药学学报

Strategy	Principle	Advantage	Challenge
Spherical nucleic acid (SNA)^[60]	Consist of hydrophobic core nanoparticles (including gold, silica, or other materials) linked by thiol bonds to hydrophilic oligonucleotides (e.g., ASO, siRNA)	They can transfect a variety of tissues and cell types non-toxic with a mild immune response, and can penetrate various biological barriers (such as skin, blood-brain barrier, and blood-tumor barrier)	① The biodistribution in vivo is greatly affected by its composition; ② The targeted delivery efficiency is low; ③ The oligonucleotides may be degraded prematurely during the delivery process
DNA nanostructures^[61]	Formed by self-assembly of complementary bases. Modular design of RNA drugs (e.g. siRNA or immunostimulatory oligonucleotides) and targeting ligands (e.g. aptamers)	The structure can be precisely and flexibly designed to tune its physicochemical properties; the small size (about 20 nm) of the designable structure may enable extrahepatic delivery	Unfunctionalized DNA nanostructures are smaller in size, and have higher renal clearance. The overall bioavailability is reduced
Metal organic frameworks (MOFs)^{[62, 63]}	Encapsulation of RNA drugs into MOFs by pore encapsulation or in situ synthesis, and targeted delivery in vivo by surface modification of MOFs (such as covering the membrane of naturally derived platelets)	MOFs have high specific surface area and porosity, enabling high-load delivery of drugs. According to the size of the RNA drug, the pore size of MOFs can be precisely designed and the physical and chemical properties can be adjusted to achieve stable and effective delivery of the RNA drug	Research on the drug loading and release kinetics of MOFs is still limited, and their preclinical evaluation performance should be optimized through systematic in vivo studies on their stability, degradation mechanism, and side effects on normal organs
Peptide-nucleic acid complex^[64]	Positively charged lysine or arginine residues can bind to nucleic acids through electrostatic interactions at physiological pH; hydrophobic amino acid residues or hydrophobic components (such as alkyl chains) facilitate their self-assembly to form nanoparticles or fibers; histidine residues enhance endosomal escape via the proton sponge effect	The secondary structure of peptides in aqueous solution and the conformational changes following peptide-nucleic acid interactions are tuned by modulating the distribution of cationic amino acids (arginine, lysine, or histidine) and hydrophobic amino acids. The complexation strength and membrane penetration ability of peptides and nucleic acids can be flexibly adjusted to improve delivery efficiency. It has good biocompatibility and low immunogenicity	There is insufficient information on the biodistribution, clearance, immunocompatibility, and specific organ toxicity of such polypeptide-nucleic acid complexes. It is necessary to develop polypeptide gene carriers with higher delivery efficiency through further optimized design and more structure-activity relationship studies to meet the needs of clinical applications

Strategy	Principle	Advantage	Challenge
Spherical nucleic acid (SNA)^[60]	Consist of hydrophobic core nanoparticles (including gold, silica, or other materials) linked by thiol bonds to hydrophilic oligonucleotides (e.g., ASO, siRNA)	They can transfect a variety of tissues and cell types non-toxic with a mild immune response, and can penetrate various biological barriers (such as skin, blood-brain barrier, and blood-tumor barrier)	① The biodistribution in vivo is greatly affected by its composition; ② The targeted delivery efficiency is low; ③ The oligonucleotides may be degraded prematurely during the delivery process
DNA nanostructures^[61]	Formed by self-assembly of complementary bases. Modular design of RNA drugs (e.g. siRNA or immunostimulatory oligonucleotides) and targeting ligands (e.g. aptamers)	The structure can be precisely and flexibly designed to tune its physicochemical properties; the small size (about 20 nm) of the designable structure may enable extrahepatic delivery	Unfunctionalized DNA nanostructures are smaller in size, and have higher renal clearance. The overall bioavailability is reduced
Metal organic frameworks (MOFs)^{[62, 63]}	Encapsulation of RNA drugs into MOFs by pore encapsulation or in situ synthesis, and targeted delivery in vivo by surface modification of MOFs (such as covering the membrane of naturally derived platelets)	MOFs have high specific surface area and porosity, enabling high-load delivery of drugs. According to the size of the RNA drug, the pore size of MOFs can be precisely designed and the physical and chemical properties can be adjusted to achieve stable and effective delivery of the RNA drug	Research on the drug loading and release kinetics of MOFs is still limited, and their preclinical evaluation performance should be optimized through systematic in vivo studies on their stability, degradation mechanism, and side effects on normal organs
Peptide-nucleic acid complex^[64]	Positively charged lysine or arginine residues can bind to nucleic acids through electrostatic interactions at physiological pH; hydrophobic amino acid residues or hydrophobic components (such as alkyl chains) facilitate their self-assembly to form nanoparticles or fibers; histidine residues enhance endosomal escape via the proton sponge effect	The secondary structure of peptides in aqueous solution and the conformational changes following peptide-nucleic acid interactions are tuned by modulating the distribution of cationic amino acids (arginine, lysine, or histidine) and hydrophobic amino acids. The complexation strength and membrane penetration ability of peptides and nucleic acids can be flexibly adjusted to improve delivery efficiency. It has good biocompatibility and low immunogenicity	There is insufficient information on the biodistribution, clearance, immunocompatibility, and specific organ toxicity of such polypeptide-nucleic acid complexes. It is necessary to develop polypeptide gene carriers with higher delivery efficiency through further optimized design and more structure-activity relationship studies to meet the needs of clinical applications

