ABSTRACT/PROJECT SUMMARY Variants at ERAP1 modulate the risk for several forms of non-infectious uveitis in the presence of disease- associated HLA class I, strongly suggesting a so far unproven change in immunodominance with impact on disease causation and protection. The overall objective of this application is to determine through which mechanism ERAP1 allotypes cause and protect from HLA I-associated uveitis. Our long-term goal is to understand and therapeutically target HLA class I associated autoimmunity in uveitis. Our central hypothesis is that allotypic ERAP1 alters the HLA I-bound peptidome to include epitopes that are immunogenic when presented by disease-relevant HLA I, which induces or controls disease through a change in immunodominance. The rationale for this study is that mechanistic understanding of ERAP1-mediated pathogenesis in HLA I immunity will enable targeted therapy design aimed at specific ERAP1-HLA I-uveitis subsets. Based on these considerations we will implement two specific aims. In Aim 1 we will determine through which mechanism the allotype ERAP1 Hap10 initiates immune dysfunction in HLA-B*51+ Behçet’s uveitis (BU) but protects from HLA-B*27+ acute anterior uveitis (AAU) via a series of CRISPR/Cas9 genome editing experiments. These experiments will allow us to define its functional contribution to the HLA I restricted peptidomes relevant to each of these disorders and their effect on the generation of immunogenic or tolerogenic immune responses. In Aim 2 we will establish how HLA I restricted pathogenic epitopes depend on allotype- specific ERAP1 function through the exploitation of clonally expanded CD8 T cells from active BU and AAU patients for cellular cloning, genome-editing and functional assessment of immunogenicity. We expect the following outcomes 1) knowledge of the immunogenic and tolerogenic effects mediated by allotypic ERAP1 in two highly disease-relevant HLA restriction contexts: HLA-B27 and B51, 2) identity of epitopes that induce or prevent immunogenicity in these contexts, 3) proof of principle that allotypic ERAP1 regulates autoimmunity and that manipulation of its activity modulates pathogenicity providing irrefutable rationale for targeting ERAP1 enzyme activity pharmacologically, or through gene therapy. This will have a positive impact on the field through the identification of molecular targets allowing the design of therapy for patient groups defined by genotypes, and through mechanistic understanding extending beyond the scope of BU and AAU to additional MHC-I-opathies with immense impact on human health, such as IBD, psoriasis, and psoriatic arthritis.