PB0185 - Immunologic Response to AAV Directed Expression of Enhanced Hemostatic Variant FVIII-QQ in Mice

Background: We previously described a FVIII variant with two amino acid changes (FVIIIR336Q,R562Q; FVIII-QQ) and enhanced hemostatic function (Wilhelm et al. Blood 2021) that may be useful translationally for HA gene therapy. As with any variant protein, there is a risk of enhanced immunogenicity for (h)uman FVIII-QQ relative to hFVIII-WT.

Aims: We sought to evaluate the risk of anti-FVIII antibody development to hFVIII-QQ relative to hFVIII-WT in the setting of recombinant protein administration and expression post gene therapy, particularly potential differences in inhibitor formation, induction of tolerance, or break in tolerance.

Methods: Recombinant hFVIII-WT and hFVIII-QQ were administered intravenously to HA/C57Bl6 (0.2µg) and HA/pF8 (1µg) mice 6 times weekly followed by a 5µg dose. HA/pF8 transgenic mice express hFVIII in megakaryocytes, with the possibility of a lesser immune response and/or central tolerance to hFVIII. Naïve mice and mice pre-treated with rFVIII-WT (1µg 4 times weekly) were treated with AAV vector for hepatocyte expression of hFVIII-WT or hFVIII-QQ; experiments were conducted in HA/C57Bl6 or HA/pF8 mice. Total anti-FVIII IgG and Bethesda titers were determined.

Results: Following recombinant protein challenge or gene therapy expression of hFVIII-QQ versus hFVIII-WT, there was no significant difference in Bethesda or total anti-FVIII IgG titers for FVIII-QQ versus FVIII-WT exposure in either HA/C57Bl6 or HA/pF8 treated mice. Relative to HA/C57Bl6, HA/pF8 developed significantly lower or no anti-FVIII immune response, suggesting the model has some degree but not complete FVIII central tolerance. HA/C57Bl6 and HA/pF8 mice challenged with recombinant hFVIII-WT to induce inhibitor formation were treated with vector to express hFVIII-QQ or hFVIII-WT. Thusfar, HA/pF8 developed no anti-FVIII immune response and tolerance induction studies in HA/C57Bl6 are ongoing.

Conclusion(s): Collectively, our preliminary murine studies support no increased risk of inhibitor formation or break in tolerance for hFVIII-QQ. Studies are ongoing to assess tolerance induction by hFVIII-QQ versus hFVIII-WT expression post gene transfer.