合成的gRNA/Cas9核糖蛋白向HBVDNA抑制病毒复制
Jinyu Zhang1,2, Yi Zhang1,2, Sushant Khanal1,2
1Center of Excellence in Inflammation, Infectious Disease and Immunity, Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee, USA.
Journal of medical virology
|July 17, 2023
概括
这项研究引入了一种新的CRISPR/Cas9基因疗法,使用合成导向RNA (gRNA) /Cas9-核核蛋白 (RNP) 向并消除乙型肝炎病毒 (HBV) DNA,为慢性HBV感染提供潜在的治疗方法.
科学领域:
- 肝病学和病毒学.
- 基因治疗 基因治疗
- 分子生物学分子生物学
背景情况:
- 慢性乙型肝炎病毒 (HBV) 感染是由 HBV cccDNA 维持的,目前的疗法无法消除.
- 现有的核胺类同类 (NA) 治疗抑制HBV复制,但不能根除集成的HBVDNA或插件cccDNA.
- 克里斯普尔/Cas9基因编辑显示出希望,但由于病毒载体的传递,面临安全问题.
研究的目的:
- 开发一种非病毒性CRISPR/Cas9基因疗法,用于根除慢性HBV感染.
- 评估合成指导RNA (gRNA) /Cas9-核糖蛋白 (RNP) 配方对抗HBV的疗效和安全性.
- 通过向基因编辑来研究HBV DNA,转录和抗原的减少.
主要方法:
- 设计并测试了多个HBV特异性gRNA与Cas9复合成RNP.
- 使用人类肝瘤细胞系 (HepG2.2.15) 稳定感染HBV进行体外实验.
- 通过测量HBV DNA,RNA和抗原来评估抗病毒疗效,并通过T7E1测定和DNA测序确认了基因编辑.
主要成果:
- 合成的gRNA/Cas9 RNP显著降低了HBV的ccDNA,HBV的总DNA,基因前RNA和HBV抗原 (HBsAg,HBeAg).
- 通过T7E1测定和DNA测序,在目标部位确认了特定的HBV基因分裂和突变.
- 基因编辑系统没有对细胞活力或增殖产生不良影响.
结论:
- 合成gRNA/Cas9 RNP配方代表了一种有希望的非病毒基因编辑治疗策略,用于根除慢性HBV感染.
- 这种方法提供了诸如快速DNA裂变,低非目标效应和减少插入性突变发生风险等优势.
- 这些发现支持基于RNP的基因编辑对HBV的功能治愈的潜在临床应用.
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