The formation of antibodies (or inhibitors) to FVIII is a major complication in the treatment of humans with hemophilia A (HA), affecting up to 30% of individuals with severe or moderate disease. Inhibitors develop mainly in young boys, and render the treatment with infused protein suboptimal or completely ineffective. Inhibitor patients exhibit significant delays in their maturation and physical development. Thus, inhibitor formation negatively affects both hematological and systemic outcomes in these patients and, therefore the prevention or eradication of inhibitors is of fundamental importance. To eliminate the presence of inhibitors in hemophilia, an immune tolerance induction (ITI) is followed consisting of large amounts of FVIII injected on a daily basis for long periods of time (months to years) with variable success rates. Although liver-directed gene therapy has been used to deliver therapeutic transgenes and can induce tolerance to the expressed protein, to date there have been no large animal studies using liver gene therapy to eradicate inhibitors to FVIII. The HA dog colonies are an excellent model for studying tolerance induction as both colonies have the same mutation found in the majority of the human population (inversion of intron 22) resulting in severe hemophilia. We hypothesize that sustained expression of cFVIII could mimic the effects of ITI in eradicating inhibitors to canine FVIII in dogs that have a history of inhibitors, thus demonstrating a potential alternative to the current ITI protocol. We injected AAV-8 vectors encoding the light and heavy chain of canine FVIII (cFVIII) (two separate vectors) driven by a liver specific promoter (2.5 e13 vg/kg per vector) into the peripheral vein of four adult (7–36 months old) HA dogs with inhibitors. The first dog treated (K03, 12 months old) was from a new strain from the Chapel Hill (UNC) HA dog colony prone to inhibitor formation. K03 had a historical maximum inhibitor titer of 12 B.U. and a titer of 3 B.U. at the time of vector infusion. Post AAV injection, we documented a slow shortening of whole blood clotting time (WBCT) to normal values (13 min) and inhibitors decreased to undetectable levels by week 3. K03 has demonstrated cFVIII antigen levels (~30 ng/mL) that are stable out to 250 days post treatment. When challenged with 4 weekly injections of 125 μg of highly purified recombinant cFVIII he failed to generate an inhibitor, indicating induction of immune tolerance. K01 (UNC, 20 months old) had a historical maximum inhibitor titer of 12–13 B.U. and a titer of 3 B.U. at the time of vector infusion. We observed an initial transient rise in cFVIII antigen, followed by a decrease to baseline that corresponded to an increase in inhibitor titer to 5–6 B.U. before decreasing to undetectable levels within 4 weeks of gene transfer. cFVIII antigen levels have slowly increased over time, and have reached 14 ng/mL by day 150. WBCT has decreased corresponding with decreasing inhibitor titers and has normalized to 13.5 min. L44 (UNC, 8 months old) had a historical maximum inhibitor titer of 3 B.U. and a titer of 1.5 at the time of infusion. After vector infusion we observed a rapid onset of cFVIII expression (peaking at 50 ng/mL) that decreased to near baseline by day 8 before increasing and stabilizing at 15–20 ng/ml (out to 40 days) and inhibitor titers have decreased while WBCT has decreased to 18 min. The fourth dog (Wembley, 3 years old) is from the HA dog colony at Queens University and had previously been exposed to both canine and human FVIII and presented inhibitors to both human (10 B.U.) and canine (3.6 B.U.) FVIII. After vector infusion Wembley showed a transient increase in cFVIII levels, followed by a decrease in cFVIII expression and no indication of tolerance induction after 10 weeks, ongoing observation. Notably, this dog presented high titer IgG1 and IgG2 anti-cFVIII antibodies, while the other three dogs had mostly IgG2 (equivalent to human IgG4). No abnormalities in liver or renal functions were observed in these dogs. Factors such as exposure to xeno-antigens, the nature of the antibody response, duration of inhibitor or age may influence the outcome of the tolerance induction protocol. Collectively, these data demonstrate the potential of liver directed, AAV mediated gene delivery to no only treat genetic deficiencies such as hemophilia, but to induce immune tolerance to the transgene in the setting of pre-existing inhibitory antibodies.

Disclosures: No relevant conflicts of interest to declare.

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