Identifying Biomarkers in ALS Patients Using Neuronal Derived Extracellular Vesicles

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 suspected sporadic ALS; healthy volunteers without neurological disease. Key procedures and timeline. Baseline (M0, inpatient): ALSFRS-R, MRC, hand dynamometry, eye-movement recording (MOC); NCS/EMG (NUMIX), TMS/MEP with cortical excitability; neuropsychology; brain \& spinal MRI; pulmonary function testing; CSF (10 mL) and blood (15 mL) for clinical labs and research (NVEC immunocapture → RNA-seq; proteomics). Follow-up: M6 clinic visit (repeat clinical/electrophysiology/neuropsychology/PFTs as per care) with blood (15 mL); additional routine follow-ups at M12, M18, M24 (clinical; MOC at M12 and M24). Controls: single visit with blood (3×5 mL EDTA) and cortical excitability; brain MRI for targeting. Sample size. 60 participants total (30 ALS, 30 controls).