Angelman syndrome (AS) is a neurogenetic disorder caused by the deficiency of the maternal UBE3A and characterized by severe developmental delay. Current treatment only helps manage symptoms and cannot meet clinical need to effectively treat AS. The expression of Ube3a in neurons can be activated through inhibition of non-coding antisense RNA of UBE3A (UBE3A- ATS). Genome editing strategy targeting UBE3A-ATS is a promising therapeutic approach for AS. AAV is the most widely used vector for genome editing. However, AAV presents potential safety concerns of immunogenicity and off-target effects. Thus, there is a clear unmet need for novel nonviral delivery technologies for safe and efficient delivery of the CRISPR machinery to neurons for AS treatment. In this application, Couragene proposes to develop a novel, chemically modified ribonucleoprotein (cRNP) complexed with Cas9 protein and gRNA for delivery of CRISPR mediated gene editing to the brain for treatment of AS. cRNPs have been previously shown to enable brain-wide genome editing and achieve long-term persistent therapeutic benefits in AS mice through single intrathecal (IT) administration. The proposed project will focus on formulation optimization of the cRNP for delivery of genome editing to neuronal cells in vitro in Aim 1, seeking to achieve over 70% editing efficiency in primary AS neuronal cells. The leading cRNP formulation will then be characterized in Aim 2 to determine cRNP-based genome editing delivery efficiency and safety in Ube3a-YFP AS reporter mice. We aim to achieve brain-wide genome editing with over 50% neuronal editing efficiency without significant toxicity through single intrathecal injection in AS mouse. The successful completion of the proposed work will build a solid foundation for Phase II projects where further biodistribution and phenotypic rescue evaluation in Ube3a maternal deficiency mice, off-target study and IND-enabling studies with potential partners will take place. The projects will eventually result in a nonviral genome editing therapy with Couragene’s innovative chemically modified ribonucleoprotein cRNP technology, greatly enhancing the clinical treatment for patients with AS.