Type 1 diabetes (T1D) is an autoimmune disease resulting in complete destruction of insulin-producing pancreatic β cells. In T1D in human and in the non-obese diabetic (NOD) mouse, the spontaneous murine model of T1D, auto-reactive T cells target islet-associated antigens. Induction of antigen (Ag)-specific tolerance could cure Type 1 Diabetes (T1D) but it has not been achieved yet. We previously showed that lentiviral vector (LV)-mediated gene expression in hepatocytes induces active tolerance toward the encoded-Ag. Systemic administration of a single dose of Integrase competent (IC) or integrase defective (ID) LV.ET.InsB9-23.142T, enabling stable and transient expression of InsB9-23 in hepatocytes, respectively, arrests β cell destruction in NOD mice at advanced pre-diabetic stage by generating InsB9-23-specific FoxP3+ T regulatory cells (Tregs). In the present study we tested the efficacy of hepatocytes-directed LV.ET. InsB9-23.142T gene transfer in protecting from disease progression at later stages and in reversing T1D.Treatment with LV.ET.InsB9-23.142T in NOD mice with glucose levels ranging from 200mg/mL to 250mg/mL blocked T1D progression in only 27% of the mice. Co-expression of the late auto-Ags-derived epitopes GAD206-220 and IGRP195-214 in hepatocytes did not improve the efficacy of LV.ET.InsB9-23.142T treatment. LV.ET.InsB9-23.142T treatment in diabetic NOD mice with blood glucose levels ranging from 250mg/mL to 300mg/mL did not result in reversion to normoglycemic levels in any of the treated mice.We next combined InsB9-23 gene transfer with anti-CD3 monoclonal antibody (mAb) treatment. Treatment with anti-CD3 mAb at optimal doses is able per se to reverse T1D in NOD mice. Therefore, we tested decreasing doses of anti-CD3 mAb in diabetic NOD mice with blood glucose levels ranging from 250mg/mL to 300mg/mL to identify the sub-optimal dose unable to revert T1D. We found that a single administration of anti-CD3 mAb at 5μg instead of 10μg results was not effective. This sub-optimal dose of anti-CD3 mAb (1X 5μg) was administered together with LV.ET.InsB9-23.142T to NOD mice with blood glucose levels ranging from 250mg/mL to 300mg/mL. Results showed T1D reversal in 75% of ICLV-treated and 40% of the IDLV-treated mice. These data indicate that the LV.ET. InsB9-23.142T treatment combined with sub-optimal anti-CD3 mAb treatment is able to reverse overt diabetes.