Emerging data from early phase clinical studies of AAV gene therapy for hemophilia B (HB) (factor IX [FIX] deficiency) show sustained expression of therapeutic levels of FIX and phenotypic improvement. However, the safety and efficacy of in vivo gene therapy is limited by the vector dose. Recently, we reported a naturally occurring, hyperfunctional FIX (FIX Padua) caused by a single amino acid change of arginine 338 to leucine that exhibits an 8-fold increase in specific activity in humans (N Engl J Med 2009), making it a potential candidate for HB gene therapy with reduced vector doses. However, to take advantage of FIX Padua for HB gene therapy, it is critical to first define the risk of immunogenicity of this variant in preclinical models of severe HB.

We have previously shown that delivery of AAV-cFIX-Padua to skeletal muscle in HB dogs with a missense mutation in the canine (c) F9 gene resulted in no anti-FIX neutralizing antibodies (inhibitors), non-neutralizing antibodies (IgG) or FIX-specific T-cell response (Blood 2012). While promising, these dogs express FIX RNA and have a pre-existing tolerance to cFIX due to the nature of their mutation, and so do not represent the most rigorous model for immunogenicity studies.

Here, we tested the efficacy and immunogenicity of cFIX Padua in a severe HB dog colony with an early stop codon mutation. This mutation results in no FIX RNA transcript, and the dogs are prone to develop cFIX inhibitors upon exposure to protein concentrates. Three dogs were infused peripherally with a liver-specific AAV8-cFIX-Padua at two different doses, and monitored for cFIX antigen and activity levels and inhibitors. The first dog, which received 3 x 1012 vg/kg, showed average plateaued expression levels of 3.98 ± 1.44% antigen and 24.5 ± 4.1% activity, with no development of anti-cFIX inhibitors or IgG antibodies. Whole blood clotting time (WBCT) and aPTTs returned to normal by day 3 post-vector administration and have remained stable for >20 months (ongoing observations). A second dog was treated with a lower dose of 1 x 1012 vg/kg and showed average plateaued expression levels of 2.41 ± 0.05% antigen and 22.0 ± 0.4% activity, with no development of anti-cFIX inhibitors or IgG antibodies. WBCT and aPTTs returned to normal by day 3 post-vector administration and have remained stable for >3 months.

An additional dog, upon previous exposure to recombinant human (h) FIX protein, had developed inhibitors that cross-reacted with cFIX. This immune response was ongoing at the time of vector administration (3 x 1012 vg/kg). Anti-cFIX antibodies peaked at day 14 post-AAV, with 4.7 BUs and 3643 ng/mL IgG2, but dropped to undetectable levels by day 70. There was a concurrent rise in cFIX Padua expression levels, suggesting successful tolerization to the cFIX Padua. Antigen levels plateaued at 14.6 ± 4.3% and activity at 51.7 ± 23.5%, with ongoing normalization of WBCT and aPTTs for >18 months. In all three dogs, cholesterol, albumin and total protein were within normal limits with no clinical or laboratory evidence of nephrotic syndrome (a potential complication in FIX inhibitor patients that have undergone immune tolerance induction with frequent FIX protein injections).

The safety of FIX Padua was further confirmed using a mouse model of HB. Mice (n=8-12/group) were treated with 5 x 1010 vg/kg liver-directed AAV8-hFIX-WT or AAV8-hFIX-Padua, resulting in expression levels of 1076 ± 343 ng/mL (21.5 ± 6.9% antigen, 67.5 ± 10.1% activity) and 797 ± 255 ng/mL (15.9 ± 5.1% antigen, 274.8 ± 73.8% activity), respectively. In cross-over experiments, the mice were then were immunologically challenged 10-15 weeks after gene delivery with 100 ug/kg of the reciprocal recombinant protein (ie mice expressing hFIX Padua were challenged with hFIX WT, and visa versa). Challenges were administered subcutaneously alone or with adjuvant (CFA) weekly for 4 weeks. In no instance did mice develop antibodies to either FIX, suggesting that tolerance was successfully induced in all cases.

Together, these date show that FIX Padua shows no increase in immunogenicity compared to FIX WT and is capable not only of preventing inhibitor formation, but also of eradicating pre-existing inhibitory antibodies to FIX in an inhibitor-prone HB dog model. Thus, FIX Padua is an attractive transgene that will allow for decreased vector doses in human HB gene therapy, improving the safety profile of AAV liver gene therapy without increased immunogenicity.


High:Alnylam Pharmaceuticals: Consultancy; BioMarin: Consultancy; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; Genzyme, Inc.: Membership on an entity’s Board of Directors or advisory committees; Intrexon: Consultancy; Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Shire : Consultancy; Benitec: Consultancy.

Author notes


Asterisk with author names denotes non-ASH members.

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