RNA药物递送研究进展

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李丹 , 黄宇坤 , 高小玲 ^*

药学学报 | 专题报道: 基于智能化递药系统的疾病精准治疗研究 2023,58(3): 469-482

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药学学报 | 专题报道: 基于智能化递药系统的疾病精准治疗研究 2023, 58(3): 469-482

RNA药物递送研究进展

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李丹, 黄宇坤, 高小玲^*

作者信息

上海交通大学医学院, 上海 200025

通讯作者:

*高小玲， Tel: 86-21-63846590-776580, E-mail: shellygao1@sjtu.edu.cn

Research progress of RNA drugs delivery

Dan LI, Yu-kun HUANG, Xiao-ling GAO^*

Affiliations

School of Medicine, Shanghai Jiaotong University, Shanghai 200025, China

出版时间: 2023-03-12 doi: 10.16438/j.0513-4870.2022-0500

文章导航

摘要

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核糖核酸(ribonucleic acid, RNA) 药物可基于碱基互补配对原则, 针对目的基因的碱基序列进行设计, 精准调控基因表达, 干预遗传病、罕见病和肿瘤等多种疾病, 具备强大治疗潜力。然而, 当前RNA药物在成药性方面仍受到众多因素制约, 面临包括稳定性、半衰期、免疫原性、组织靶向性、细胞摄取和内涵体逃逸等多方面的挑战。本文介绍了近年来受到广泛关注的几种RNA药物, 主要包括信使核糖核酸(messenger RNA, mRNA)、小干扰核糖核酸(small interfering RNA, siRNA)、小分子核糖核酸(microRNA, miRNA) 和反义寡核苷酸(antisense oligonucleotide, ASO), 总结了其体内作用机制和面临的挑战, 概述了包括化学修饰、配体偶联、纳米载体在内的多种克服RNA体内递送瓶颈的递送策略; 从制备及应用等方面, 详述了RNA纳米递送载体中的脂质纳米粒、高分子聚合物载体及外泌体。本文旨在为发展高效安全的体内RNA递送技术提供理论依据和设计思路, 促进RNA药物临床转化。

关键词

信使核糖核酸 / 小干扰核糖核酸 / 小分子核糖核酸 / 反义寡核苷酸 / 药物递送系统

Abstract

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Ribonucleic acid (RNA) medicines have strong therapeutic potential for numerous rare genetic illnesses and malignancies because of its exact programmability based on Watson-Crick base pairing principle and unique ability to regulate gene expression. However, RNA medicines still have limitations in many areas, including stability, half-life time, immunogenicity, organ selectivity, cellular uptake and endosomal escape efficiency despite their great therapeutic potentials. This review briefly introduced numerous RNA medications [mostly messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA) and antisense oligonucleotide (ASO)] that have intrigued of researchers in recent years, as well as their action mechanism in vivo. A number of delivery techniques, such as chemical modification, ligands coupling and nanocarriers have been proposed. The manufacture and applications of lipid nanoparticle, polymer nanoparticle and exosomes were discussed in depth. The goal of this work is to give a theoretical foundation and design concepts for the development of effective and safe RNA delivery technology, as well as to facilitate RNA therapeutic clinical translation.

