Cas9 Protein Outperforms mRNA in Lipid Nanoparticle-Mediated CFTR Repair

Abstract

Lipid nanoparticles (LNPs) are currently one of the most clinically advanced delivery systems for nucleic acid cargo and hold great potential for clinical applications in gene editing and the treatment of genetic diseases. LNP-mediated delivery of Cas9 with single guide RNA (sgRNA) and homology-directed repair DNA template (ssDNA) enables efficient and precise editing in vitro and in vivo. Comparative analysis of LNP delivery of Cas9 as protein or mRNA for relevant clinical targets, such as cystic fibrosis (CF), which is caused by mutations in the CFTR gene, is imperative in the design of corrective therapeutics for genetic diseases. Here, we show that delivery of Cas9 protein LNPs outperforms Cas9 mRNA LNPs when evaluated for in vivo lung editing as well as corrective CRISPR/Cas9 editing and functional recovery of the CFTR protein. These results demonstrate the ability to optimize the use of CRISPR/Cas9 LNPs for cystic fibrosis applications.

Keywords: CRISPR/Cas9 editing; cystic fibrosis; lipid nanoparticles; lung delivery.