PROJECT SUMMARY Infants with Rett syndrome (Rett) are born with loss-of-function mutations in the gene encoding MeCP2, a global regulator of gene expression. MeCP2 dysfunction in the brain severely affects neurons, leading to developmental deficits of varying severity that manifest after 6 months of age. Current treatments can manage some symptoms, but correcting MECP2 mutations would more effectively restore patients’ quality of life. CRISPR gene editing has made this approach conceivable. Among CRISPR technologies, prime editing is the most flexible, utilizing an RNA-guided Cas9 nuclease fused to reverse transcriptase to “search and replace” mutations in post-mitotic cells. Thus, prime editing is a strong candidate for Rett treatment. Yet, prime editor has only been delivered to neurons via lentivirus (not clinically relevant), and its editing efficiency in cells is low. Previous work demonstrates that delivery of Cas9 mRNA encapsulated in lipid nanoparticles (LNP) is simple, safe, and supports robust editing in mouse liver. LNP-encapsulated mRNA also delivers to brain, but delivery of prime editor mRNA and efficiency of prime editing in neurons remains untested. In addition, chemically modifying the guide RNA of other CRISPR systems can protect against nuclease-mediated degradation and improve gene editing rates in cells. The Watts lab recently developed a method to synthesize chemically modified prime editing guide RNA (pegRNA), something that was considered unfeasible due to the length of pegRNA (~150 nt). The effect of pegRNA modification on prime editing efficiency has not yet been tested. With support from Drs. Jonathan Watts (nucleic acid chemistry), Michael Green (Rett neurobiology), Erik Sontheimer (prime editor biology), Scot Wolfe (gene regulation), and Athma Pai (bioinformatics), this project seeks to establish and chemically optimize mRNA-based prime editors to correct MECP2 mutations and reverse their phenotypes in neurons. Aim 1 will establish baseline effectiveness of mRNA-delivered prime editor (vs. lentiviral) against the most common Rett mutation (a missense mutation) and two clinically severe nonsense mutations in HEK cells expressing each mutant MeCP2, patient-derived induced pluripotent stem cells (iPSCs), and iPSC-derived neurons. This Aim will also probe neurons with and without editing to understand the molecular phenotypes of each MECP2 mutation and extent to which editing reverses them. Aim 2 will iterate on the Watts lab’s pegRNA assembly method to optimize pegRNA yield and synthesis time, and identify editing-compatible pegRNA modification patterns using in vitro and in cellulo assays. The effect of pegRNA modifications on MECP2 editing will be tested and optimized in HEK cells, iPSCs, and iPSC-derived neurons, as in Aim 1. Molecular phenotypes of prime edited vs. unedited neurons will also be characterized as in Aim 1. This work will offer insight into how MeCP2 mutants affect severity of Rett phenotypes in neurons and inform development of a prime-editing platform to treat any form of Rett as well as other neurological disorders. The training provided from this research will prepare the fellow for a productive career in the gene editing and neuro-therapeutics field.

PROJECT NARRATIVE Rett syndrome (Rett) is a severe, genetically diverse neurodevelopmental disease with no curative treatments. This study seeks to develop a therapeutic approach to correct the underlying genetic cause of Rett in neurons. If successful, this work will inform the development of a gene-editing platform to treat almost any case of Rett as well as other neurological disorders.