Key words

messenger RNA / small interfering RNA / microRNA / antisense oligonucleotide / drug delivery system

引用本文

李丹, 黄宇坤, 高小玲. RNA药物递送研究进展. 药学学报, 2023 , 58 (3) : 469 -482 . DOI: 10.16438/j.0513-4870.2022-0500

Dan LI, Yu-kun HUANG, Xiao-ling GAO. Research progress of RNA drugs delivery[J]. Acta Pharmaceutica Sinica, 2023 , 58 (3) : 469 -482 . DOI: 10.16438/j.0513-4870.2022-0500

正文

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核糖核酸(ribonucleic acid, RNA) 药物依赖中心法则和碱基互补配对原则, 在翻译水平特异且高效地调控靶标基因及相关下游通路, 产生治疗效果。自1998年全球首款RNA药物Fomivirsen获得美国食品药品监督管理局(Food and Drug Administration, FDA) 批准上市用于治疗巨细胞病毒性视网膜炎, 至2020年多款信使核糖核酸(messenger RNA, mRNA) 疫苗被FDA紧急授权用于新型冠状病毒肺炎(coronavirus disease 2019, COVID-19) 防治, RNA药物已获得了广泛研究, 临床应用大幅拓宽^[1]。现今RNA药物已被用于多种传染病、慢性代谢性疾病和罕见遗传病的临床治疗, 有望开启继小分子、抗体药物之后的第三次新药创制浪潮。

化学小分子和蛋白大分子占据了现今药学研究的主导地位。小分子药物(分子质量小于900 Da) 易于生产使用但特异性低^[2]。蛋白药物可通过蛋白质工程技术精准设计和改造结构实现体内的高特异性并降低脱靶毒性, 但其分子结构复杂、生产成本高、患者依从性较差^[3]。RNA药物基于中心法则和碱基互补配对原则, 可针对目标基因精确设计碱基序列, 沉默、恢复或上调特定基因的表达, 实现全细胞蛋白的可控调节。RNA药物候选靶点丰富, 开发过程简单高效, 比化学小分子特异性强, 比蛋白大分子生产更为简便。然而, RNA药物在生物体复杂的内环境中仍面临着许多挑战, 包括稳定性差、靶向性弱和递送效率低等。只有克服上述挑战, RNA药物才能最终大规模应用于临床治疗。

本文介绍了mRNA、小干扰核糖核酸(small interfering RNA, siRNA), 小分子核糖核酸(microRNA, miRNA) 和反义寡核苷酸(antisense oligonucleotide, ASO) 的作用机制及应用现状, 总结并阐释了包括化学修饰、偶联配体和纳米载体在内的多种体内递送策略, 以期为发展更高效安全的RNA体内递送技术提供理论依据和设计思路。

1 RNA的作用机制

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图 1展示了不同类型RNA的细胞内作用机制^[4]。mRNA直接于胞质内翻译为特定蛋白, 可应用于疫苗和蛋白替代疗法; 寡核苷酸药物包含siRNA、miRNA和ASO等, 可直接沉默或间接调控mRNA的转录和翻译, 现主要用于治疗罕见遗传性疾病。本文将重点关注mRNA (分子质量300~5 000 kDa) 和寡核苷酸(分子质量4~14 kDa) 药物。

1.1 mRNA

mRNA是由脱氧核糖核酸(deoxyribonucleic acid, DNA) 的一条链作为模板转录而来的、携带氨基酸编码信息的、在核糖体中指导蛋白质合成的一类单链核糖核酸。mRNA包含5个部分(图 1a): 5'端帽子结构(CAP)、5'端非编码区(untranslated region, UTR)、开放阅读框(open reading frame, ORF)、3'端UTR、多聚腺苷酸尾巴[polyadenylation tail, poly(A)]。CAP即7-甲基鸟苷(7-methylguanosine, m7G) 通过三磷酸桥连接至mRNA的5'端形成的修饰结构, 有助于结合核糖体, 减少细胞对外源性mRNA的识别和清除; 编码区两侧的UTR区域可调节翻译效率、协调亚细胞定位和增强mRNA稳定性; ORF是编码特定蛋白质的序列; poly(A)可在翻译中募集聚腺苷酸结合蛋白[poly(A) binding protein, PABP] 增强翻译效率并减少核酸外切酶的降解^[5]。mRNA可在细胞质中翻译为抗体、激素和细胞因子等特定功能蛋白, 治疗或杀伤病变细胞^[5]。mRNA疗法的概念最早由Wolff等^[6]提出, 其将体外转录mRNA (in vitro transcribed mRNA, IVT mRNA) 直接注射入小鼠骨骼肌, 成功在注射部位表达了目的蛋白。随着mRNA研究的迅速进展, 当前已有60多种mRNA制剂进入临床研究阶段^[7]。2019年底COVID-19的暴发, 使基于mRNA的疫苗受到极大关注。2020年12月, Pfizer-BioNTech和Moderna等公司研制的mRNA疫苗先后获FDA紧急使用授权用于COVID-19防治^[8], 这是mRNA药物的首次超大规模临床应用, 对于RNA药物具有里程碑意义。

