Summary/Abstract Functional Piezo1 is enriched in the Schwann cells (SCs) of the peripheral nervous system; however, whether SCs-Piezo1 contributes to pain pathology is generally unknown. The overall goal of this exploratory/innovative proposal is to investigate the possible role of SCs-Piezo1 in pain sensation by adeno-associated virus (AAV)- mediated selective SCs-Piezo1 inhibition in vivo. We 1) Verify physical and functional Piezo1 expression in SCs, 2) Verify that sciatic nerve injection of Piezo1 agonist Yoda1 induces hypersensitivity of mechanical response in rats, which is reversed by co-injection of GsMTx4, a non-specific Piezo1 inhibitor, 3) Confirm a novel AAV capsid (olig001) with primary Schwann cell tropism via intrasciatic delivery, 3) Optimized sciatic nerve injection technique for successfully applying injecta into subepineural space avoiding severe injection injury, 4) Develop a short hairpin Piezo1-shRNA (PZ1shRNA) that efficiently silences physical and functional Piezo1 expression, and 5) Develop a potent Piezo1 inhibitory peptide aptamer (PZ1iPA) derived from the human Piezo1 protein. These preliminary data support the feasibility of testing the hypothesis that AAVolig001-mediated selective inhibition of Schwann cell Piezo1 will mitigate nerve injury-induced hypersensitivity in rat model of neuropathic pain. To accomplish the goal, the following Specific Aims are proposed. Aim 1 will develop and produce the test- AAVolig001s (AAVolig001-PZ1shRNA and AAVolig001-PZ1iPA) for selective Schwann cell transduction in vivo and validate the efficacy of the test-AAVolig001s for physical/functional inhibition of SCs-Piezo1 in primary cultured SCs. Aim 2 will determine the sensory biological effects after sciatic nerve delivery of the test- AAVolig001s in naïve and neuropathic pain rats. Completion of these Aims will verify the role of SCs-Piezo1 in pain sensation and lay a foundation to explore the physiology and pathophysiology of SCs-Piezo1 in future studies.