Targeting FVIII Expression to Platelets for Hemophilia A Gene Therapy Does Not Bare an Apparent Thrombosis Risk

Abstract Our group has previously developed a gene therapy approach for hemophilia A in which FVIII expression is targeted to platelets. Platelet expressed FVIII successfully restores hemostasis in hemophilic mice even in the presence of high titer inhibitory anti-FVIII antibodies and induces immune tolerance to FVIII. Therapeutic efficacy was achieved in our original transgenic mouse line at a level of only 0.75mU FVIII per 108 platelets, which corresponded to an equivalent of 1.25% FVIII in plasma of wild-type (WT) mice. FVIII is not normally expressed in platelets but with platelet-FVIII gene therapy FVIII levels at the site of injury might dramatically increase due to platelet aggregation and activation, and consequent local FVIII release from platelets together with VWF. Furthermore, a higher embolism rate has been suggested in transgenic mice expressing FVIII in platelets on an otherwise FVIII deficient background. Thus, although this approach is very successful in restoring hemostasis, evaluating potential pathological consequences in conjunction with platelet-FVIII is of great importance. Here we explored whether a pro-thrombotic state was induced by platelet expressed FVIII in an attempt to define the breadth of the therapeutic window. To examine this, we analyzed high platelet-FVIII expressing mice using five techniques including 1) a native whole blood thrombin generation assay, 2) ex vivo clot formation using thomboelastometry (ROTEM), 3) assessment of plasma parameters linked with an increased thrombosis risk (D-Dimer, thrombin anti-thrombin complexes (TAT), fibrinogen), 4) in vivo clot formation using a ferric chloride carotid artery injury model, and 5) tissue fibrin deposition. In addition to steady state, mice were subjected to an inflammatory challenge to induce pro-thrombotic conditions. We generated transgenic mice, LV17/18tg, that expressed 18mU FVIII per 108 platelets on a FVIII deficient background. This platelet-FVIII level was 24-fold higher than in our originally described mouse line. While FVIII deficient mice had negligible thrombin generation, comparing WT control to LV17/18tg mice, neither thrombin generation nor ex vivo clot formation was increased above WT levels in the transgenic mice. In WT and LV17/18tg, respectively, peak thrombin was 216 ± 16 and 195 ± 13 nM and endogenous thrombin potential was 1718 ± 41 and 1642 ± 56 nM. Clotting time determined by ROTEM in WT and LV17/18tg, respectively, was 424 ± 20 and 705 ± 37 seconds and maximum clot firmness was 51.3 ± 1.4 and 54.0 ± 1.7 mm. Fibrinogen and D-Dimer levels were similar in WT and LV17/18tg mice. While TAT levels were significantly higher in LV17/18tg (11.6 ± 1.1 ng/ml) than in WT mice (6.0 ± 0.5 ng/ml), interestingly this increase was also observed in FVIIInull mice (11.0 ± 1.2 ng/ml). Therefore, we attributed the elevation of TAT levels to the FVIII deficient background. Why the FVIII deficient mice have elevated TAT levels is not clear and may reflect an abnormality in the FVIII deficient line that we are further investigating. When we monitored time to occlusion in an in vivo thrombus formation model using ferric chloride induced carotid artery injury, vessels in LV17/18tg mice did not occlude faster than in WT mice (time to occlusion: 14.5 ± 0.5 versus 5.9 ± 0.5 min, respectively). Thus, under steady state, platelet-FVIII did not seem to be pro-thrombotic. This led us to evaluate the thrombosis risk under pro-thrombotic conditions. Because inflammation induces a pro-thrombotic state, we challenged mice with 40mg/kg LPS and analyzed the animals 16 hours later. WT and LV17/18tg mice treated with LPS showed a similar significant increase in fibrinogen, D-Dimer and TAT levels over PBS treated control mice. While no fibrin deposition was observed in the liver of WT or LV17/18tg mice, LPS challenge did induce fibrin deposition, but there was no difference between WT and LV17/18tg mice. In conclusion, 24-fold higher platelet-FVIII levels than in our originally described mouse line in which hemostasis was fully restored, did not show any signs of thrombosis in steady state or under inflammatory conditions. Therefore, in this regard, platelet targeted FVIII gene therapy can be considered a safe therapy with a relatively wide therapeutic window that is in excess of 24-fold. Disclosures Baumgartner: Novo Nordisk: Research Funding. Shi: BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETED GENE EXPRESSION. . Montgomery: Biogen: Consultancy; Bayer: Consultancy; CSL Behring: Consultancy; Baxter: Consultancy; Octapharma: Consultancy; Grifols: Consultancy.