Silencing of the beta7 Chain Using Antisense Technology to Ameliorate Gvhd

Acute graft-versus-host disease (GVHD), one of the major complications associated with allogeneic blood and marrow transplantation (BMT), develops primarily in the skin, liver, gastrointestinal tract and lymphoid tissues of patients who receive this therapy for various hematological malignancies. The homing of T cells to GVHD target organs and their regulation via integrins, selectins and chemokine receptors have been recognized as potential novel realms for intervention to ameliorate or prevent GVHD while still allowing the beneficial graft-versus-tumor effects. A number of studies have shown that transplantation of donor T cells lacking beta7; a chain of the intestinal homing receptor alpha4beta7 integrin, was effective at ameliorating GVHD in BMT murine models. Likewise the use of monoclonal antibody against the alpha4 or the beta7 subunits have been alluded to as potential treatments for intestinal bowel disease and GVHD, and trials, at least for the former, have been initiated in the clinic. Here we investigated an alternative method to block alpha4beta7 expression on donor T cells in order to impair their capacity to infiltrate gut tissue in a murine BMT model, using antisense morpholino oligonucleotide (AMO) technology (Gene Tools, LLC). Electroporation of donor T cells permitted a greater than 90% transfection of AMO and an average 82.26±5.78% knockdown on alpha4beta7 expression, just 24 h after treatment with translational-blocking AMO targeting the mouse beta7 chain. Beta7 expression = 84.35±5.30% in electroporation control cells vs. 41.65±6.77% in AMO treated cells (50.62±7.85% decrease), and a4b7 expression = 23.0±7.40% in electroporation controls cells vs. 4.1±1.91% after AMO treatment. In the MHC-haploidentical C57BL/6 (B6) into (B6xDBA/2)F₁ [H2^b/d; B6D2] BMT murine model, B6D2 mice challenged with 3x10⁶ AMO-treated (50 mM) B6 T cells had a MST of 20 days vs. a control group  given untreated/electroporated donor T cells (MST 14 days; p<0.01). This significant prolongation of survival correlated with a significant decreased infiltration of donor eGFP⁺B6 T cells into intestinal tissues as measured by histological analysis (p = 0.024). This data strongly suggested that silencing beta7 on donor T cells with AMO technology, albeit transiently as done here, could be utilized as an effective immunotherapeutic intervention to ameliorate GVHD.