1.2 寡核苷酸

寡核苷酸包含了siRNA、miRNA和ASO等, 可特异性地结合胞质内前体mRNA (pre-mRNA)、mRNA、miRNA、核糖核蛋白等核酸和蛋白^[9], 调控特定基因及蛋白的表达。

siRNA也称沉默RNA, 是一类长度为20~25 bp的非编码双链RNA。siRNA既可在体外合成后直接导入细胞, 也可通过胞内Dicer酶切割外源双链RNA形成。siRNA首先在细胞内与AGO (Argonaute) 蛋白结合形成RNA诱导的沉默复合物(RNA-induced silencing complex, RISC)。随后siRNA双链的有义链被切割并降解, 反义链被激活。siRNA基于碱基互补配对原则驱动RISC识别并剪切mRNA中的靶标序列, 促进mRNA降解, 下调特定蛋白翻译水平进而产生治疗效果(图 1b)^[10]。Patisiran作为全球首个siRNA药物, 于2018年8月10日经FDA批准上市, 用于治疗成人患者因转甲状腺素蛋白淀粉样变性(hATTR, 也称家族性淀粉样多发性神经病变) 引起的神经和心肌损伤。Patisiran中的siRNA可通过靶向诱导mRNA降解, 特异性敲低转甲状腺素蛋白水平, 减少肝脏中异常淀粉样蛋白沉积, 有效阻止或减缓hATTR进展^[11]。

miRNA是一类长度约为22 nt的非编码单链RNA, 主要功能为负调控靶基因的mRNA。miRNA的调控机制比siRNA更为复杂, 通常一种miRNA能同时调控多种mRNA, 而一种mRNA也可受多种miRNA调控, 其作用机制取决于miRNA的核酸序列及其与mRNA的结合位点。例如miRNA可与mRNA的3'端UTR结合促进mRNA去腺苷酸化, 减少mRNA与PABP的结合从而抑制翻译; 也可通过促进AGO蛋白激活RISC引起mRNA切割(图 1c)。此外, miRNA还具有直接激活转录、调控其他非编码RNA的功能。基于miRNA丰富的生物学功能, miRNA具备治疗肿瘤、代谢、免疫等多种疾病的潜力^[1]。目前miRNA药物研究热度持续走高。在研的miRNA药物主要包括miRNA模拟物和抗miRNA (antimiRs), 分别发挥增强或抑制内源性miRNA的作用^[12]。

ASO的长度通常为13~25 nt, 其调控机制分为核糖核酸酶H (ribonuclease H, RNase H) 依赖型和非RNase H依赖型(图 1d)。前者通过ASO与mRNA互补结合, 募集RNase H剪切mRNA阻断靶基因翻译。后者主要利用ASO的空间位阻效应调控转录, 使pre-mRNA发生特异性剪切, 抑制蛋白质翻译; 此外, ASO也可通过抑制mRNA与关键酶之间的相互作用阻止mRNA翻译^[1]。自1978年ASO的作用被发现以来, 已有多个ASO药物上市并展现出良好的治疗效果。Etepliren作为第一个获批用于治疗杜氏肌营养不良症(duchenne muscular dystrophy, DMD) 的药物, 是一种磷酰二胺吗啉代寡核苷酸(phosphoramidite morpholino oligonucleotides, PMO), 通过靶向结合pre-mRNA的外显子51并促进其发生跳跃剪接, 将高致病性的移框缺失转变为低致病性的同框缺失, 让细胞得以产生整体结构缩短但仍保存部分功能的抗肌萎缩蛋白来发挥治疗作用^[13]。

2 RNA药物的体内命运及挑战

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由于RNA药物在胃肠道内易降解且吸收效率低, 目前均采取静脉滴注、皮下注射或局部给药等肠外给药途径。RNA药物自注射处进入目标部位将经历若干复杂的生物学阶段, 主要可分为系统阶段和靶细胞阶段, 而RNA药物在各阶段中都将面临不同的挑战^[14] (图 2)。

