Development of ionizable lipid nanoparticles and a lyophilized formulation for potent CRISPR-Cas9 delivery and genome editing

• Published in: International journal of pharmaceutics 2024-03, Vol.652, p.123845-123845, Article 123845
• Main Authors: Sun, Qian, Zhang, Hongqian, Ding, Feng, Gao, Xue, Zhu, Zongwei, Yang, Chuanxu
• Format: Article
• Language: English
• Subjects: CRISPR-Cas9; Genome editing; Lipid nanoparticle; Lyophilization; mRNA delivery
• ISSN: 0378-5173; 1873-3476
• DOI: 10.1016/j.ijpharm.2024.123845

A potent ionizable lipid nanoparticle and its lyophilization formulation was developed to efficiently co-delivery of Cas9 mRNA and sgRNA for genome editing. [Display omitted] CRISPR-Cas genome editing technology holds great promise for wide-ranging biomedical applications. However, the development of efficient delivery system for CRISPR-Cas components remains challenging. Herein, we synthesized a series of ionizable lipids by conjugation of alkyl-acrylate to different amine molecules and further assembled ionizable lipid nanoparticles (iLNPs) for co-delivery of Cas9 mRNA and sgRNA. Among all the iLNP candidates, 1A14-iLNP with lipids containing spermine as amine head, demonstrated the highest cellular uptake, endosomal escape and mRNA expression in vitro. Co-delivery of Cas9 mRNA and sgRNA targeting EGFP by 1A14-iLNP achieved the highest EGFP knockout efficiency up to 70% in HeLa-EGFP cells. In addition, 1A14-iLNP displayed passive liver-targeting delivery of Cas9 mRNA in vivo with good biocompatibility. Moreover, we developed a simple method of lyophilization-mediated reverse transfection of CRISPR-Cas9 components for efficient genome editing. Therefore, the developed 1A14-iLNP and the lyophilization formulation, represent a potent solution for CRISPR-Cas9 delivery, which might broaden the future of biomedical applications of both mRNA and CRISPR-based therapies.