Extrahepatic Gene Editing In Vivo Using Organic Solvent-Free Lipid Nanoparticles

Targeted therapy, which modifies genes and their expression, holds great promise for treating a variety of diseases, including cancer, inborn errors of metabolism, and acute and chronic inflammatory and infectious conditions. However, it also presents challenges related to RNA delivery, immune responses, side effects of delivery vectors, and the need for individualized formulations. To overcome these limitations, the choice of lipids and formulation processes might be re-evaluated, with a focus on eliminating critical components, such as poly(ethylene glycol) (PEG) and ethanol. Thus, a purely water-based formulation for lipid nanoparticles was developed, offering a material-efficient, time-saving process with high reproducibility. Initially, a stealth lipid containing poly(2-methyl-2-oxazoline) (PMeOx) was used, and the formulation was later expanded to include approved lipids. These nanoparticles not only efficiently transfect primary human immune cells but also effectively deliver multiple nucleotides in CRISPR-Cas9 applications. Moreover, an in vivo comparison revealed that the nanoparticles exhibited preferential transfection in extrahepatic tissues. This distinguishes them from conventional cholesterol-rich lipid nanoparticles, which primarily target the liver regardless of the application route.