Eradication of neutralizing antibodies to factor VIII in canine hemophilia A after liver gene therapy

Abstract

Inhibitory antibodies to factor VIII (FVIII) are a major complication in the treatment of hemophilia A, affecting approximately 20% to 30% of patients. Current treatment for inhibitors is based on long-term, daily injections of large amounts of FVIII protein. Liver-directed gene therapy has been used to induce antigen-specific tolerance, but there are no data in hemophilic animals with pre-existing inhibitors. To determine whether sustained endogenous expression of FVIII could eradicate inhibitors, we injected adeno-associated viral vectors encoding canine FVIII (cFVIII) in 2 strains of inhibitor hemophilia A dogs. In 3 dogs, a transient increase in inhibitor titers (up to 7 Bethesda Units [BU]) at 2 weeks was followed by continuous decline to complete disappearance within 4-5 weeks. Subsequently, an increase in cFVIII levels (1.5%-8%), a shortening of clotting times, and a reduction (> 90%) of bleeding episodes were observed. Immune tolerance was confirmed by lack of antibody formation after repeated challenges with cFVIII protein and normal protein half-life. A fourth dog exhibited a strong early anamnestic response (216 BU), with slow decline to 0.8 BU and cFVIII antigen detection by 18 months after vector delivery. These data suggest that liver gene therapy has the potential to eradicate inhibitors and could improve the outcomes of hemophilia A patients. (Blood. 2010;116(26): 5842-5848) IntroductionThe development of neutralizing antibodies to replacement protein is a major complication of protein and enzyme replacement therapies for several genetic diseases. Hemophilia A is an X-linked bleeding disorder characterized by deficiency in the activity of factor VIII (FVIII), a key component of the coagulation cascade. The disease occurs in approximately 1 in 10 000 live births worldwide, and Ͼ 40% of these patients have severe disease, with FVIII activity Ͻ 1% of normal. 1 Infusion of plasma-derived or recombinant FVIII is the standard treatment. Alloantibodies (inhibitors) that neutralize the protein-replacement therapy develop in 20% to 30% of young patients with severe and moderate hemophilia A, resulting in high morbidity and mortality, 2,3 and this is a growing problem for adults as well. 4,5 Risk factors for inhibitor formation include both genetic and environmental factors. Underlying mutations in the FVIII gene, such as large gene deletions, nonsense mutations, and the most common mutation in severe hemophilia A patients, the inversion of intron 22, are all associated with inhibitor formation; however, it is not possible to predict with certainty which patients will develop inhibitors. For this reason, preventive strategies are not currently feasible. [6][7][8] Patients with high titers of inhibitors, defined as Ͼ 5 Bethesda units (BU), cannot usually be treated with FVIII replacement, necessitating the use of products that bypass the procoagulant effect of FVIII and are extremely expensive. 1 Thus, strategies for the eradication of inhibitors are of fundamental clinical relevance.Currently, the only pro...

AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice

“…22 The expression cassette contained canine FIX-R338L (cFIX-Padua), human FIX-R338L (hFIX-Padua), or human FIX wild-type (hFIX-WT) behind a liver-specific promoter. 23 …”

“…22 Coating with serially diluted dog reference serum with known quantities of IgG1, IgG2, and total IgG (Bethyl Laboratories, Montgomery, TX) served as a standard. 27 Immunological challenges with canine FIX-WT protein concentrate Dogs were challenged with 0.5 mg of pooled plasma-derived, purified cFIX concentrate (Enzyme Research Laboratory, South Bend, IN) by intravenous injection.…”

“…22 Despite the fact that detectable anti-FIX IgG2 resolved in 70 days, FIX activity took nearly 800 days to plateau. This suggests there were anti-FIX antibodies that our assay was unable to detect, which likewise reached the limit of detection and plateau around day 800.…”

Toward optimal therapy for inhibitors in hemophilia

“…56,57 In a canine hemophilia A model, liver-directed gene therapy led to tolerance in 3 of 4 dogs. 58 In addition, transplantation of hematopoietic stem cells engineered to express fVIII has been shown to induce fVIII-specific tolerance and eradicated fVIII inhibitors in mouse models.…”

The search for the origin of factor VIII synthesis and its impact on therapeutic strategies for hemophilia A

“…The fact that factor VIII is not normally secreted from human hepatocytes does not prevent the use of gene delivery to the hepatocyte, as promising data from preclinical studies on factor VIII expression in large animals support the concept that targeting hepatocytes has potential for translational studies. 19,20 However, this strategy may be limited by the intrinsic inability of the human hepatocyte to fully synthesize and secrete factor VIII, despite the presence of mRNA. 6 This may explain, at least in part, the higher dose of vectors required for expression of factor VIII compared to factor IX.…”