Dependence of Vector Dose and Age of Mice in the Induction of Immune Response Against Factor VIII Following Liver-Directed Nonviral Gene Transfer.

Formation of inhibitory antibodies to transgene product may limit the success of gene therapy especially for the treatment of hemophilia A. The risk of forming inhibitory antibodies against factor VIII depends on multiple factors. Previously we have shown that following naked gene transfer of fifty micrograms of a liver-specific, high-expressing factor VIII plasmid, pBS-HCRHPI-FVIIIA into hemophilia A mice (at least 60 days old), a robust humoral response was induced two weeks post plasmid injection despite of initial high-level gene expression of factor VIII (Ye et al. (2004) Mol. Ther. 10, 117–126). This response completely inhibited the activity of circulating factor VIII although factor VIII was persistently expressed in the liver. In this study, the cytokine production was characterized in human factor VIII-activated T cells from plasmid-treated and untreated hemophilic A mice, consistent with a response predominantly mediated by Th2-induced signals. Injection of plasmid DNA into 4 groups of hemophilia A mice (n=5, 60 days old) with 4 different doses (0.4, 2, 10, & 50 microgram per animal) resulted in vector dose-dependent expression of factor VIII. In addition, the two groups of mice with lower doses of plasmid DNA (0.4 & 2 microgram per animal) did not elicit any antibody response against factor VIII, whereas the two groups of mice with higher doses of plasmid DNA (10 & 50 microgram per animal) induced inhibitory antibody formation. Nevertheless, when the two groups of animals (n=4) with lower doses were treated with second injection of fifty microgram of factor VIII plasmid 180 days post plasmid delivery, all mice developed inhibitors suggesting no immune tolerance was induced by first injection of plasmids. Furthermore, fifty micrograms of factor VIII plasmids were injected into 4 groups of hemophilia A mice (n=5) of 4 different age groups (36, 48, 60 & 72 days). It was found that none of the mice with age 36 days at the time of plasmid injection developed inhibitors, 1/5 mice with age 48 days developed inhibitors, whereas the two groups of mice with age 60 & 72 days all developed high-titer inhibitors. These results indicate that induction of anti-factor VIII antibody following gene therapy is strongly dependent upon the vector dose and age of the animals, which has important implication for developing immunomodulation or other strategies to avoid/eliminate antibody responses.