Prophylactic Administration of Glucocorticoids and Tacrolimus in AAV8-F8 Gene Therapy of Severe Haemophilia-a Achieved a Significant Long-Term Efficacy

Following adeno-associated virus (AAV) gene therapy, administering immunosuppressants can enhance therapeutic efficacy by suppressing induced immune responses. GS001, a novel vector constructed with AAV8 and a liver-specific expression cassette for B domain-deleted factor VIII (FVIII), was evaluated in an open-label, phase 1 dose-escalation pilot study (NCT04728841) with 12 adult severe haemophilia A (HA) patients. Patients received either 2×10¹² or 4×10¹² vector genomes (vg)/kg of GS001 following one-week prophylactic treatment with prednisone alone or with tacrolimus. Median follow-up was 123.5/58.5 weeks (2×10¹²/4×10¹² vg/kg). Transgene expression peaked at 5-7 weeks post-infusion. At week 52, mean (±SD) FVIII activity (FVIII:C) levels were 14.6 ± 20.2 IU/dL (2×10¹² vg/kg) and 71.0 ± 24.0 IU/dL (4×10¹² vg/kg). Both groups showed significant reductions in annualized bleeding rate and FVIII consumption with grade 1-2 adverse events. Prophylactic prednisone and tacrolimus increased FVIII expression in the 2×10¹² vg/kg group. Immune responses, including AAV8-specific interferon-secreting T cells and antibodies, did not differ between dose groups. Single-cell RNA sequencing of peripheral blood immune cells before and after infusion revealed major inflammatory pathways not activated in CD8+ T cells in either dose group, while interferon-α-related genes exhibited a slight increase. Tacrolimus effectively inhibited patient-derived activated CD8+ T cell proliferation in vitro. Overall, GS001 infusion was safe, well-tolerated, achieving a significant and durable increase in FVIII. Pre-treatment with glucocorticoids and tacrolimus may benefit by suppressing induced immune responses, thereby enhancing sustained transgene expression.