Project Summary / Abstract MS is a chronic neuro-inflammatory autoimmune disease that leads to demyelination and damage of axons. A breakdown of immune tolerance mechanisms that permits activation of naive myelin-specific T cells is considered an initial step in the pathogenesis. A number of pivotal studies have demonstrated that antigen Ag- specific Tregs not only have a significant role in modulating autoimmune CNS disease, but can also be highly effective at treating MS.2-6 Using the clinically proven adeno-associated virus (AAV) platform, we have developed an immunotherapy that restores Ag-specific tolerance in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, that is efficient at preventing disease.1 Importantly, we demonstrate the AAV immunotherapy is capable of suppressing both clinical disease and inflammatory response in mice with established disease ranging from mild to severe. In addition to primary pathogenic antigens implicated in disease initiation, neo-epitopes can emerge as result of intra- and/or intermolecular epitope spreading during progression of EAE, and MS. The potential for increasing multiplicity of anti-myelin autoreactivities imposes major difficulties in developing antigen-specific therapeutic agents for MS. Not only because of the possibility that the primary target antigen and/or emerging neo-reactivities may differ in different patients, but the that the potentially pathogenic autoreactivities to several myelin antigens may occur in any one patient at any one time.7 Studies aimed at inhibiting the development or progression of EAE associated with more than one pathogenic autoreactivity by antigen-specific immunomodulation targeting have suggested that all relevant autoreactivities should be targeted for maximal therapeutic effect. Unlike other approaches that require ex vivo T cell engineering and/or expansion of TCR/Ag-specific T cells, by designing the AAV vectors to express the complete protein coding sequence (not just the immunodominant epitope) of the autoreactive protein, our approach induces Ag-specific Tregs in vivo and eliminates the need to identify HLA/MHC specific epitopes, making this approach unique and universally applicable. Building on our previous results, the continuing hypothesis to be tested is that stable hepatic expression of AAV-delivered neuroantigens will establish a robust and persistent immunological tolerance, mediated by the in vivo induction and expansion of Ag-specific Tregs, can significantly reduce or stop disease progression in the EAE model of MS.