脂質核糖蛋白-寡核苷酸複合物負載的脂質納米顆粒，使用微

StephenW

Lipid nanoparticles loaded with ribonucleoprotein-oligonucleotide complexes synthesized using a microfluidic device exhibit robust genome editing and hepatitis B virus inhibition
Yuichi Suzuki  1 , Haruno Onuma  2 , Risa Sato  2 , Yusuke Sato  3 , Akari Hashiba  1 , Masatoshi Maeki  4 , Manabu Tokeshi  5 , Mohammad Enamul Hoque Kayesh  6 , Michinori Kohara  7 , Kyoko Tsukiyama-Kohara  8 , Hideyoshi Harashima  9
Affiliations
Affiliations

    1
    Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan.
    2
    Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan; Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan.
    3
    Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan; Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan. Electronic address: [email protected].
    4
    Division of Applied Chemistry, Faculty of Engineering, Hokkaido University, Hokkaido, Japan; JST PRESTO, Saitama, Japan.
    5
    Division of Applied Chemistry, Faculty of Engineering, Hokkaido University, Hokkaido, Japan; Innovative Research Center for Preventive Medical Engineering, Nagoya University, Nagoya, Japan; Institute of Nano-Life Systems, Institutes of Innovation for Future Society, Nagoya University, Nagoya, Japan.
    6
    Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Department of Microbiology and Public Health, Patuakhali Science and Technology University, Patuakhali, Bangladesh.
    7
    Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
    8
    Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
    9
    Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan. Electronic address: [email protected].

    PMID: 33333121 DOI: 10.1016/j.jconrel.2020.12.013

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has considerable therapeutic potential for use in treating a wide range of intractable genetic and infectious diseases including hepatitis B virus (HBV) infections. While non-viral delivery technologies for the CRISPR/Cas system are expected to have clinical applications, difficulties associated with the clinically relevant synthesis of formulations and the poor efficiency of delivery severely hinder therapeutic genome editing. We report herein on the production of a lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery nanoplatform synthesized using a clinically relevant mixer-equipped microfluidic device. DNA cleavage activity and the aggregation of Cas enzymes was completely avoided under the optimized synthetic conditions. The optimized formulation, which was identified through 2 steps of design of experiments, exhibited excellent gene disruption (up to 97%) and base substitution (up to 23%) without any apparent cytotoxicity. The addition of negative charges to the RNPs by complexing single-stranded oligonucleotide (ssON) significantly enhanced the delivery of both Cas9 and Cpf1 RNPs. The optimized formulation significantly suppressed both HBV DNA and covalently closed circular DNA (cccDNA) in HBV-infected human liver cells compared to adeno-associated virus type 2 (AAV2). These findings represent a significant contribution to the development of CRISPR/Cas RNP delivery technology and its practical applications in genome editing therapy.

Keywords: Design of experiment; Genome editing; Hepatitis B virus; Lipid nanoparticles; Microfluidic device; Ribonucleoprotein.

Copyright © 2020. Published by Elsevier B.V.

StephenW

脂质核糖蛋白-寡核苷酸复合物负载的脂质纳米颗粒，使用微流控设备合成，显示出强大的基因组编辑和乙型肝炎病毒抑制作用
铃木雄一1，沼野大雄2，佐佐木佑佐2，佐藤佑介3，桥司明里1，前任正敏4，真锅笃志5，穆罕默德·埃纳穆尔·霍克·加耶什6，沟内真纪子7，月山小原恭子8，原岛秀吉9
隶属关系
隶属关系

    1个
    日本北海道北海道大学药学院分子药物设计实验室。
    2
    日本北海道北海道大学药学院分子药物设计实验室日本北海道北海道大学药学院分子药物设计实验室。
    3
    日本北海道北海道大学药学院分子药物设计实验室日本北海道北海道大学药学院分子药物设计实验室。电子地址：[email protected]。
    4
    日本北海道北海道大学工程学院应用化学系；日本Sa玉市的JST PRESTO。
    5
    日本北海道北海道大学工程学院应用化学系；名古屋大学预防医学工程创新研究中心，日本名古屋；名古屋大学纳米生命系统研究所，未来社会创新研究所，日本名古屋。
    6
    鹿儿岛大学动物医学联合学院动物卫生学实验室，日本鹿儿岛；鹿儿岛大学动物医学联合学院跨界动物疾病中心，日本鹿儿岛；孟加拉国Patuakhali科技大学微生物学与公共卫生系。
    7
    日本东京都医学科学研究所微生物学和细胞生物学系。
    8
    鹿儿岛大学动物医学联合学院动物卫生学实验室，日本鹿儿岛；鹿儿岛大学动物医学联合学院跨界动物疾病中心，日本鹿儿岛。
    9
    日本北海道北海道大学药学院分子药物设计实验室。电子地址：[email protected]。

    PMID：33333121 DOI：10.1016 / j.jconrel.2020.12.013

抽象

簇状规则间隔的短回文重复序列（CRISPR）相关（Cas）系统具有可观的治疗潜力，可用于治疗包括乙型肝炎病毒（HBV）感染在内的各种顽固性遗传和传染病。尽管预期用于CRISPR / Cas系统的非病毒传递技术将在临床上应用，但与临床相关的制剂合成相关的困难以及传递效率差严重阻碍了治疗性基因组编辑。我们在此报告使用临床相关的配备混频器的微流控设备合成的基于脂质纳米颗粒（LNP）的CRISPR / Cas核糖核蛋白（RNP）交付纳米平台的生产。在优化的合成条件下，完全避免了DNA裂解活性和Cas酶的聚集。经过2个实验设计步骤鉴定出的优化配方，具有出色的基因破坏（高达97％）和碱基取代（高达23％），没有任何明显的细胞毒性。通过复合单链寡核苷酸（ssON）向RNP添加负电荷显着增强了Cas9和Cpf1 RNP的传递。与2型腺相关病毒（AAV2）相比，优化的制剂可显着抑制HBV感染的人肝细胞中的HBV DNA和共价闭合环状DNA（cccDNA）。这些发现代表了CRISPR / Cas RNP递送技术的发展及其在基因组编辑疗法中的实际应用的重大贡献。

关键字：实验设计；基因组编辑；乙型肝炎病毒;脂质纳米颗粒；微流体装置；核糖核蛋白。

版权所有©2020。由Elsevier B.V.发布。