PROJECT SUMMARY Optic neuritis (ON) is an acute autoimmune disease caused by immune attack on the myelin that protects the optic nerve leading to vision loss. Steroid treatments accelerate recovery of visual acuity in some patients but have no effect on other visual functions such as contrast sensitivity that are important for activities of daily life. Even with steroid treatments, 13,000 ON patients per year fail to fully recover visual acuity and 50% of ON patients eventually convert to multiple sclerosis. New therapies are needed to improve ON patient outcomes and quality-of-life. Aberrant activation of T and B lymphocytes drives ON pathologies. Targeting these pathogenic cells is a potential therapeutic strategy. Our company, Trethera, has conducted extensive preclinical studies to develop a small molecule drug, TRE-515, that has the potential to selectively block lymphocyte activation in ON by inhibiting deoxycytidine kinase (dCK), a key rate-limiting enzyme in the deoxyribonucleoside salvage pathway. Our preliminary studies show (i) that cells of the immune system activate dCK during all phases of disease in the C57Bl/6 MOG35-55 experimental autoimmune encephalomyelitis (EAE) mouse model of ON, (ii) that TRE-515 blocks dCK activity in immune cells in this model, (iii) that TRE-515 blocks phenotypes of CNS demyelination in this model, (iv) that TRE-515 blocks inflammation of the optic nerve in this model, (v) that TRE-515 blocks T cell activation in culture, (vi) that TRE-515 blocks B and T cell activation in this model, and (vii) that TRE-515 treatments and dCK knockout are not associated with significant toxicities. Collectively, these data strongly suggest that TRE-515 could be an important new therapy for ON. In support of this, the FDA recently awarded TRE-515 Orphan Drug Status for ON. In the proposed Fast-Track project, we will conduct critical preclinical studies to confirm the safety properties of TRE-515 as a therapy for ON, to study the ON disease stage that TRE-515 affects, to identify the appropriate dosage regimen, and to identify potential biomarkers of target engagement. In Phase I, we will study whether TRE-515 administered therapeutically can block ON symptoms (Aim 1) and evaluate the genotoxicity of TRE-515 (Aim 2). In Phase II, we will examine the dose-response relationship between TRE-515 and ON symptoms in the MOG35-55 EAE mouse model of ON (Aim 3), evaluate the effect of TRE-515 in an additional ON model (Aim 4), study the mechanisms through which TRE-515 blocks lymphocyte proliferation (Aim 5), and evaluate whether plasma deoxycytidine and deoxyuridine levels could serve as biomarkers of TRE-515 target engagement (Aim 6). This IND-enabling work will be critical for moving TRE-515 into the clinical for ON patients and for designing clinical trials with the highest chance of success.

PROJECT NARRATIVE Optic Neuritis (ON) patients need new, effective, and well-tolerated therapies to improve treatment outcomes and reduce side effects, and for these patients, reducing aberrant lymphocyte activation with targeted drugs is a promising therapeutic strategy. Our company, Trethera, is developing a novel small-molecule drug, TRE-515, to treat ON by targeting a pathway required for lymphocyte activation. In the proposed project, we will conduct critical preclinical work to further evaluate the safety of TRE-515 as a drug for ON and to further study how TRE- 515 affects ON relevant cells and disease stages.