Genome editing holds great promise for the treatment of many genetic diseases; however its application in the clinic has been slow due to the lack of the safe delivery tools and significant cost and time investment required to custom-develop individual therapies. In our SCGE program phase 1 study, we developed a chemically modified ribonucleoprotein (cRNP)-based gene delivery system that specifically targets neuronal cells throughout the brain after intrathecal (IT) administration. The overarching goal of this application is to apply this novel gene editing technology towards the treatment of two severe neurodevelopmental disorders (NDD): Angelman syndrome (AS) and H1-4 syndrome (HIST1H1E syndrome). AS is a devastating neurodevelopmental disease caused by the deficiency of the maternal and brain specific imprinting UBE3A gene in human chromosome 15q11-q13 region. The structure of UBE3A is intact in the paternal chromosome in all AS cases but transcriptionally repressed by a non-coding and antisense RNA of UBE3A (UBE3A-ATS) mediated mechanism. It has been shown convincingly that reduction of UBE3A-ATS by antisense oligo (ASO), topoisomerase inhibitors, and virus delivered Cas9 gene editing can de-repress the expression of UBE3A and correct the abnormal neurological phenotypes in AS mouse models. H1-4 syndrome is caused by a gain of function mechanism due to a mutated protein with aberrant C-terminal frameshift tail (CFT). H1-4 syndrome has similar but milder clinical features than AS. There is no effective intervention for H1-4 syndrome. Thus, a long- term molecular therapy for AS and H1-4, as well as other NDDs is urgently needed. In our preclinical study using a well validated AS mouse model, we demonstrated that a single IT delivery of Ube3a antisense-targeting RNP/Cas9efficiently de-represses the expression of Ube3a from the paternal chromosome, leading to correction of neurobehavioral phenotypes. Similarly, the knockdown of H1-4 CFT rescue the abnormal phenotypes in H1- 4 humanized mice. We propose our cRNP-based platform for the treatment of AS and H1-4 syndrome utilizing the same genome editor (CRISPR), delivery system (cRNPs), route (IT), target cell type (neurons), therapeutic mechanism (genetic inactivation) and overall trial design. We have assembled an outstanding team from Yale and Rush University with strong and complementary expertise in the areas of preclinical, IND enabling studies, and clinical trials. The success of this study will lead to the first ever gene editing based therapy for AS and H1- 4. More importantly, it will support a paradigm shift for genome editing; rapidly expanding the number of neurogenetic diseases treated by in vivo gene editing and accelerating the transition of genome editing technology into clinical applications.

We propose a new ribonucleoprotein (RNP)-gRNA CRISPR genome-editing strategy that will significantly increase efficiency of genome-editing clinical trials for neurogenetic diseases. We will specifically target two neurogenetic diseases with our RNP-based platform: Angelman Syndrome (AS) and H1-4 syndrome. Based on strong proof-of-concept and IND-enabling data and our extensive experience with clinical treatment and trials relating to these diseases, we propose a Phase 1/2a clinical trial to assess safety and tolerability in adults and children.