RNA体内稳定性差, 半衰期短。游离RNA进入血液后, 一部分被人体血液和组织液中大量存在的核糖核酸酶降解, 另一部分则被肾脏直接过滤清除。外源RNA具有免疫原性, 可被宿主免疫系统的模式识别受体(pattern recognition receptor, PRR) 识别为病毒信号并产生应答。目前认为可产生抗病毒反应的PRR主要有3种, 分别为Toll样受体(Toll-like receptors, TLRs)、维甲酸诱导基因I (retinoic acid inducible gene I, RIG-I) 样受体(RLRs) 和NOD样受体(NOD-like receptors, NLRs)。TLRs存在于免疫细胞的内体中, 可诱导产生干扰素, 其中TLR3识别双链RNA; TLR7和TLR8识别单链RNA; TLR9识别含有非甲基化的胞嘧啶鸟嘌呤二核苷酸^[15]; 而RLRs和NLRs存在于免疫细胞的细胞质中, 识别细胞质中的双链RNA^[16]。PRRs感知RNA药物激活信号转导级联反应, 促进细胞因子分泌, 激活免疫系统, 诱导细胞死亡^[17]。RNA不易结合血浆蛋白, 通常不具备特定器官和组织的靶向性, 难以单独成药。药用RNA通常是负电大分子, 必须依赖细胞表面蛋白介导的内吞进入细胞。当RNA被细胞内吞后, 还需穿过内体囊泡的脂质双分子层方能进入细胞质发挥药效。研究表明, RNA药物主要从次级内涵体或多泡体释放, 但对于未修饰的RNA药物只有相当少部分(小于1%) 能从内体/溶酶体中逃逸进入细胞质, 绝大部分仍滞留在内体/溶酶体中^[18]。因此RNA药物在胞内的效力强度取决于能否高效逃逸内体/溶酶体。

3 RNA药物的体内递送策略

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如上文所述, RNA分子的药用瓶颈主要包括稳定性差、半衰期短、免疫原性高、靶向性弱、进入胞质少等。因此, 除通过化学修饰改善RNA稳定性和免疫原性外, 还需为其提供高效安全的体内递送系统。当前RNA分子的药用开发可分为化学改造和物理改性两大方向, 二者可单独或共同提升RNA分子的体内外稳定性和特定组织、细胞内的翻译效率。目前已开发出多种RNA改造和递送技术, 主要包括化学修饰、配体偶联和纳米递送载体。

3.1 化学修饰

RNA是由磷酸骨架、核糖和碱基构成的大分子(图 3a), 对其各部分进行化学修饰可增加其酶稳定性、强化核酸与蛋白的结合、减弱免疫系统的识别^[19-21] (图 3b~e)。

RNA的磷酸骨架(图 3a) 可被核酸酶快速降解, 是其药用发展的主要限制因素。第一代RNA改造策略聚焦于磷酸骨架改造, 即采用部分基团取代磷酸骨架中的非桥氧。硫代磷酸(phosphorothioate, PS) 改造是目前最常用的化学修饰(图 3b)^[20]。PS改造能提高RNA的核酸酶抗性, 增强其血液稳定性, 并可通过提高其与血浆蛋白的结合力降低肾脏清除, 延长RNA的体内循环时间^[22]。但也有研究表明, 以PS改造后的核苷酸合成的siRNA相较于等量的未经改造的siRNA, 活性降低。因此需根据具体情况调整PS修饰核苷酸的比例以达到RNA药物的活性及体内稳定性^[23]。此外, PS改造也会阻碍外源RNA与其互补靶标序列的结合, 因此仍需通过其他类型化学修饰来改善^[20]。

第二代RNA改造策略主要围绕核糖2'-羟基位点进行修饰。常见方案包括2'-氧-甲氧基乙基(2'-oxy-methoxyethyl, 2'-MOE)、2'-甲氧基(2'-methoxy, 2'-OMe)、2'-氟(2'-fluorine, 2'-F) 等^[19] (图 3c)。这些修饰不但能增加RNA的核酸酶耐受, 进而延长半衰期, 且相较于第一代的PS修饰, 经2'-羟基位点修饰后的小核酸药物具有更小的毒性和更强的互补核苷酸链结合能力^[19]。另一方面, 有研究报道, 在siRNA的特定位置进行2'-核糖修饰可消除非目标mRNA转录本的RISC加载及沉默活动, 减少siRNA脱靶导致的毒副作用^[24]。此外有报道称, 对核糖进行2'-OMe改造可减弱宿主的免疫识别, 促进外源性RNA的免疫逃逸^[25]。

第三代RNA改造策略是通过改造核糖结构合成核酸类似物, 常见修饰方案包括锁核酸(locked nucleic acid, LNA)、肽核酸(peptide nucleic acid, PNA) 和PMO等(图 3d)^[19]。LNA是含有桥接双环糖基的合成核酸类似物。LNA核糖结构的2'-O位和4'位通过氧亚甲基桥、硫亚甲基桥或胺亚甲基桥连接成环。由于双环糖骨架的限制, LNA只会与靶核酸链结合形成热力学稳定的A型双链体, 因此其更易识别互补序列形成高亲和力的杂交物, 并能提高形成的互补双链的热稳定性和抗核酸酶活性^[26]。PNA是一种以多肽骨架模拟并取代RNA糖磷酸主链的核酸类似物, 其中嘌呤和嘧啶碱基均通过亚甲基羰基与主链相连^[27]。PMO则是以六元吗啉环取代五元核糖杂环的核酸类似物^[28]。PNA和PMO改造都可改善核酸的靶标亲和力、核酸酶抗性和药代动力学性质。但由于PNA和PMO为电中性, 与血浆蛋白结合能力弱而易被肾脏清除, 半衰期较短, 因此可进一步通过与脂质、多肽、适配体或抗体等带电基团共价结合以提高其入胞效率。

