441. IL2 Plasmid Treated Hemophilia A Mice Show an Increase in Regulatory T Cell Population and Initial Tolerance to FVIII
Authors: Meghan J. Lyle, Richard Y. Fu, Carol H. Miao
Journal: Molecular Therapy (2016)

Hemophilia A is an inherited X chromosomal linked recessive disease. Hemophilia A patients lack or possess low levels of functional FVIII protein, which results in an inability for the blood to clot when injury occurs. This inability to clot can result in major blood loss and potential death. The current treatment for patients with hemophilia A is FVIII protein replacement, but this treatment is expensive and often results in anti-FVIII immune responses, neutralizing the clotting effect. In order to overcome this potential immune response patients undergo a high dose regimen of FVIII to induce tolerance. However, a third of patients still develop an immune response. Regulatory T (Treg) cells help balance T effector (Teff) cells through their suppressive function during an immune response and keep autoimmunity in check. Both of these cell types bind to interleukin 2 (IL2), a cytokine which promotes the differentiation, expansion and activation of these cell populations. Treg cells possess a high affinity epitope of the IL2 receptor. With a low dose of IL2, the Treg population outcompetes the Teff cells, leading to an increase in activation and number. The increased suppressive activity may induce tolerance in hemophilia A mice treated with FVIII plasmid. Hemophilia A mice were hydrodynamically injected with either 2 µg or 5 µg IL2 plasmid and 50 µg FVIII plasmid, either sequentially (1 week apart) or simultaneously. Both plasmids were driven by a liver-specific promoter (hAAT-HCR). Cell staining data showed a marked increase in Treg cell population and activation, demonstrating that a small amount of IL2 plasmid incorporated into liver cells is enough to dramatically increase the Treg population. A larger increase of Teff cells were observed in mice treated with 5 µg IL2 plasmid than in mice treated with 2 µg IL2 plasmid. Importantly, Treg/Teff ratio was significantly increased in the 2 µg IL2 plasmid group in the first 3 weeks and maintained at increased levels over several weeks afterwards. ELISA showed an initial higher level of IL2 production; IL2 expression dropped and maintained at low levels after one week. On Day 28-post FVIII plasmid injection, control mice had started to develop inhibitors associated with significant decrease of FVIII expression, while mice treated with IL2 plasmid showed no inhibitor development with persistent FVIII gene expression. The treated mice are monitored to determine the potential long term tolerance effects induced by low dose IL2 plasmid. Using gene therapy to slightly increase the amount of a cytokine in patients could provide a better treatment option than existing drug regimens. FVIII protein is also extremely expensive, especially in the amounts needed for treating hemophilia A patients. Combined gene transfer of FVIII and IL2 plasmids has the potential to produce therapeutic FVIII and simultaneously prevent inhibitory antibody formation, therefore reducing the amount of expensive reagents needed, the number of doctor visits required, and the overall cost, potential morbidity and stress to the patient.