Figure 6: Correction of the canine haemophilia A phenotype with platelet BDDFVIII.
A Coatest SP4 FVIII chromagenic analysis assay was performed on lysates of 1 × 108 washed peripheral blood platelets resuspended in 1 ml of lysis buffer from samples collected periodically for at least 2.5 years after transplant (x axis). The mean value of FVIII activity (mU ml per 108 platelets) is represented by the plotting of data points recorded from two independent samples (each measured in duplicate) with error bars showing ±s.d. for at least 12 time points (y axis). Analysis of platelet FVIII:C activity from a FVIII-deficient dog served as a negative control (black, solid line). Interestingly, the level of FVIII:C activity was detected above background levels at regular intervals (blue dashed line) for ≈2.5 years after transplant of BDDFVIII-transduced G-PBC (F20, top; I42, middle; M64, bottom panel). Note, each dog was administered daily injections of FVIII in the form of canine blood transfusions or cFVIII (red bracket) and EACA (green bracket) for uncontrolled bleeding for a short time after transplant (until haemocult tests were negative). As anticipated, each dogs required cFVIII supplements (red arrows) to help resolve severe bleeding episodes incurred before G-PBC transplant. Remarkably, only F20 (which had the lowest steady state level of FVIII:C activity) required cFVIII supplements after G-PBC transplant demonstrating platelet FVIII:C activity reached therapeutic levels in I42 and M64.