虽然RNA改造已历经多次迭代, 其体内耐受仍不理想, 还需进一步开发新策略。Karikó等^[21]在2005年提出将胞苷(C) 替换为5-甲基胞苷(m5C) 或将尿苷(U) 替换为假尿苷(ψ) 以逃脱免疫系统识别(图 3e)。m5C、ψ等化学修饰碱基是人体内RNA的天然成分, 在外源性RNA中引入m5C、ψ等化学修饰碱基可减少人体免疫系统中PRRs的识别和后续应激反应降低树突状细胞内细胞因子和生物标记物的活性^[21]。除了对核苷酸的戊糖环和碱基进行改造外, 研究者也提出对核酸链整体结构进行优化改造以提升其生物学功能。以mRNA为例, 可在融合上述化学修饰改造策略的基础上, 进一步优化mRNA序列和结构以增强其稳定性并提高蛋白质翻译效率, 主要技术包括5'CAP修饰和优化, 优化poly(A)尾、UTR和ORF序列等。其中5'CAP作为翻译起始因子(eukaryotic initiation factor, eIF) 4E的主要识别和结合位点, 其结构是当前研究热点。经过加CAP后的mRNA, 其翻译效率提高, 免疫原性下降。根据mRNA 5'末端核苷酸甲基化的位点差异可形成3种CAP结构: CAP-0 [m7G(5')pppN1pN2p]、CAP-1 [m7G(5')pppN1mpNp]和CAP-2 [m7G(5')pppN1mpN2mp], 其中真核生物mRNA主要包含CAP-1或CAP-2而不包含CAP-0, 可通过在IVT mRNA添加1个CAP-1或CAP-2减少免疫刺激。临床试验中较常用的mRNA帽是m7帽类似物(m7GpppG), RNA聚合酶通过催化鸟苷上3'位的羟基亲核进攻核苷三磷酸上的α-磷酸来启动转录, 但m7G上的3'位的羟基也可发动亲核进攻, 产生反向加帽的情况, 导致部分mRNA不被核糖体识别, IVT mRNA整体活性降低^[1]。为减少上述情况, 研究者合成了抗反向帽类似物(anti-reverse cap analog, ARCA; m₂^{7, 3'-O}GpppG), 将m7G的3'位的羟基基团替换为甲氧基, 迫使ARCA只能以正向掺入合成mRNA。相较于传统CAP类似物, ARCAs可提高RNA翻译效率, 增加相关蛋白质的表达水平^[29]。此外, PS^[30]、LNA^[31]等新型化学修饰也已被证实可增强mRNA对脱帽酶的抵抗, 延长mRNA的半衰期。

3.2 偶联配体

RNA药物可通过共价偶联脂质、半乳糖、蛋白/多肽或适配体等来延长体内循环时间, 增加在特定组织和细胞的蓄积与摄取。利用特定的偶联配体不但能调节RNA药物与血浆蛋白的结合, 改善药物的组织分布, 还能通过配体靶向细胞表面受体实现特定组织或细胞的递送。同时, 还可通过设计配体与RNA药物之间的连接键, 如采用酸敏感的酰胺键、在胞质中易被还原断开的二硫键或Dicer敏感键等^[32], 使RNA在进入特定生理环境后与偶联物分离, 有效适应特定的治疗机制。

3.2.1 脂质偶联

寡核苷酸共价结合脂质及其衍生物形成的缀合物可增强寡核苷酸的体内递送效率。例如, 在3'末端共价键合胆固醇的siRNA-胆固醇可有效沉默肌肉生长抑素诱导肌肉生长^[33]; 共价键合α-生育酚的siRNA可沉默小鼠肝脏中ApoB^[32]。偶联胆固醇的RNA可通过与高密度脂蛋白(high-density lipoprotein, HDL) 或低密度脂蛋白(low-density lipoprotein, LDL) 的预组装, 增加特定组织和细胞的摄取, 改变siRNA的分布(图 4a)^[34]。与游离siRNA-胆固醇相比, 与HDL预组装的siRNA-胆固醇(HDL siRNA) 可在肝脏和空肠中产生更强的沉默效应。进一步研究发现, 与LDL预组装的siRNA-胆固醇(LDL siRNA) 颗粒几乎只在肝脏中吸收, 而HDL siRNA颗粒主要由肝脏、肾上腺、卵巢、肾脏和小肠吸收。因此, 研究者提出siRNA-胆固醇的内吞作用分别由HDL和LDL相关的B类I型清道夫受体和LDL受体介导^[35]。还有研究发现siRNA-脂质缀合物与不同类别脂蛋白的体内结合效力受其疏水性影响, 疏水性更强的缀合物优先结合LDL, 而疏水性弱的缀合物优先结合HDL^[34]。因此可以通过偶联不同疏水性的脂质实现RNA药物在不同组织的靶向递送。

