic liver injury did not develop in any participant. Two cancers were identified but were deemed by the investigators, together with an expert multidisciplinary team, as being unrelated to the vector. A liver biopsy that was conducted in 1 participant 10 years after the infusion revealed transcriptionally active transgene expression in hepatocytes without fibrosis or dysplasia. Levels of neutralizing antibodies to AAV8 remained high throughout follow-up, thus indicating potential barriers to readministration of the vector. CONCLUSIONS: A single administration of scAAV2/8-LP1-hFIXco gene therapy resulted in durable factor IX expression, sustained clinical benefit, and no late-onset safety concerns over a period of 13 years. These data support the long-term efficacy and safety of AAV gene therapy for severe hemophilia B. (Funded by the U.K. Medical Research Council and others; ClinicalTrials.gov number, NCT00979238; EudraCT number, 2005-005711-17.).

Ten men with severe hemophilia B received a single dose of scAAV2/8-LP1-hFIXco vector via peripheral vein in one of three dose cohorts (low, 2x1011 vector genome copies (vg) per kilogram, N=2; intermediate, 6x1011 vg per kilogram, N=2; or high, 2x1012 vg per kilogram, N=6) between March 2010 and December 2012 (Table 1) as described before and detailed in the Supplementary Appendix (Table S1).2,3 The scAAV2/8-LP1-hFIXco vector has been described previously.7,8 Safety and efficacy assessments included routine laboratory studies, factor IX activity, annualized bleeding rate (ABR), factor IX concentrate usage, and immune responses. Median follow-up, as of December 31, 2023, has been 13.0 years (range 11.1-13.8 years). A liver biopsy was performed on one participant 10 years post-gene therapy for molecular analysis under a separate protocol (Eudract 2018-001333-40).

annualized bleeding rate (ABR), factor IX concentrate usage, and immune responses. Median follow-up, as of December 31, 2023, has been 13.0 years (range 11.1-13.8 years). A liver biopsy was performed on one participant 10 years post-gene therapy for molecular analysis under a separate protocol (Eudract 2018-001333-40). This study was sponsored by St. Jude Children’s Research Hospital (SJCRH). The protocol, developed jointly by the authors and sponsor, was overseen by a trial steering committee, an independent data and safety monitoring committee, and a trial management group. A confidential disclosure agreement between sponsor and study sites was maintained throughout the study. Principal investigators collected data, which were analyzed in collaboration with the statisticians at SJCRH. The authors affirm the completeness, accuracy, and protocol fidelity of the data (protocol available at NEJM.org). The manuscript was drafted by a working group including the first two and corresponding authors, then revised and approved by all authors. The trial management group approved submission.

This study was sponsored by St. Jude Children’s Research Hospital (SJCRH). The protocol, developed jointly by the authors and sponsor, was overseen by a trial steering committee, an independent data and safety monitoring committee, and a trial management group. A confidential disclosure agreement between sponsor and study sites was maintained throughout the study. Principal investigators collected data, which were analyzed in collaboration with the statisticians at SJCRH. The authors affirm the completeness, accuracy, and protocol fidelity of the data (protocol available at NEJM.org). The manuscript was drafted by a working group including the first two and corresponding authors, then revised and approved by all authors. The trial management group approved submission.

Since 2010, 354 adverse events were reported (Table S2, Supplementary Appendix). No cases of factor IX inhibitor, thrombosis, recurrent transaminitis, or death were observed. Fifteen adverse events were linked to AAV gene therapy, including transient liver transaminase elevations (Grade 1-2) in 4 of 6 participants treated with the high vector dose, leading to transgene expression loss in two cases due to delayed glucocorticoid treatment.2,3 By month 5, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels normalized. Liver ultrasounds and chest CT scans in compliant participants showed no ongoing liver damage, fibrosis, malignancy, or lung pathology. Per protocol, two malignancies were reported to the regulators as serious adverse events: (1) lung adenocarcinoma in situ, detected incidentally after bullectomy for recurrent pneumothorax 5 years post-therapy in a 44-year-old with an approximately 10 pack-year smoking history over 27 years, and (2) prostate adenocarcinoma in a 74-year-old 11.6 years post-therapy. Molecular analyses and expert multidisciplinary team review suggested both were likely unrelated to gene therapy. Further details are in the Supplementary Appendix.

