Defining the Risk of Suboptimal AAV8-F9 Vector Delivery in Inhibitor Positive Hemophilia B Mice

Abstract Hemophilia B (HB) is a coagulation disorder where patients lack functional coagulation factor IX (FIX) protein. The recommended treatment is prophylactic infusion of plasma derived or recombinant FIX protein. 3-5% of HB patients develop anti-drug antibodies, termed inhibitors. Patients with high titer inhibitors (> 5 BU) require bypassing agents for management of hemostasis. Immune tolerance induction therapy (ITI), while effective in hemophilia A patients (factor VIII deficiency), is less effective in HB patients due to the development of severe anamnestic responses. Over the last 25 years, gene therapy has emerged as an effective therapy for HB patients. Adeno-associated virus (AAV) vectors have had the best translational success in the clinic. Early clinical studies revealed an unpredicted risk of a vector dose dependent immune response targeting the AAV capsid presented by transduced hepatocytes, resulting in the loss in FIX protein expression. Successive clinical trials have adapted to now include transient immune suppression, alternative capsids, and a naturally occurring hyperactive FIX variant, to reach therapeutic FIX activity with reduced vector doses. Importantly, there has been no report of inhibitors in AAV treated HB patients, with the caveat that patients with a high risk for inhibitors have not been treated. Studies in HB mice and dogs have shown that AAV-F9 gene delivery to and expression in hepatocytes induces immunological tolerance to FIX protein, dependent on a threshold level of FIX protein expression and regulatory T cells (Treg). Therefore, we hypothesized that AAV-F9 liver gene transfer may be an effective ITI therapy. Using an inhibitor and anaphylaxis prone HB mouse strain (C3H/HeJ-F9-/Y), we demonstrated that AAV-F9 gene therapy rapidly eliminated high titer anti-FIX IgE and IgG1 antibodies, provided long-term correction of hemostasis, and protected mice from anamnestic responses following supplemental FIX protein therapy (Markusic et al. EMBO Mol Med 2013). The average reported sustained levels of FIX protein expression in ongoing clinical studies is ~5% normal, at the threshold level for ITI in our model (Markusic et al. EMBO Mol Med 2013), raising concerns regarding the efficacy and safety of gene therapy for inhibitor positive patients. To address this, we set out to identify a suboptimal AAV-F9 vector dose (2x109 - 1x1011 vg) in C3H/HeJ-F9-/Y mice with the following criteria (1) measurable expression of FIX protein, (2) absence of spontaneous inhibitors, and (3) susceptibility to inhibitor formation following FIX protein challenge.Vector treated mice were followed for 4-8 weeks and were then immunized with FIX protein in adjuvant and followed out 20 weeks. As previously shown mice receiving 1x1010 and 1x1011 vg had stable FIX expression that persisted following FIX-adjuvant challenge. We identified a suboptimal vector dose of 6x109 vg with an average FIX level of ~75 ng/mL (1.5% normal) over 8 weeks which became undetectable following adjuvant-FIX challenge. Next we generated three groups of inhibitor positive mice (1) no treatment control, (2) 6x109 vg AAV8-F9, and (3) 1x1011 vg AAV8-F9 treated to determine the impact of suboptimal vector on inhibitor levels and potential risks of anaphylaxis. Mice receiving the suboptimal vector dose were monitored daily for signs of anaphylaxis out to two weeks post vector delivery. No animals in the 6x109 vg group displayed any physical symptoms of anaphylaxis. Positive control mice receiving 1x1011 vg demonstrated a rapid reduction in anti-FIX titers at two weeks post gene delivery and have had sustained FIX levels ~100% of normal. Whereas mice in the 6x109vg treated group have had undetectable FIX levels and sustained and elevated anti-FIX IgG1 levels compared to untreated control inhibitor mice. The study is still ongoing and we have now followed mice out to 12 weeks post vector delivery. This study raises some potential concerns regarding gene therapy in HB patients with inhibitors. The present adoption of hyperactive FIX protein in gene therapy vectors, inefficient human hepatocyte transduction, and immune responses against AAV capsids may compromise tolerance induction by reducing circulating FIX protein levels, as modeled in our present study. These studies were supported by an ASPIRE Hemophilia Research Award from Pfizer. Disclosures Markusic: Pfizer: Research Funding.