CES1‐Triggered Liver‐Specific Cargo Release of CRISPR/Cas9 Elements by Cationic Triadic Copolymeric Nanoparticles Targeting Gene Editing of PCSK9 for Hyperlipidemia Amelioration

The broad application of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing tools is hindered by challenges in the efficient delivery of its two components into specific cells and intracytoplasmic release. Herein, a novel copolymer for delivery of Cas9‐mRNA/ single‐guide RNA (Cas9‐mRNA/sgRNA) in vitro and vivo, using carboxylesterase‐responsive cationic triadic copolymeric nanoparticles targeted proprotein convertase subtilisin/kexin type 9 (PCSK9) for hyperlipidemia amelioration is reported. A dimethyl biguanide derivative is designed and synthesized to form cationic block, and copolymerization onto prepolymer with propyl methacrylate, to fabricate a triadic copolymer mPEG‐b‐P(Met/n‐PMA). The copolymer can self‐assemble with Cas9‐mRNA/sgRNA, indicating the excellent potential of nanoparticles to form a delivery carrier. This vehicle can efficiently release RNA in response to the hepatocytes carboxylesterase for genome editing. It was demonstrated that the mPEG‐b‐P(Met/n‐PMA)/Cas9 mRNA/sgRNA nanoparticles effectively accumulated in hepatocytes, lead to the inhibition of PCSK9, and lowered the levels of Low‐density lipoprotein cholesterol and total cholesterol in mouse serum down 20% of nontreatment. Interestingly, the nanoparticles even enable multiple functions in the regulation of blood glucose and weight. This study establishes a novel method to achieve complex CRISPR components stable loading, safe delivery, and fixed‐point release, which expand the application of CRISPR delivery systems. This work reports a novel triadic copolymer‐based delivery vehicle to deliver Cas9‐mRNA and sgPCSK9 for gene editing both in vitro and vivo. Carboxylesterase‐responsive motifs are introduced in the vehicle, endowing the delivery vehicle enzymatic responsiveness in hepatocyte and disrupt the target PCSK9 gene and achieve a potent therapeutic efficacy for hyperlipidemia.