n a 44-year-old with an approximately 10 pack-year smoking history over 27 years, and (2) prostate adenocarcinoma in a 74-year-old 11.6 years post-therapy. Molecular analyses and expert multidisciplinary team review suggested both were likely unrelated to gene therapy. Further details are in the Supplementary Appendix. At a median of 3.2 years after infusion of scAAV2/8-LP1-hFIXco, all participants exhibited dose-dependent increases in factor IX coagulant activity to a mean of 1.8 ± 0.7 IU/dL, 2.5 ± 0.9 IU/dL, and 5.1 ± 1.7 IU/dL for the low, intermediate, and high-dose cohorts, respectively (mean of all levels after month 4).2 Over a median study period of 13 years, mean factor IX activity remained stable, with values of 1.7 ±0.3, 2.3 ±0.5, and 4.8 ±1.7 IU/dL. This resulted in a 3.3 IU/dL (high dose median: 5.18, IQR, 3.5 to 5.7; low and intermediate dose median:1.9, IQR, 1.8 to 2.1) difference in steady-state factor IX activity levels between the high-dose group and the combined low and intermediate-dose groups as of December 31, 2023 (Figure 1). Three participants with severe hemophilic arthropathy (Participants 2, 3, and 5, median number of target joints = 10) resumed factor IX prophylaxis within four years of gene therapy due to recurrent spontaneous joint bleeds. Factor IX levels of 1–3 IU/dL in these individuals measured at least three days post-infusion, proved insufficient to prevent such bleeding, highlighting the impact of joint health and other biological factors on outcomes post-gene therapy. In contrast, seven participants (Participants 1, 4, 6, 7, 8, 9, and 10) sustained factor IX levels of 2–7 IU/dL and remained off prophylaxis during long-term follow-up.

sion, proved insufficient to prevent such bleeding, highlighting the impact of joint health and other biological factors on outcomes post-gene therapy. In contrast, seven participants (Participants 1, 4, 6, 7, 8, 9, and 10) sustained factor IX levels of 2–7 IU/dL and remained off prophylaxis during long-term follow-up. Before gene therapy, the median ABR was 14 episodes (interquartile range [IQR], 12 to 21.5) for all 10 participants, including 3 participants receiving on-demand treatment. With nearly 13 years of follow-up, the median ABR for the 10 participants was 1.5 episodes (IQR, 0.7 to 2.2) (Figure S1, Supplementary Appendix), representing a median 9.7-fold (IQR, 3.7 to 21.8) reduction in bleeding events compared to the pre-treatment period. In the 6 high-dose participants, the ABR decreased by a median of 16.4-fold (IQR, 9.7 to 31.3) to 1 bleeding episode annually (IQR, 0.4 to 2.0) compared to a median of 21 episodes (IQR, 16.2 to 27.2) pre-gene therapy. Post-hoc assessment of bleeding events showed a decline from a median of 5.26 (IQR, 3.27 to 9) at 1-year post-gene therapy to a median of 1.5 (IQR, 0.6 to 2.3) bleeds at 11 years post-gene therapy. These reductions (median fold change=3.9, IQR, 2.1 to 8.2) in ABR over a decade following infusion suggest that sustained, albeit low-level, factor IX expression may attenuate pre-existing synovitis and inflammation, potentially providing long-term protection against bleeding and mitigating further joint damage. These findings align with observations from hemophilia A gene therapy studies, warranting further investigation.9,10

t sustained, albeit low-level, factor IX expression may attenuate pre-existing synovitis and inflammation, potentially providing long-term protection against bleeding and mitigating further joint damage. These findings align with observations from hemophilia A gene therapy studies, warranting further investigation.9,10 The median annual factor IX concentrate usage before gene therapy was 2613 IU per kilogram (IQR, 1671 to 4513). Over 13-years following gene therapy, factor IX concentrate usage decreased a median 12.4-fold (IQR, 2.21 to 27.1) to 367 IU per kilogram (IQR: 60 to 1597) at 13 years (Figure S2, Supplementary Appendix). In the high-dose group, usage dropped from 2613 IU/kg (IQR, 1627–3487) to 171 IU/kg (IQR, 60–432), a median 14.7-fold (IQR, 11.9 to 27.1) reduction. A transjugular liver biopsy in participant 8 from the high-dose group, 10 years post-gene therapy, showed preserved lobular architecture with no necrosis, fibrosis, or dysplasia. In situ hybridization (ISH) detected hFIXco DNA in 10.3 ±3.4% of hepatocytes, while RNA in situ hybridization (RISH) revealed active transcription in 5.5 ±2.3%, indicating that just over half of the transduced hepatocytes were transcriptionally active in transgene-positive cells. No segregation between active and inactive hepatocytes was observed (Figure S3, Supplementary Appendix). This single liver biopsy provides data limited to the sampled region and may not fully reflect transgene expression across the entire liver. Nevertheless, it demonstrates persistent transgene transcription within a subset of transduced hepatocytes.

