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Rationale. ENGRAILED1 (EN1) is under consideration as a therapeutic approach for amyotrophic lateral sclerosis (ALS). To assess EN1 target engagement in patients, we aim to identify EN1-responsive biomarkers suitable as Prentice-style surrogate endpoints. We will discover candidates by RNA-seq of neuron-derived extracellular vesicles (NVEC) immuno-isolated from blood. Establishing such biomarkers would enable and de-risk early-phase (I/II) EN1 trials. Primary endpoint. Discovery: RNA-seq identification of circulating NVEC-borne biomarkers that differ between sporadic ALS patients and healthy controls. EN1 modulation: Demonstration that these biomarkers are modulated by EN1 in En1+/- mouse models and in ALS patient iPSC-derived motor neurons. Design. Prospective cohort, N=60 (30 sporadic ALS; 30 healthy controls matched on age/sex). Population. Adults undergoing diagnostic work-up for
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