78. Regulation of Immune Responses Against Factor VIII Following Nonviral Gene Transfer in Hemophilia A and FOXP3 Transgenic Mouse Models
Authors: Benjamin R. Harmeling, Steven Ziegler, Troy Torgerson, Liping Chen, Hans D. Ochs, David J. Rawlings, Carol H. Miao
Journal: Molecular Therapy (2006)

The success of naked DNA gene transfer as a novel approach to the treatment of hemophilia A has been limited by the formation of inhibitory antibodies against factor VIII (FVIII). Approximately 25–30% of hemophilia A patients produce inhibitory antibodies in response to FVIII protein replacement therapy. Potential gene therapy techniques used to treat hemophilia A have also resulted in a significant humoral immune response in murine models (Ye et al., 2004). The critical role of CD4+CD25+ regulatory T (Treg) cells in controlling autoimmune and alloimmune responses has recently been recognized. We hypothesize that regulatory T cells could modulate the formation of inhibitory antibodies against FVIII. The development of CD4+CD25+ Treg cells is controlled by a transcriptionfactor encoded by FOXP3. Increased FOXP3 expression directly correlates with the suppressive activity of Treg cells. An adoptive transfer of antigen-specific FOXP3 Treg cells could potentially suppress the FVIII-specific immune response in hemophilia A patients. In order to evaluate whether FOXP3 expressing Treg cells can modulate the antigen-specific immune response in the Hemophilia A (HemA) mouse model, we have developed transgenic mice of a HemA phenotype with overexpressed FOXP3 (HemA/FOXP3-Tg). The mice were created by crossbreeding HemA mice and transgenic FOXP3 (FOXP3-Tg) mice to produce constitutively high levels of human FOXP3 in addition to their endogenous murine Foxp3 encoded protein. Four different groups of genetically altered C57BL/6 mouse strains including HemA, FOXP3-Tg, HemA/ FOXP3-Tg, and wild-type control, were injected intravenously with a 100 μg dose of FVIII plasmid (pBS-HCRHPI-FVIIIA) using a hydrodynamics-based gene delivery method. Clotting time of treated mouse plasma was significantly shortened one-day post treatment in all cohorts, as evaluated by a modified APTT assay, indicating similar responses to the plasmid treatment. Approximately two weeks following infusion, HemA mice developed a high-titer FVIII inhibitory antibodies and a concomitant increase in APTT clotting time. Plasmid-injected wild-type mice responded with a considerably lower titer of inhibitory antibodies, likely due to the presence of tolerizing endogenous murine FVIII. In contrast, both groups of FOXP3-Tg mice and HemA/ FOXP3-Tg mice failed to develop FVIII specific inhibitory antibodies, and retained a shorter APTT clotting time. These results strongly suggest that Treg cells induced by FOXP3 gene expression can regulate the formation of inhibitory antibodies against human FVIII. We are currently assessing the potential of adoptively transferred antigen-specific Treg cells to modulate this inhibitory immune response to gene transfer in HemA mice. If transferred Treg cells from FVIII exposed HemA/FOXP3- Tg mice are as successful in controlling immune responses in recipients, then FOXP3 expressing FVIII-specific Treg cells could provide an effective therapeutic approach of regulating FVIII-specific inhibitory antibodies.