The success of a drug delivery method cannot be separated from the quality of the drug it is delivering. A cargo can only go as far as its delivery vector enables, and a delivery vector can only be efficient if its cargo is stable and potent. For in vivo genome editing, lipid nanoparticle (LNP) delivery of CRISPR machinery in RNA format has seemingly addressed the challenge of liver editing. Significant efforts have now turned to LNP formulations that target other organs. We set out to develop local delivery to the central nervous system (CNS) to maximize editing efficiency and minimize systemic toxicity. To understand how LNPs deliver gene editor mRNA in the CNS, we used a Cre-reporter model and examined patterns of editing following LNP-Cre mRNA injection into the cerebrospinal fluid and into different areas in the brain. We found that certain formulations led to editing signal in the hippocampus after intracerebroventricular injection. Cre editing signal from direct brain injections suggested that LNPs rely on functional connectivity of the brain to reach distant brain regions. These findings will inform future efforts to formulate LNPs for local injection of RNA cargoes including CRISPR therapeutics targeting diseases affecting the CNS.

We also present our work on enhancing CRISPR editing outcomes through engineering single guide RNA (sgRNA) designs. When co-delivered with Cas9 mRNA in LNPs, chemically modified sgRNAs increased in vivo nuclease editing efficacy and showed promising results in clinical trials. Building on the insight that cyclic antisense oligonucleotides can improve gene silencing, we extended the 3′-end of sgRNAs with inverted nucleotides complementary to the 5′-end, producing compounds that we term circularly structured guide RNAs (csgRNA). We demonstrated that csgRNA improved editing potency across different genome editing systems in vitro and consistently led to higher in vivo editing efficiency, highlighting its potential for clinical translatability. This thesis work contributes to ongoing efforts to develop safe and effective CRISPR therapeutics.