聚合物-脂质纳米粒用于增强酪氨酸血症I型治疗性碱基编辑器质粒的肝靶向递送研究

doi:10.5246/jcps.2024.03.015

中国药学(英文版) ›› 2024, Vol. 33 ›› Issue (3): 189-200.DOI: 10.5246/jcps.2024.03.015

• 【研究论文】 • 下一篇

聚合物-脂质纳米粒用于增强酪氨酸血症I型治疗性碱基编辑器质粒的肝靶向递送研究

高达同¹, 林萌¹^,², 彭祎玮¹, 李嘉嘉¹, 杨宜靓¹, 滕雨璐¹, 陈思宇¹, 孙雯¹, 吴子楠¹, 袁泉¹, 仰浈臻¹^,³, 周艳霞¹, 李馨儒¹, 齐宪荣¹^,^*()

^1. 北京大学药学院药剂学系, 分子药剂学与新释药系统北京市重点实验室, 北京 100191
^2. 四川大学华西医院药剂科, 四川成都 610041
^3. 北京大学第三医院医学创新研究所, 药物临床试验中心, 北京 100191

收稿日期:2023-11-14 修回日期:2023-12-08 接受日期:2024-01-13 出版日期:2024-03-31 发布日期:2024-03-31
通讯作者: 齐宪荣

Polymer-lipid nanoparticles enhance liver-targeted delivery of therapeutic base editor plasmid for the treatment of hereditary tyrosinemia type 1 (HT-1)

Datong Gao¹, Meng Lin¹^,², Yiwei Peng¹, Jiajia Li¹, Yiliang Yang¹, Yulu Teng¹, Siyu Chen¹, Wen Sun¹, Zinan Wu¹, Quan Yuan¹, Zhenzhen Yang¹^,³, Yanxia Zhou¹, Xinru Li¹, Xianrong Qi¹^,^*()

¹ Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery System, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
² Department of Pharmacy, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China
³ Drug Clinical Trial Center, Institute of Medical Innovation and Research, Peking University Third Hospital, Peking University, Beijing 100191, China

Received:2023-11-14 Revised:2023-12-08 Accepted:2024-01-13 Online:2024-03-31 Published:2024-03-31
Contact: Xianrong Qi
Supported by:
Beijing Natural Science Foundation (Grant No. L202044), National Natural Science Foundation of China (Grants No. 82373806, 82373806).

摘要/Abstract

摘要：

酪氨酸血症I型是一种罕见的常染色体隐性遗传病, 目前尚无有效的治疗方法。近年来, 以碱基编辑器为代表的基因编辑技术已被报道用于酪氨酸血症I型的治疗。然而, 由于生理屏障的存在, 碱基编辑器递送困难。在本研究中, 我们构建了一种靶向去唾液酸糖蛋白受体的聚合物-脂质纳米递送系统, 用于改善酪氨酸血症I型治疗性核酸药物的递送效率。我们首先合成了一种生物可降解性丙烯酸酯-氨基醇共聚物用于递送碱基编辑器质粒, 其转染效率显著优于市售转染试剂Hieff Trans^TM。随后, 共聚物纳米粒与DOPE-PEG-GalNAc自组装形成聚合物-脂质纳米粒, 用于增强纳米粒的肝脏递送效率。在体外转染实验中, 包载Fah-pCMV-ABE6.3-EGFP碱基编辑器质粒的聚合物-脂质纳米粒表现出了良好的肝细胞选择性, 其转染效率是游离质粒的70倍以上。研究表明, 携带肝靶向配体的聚合物-脂质纳米递送系统能够有效增强治疗性碱基编辑器质粒的肝靶向递送效率并为酪氨酸血症I型的基因治疗提供了一种潜在的递送载体。

关键词: 酪氨酸血症I型, 碱基编辑器, 聚合物-脂质纳米粒, 转染效率, 基因递送

Abstract:

