GO-8: Stable Expression of Factor VIII over 5 Years Following Adeno-Associated Gene Transfer in Subjects with Hemophilia a Using a Novel Human Factor VIII Variant

Background: GO-8 (ClinicalTrials.gov: NCT03001830) is a study of liver-directed adeno-associated virus (AAV) gene therapy for haemophilia A (HA) that uses a factor VIII (FVIII) variant containing a 17 amino-acid peptide comprising six N-linked glycosylation motifs from the human FVIII B-domain (AAV-HLP-hFVIII-V3). In preclinical studies, AAV-HLP-hFVIII-V3 mediated a 3-fold higher FVIII expression when compared to an identical AAV construct encoding the hFVIII-SQ variant used in most HA gene therapy trials. Methods: In a multi-centre, open-label, non-randomised, phase I/II clinical trial, we assessed the safety and efficacy of escalating doses of AAV-HLP-hFVIII-V3 pseudotyped with an AAV8 capsid in adults with severe haemophilia A (FVIII activity ≤1%). All participants received prophylactic glucocorticoids, with or without tacrolimus, with the aim of reducing the risk of vector-related transaminase elevation. The primary endpoints were safety and efficacy. Efficacy was assessed by measuring FVIII activity (FVIII: C) using both chromogenic and one-stage clotting assays and factor consumption pre and post-gene therapy. Results: As of May 31 2023, 12 participants were enrolled sequentially into one of four vector doses: 6×1011 vector genomes (vg)/kg body weight (n=1), 2×1012 vg/kg (n=3), 4×1012 vg/kg (n=3), or 6×1012 vg/kg (n=5). All participants were on FVIII prophylaxis prior to gene therapy. The most common vector-related adverse event was an elevation in liver aminotransferase levels, which occurred in 10 of 12 participants. In 7 of the 8 participants treated at doses ≥4×1012 vg/kg, recurrent elevation in aminotransferase levels was observed during the first 12 months, often associated with tapering of immunosuppression. This resulted in a reduction in transgene expression from peak levels in all participants, with a complete loss of transgenic protein in one participant. Vector-related elevation in aminotransferase was not observed after the 12-month time point in long-term follow-up. Mean chromogenic FVIII: C levels at 12 months after gene therapy were 3 IU/dL in the 6×1011 vg/kg cohort, 13±9IU/dL (range: 2-19 IU/dl) in the 2×1012 vg/kg cohort, 8±1IU/dl in the 4×1012 vg/kg cohort (range: 7-9 IU/dl) and 22±34 IU/dl in the 6×1012 vg/kg cohort (range 1-82 IU/dl). Transgene expression was then stably maintained over a median follow-up of 3 years (range: 0.2-5 years) from the level achieved 1-year post-infusion, best illustrated by the data from the 2×1012 and 4×1012 vg/kg cohorts shown in Figure 1. FVIII: C was, on average 2-fold higher when measured using a one-stage clotting assay compared to the chromogenic method. Nine of the 12 participants remained off prophylaxis after gene therapy for the duration of the follow-up period. Baseline mean and median annualised factor VIII use was 4097 and 4657 IU/kg per year before gene therapy. Following gene therapy, the mean and median annualised factor VIII concentrate use reduced across all participants to 1186 and 61 IU/kg (One sample t-test p=0.0009), respectively. No FVIII inhibitors or thrombotic events were reported for the duration of the study. Conclusion: A single infusion of AAV-HLP-hFVIII-V3 resulted in stable FVIII expression over a follow-up period of up to 5 years in participants with severe haemophilia A. A high rate of liver aminotransferase elevation following gene transfer impacted transgene expression. However, 9 of the 12 participants were able to discontinue FVIII prophylaxis over the duration of the study, resulting in a significant reduction in FVIII concentrate usage.