66. Gene Transfer-Mediated Expression of Murine Factor VIIa Improves Clot Formation in a Mouse Model for Bernard-Soulier Syndrome

Moderate to severe bleeding occurs in Bernard-Soulier syndrome (BSS) patients due to a rare inherited dysfunction of their platelet GPIb|[alpha]|/|[beta]|-IX-V receptor, a four-gene encoded protein complex. Defective receptor disrupts the interaction between platelets and vascular wall and form abnormal macrothrombocytopenia, in addition to causing impaired thrombin generation and defective platelet plug at the tissue injury site. While platelet transfusion is the conventional therapy, treatment with a bolus injection of recombinant human factor VIIa (rhVIIa, NovoSeven|[reg]|) offers an alternative strategy especially when alloimmunization develops against administered platelets. At pharmacological plasma levels, rhVIIa led to cessation of mild and severe bleeding episodes in a number of BSS patients (Ozelo et al., Ann Hematol, 84(12), 2005 and Almeida et al, Br J Haematol, 121(3) 2003). However, the short half-life of the protein and its high expense remain as two major disadvantages of a rhVIIa regimen. We here report a gene therapy-based approach resulting in continuous expression of activated murine factor VII (mVIIa) in the plasma of a mouse model for BSS. Male BSS mice were tail-injected with three doses of AAV8-mVIIa vector ranging from 0.3 to 4.8e12 vg/mouse. The liver specific expression of mVIIa resulted in a dose- dependent increase in plasma mVIIa levels and shortening of prothrombin time (PT) with a plateau at four weeks after injection. At mVIIa levels >500 ng/ml (n=5), there was a significant increase in the rate of initial and progressing clot formation in whole blood measured by a thromboelastogram (ROTEM|[reg]|) compared to untreated age- and gender-matched control mice (n=5); CT (266|[plusmn]|44 vs 387|[plusmn]|59, P<0.01), CFT (125|[plusmn]|19 vs. 222|[plusmn]|76, P<0.03) and a angle (66.4|[plusmn]|3.6 vs. 52.7|[plusmn]|9.5, P<0.02), respectively. Moreover, tail clip assay also showed a dose-dependent decrease in blood loss, which was lower in mice receiving doses of 1.2 (n=12) and 4.8e12 (n=5) vg/mouse; OD measurement of hemoglobin released after lysis of RBC (1.0|[plusmn]|0.5, P<0.005 and 0.4|[plusmn]|0.3, P<5e-5) in the two doses vs. (1.5|[plusmn]|0.1) in untreated controls (n=5), respectively. Higher thrombin- anti thrombin (TAT) levels (49|[plusmn]|36 vs 12|[plusmn]|7, P<0.05) in the highest dose vs. control, respectively, reflected the increased overall thrombin generation upon VIIa treatment. Our data suggest that the increase in both the amount and rate of thrombin generation, resulting from continuous expression of mVIIa, could contribute to a better fibrin clot and partially correct the abnormal hemostasis in BSS mice. Whereas the mechanism of VIIa action is independent of the genetic defect, a gene approach to compensate for the ineffectiveness of receptor components largely depends on the affected gene(s), hence narrowing down the number of already rare recipients. Moreover, our approach could be expanded to include other bleeding diatheses due to more common platelet defects such as Glanzmann thrombasthenia, especially with failure of transfusion therapy.