中性胞苷脂材包载部分磷硫代反义核酸的生物活性评价及作用机制初探  

Biological activity and mechanism of partial phosphorothiolated antisense oligonucleotides delivering by neutral cytidinyl lipid

作　　者：李伊丁 马元[1] 赵文婷 潘宇飞 于小桐 王舒鹤 关注[1] 王芳 张礼和[1] 杨振军[1] LI Yi-ding;MA Yuan;ZHAO Wen-ting;PAN Yu-fei;YU Xiao-tong;WANG Shu-he;GUAN Zhu;WANG Fang;ZHANG Li-he;YANG Zhen-jun(State Key Laboratory of Natural and Biomimetic Drugs,School of Pharmaceutical Sciences,Peking University,Beijing 100191,China;Beijing Increase Innovative Medicine Co.,Ltd.,Beijing 102299,China)

机构地区：[1]北京大学药学院天然药物及仿生药物国家重点实验室,北京100191 [2]北京盈科瑞创新医药股份有限公司,北京102299

出　　处：《中国药物化学杂志》2019年第2期87-95,共9页Chinese Journal of Medicinal Chemistry

基　　金：国家科技部项目(2017ZX09303013);国家自然科学基金项目(21778006,21332010)

摘　　要：目的反义核酸是一段与靶基因互补的单链寡核苷酸序列,通过与细胞内核酸杂交成双链结构抑制靶基因转录和翻译过程,从而调控基因表达。然而,无载体递送反义核酸跨膜能力较差且寡核苷酸磷酸二酯键易被核酶水解。为提高反义核酸人胞效率,并对活性作用的发挥进行进一步优化,本课题主要从寻求高效低毒的载体系统及优化修饰策略入手对其进行研究。方法本研究利用中性胞苷脂材(DNCA)混合阳离子脂质体(CLD)对反义核酸G3139包载递送,结合部分位点磷硫代(PS)修饰方式,考察不同修饰位点的序列活性与作用机制。结果与结论采用混合脂材包载能显著提高反义核酸G3139的抗MCF7/ADR细胞增殖能力,并发现9-16位(5'-端计算)区域内多位点(≥4)修饰物抗肿瘤活性较高。此外,G3139部分位点PS修饰物有效激活RNaseH降解靶mRNA,并降低杂交双链Tm(DNA的熔解温度)值。结合转录组学与蛋白组学研究,确证减少PS修饰位点数可以降低反义核酸与非特异性靶标结合,从而降低毒副作用。Antisense oligonucleotide(ASON)is a single-stranded oligonucleotide sequence complementary to its target gene,which regulates gene expression by inhibiting transcription and translation of a target gene.However,natural oligonucleotide therapeutic has inherent shortcomings,such as susceptibility to nucleases,difficulty in transmembrane and possibility of immune activation.In order to improve the permeation and bioactivity of ASONs,neutral cytidinyl lipid DNCA and cationic lipid CLD was used for the transfection of phosphorothiolated(PS)ASONs.The results showed that the anti-MCF7/ADR cell proliferation ability of G3139 was increased significantly and the multiplesites(≥4)PS modificationaround 3'-terminus showed higher antitumor activity.Transcriptomics and proteomics analysis confirmed that reducing the number of PS modification could reduce non-specific binding of the antisense oligonucleotide to other targets,thereby reducing toxicity.A combination of delivery system and partial phosphorothioation effectively spread ASONs application category,which exhibited bright clinical potentials in the future.

关 键 词：核苷碱基脂质材料 阳离子脂质材料 反义核酸 磷硫代修饰 

分 类 号：R914[医药卫生—药物化学]