3.2.2 N-乙酰氨基半乳糖(N-acetylgalactosamine, GalNAc) 偶联

GalNAc是一种与去唾液酸糖蛋白受体(asialoglycoprotein receptor, ASGPR) 高度亲和的二糖类化合物(K_d = 2.5 nmol·L^-1)。ASGPR在肝脏中高度特异性表达, 采用GalNAc偶联的RNA药物有望实现肝脏的高效特异性递送(图 4b)^[36]。2019年, 第1个靶向δ-氨基乙酰丙酸合酶1的GalNAc偶联siRNA药物Givosiran获批用于治疗急性间歇性卟啉症患者^[37]。2020年底, 两种GalNAc偶联siRNA药物lumasiran和inclisiran也分别获批临床使用。Lumasiran是一种用于治疗1型原发性高草酸尿症的siRNA药物, 可降低肝细胞中羟基酸氧化酶1的mRNA的翻译^[38]。Inclisiran是一种用于治疗动脉粥样硬化性心血管疾病的siRNA药物, 可降低前蛋白转化酶枯草杆菌蛋白酶/kexin 9型的mRNA的翻译^[39]。然而ASGPR只在高度分化的肝细胞中高表达, 而在低度分化的肝癌细胞系中表达水平较低^[36], GalNAc偶联用于肝癌治疗的策略仍有待改进。此外, 由于肝外组织缺乏ASGPR的表达, 尚无法通过GalNAc偶联技术实现高效的RNA药物递送。

3.2.3 ‍多肽偶联‍

细胞穿透肽(cell penetrating peptide, CPP) 一般是由5~30个氨基酸组成的两亲性或阳离子肽片段, 包括HIV-1 (human immunodeficiency virus-1) 反式激活因子(HIV-Tat)、果蝇的触角足同源异形域的DNA结合结构域第3片段(peneytatin 1)、由神经肽甘丙肽和乳突蛋白合成的嵌合肽(transportan) 或基于碱性氨基酸(如精氨酸和赖氨酸) 的聚合物等^[40]。CPP与负电核酸或电中性的改造核酸偶联后, 可增加偶联物整体的正电性, 促进核酸的细胞摄取^[40]。当前已有多个多肽-PMO (peptide-PMO, PPMO) 缀合物处于临床前研究阶段。PPMO M23D-B能增强肌营养不良蛋白外显子23的跳跃, 实现疾病模型小鼠肌肉中肌营养不良蛋白的持续表达^[41]; 富含精氨酸的Pip6a肽与PMO的缀合物(Pip6a-PMO) 可直接携带核酸进入脊髓性肌萎缩模型动物的中枢神经系统, 通过靶向CUG重复扩增的转录本, 增强运动神经元存活蛋白的表达, 治疗I型强直性营养不良(myotonic dystrophy 1, DM1) (图 4c)^{[42, 43]}。

除CPP外, 靶向胰高血糖素样肽1受体(glucagon-like peptide 1 receptor, GLP1R) 的GLP1样肽和多价环状精甘天冬氨酸肽(cyclic arginylglycylaspartic acid, cRGD) 也已证明可介导RNA递送。研究者将由40个氨基酸构成的GLP1样肽修饰于ASO羧基末端, 可在胰岛β细胞中沉默靶基因^[44]。cRGD修饰的siRNA可特异性递送至高表达αvβ3整合素的黑色素瘤细胞, 敲低靶基因^[45]。上述研究表明多肽偶联可改善RNA的组织分布和细胞摄取。然而需注意的是, 多肽与RNA之间也可能因异种电荷而相互吸引而导致团聚, 进而影响RNA的递送效力。