Hereditary tyrosinemia type 1 (HT-1) is a rare autosomal recessive genetic disease with no effective cure at present. In recent years, gene-editing techniques such as base editor (BE) have been explored for treating HT-1. However, the delivery of nucleic acids faces challenges due to existing physiological barriers. In the present study, we constructed an asialoglycoprotein receptor (ASGPR)-targeted polymer-lipid nanoparticle to enhance the delivery efficiency of therapeutic nucleic acids for HT-1. To deliver BE plasmids, a biodegradable cationic polymer, acrylate-amino alcohol copolymer was synthesized and demonstrated superior transfection efficiency compared to the commercially available transfection reagent, Hieff Trans^TM. Subsequently, DOPE-PEG-GalNAc was combined with copolymer nanoparticles to enhance the hepatocyte delivery of the nanoparticles. Upon loading the recombinant BE plasmid, Fah-pCMV-ABE6.3-EGFP, the polymer-lipid nanoparticles exhibited remarkable hepatocyte selectivity, with a transfection efficiency over 70-fold higher than that of the free plasmid. The study suggested that polymer-lipid nanoparticles, in combination with liver-targeted ligands, could effectively enhance the liver-targeted delivery of therapeutic BE plasmids, providing a promising vector for gene therapy of HT-1.

Key words: Hereditary tyrosinemia type 1, Base editor, Polymer-lipid nanoparticles, Transfection efficiency, Gene delivery

Supporting:

高达同, 林萌, 彭祎玮, 李嘉嘉, 杨宜靓, 滕雨璐, 陈思宇, 孙雯, 吴子楠, 袁泉, 仰浈臻, 周艳霞, 李馨儒, 齐宪荣. 聚合物-脂质纳米粒用于增强酪氨酸血症I型治疗性碱基编辑器质粒的肝靶向递送研究[J]. 中国药学(英文版), 2024, 33(3): 189-200.

Datong Gao, Meng Lin, Yiwei Peng, Jiajia Li, Yiliang Yang, Yulu Teng, Siyu Chen, Wen Sun, Zinan Wu, Quan Yuan, Zhenzhen Yang, Yanxia Zhou, Xinru Li, Xianrong Qi. Polymer-lipid nanoparticles enhance liver-targeted delivery of therapeutic base editor plasmid for the treatment of hereditary tyrosinemia type 1 (HT-1)[J]. Journal of Chinese Pharmaceutical Sciences, 2024, 33(3): 189-200.

图/表 3

Figure 1. Characterization of PDATAA nanoparticles loading Fah-pCMV-ABE6.3 plasmid. (A) Synthesis of PDATAA via Michael addition reaction; (B) 1H NMR spectra analysis of PDATAA; (C) GPC trace of PDATAA; (D) Representative TEM image of PDATAA nanoparticles. Scale bar: 200 nm; (E) Hydrodynamic size and zeta potential of PDATAA nanoparticles stored at 4 °C for 7 d; (F) DNA condensation ability of PDATAA nanoparticles determined via agarose gel retardation assay; (G) Cytotoxicity of PDATAA nanoparticles in HepG2 cells determined via CCK-8 assay (n = 5); (H) EGFP expression in Hela cells transfected with different preparations observed via an inverted fluorescence microscope at 24 h; and (I, J) quantified via flow cytometry at 48 h (n = 3). **P < 0.01, ***P < 0.001. Scale bar: 200 μm.

Figure 2. Characterization and evaluation of PDATAA/GalNAc nanoparticles. (A) Synthesis of DOPE-PEG-GalNAc via a two-step reaction; (B) 1H NMR spectra analysis of GalNAc-NH2; (C) TOFMS of DOPE-PEG-GalNAc. The peak was 3555.7; (D) TEM image of PDATAA/GalNAc nanoparticles. Scale bar: 50 nm; (E) Hydrodynamic size and zeta potential of PDATAA/GalNAc nanoparticles; (F) EGFP expression in Hela and HepG2 cells observed via an inverted fluorescence microscope at 24 h; and (G) quantified via flow cytometry at 48 h (n = 3). **P < 0.01, ***P < 0.001. Scale bar: 200 μm.

Figure 3. Conduction and delivery of Fah-pCMV-ABE6.3-EGFP plasmid. (A) Conduction scheme of Fah-pCMV-ABE6.3-EGFP plasmid; (B) Results of agarose gel electrophoresis of linear vector and insert fragments; (C) Representative Sanger sequence result of Fah-pCMV-ABE6.3-EGFP plasmid; (D) EGFP expression in Hepa1-6 cells observed via an inverted fluorescence microscope; and (E) quantified via flow cytometry after transfected with different preparations for 48 h. Scale bar: 200 μm.

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聚合物-脂质纳米粒用于增强酪氨酸血症I型治疗性碱基编辑器质粒的肝靶向递送研究

Polymer-lipid nanoparticles enhance liver-targeted delivery of therapeutic base editor plasmid for the treatment of hereditary tyrosinemia type 1 (HT-1)

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