PROJECT SUMMARY Tauopathies are a set of degenerative diseases that includes Alzheimer’s disease (AD) and AD-related dementias. Several passive anti-tau immunotherapies are under development and/or in clinical trials. While hope is high for therapeutic benefits, there is concern that they are not the right targets (e.g. bind total tau) and/or are not delivered to the right place (i.e. not intraneuronal), which may significantly limit therapeutic benefit. Alternatively, gene therapy delivery of antibody single chain fragment variables (scFvs), represents a more robust approach to deliver pathological tau conformation-specific antibodies (right targets) within neurons affected by tauopathy (right place). We developed a tau antibody that specifically recognize pathogenic tau conformations directly connected to mechanisms of axonal and synaptic toxicity in neurons, called Tau N- Terminal 2 (TNT2). This antibody binds tau when it undergoes modifications that cause conformation-dependent display of an N-terminal motif called the phosphatase-activating domain (PAD). PAD exposure is an early pathogenic change in human AD and other tauopathies linked with pathogenic modifications (e.g. oligomers or phospho-tau) and toxicity. Thus, adapting TNT2 as a therapeutic agent has a potentially high impact. We will use recombinant adeno-associated viruses (AAVs) to deliver the TNT2-scFv coupled to HaloTag protein and a protein degradation signal (PDS) peptide to facilitate proteasomal breakdown of PAD exposed tau species. Recent AAV advances, such as AAV CAP-B10 capsid, provide a novel non-invasive route to systemically deliver AAVs that produce widespread CNS transduction further enhancing potential translatability of this approach. We hypothesize that TNT2-scFv-Halo-PDS will effectively engage and degrade PAD exposed tau (Aim 1A) and systemic delivery of AAV-TNT2-scFv-Halo-PDS will effectively deliver the scFv intraneuronally in the CNS and subsequently rescue the phenotype of the PS19 mouse model of tauopathy (Aim 1B). In Aim 1A, we will develop and validate the novel TNT2-scFv-Halo-PDS by testing heavy chain/light chain and light chain/heavy chain configurations of the scFv and target engagement will be measured using a series of in-cell protein-protein interaction assays in a HEK cell tauopathy model. In Aim 1B, we will deliver AAV-TNT2-scFv-Halo-PDS using the AAV-CAP-B10 viral vector to allow non-invasive systemic delivery and widespread CNS expression in PS19 mice. First, we will test expression in the CNS and peripheral organs using wild-type mice. Then, we will perform a disease prevention study (early AAV delivery) and a disease modifying study (mid-disease AAV delivery) in PS19 mice. We will measure behavior/cognitive performance, scFv expression, target engagement, tau neuropathology, neurodegeneration markers (neurons, synapses, etc.) and neuroinflammation markers. Successful completion of this project will conclusively determine whether, or not, a hig...