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.

Figure 1

cFVIII expression and anti-cFVIII antibody responses in Chapel Hill hemophilia A dog K01 after liver delivery of AAV-cFVIII. One Chapel Hill dog (K01) with preexisting inhibitors to cFVIII was administered 2.5 × 10¹³ vg/kg of AAV8-TBG-cFVIII-HC and AAV8-TBG-cFVIII-LC by peripheral venous injection. (A) cFVIII antigen levels were assayed by a cFVIII-LC specific ELISA, and activity was monitored by FVIII assay. (B) Anti-cFVIII antibody responses were measured by anti-cFVIII IgG2 ELISA and Bethesda assays. Black arrows indicate 4 weekly challenges with 500 U of rBDD-cFVIII.

Figure 2

cFVIII expression and anti-cFVIII antibody responses in Chapel Hill hemophilia A dog L44 after liver delivery of AAV-cFVIII. One Chapel Hill dog (L44) with preexisting inhibitors to cFVIII was administered 2.5 × 10¹³ vg/kg of AAV8-TBG-cFVIII-HC and AAV8- TBG-cFVIII-LC by peripheral venous injection. (A) cFVIII antigen levels were assayed by a cFVIII-LC specific ELISA, and activity was monitored by FVIII assay. (B) Anti-cFVIII antibody responses were measured by anti-cFVIII IgG2 ELISA and Bethesda assays. Black arrows indicate 4 weekly challenges with 500 U of rBDD-cFVIII.

Figure 3

cFVIII expression and anti-cFVIII antibody responses in Chapel Hill hemophilia A dog K03 after liver delivery of AAV-cFVIII. One Chapel Hill dog (K03) with preexisting inhibitors to cFVIII was administered 2.5 × 10¹³ vg/kg of AAV8-TBG-cFVIII-HC and AAV8-TBG-cFVIII-LC by peripheral venous injection. (A) cFVIII antigen levels were assayed by a cFVIII-LC specific ELISA, and activity was monitored by FVIII assay. (B) Anti-cFVIII antibody responses were measured by anti-cFVIII IgG2 ELISA and Bethesda assays. Black arrows indicate 4 weekly challenges with 500 U of rBDD-cFVIII, and gray arrows indicate 4 weekly injections of 25 mL of pooled normal canine plasma per dose.

Figure 4

Whole-blood clotting time of Chapel Hill inhibitor dogs after vector administration. Whole blood clotting time (WBCT) for all 3 hemophilia A Chapel Hill dogs is shown. The WBCT range for a normal dog is shaded in light gray (8-12 minutes), and the range for a hemophilia A dog is shaded in dark gray (> 45 minutes).

Figure 5

Recovery of cFVIII after intravenous administration. rBDD-cFVIII was administered (100 IU/kg) to Chapel Hill dogs K01 and K03 by intravenous injection, and cFVIII activity was monitored over time by FVIII assay.

Figure 6

cFVIII expression and anti-cFVIII antibody response in a high-responding hemophilia A dog after AAV-mediated liver expression of cFVIII. A high-responding hemophilia A dog from the Queen's University colony (Wembley) was administered 2.5 × 10¹³ vg/kg of AAV8-TBG-cFVIII-HC and AAV8-TBG-cFVIII-LC by peripheral venous injection. (A) cFVIII antigen levels were assayed by a cFVIII-LC-specific ELISA, and activity was monitored by FVIII assay. (B) Anti-cFVIII antibody responses were measured by anti-cFVIII IgG2 ELISA and Bethesda assays.

Figure 7

Anti-AAV8 capsid humoral responses. Anti-AAV8 capsid IgG2 responses were assayed in all 4 dogs. Plasma samples were assayed for capsid-specific IgG2 by ELISA with plates coated with empty AAV8 capsid.