PROJECT SUMMARY Oligomeric tau species are a central instigator of dysfunction and degeneration in tauopathies, a group of degenerative conditions encompassing Alzheimer’s disease (AD) and related dementias (i.e. tauopathies). Several passive and active anti-tau immunotherapeutics are currently undergoing preclinical and clinical testing, and there are high hopes for efficacy. However, substantial concerns exist because of the possibility that many of these agents are not specific for the right target (e.g., bind total or physiological tau) and/or are not efficiently delivered to the right location (i.e., not intraneuronal). As an alternative strategy, we propose leveraging gene therapy for the delivery of single chain fragment variables (scFvs) derived from an anti-Tau Oligomeric Complex 1 (TOC1) antibody, will specifically target a toxic tau species (right targets) and provide robust intraneuronal delivery non-invasively into the brain (right place). TOC1 specifically recognizes a conformation-dependent epitope in tau, necessitating tau dimerization or the formation of higher-order multimeric oligomers. Tau oligomers are considered one of the earliest forms of tau pathology to emerge in human disease and are directly linked to several cellular mechanisms causing neuronal dysfunction and degeneration. Given the pivotal role of oligomeric tau in disease pathogenesis, they are a prime target for therapeutic intervention. Using recombinant adeno-associated viruses (AAVs), we will deliver TOC1-scFv coupled to HaloTag protein and a proteasome degradation signal (PDS) peptide for ubiquitin-proteasome-mediated breakdown of oligomeric tau. Advanced AAVs like the AAV CAP-B10 capsid provide a novel non-invasive route for systemic AAV delivery. We will deliver AAV-TOC1-scFv coupled to HaloTag protein and a proteasome degradation signaling (PDS) peptide for ubiquitin-proteasome system-mediated breakdown of oligomeric tau species. We will test the hypothesis that TOC1-scFv-Halo-PDS will bind and target oligomeric tau species for degradation (Aim 1A), and systemic AAV- TOC1-scFv-Halo-PDS will efficiently express the scFv intraneuronally, subsequently rescuing the phenotype in a mouse model of tauopathy (i.e. PS19 mutant tau mice; Aim 1B). Aim 1A involves the generation and validation of TOC1-scFv-Halo-PDS via assessing different scFv configurations (heavy chain/light chain and light chain/heavy chain) and measuring target engagement utilizing in-cell protein-protein interaction assays in a cell model of tauopathy. Aim 1B focuses on delivering TOC1-scFv-Halo-PDS using the AAV-CAP-B10 viral vector for non-invasive systemic delivery and widespread CNS expression in PS19 mice. Expression in the CNS and peripheral organs will be tested using wild-type mice. Subsequently, a disease prevention study (AAV delivery in early disease) and a disease-modifying study (AAV delivery in mid-disease) will be conducted in PS19 mice. Cognitive performance, scFv expression, targe...