PROPOSAL SUMMARY Hybrid Insulin Peptides (HIPs) are a new family of autoantigens in Type 1 Diabetes (T1D). These peptides form through covalent fusion of pro-insulin fragments to other beta-cell peptides. HIPs contain non-genomic amino acid sequences, making them plausible targets for autoreactive T cells. We demonstrated that HIP- reactive CD4 T cells trigger diabetes in non-obese diabetic (NOD) mice, are present in residual islets of organ donors with T1D, and can be detected at significantly elevated levels in the peripheral blood of recent-onset T1D patients. We also verified the presence of HIPs in human and murine islets by mass spectrometry. Our data indicate that the protease Cathepsin D is responsible for the generation of HIPs that are targeted by diabetes-triggering CD4 T cell clones. Here we will generate NOD mice carrying a mutation that prevents the generation of HIPs. We predict that HIP-deficient beta-cells of these mice cannot be recognized by pathogenic CD4 T cells and the mice will be protected from disease onset. We will analyze the islets of these mice to assess HIP-content and T cell infiltration. We will also study disease incidence in these mice and determine whether pathogenic CD4 T cell clones can transfer disease into these mice. Beta-cells, deficient of disease- critical autoantigens, could allow us to reverse autoimmune diabetes in NOD mice and humans without the need for immune-modulation or shielding of beta-cells through encapsulation.