inactive hepatocytes was observed (Figure S3, Supplementary Appendix). This single liver biopsy provides data limited to the sampled region and may not fully reflect transgene expression across the entire liver. Nevertheless, it demonstrates persistent transgene transcription within a subset of transduced hepatocytes. A dose-dependent increase in total IgG antibodies and neutralizing antibodies against AAV8 capsid was observed in all participants, with both measures showing a similar profile over the follow-up period (Figure 2). At one-year, neutralizing antibody levels increased a median of 3721.3-fold (IQR, 1374.7 to 6640) and remained >300-fold higher than controls (392.5-fold, control median= 43.6, IQR 5.0 to 96.5; participant median = 17100.0, IQR 13530.2 to 27125.0). By five years, neutralizing antibody levels in the high-dose cohort declined but were still >2,400-fold above baseline (median=2427.8-fold, IQR 459.1 to 3340), exceeding the predefined threshold for successful gene transfer, which is based on preclinical in-vivo experience. Cross-reactivity with AAV5 and AAV3b was dose-dependent. In murine assays, patient sera >5 years post-therapy inhibited AAV8 transduction but allowed limited AAV5 transfer, suggesting persistent NABs may hinder repeat dosing, while alternative serotypes may offer a viable option for retreatment in the future (Figure S4, Supplementary Appendix).

At a median of 3.2 years after infusion of scAAV2/8-LP1-hFIXco, all participants exhibited dose-dependent increases in factor IX coagulant activity to a mean of 1.8 ± 0.7 IU/dL, 2.5 ± 0.9 IU/dL, and 5.1 ± 1.7 IU/dL for the low, intermediate, and high-dose cohorts, respectively (mean of all levels after month 4).2 Over a median study period of 13 years, mean factor IX activity remained stable, with values of 1.7 ±0.3, 2.3 ±0.5, and 4.8 ±1.7 IU/dL. This resulted in a 3.3 IU/dL (high dose median: 5.18, IQR, 3.5 to 5.7; low and intermediate dose median:1.9, IQR, 1.8 to 2.1) difference in steady-state factor IX activity levels between the high-dose group and the combined low and intermediate-dose groups as of December 31, 2023 (Figure 1). Three participants with severe hemophilic arthropathy (Participants 2, 3, and 5, median number of target joints = 10) resumed factor IX prophylaxis within four years of gene therapy due to recurrent spontaneous joint bleeds. Factor IX levels of 1–3 IU/dL in these individuals measured at least three days post-infusion, proved insufficient to prevent such bleeding, highlighting the impact of joint health and other biological factors on outcomes post-gene therapy. In contrast, seven participants (Participants 1, 4, 6, 7, 8, 9, and 10) sustained factor IX levels of 2–7 IU/dL and remained off prophylaxis during long-term follow-up.

A dose-dependent increase in total IgG antibodies and neutralizing antibodies against AAV8 capsid was observed in all participants, with both measures showing a similar profile over the follow-up period (Figure 2). At one-year, neutralizing antibody levels increased a median of 3721.3-fold (IQR, 1374.7 to 6640) and remained >300-fold higher than controls (392.5-fold, control median= 43.6, IQR 5.0 to 96.5; participant median = 17100.0, IQR 13530.2 to 27125.0). By five years, neutralizing antibody levels in the high-dose cohort declined but were still >2,400-fold above baseline (median=2427.8-fold, IQR 459.1 to 3340), exceeding the predefined threshold for successful gene transfer, which is based on preclinical in-vivo experience. Cross-reactivity with AAV5 and AAV3b was dose-dependent. In murine assays, patient sera >5 years post-therapy inhibited AAV8 transduction but allowed limited AAV5 transfer, suggesting persistent NABs may hinder repeat dosing, while alternative serotypes may offer a viable option for retreatment in the future (Figure S4, Supplementary Appendix).

