Background: Haemophilia A (HA), the most common inherited bleeding disorder, is well suited for gene therapy because a modest increase in the plasma factor VIII (FVIII) levels to ≥1% of normal levels will substantially ameliorate the bleeding diathesis and improve quality of life. Earlier gene transfer strategies for FVIII replacement approaches using plasmid electroporation, retroviral vector, or adenoviral vector failed to achieve persistent phenotypic correction of bleeding. We have recently shown that a single peripheral vein administration of adeno-associated viral (AAV) vectors expressing the FIX transgene results in stable long-term expression of transgenic FIX at therapeutic levels without long term toxicity in patients with severe haemophilia B ( However, the use of AAV vectors for HA gene therapy has been limited by inefficient expression of transgenic FVIII and the large size of the FVIII cDNA. To overcome these obstacles, we developed two AAV-FVIII expression cassettes containing a small synthetic liver specific promoter (HLP) driving the expression of codon optimized FVIII variants. These vectors mediated therapeutic expression of FVIII in murine and non-human primate models (McIntosh et al 2013). The first of these constructs, AAV-HLP-hFVIII-SQ, encoding a B-domain deleted FVIII variant, was recently shown (Rangarajan et al, 2017) to mediate sustained (>1 year) normalisation of factor VIII activity in six of seven participants following a single intravenous infusion of AAV serotype 5 pseudotyped vector. However, high vector doses (6x1013 vector genomes/kg [vg/kg]) were required for efficacy, possibly because this product was manufactured using the insect cell/baculovirus system. In this report we describe the preliminary results of our on-going Phase I/II clinical trial (GO-8) evaluating the second FVIII cassette (AAV-HLP-hFVIII-V3), which contains a 17 amino-acid peptide comprising six N-linked glycosylation motifs from the human FVIII B-domain that are highly conserved through evolution. In murine studies, AAV-HLP-hFVIII-V3 mediated expression of FVIII at 3-fold higher levels when compared to AAV-HLP-hFVIII-SQ.

Methods: The safety and efficacy of a single intravenous infusion of AAV8-HLP-hFVIII-V3, pseudotyped with AAV serotype 8 capsid was assessed in three adult men with severe hemophilia A (FVIII activity levels ≤1% of normal) in the context of an Investigator led, Phase I/II, open-label, non-randomized, dose-escalation trial ( NCT03001830GO-8). The first subject received a dose of 6x1011vg/kg and the subsequent two patients each received a dose of 2x1012vg/kg. AAV8-HLP-hFVIII-V3 was manufactured in mammalian HEK 293T cells. The subjects have been followed up for 13-47 weeks after vector administration.

Results: Peripheral vein administration of AAV8-HLP-hFVIII-V3 was well tolerated in all patients with no infusion-related reactions. Transgenic FVIII was detectable within two weeks and was more than 5 IU/dl by 6 weeks of gene transfer in all three subjects. Factor VIII activity (one stage clotting assay) levels have remained stable at 7±1IU/dl in patient 1 over a period of 47 weeks. The second participant is 20 weeks following administration of 2x1012 vg/kg of AAV8-HLP-hFVIII-V3 and has steady-state FVIII activity of 6±2IU/dl. In the third subject, who was also treated at a dose of 2x1012 vg/kg, the steady state FVIII activity is almost 10 times higher at 69±7 IU/dl. Elevation of serum alanine aminotransferase was observed in patients 1 and 3 at between weeks 4-6 after gene transfer, reaching peak levels that were 1.5 X upper limit of the normal range. Both patients were treated with corticosteroids within 48 hours of the onset of transaminitis with no loss of transgene expression. No participant has developed a FVIII inhibitor.

Conclusion: Our preliminary results from the ongoing Phase I/II study demonstrate FVIII activity levels >5% in all three subjects with normalization of FVIII:C levels in one patient. These levels are sufficient to reduce/prevent spontaneous hemorrhage and have been achieved using relatively lower doses of AAV8-HLP-FVIII-V3 than reported previously with a related FVIII expression cassette. No Grade III (CTCAE v4.03) or greater adverse events have been observed over a period of 47 weeks after administration of AAV8-HLP-hFVIII-V3.


Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tuddenham:Freeline: Consultancy; BioMarin: Consultancy, Patents & Royalties. Chowdary:Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Swedish Orphan Biovitrum AB (Sobi): Honoraria; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Freeline: Consultancy. McIntosh:Freeline: Consultancy.

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Asterisk with author names denotes non-ASH members.

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