Lipid nanoparticles (LNPs) are the most advanced RNA delivery technology and are used with CRISPR-RNA in multiple clinical in vivo genome editing trials. By contrast, systemic delivery of Cas9 ribonucleoproteins (RNPs) - despite their high intrinsic efficiency - has lagged, largely due to a lack of mature delivery systems, and RNA‑optimised LNPs cannot readily be translated to RNPs. Differences arise from the pH-sensitive protein and cargo-specific optimal lipid compositions. With regard to the fundamental ionisable lipid component, comparatively less optimisation has been carried out for Cas9-RNPs than for RNA. In this work, C12-200, developed as a potent ionisable lipidoid for RNA-LNPs and also well-suited for Cas9-RNPs, served as the lead structure. Using an analogue synthesis strategy, 17 alternative C12-lipidoids were generated from different oligoamine precursors with structural differences, including the number of nitrogens (2 to ≈40), architecture (linear, branched or containing an N-heterocycle), and separating alkyl spacers (ethyl, propyl) between ionisable groups. Employing the different lipidoids in analogous LNP formulations enabled a systematic assessment at relevant stages of Cas9-RNP delivery and the identification of structure-activity relationships. Two C12-lipidoids with piperazine ring, ethyl spacers and three (C12-AEP) or four nitrogens (C12-BAEP) were identified as the most effective, exhibiting potencies comparable to or exceeding C12-200 in the in vitro knockout model. This study reports a systematic evaluation of ionisable oligoamine-lipidoids in Cas9-RNP-LNP formulations, highlights critical delivery bottlenecks, and provides recommendations for the design of potent candidates.
