Abstract Number: PB1097
Meeting: ISTH 2020 Congress
Background: Hemophilia A (HA) dogs remain a valuable large animal model for the study of long-term efficacy and safety of gene therapy, especially for translation of novel human factor VIII (hFVIII) variants. Due to differences between the human and canine (c) FVIII proteins, hFVIII provokes an anti-hFVIII immune response when tested in HA dogs, while using cFVIII in dogs would not be predictive of AAV dosage of a novel hFVIII variant in humans.
Aims: HA dogs were tolerized to hFVIII to evaluate a novel hFVIII variant with enhanced secretion in this model after AAV administration.
Methods: Puppies (d2) were treated with a retroviral (RV) vector expressing hFVIII-BDD. At 6 months old, the RV-treated dogs express 1-2% hFVIII and were challenged with hFVIII-BDD protein (25U/kg, 6 weekly I.V) and exhibited tolerance. Our novel variant lacks the first three amino acids (Δ3) of the furin cleavage site (1645-RHQR-1648) and contains modifications at the second major processing site in acidic region 3 (S1657P/D1658E)(SP/DE). The variant was incorporated into a liver-specific codon-optimized (CO) expression cassette with an enhanced promoter, TTRm.
Results: After AAV delivery in HA-CD4KO mice, TTRm-CO-BDD-Δ3-SP/DE exhibits a 2-fold increase in expression over TTRm-CO-BDD at both vector doses (Figure 1). The Δ3-SP/DE variant with altered processing sites is secreted more efficiently and almost exclusively in a single chain form both in vitro and in vivo, while hFVIII-BDD forms a heterodimer. One tolerized dog was treated with TTRm-CO-BDD (2e12vg/kg) and expressed around 4% hFVIII. A tolerized littermate was administered TTRm-CO-Δ3-SP/DE at the same AAV dose and expressed ~12% hFVIII
(Figure 2). Both dogs exhibit no increase in anti-hFVIII IgG1, IgG2, or total IgG.
Conclusions: These studies suggest that Δ3-SP/DE leads to ~3-fold increase in expression compared to hFVIII-BDD in the HA dog, providing a unique approach to lower the AAV dose for HA gene therapy.
To cite this abstract in AMA style:Nguyen G, Merricks E, Long T, Ponder K, Nichols T, Sabatino D. A Novel Human Factor VIII Variant Exhibits Increased Factor VIII Expression after AAV Gene Therapy in a Unique Hemophilia A Dog Model that is Tolerant to Human Factor VIII [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/a-novel-human-factor-viii-variant-exhibits-increased-factor-viii-expression-after-aav-gene-therapy-in-a-unique-hemophilia-a-dog-model-that-is-tolerant-to-human-factor-viii/. Accessed October 26, 2020.
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