Interpretations of Experiment 2:
What conclusions can you draw from the graphs?
The ELISA results just show the level of hFIX.
The result of the bleeding times for the mice depicted in graphs b. and d. are very promising. The normal control mouse has a bleeding time of 3 to 5 minutes. The hemophilia B mice treated with the control vector, rAAV-TH, needed to be cauterized after 30 minutes because it's blood had not yet clotted. One mouse died at 54 weeks due to unknown causes. The other mice included in this study were the rAAV-hFIX transduced hemophilia B mice. These mice show a correction in their bleeding times ranging from 3 to 20 minutes. They also show that with 16-25% or more biological activity, the bleeding times of these mice were in normal range.
How was the biological activity determined?
The biological activity of hFIX in the transduced mice was calculated. A standard curve was derived by running an invitro clotting time assay using plasma deficient in Factor IX, adding known concentrations of Factor IX and measuring the clotting time. They could then repeat the assay using serial dilutions of the unknown samples and plot the biological activity levels by comparison to the standard curve.
What is the clinical significance of looking at the biological activity of the transfected hFIX, as opposed to the concentrations of hFIX?
Looking at only the concentration of hFIX tells you the amount that is there but doesn't tell you if the protein generated from the transfection is functional. So biological activity would be more clinically significant when comparing the correction of bleeding times in the mice.
To view experiment 3, click here
