Membrane destabilizing ionizable phospholipids for organ selective mRNA delivery and CRISPR/Cas gene editing

Endosomal escape remains a fundamental barrier hindering advancement of nucleic acid therapeutics. Taking inspiration from natural phospholipids that comprise biological membranes, we report the combinatorial synthesis of multi-tailed ionizable phospholipids (iPhos) capable of delivering mRNA or mRNA/sgRNA for gene editing in vivo. Optimized iPhos lipids are composed of one pH-switchable zwitterion and three hydrophobic tails, which adopt a cone shape in endosomal acidic environment to facilitate membrane hexagonal transformation and subsequent cargo release from endosomes. Structure-activity relationships reveal that iPhos chemical structure can control in vivo efficacy and organ selectivity. iPhos lipids synergistically function with various helper lipids to formulate multi-component lipid nanoparticles (iPLNPs) for Selective Organ Targeting (SORT). Zwitterionic, ionizable cationic, and permanently cationic helper lipids enable tissue-selective mRNA delivery and CRISPR/Cas9 gene editing in spleen, liver, and lungs (respectively) following intravenous administration. This rational design of functional phospholipids demonstrates significant value for gene editing research and therapeutic applications.