Immunomodulation of Transgene Responses Following Naked DNA Transfer of Factor VIII into Hemophilia A Mice.

Naked DNA transfer of liver-specific, high-expressing plasmid pBS-HCRHPI-FVIIIA in Rag2(−/ −) SCID mice produced persistent high-level gene expression of human factor VIII (hFVIII) (Miao, Hum. Gene Ther. 2003). However, in immunocompetent hemophilia A mice, a robust humoral immune response against FVIII that followed gene transfer led to complete inhibition of circulating FVIII activity (Ye, Mol. Ther. 2004). Transient immunomodulation strategies were explored to prevent the formation of inhibitory antibody formation. Eight groups of mice (n=8) were treated by naked DNA transfer of plasmid pBS-HCRHPI-FVIIIA. Each group were subjected to treatment with single or combined immunosuppressive regimen: CyclosporineA (CSA) daily for 14 days; Rapamycin daily for 14 days; Mycophenylate mofetil (MMF) daily for 14 days; combination of CSA and MMF; combination of Rapamycin and MMF; a monoclonal antibody (MR1) against murine CD40 ligand on days -1, 1, 2, 7, & 14; recombinant murine Ctla4Ig on days 1 & 2; and combination of MR1 and Ctla4Ig. Combination regimens were given using the same combined schedule and dosages. All animals treated with immunosuppression had delayed or no immune responses against hFVIII except the group treated with CSA only. The most effective treatment was observed in animals treated with the combination of Ctla4Ig and MR1. Seven of 8 animals failed to develop detectable inhibitors. One animal developed transient low-titer antibodies. This group of animals produced persistent, therapeutic levels of hFVIII gene expression for over 6 months. Tolerized animals were subsequently challenged by the T dependent antigen, bacteriophage Φx174, and exhibited a normal primary and secondary response including amplification and isotype switch. These results strongly suggest that transient immunomodulation strategies to disrupt B- and T- cell interactions at the time of plasmid injection is effective to promote long-term immune tolerance that is specific for FVIII without altering subsequent immune responses to other T cell dependent antigens.