Abstract Induction of peripheral immune tolerance requires regulatory T cells (Tregs). Protocols designed to manipulate antigen (Ag) specific-Treg populations in vivo, represent a powerful approach for treating autoimmune disease such as Multiple Sclerosis (MS). It has been shown that Ag-specific Tregs can play a critical role in the protection and recovery of MS, and its animal model, experimental autoimmune encephalomyelitis (EAE). Unfortunately, methods to induce endogenous CNS-targeting Ag-specific Tregs that achieve therapeutic efficacy are lacking. To address this, we developed an immune tolerance induction therapy using an AAV vector expressing the full coding sequence for the neuro-protein MOG under the control of a strong liver specific promoter. Our data demonstrates that peripheral injection of this vector induces functionally suppressive transgene-specific Tregs, and mice pre-treated with this vector are protected from developing EAE. More notably, when vector is administered during mild to moderate disease, there is a nearly complete reversal of EAE, both clinically and histopathologically. When treatment is withheld until mice exhibit moderately severe EAE, our therapy still significantly reduced the neurological symptoms and clinical score, although to a lesser degree. Thus, to increase efficacy of the vector induced Tregs, a short course of rapamycin treatment was given concomitantly which had a synergistic affect, resulting in a long-term significant reversal of disease. Using our vector platform to deliver full-length proteins offers a superior MHC/HLA independent approach for in vivo induction of Ag-specific Tregs compared to other ex vivo or epitope restricted Treg mediated therapies being investigated.