M. Assadian, A. Armstead, A. Thomas, R. De Souza, I. Backstorm, A. Brown, E. Ouellet, S. Garg, G. Tharmarajah, K. Marshall, S. Chang, T. Leaver, A. Wild, O. Seira, J. Liu, W. Tetzlaff
Precision NanoSystems Inc,
Keywords: CRISPR, Cas9, lipid nanoparticles, primary neurons, gene editing, siRNA, mRNA, pDNA, plasmid
Summary:Advances in the gene editing arena, specifically with CRISPR-Cas9, has pushed the demand for efficiently delivering payloads even further. Of the tools available, developments in the field of lipid nanoparticles (LNPs) has allowed for the reliable and efficient delivery of CRISPR components, both in research and clinical settings. Here, we bridge that gap by describing the development of an LNP delivery system for CRISPR components, robustly manufactured with clinical-grade materials using microfluidic technology at scales for screening applications, in vitro experiments and research in animals. We describe the use of lipid-based nanoparticles for highly efficient encapsulation and delivery of payloads, such as siRNA, mRNA and plasmid. In this proof of concept, we show that representative small RNAs, mRNAs and plasmids can be successfully delivered to primary neurons. LNPs manufactured to encapsulate various nucleic acids can do so with high efficiency, encapulating more than 95% of the payload, minimizing payload loss. Transfection efficiency of the LNPs is >95%, quantified using a flourescent dye. The biological endpoint assays used to determine the accessiblility of the payloads delivered varies for siRNA, mRNA and plasmid. Using doses of 1g per mL of media, we achieved >90% knockdown with siRNA delivery, >90% of the primary neurons are GFP+ with GFP mRNA delivery and >60% of the primary neurons are GFP+ with GFP plasmid delivery. The LNPs are well tolerated, such that 5x the required doses have no oberservable cytoxicity. We show that the LNPs can also be used to deliver payloads into various regions of the animal brain. The localized injections into the cortex and the striatum are well tolerated and have extensive distribution. These validation studies provide suitable insights in establishing strategies for efficiently delivering CRISPR components into primary cultures and into the animal. The use of LNPs can be extrapolated to CRISPR components with a simple change in payload.