This 13-year longitudinal study provides long-term observation data in patients who had successful AAV gene therapy for severe hemophilia B. Beyond transient liver transaminase elevations, no long-term or new AAV-related adverse events were observed. Factor IX expression remained stable with seven of ten participants remaining off factor IX prophylaxis. Clinically, AAV-gene transfer resulted in an >9-fold reduction on in ABRs and factor IX concentrate usage, significantly alleviating the disease burden. These findings support the long-term safety and efficacy of AAV gene therapy for hemophilia B, offering this group of patients a promising and durable treatment option through recently licensed gene therapy products. Two participants developed neoplastic lesions, which were reported as serious adverse events possibly related to AAV per protocol. However, subsequent molecular investigations and expert multidisciplinary review suggested these events were likely unrelated to AAV gene therapy, attributing them instead to age-related or environmental risk factors prevalent in the general population, as previously described.4 It is important to note, however, that long-term studies in hemophilia A dogs revealed clonal expansion of hepatocytes with AAV vector insertions near genes associated with human cancers.11 While no overt nodule formation or transformation was observed in these dogs, this finding highlights the importance of long-term surveillance and further investigation into the potential long-term effects of AAV-mediated gene therapy.

f hepatocytes with AAV vector insertions near genes associated with human cancers.11 While no overt nodule formation or transformation was observed in these dogs, this finding highlights the importance of long-term surveillance and further investigation into the potential long-term effects of AAV-mediated gene therapy. As reported in other studies, an asymptomatic, vector-related, rise in liver transaminases (Grade 1-2) occurred in some participants but did not result in lasting impairment of liver function.2,3,12 Glucocorticoids appeared to effectively manage the liver transaminases. While the precise mechanism of hepato-cellular injury remains unclear, close monitoring is crucial due to the potential for late liver enzyme elevations as well as fulminant liver failure. 13 14, 15, 16,17 A liver biopsy from a participant 10-years post-gene transfer demonstrated that the sustained transgenic factor IX expression and hemostatic protection in this individual was driven by a small subset of transcriptionally active hepatocytes, with no evidence of active inflammation or histological abnormalities. While integration analysis was not feasible due to lack of sufficient DNA, we hypothesize that both episomal and integrated forms of the AAV vector contribute to factor IX expression in this individual based on the current understanding of the AAV lifecycle in humans and animal models.6,18 This likely occurs within the context of natural hepatocyte turnover (~12 months in adults), which may contribute to the gradual loss of episomal genomes.19,20

of the AAV vector contribute to factor IX expression in this individual based on the current understanding of the AAV lifecycle in humans and animal models.6,18 This likely occurs within the context of natural hepatocyte turnover (~12 months in adults), which may contribute to the gradual loss of episomal genomes.19,20 Durable AAV-mediated transgene expression with concomitant clinical improvement has also been observed in other disorders, including spinal muscular atrophy and inherited retinal dystrophy21,22 However, decline or loss of transgene expression has been reported in both animal models and humans.23,15 This suggests that durability of transgene expression is complex and multifactorial, involving a dynamic interplay between the AAV genome, target disease, immunological and non-immunological factors encompassing vector design, epigenetics, as well as patient-specific considerations.4

both animal models and humans.23,15 This suggests that durability of transgene expression is complex and multifactorial, involving a dynamic interplay between the AAV genome, target disease, immunological and non-immunological factors encompassing vector design, epigenetics, as well as patient-specific considerations.4 Humoral immune responses to AAV have been observed across various diseases, delivery routes, and serotypes, but their long-term persistence in humans remains underexplored. In our study, high-titer neutralizing antibody against AAV8 persisted for at least 10 years post-gene therapy, with broad cross-reactivity, consistent with other reports.24 While neutralizing antibody levels after environmental exposure are comparable between humans and non-human primates, the response to intravenous recombinant AAV administration was magnitudes higher in humans.25 This suggests species-specific differences in immune responses. Although alternative serotypes may offer potential for vector re-administration, their efficacy in fully overcoming persistently high levels of cross-reactive neutralizing antibody in humans remains uncertain, suggesting that AAV-mediated gene therapy for most patients is a one-time opportunity. In summary, this 13-year longitudinal follow-up of men with severe hemophilia B confirms the long-term safety of AAV gene therapy associated with durable factor IX expression, accompanied by lasting improvement in hemostasis and reduction in the need for factor IX prophylaxis.