PROJECT SUMMARY Hearing loss during early childhood significantly affects learning and acquisition of social skills, while hearing loss in adults can often result in social isolation and inability to perform many routine social functions. A leading cause of sensorineural hearing loss is the loss of sensory hair cells of the inner ear. A lifetime exposure to aminoglycoside and loud sounds will result in an estimated 15% of adult Americans (~36 million) having some form of hearing loss. A promising approach to mitigate hearing loss and deafness is a cell replacement therapy by transdifferentiating supporting cells into hair cells. Unfortunately, current approaches for transdifferentiation rely on viral delivery may be unsafe and impractical for clinical translation. Therefore, there is a critical need to develop alternative platforms to regulate gene expression and induce transdifferentiation in an efficient, non-viral manner suitable for hearing restoration. To this end, our long-term goal is to develop NanoScript, an innovative, tunable nanoparticle-based artificial transcription factor platform capable of effectively regulating gene expression in a non-viral manner. Using NanoScript, we will transdifferentiate supporting cells into functional hair cells. NanoScript consists of a nanoparticle functionalized with specific small molecules and peptides that are designed to mimic the individual domains of natural transcription factor (TF) proteins. TFs are endogenous, multi-domain proteins that orchestrate many cellular functions including differentiation. Since NanoScript is a functional replica of TF proteins, it can replace virally-delivered TFs for regenerative medicine-based applications. The overall objective of this proposal is to design three NanoScripts that mimic three TFs essential for hair cell differentiation (Gfi1, Pou4f3, and Atoh1; GPA). We will test whether GPA-NanoScript binds to the same DNA sequence and activate gene expression in vitro. Next we will determine if addition of epigenetic modulators to GPA-NanoScript will bind to the same targets as the TF proteins, locally alter the chromatin structure and enhance gene expression. Finally, we will use cochlear explants to determine whether GPA-NanoScript promotes transdifferentiation of supporting cells into hair cells. Generation of nascent hair cells using an ex vivo model will serve as a springboard to test NanoScript technology for regenerative medicine. It will also establish NanoScript as an effective and non-viral tool for researchers to generate functional cells via direct reprogramming.