Single Nuclei RNA Sequencing of Mice Hippocampus to Evaluate in vivo Gene Editing by a Biodegradable Nanocage for CRISPR/Cas9 Ribonucleoproteins

In recent years, CRISPR-Cas9 technology has shown promise as a versatile gene editing tool that enables precise nucleotide base-editing (Sander & Joung, 2014), and are anticipated to drive the next wave of gene therapies for a range of health conditions, including neurodegenerative diseases. However, a major challenge in gene therapy development is in the generation of safe and efficient non-viral delivery devices for in vivo Cas9-mediated gene editing. To date, very few safe and effective non-viral Cas9 in vivo delivery methods have been reported, with most in vivo Cas9 delivery methods utilizing viral vectors that require extended manufacturing times with questionable safety profiles and limited packing capacity (Miller et al., 2017). To address these challenges, we have applied a previously developed nanocage (NC) design for non-viral in vivo Cas9 ribonucleoprotein (RNP) delivery in mice brain. In vivo editing efficiency of RNPs delivered with this NC (NC-RNPs) was evaluated in the hippocampi of Ai14 tdTomato reporter mice using single nuclei RNA sequencing. Seurat integrated differential gene expression analyses between NC-RNP Ai14 targeting experimental and NC-RNP scramble control hippocampi conditions revealed a 2.8 fold increase in tdTomato expression in Ai14 targeting NC-RNP, indicating targeted editing at the Ai14 locus. Upregulated tdTomato expression was observed in all cell types in the hippocampal injection area, suggesting high penetrance of our NC-RNP formulation. Further cluster annotation and gene expression analyses revealed no new immune cell activity between experimental and control conditions, thereby supporting the safety of the NC for in vivo Cas9-RNP delivery. While future RNAscope assays are necessary to confirm gene signatures we found through snRNA-seq analyses, our data indicates a safe and robust non-viral Cas9-RNP delivery method for in vivo brain editing