Peiqing Ye

Transient blockade of the inducible costimulator pathway generates long-term tolerance to factor VIII after nonviral gene transfer into hemophilia A mice

Formation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII (hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid-treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS(+)CD4(+) T cells and activation of CD25(+)Foxp3(+) Tregs in the peripheral blood, spleen, and lymph nodes. CD4(+) T cells from anti-ICOS-treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-beta. Moreover, CD4(+)CD25(+) Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIII plasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS-treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Phix 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.

611. Nonviral Gene Transfer of Factor VII as Alternative Treatment for Hemophilia A

A major problem for clinical treatment of hemophilia A using factor replacement therapy is the high frequency formation of inhibitory antibodies against factor VIII. This problem is also predicted to occur following strategies currently aimed at targeted genetic correction of this disease. Recombinant activated factor VII (rFVIIa) has been successfully used as an effective alternative treatment for hemophilia patients who developed inhibitors. In order to decrease the cost and fluctuation of FVIIa levels associated with frequent infusions of rFVIIa, nonviral gene transfer of factor VII (FVII) was attempted in a hemophilia A mouse model. To investigate the potential thrombotic risks associated with prolonged, high level of FVIIa expression following gene transfer, we compared the effects of gene transfer vectors encoding zymogen FVII and an engineered secreted FVIIa in hemophilia A mice. We have first inserted human FVII cDNA into a liver-specific vector developed recently in our lab to make pBS-HCRHPI-hFVIIA. The hFVII cDNA sequence was then modified by site-directed mutagenesis to insert a protease cleavage site in between Arg152-Ile153. The resulting construct, pBS-HCRHPI-hFVIIaA encodes a modified protein which can be cleaved by intracellular proteases of the furin family to secrete the activated form of FVII. Fifty mg of the plasmids were delivered into the hemophilia A mouse livers by a rapid, high volume (hydrodynamics-based) infusion method (n=8 mice/group). Both constructs produced supra-physiological levels (800-1200ng/ml) of hFVII or hFVIIa at days 1 and 4 post plasmid administration. Phenotypic correction was evaluated using bleeding assays (normal mice |[sim]|2-5mins, hemophilia A mice >30mins). The hemophilia A mice treated with pBS-HCRHPI-FVIIA had reduced bleeding times of 20-25 mins, although no significant reduction in clotting times by both prothrombin time (PT) and activated partial thromboplastin time (APTT) assays was observed. The normal mice treated with pBS-HCRHPI-FVIIaA had bleeding times of 5-10 mins with more significant reduction in clotting times by PT, and minor reduction by APTT. These results indicate that the evaluation of PT and APTT does not completely reflect the ability to achieve hemostasis in vivo upon injury under these experimental conditions. The levels of hFVII and hFVIIa dropped precipitously at day 14 post treatment possibly due to formation of species-specific antibodies against hFVII. Since interaction of human FVII with murine tissue factor is much weaker than with the human tissue factor, we next constructed vectors to transfer murine FVII or FVIIa to test their efficacy for correcting hemophilia A. Preliminary results in a tail-clip bleeding assay indicated that vectors carrying either mFVII or mFVIIa can partially correct clotting following gene transfer. Various assays including bleeding assays, PT, APTT, a plasma-based thrombin generation (TG) assay and a platelet-based TG assay with more treated animals will be used to assess their full function in rescuing bleeding.