Engineering branched ionizable lipid for hepatic delivery of clustered regularly interspaced short palindromic repeat-Cas9 ribonucleoproteins
Haruno Onuma,
Rina Shimizu,
Yuichi Suzuki,
Mina Sato,
Hideyoshi Harashima,
Yusuke Sato
Affiliations
Haruno Onuma
Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
Rina Shimizu
Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
Yuichi Suzuki
Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
Mina Sato
Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
Hideyoshi Harashima
Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
Yusuke Sato
Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan; Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan; Corresponding author
Summary: The delivery of the CRISPR/Cas ribonucleoprotein (RNP) has received attention for clinical applications owing to its high efficiency with few off-target effects. Lipid nanoparticles (LNPs) are potential non-viral vectors for the delivery of RNPs. Herein, we report the engineering of a branched scaffold structure of ionizable lipids for the hepatic delivery of RNPs. Both the total carbon number and branching position were critical for the functional delivery of RNPs. The optimal ionizable lipid exhibited a more than 98% reduction in transthyretin protein after a single dose with no obvious signs of toxicity. The mechanistic study has revealed that optimal LNPs have a unique “flower-like structure” that depends on both the lipid structure and the payload and that these LNPs accumulate in hepatocytes in an apolipoprotein E-independent manner. These results represent a major step toward the realization of in vivo genome editing therapy via RNP delivery using chemically synthesizable LNP formulations.