3.2.4 抗体/适配体偶联

目前已有3种非结合型PMO药物eteplirsen、golodirsen和viltolarsen获批用于治疗杜氏肌营养不良症, 然而, 其改善肌肉的效果仍有待提高。研究者尝试通过受体介导的方法增强用于肌肉疾病的寡核苷酸药物递送, 如偶联转铁蛋白受体(transferrin receptor, TfR) 的抗体加强肌肉组织递送效率(图 4d)^[46]。TfR作为铁结合转铁蛋白的主要入口, 在高代谢活性的骨骼肌细胞、心肌细胞、脑内皮细胞和增殖性肿瘤细胞中高表达, 可在网格蛋白介导下将偶联物内化。通过此途径, TfR能促使siRNA-抗体/适配体偶联物进入肌肉或跨越血脑屏障进入中枢神经系统, 沉默特定基因的表达^{[47, 48]}。基于受体-配体的相互作用, 已有多种受体被证实可提高siRNA和ASO的靶向递送效率, 包括人类表皮生长因子受体2^[49]、T细胞标记物CD7^[50]、转铁蛋白受体CD71^[48]、CD44、A型肝配蛋白受体-2和表皮生长因子受体等^[51]。

核酸适配体是长度为20~100 nt的单链DNA或RNA, 能形成特定且稳定的三级结构, 从而与靶分子高特异性稳定结合, 因此也被称为“化学抗体”。相较于蛋白抗体, 核酸适配体制造简单、成本低廉、尺寸小、免疫原性低。通过将核酸适配体与特定RNA药物偶联, 可增强目标RNA的体内靶向递送^[52]。前列腺特异性抗原(prostate specific membrane antigen, PSMA) 适配体A10是第一代靶向PSMA阳性细胞的核酸适配体。经A10偶联的siRNA可显著抑制PSMA阳性细胞内PLK1和BCL2基因的表达, 实现核纤层蛋白A/C基因沉默^{[53, 54]}。类似地, gp120的RNA适配体可通过由2'-OMe和2'氟嘧啶修饰的富含GC序列的核酸短链结构连接siRNA形成复合物, 抑制模型小鼠体内的HIV-1复制(图 4e)^[55]。

3.3 纳米递送载体

除了上述策略, 研究者还设计开发了多类递送系统用以携带核酸进入靶组织及细胞。递送系统可分为病毒载体和非病毒载体。病毒载体核酸递送效率高, 但局限明显^[56], 如: ①部分病毒负载的核酸只能整合到分裂期细胞; ②核酸荷载能力有限(< 10 kb); ③免疫原性强, 本身具有一定的毒性; ④可产生野生型病毒或辅助型病毒, 具有致畸致突变风险。由于病毒载体已有广泛深入的综述, 本文将重点综述脂质纳米粒(lipid nanoparticle, LNP)、聚合物类纳米粒、外泌体等非病毒类纳米载体^[57-64] (图 5), 介绍相关纳米递送载体的化学组成、物理特性及其体内RNA递送机制与特点。

3.3.1 LNP

LNP是由正电/可离子化脂质、辅助磷脂、胆固醇和PEG化脂质基于分子间相互作用自组装形成的纳米结构, 可通过静电作用与负电荷的核酸结合^[57]。LNP粒径通常为20~200 nm, 该大小可帮助LNP克服体液(如血液和淋巴液) 的剪切并穿过组织间隙^[65]。经典LNP处方通常包括4种成分: 阳离子脂质(cationic lipids, CLs)/可离子化脂质(ionizable lipids, ILs)、胆固醇、辅助磷脂和聚乙二醇化脂质^[57] (图 5a)。研究表明, LNP在体内主要通过与载脂蛋白E (apolipoprotein E, ApoE) 形成复合物, 在低密度脂蛋白受体介导的内吞后由CLs/ILs介导的内体逃逸进入细胞质。LNP的体内分布与处方及相关受体密切相关, LNP的处方研究是当前RNA递送的研究热点和难点。

CLs/ILs通常占LNP配方总脂质的30%~50%。CLs具有一定毒性, 可引发细胞凋亡及炎症^[66], 临床应用有限。ILs于生理pH下呈电中性, 使用ILs处方可显著增加LNP的体内循环时间和安全性。ILs由头基、连接键和烷基尾链组成, 通过调节这3个部分可改变LNP特性以调整递送效率^[67]。ILs的头基通常是在低pH下带有正电荷的各类含胺基的基团。当LNP经内体途径入胞后, ILs的头基在内体酸性环境中质子化, 促进LNP与负电内体膜的吸附与融合, 帮助荷载RNA逃逸内体。头基和尾链之间的连接键影响LNP的稳定性、生物可降解性、细胞毒性和转染效率, 选择可生物降解的接头(如酯、酰胺、硫醇等) 可促进LNP的体内清除, 降低毒副作用。ILs通常包含1~4个含有8~20个碳原子(饱和或不饱和) 的烷基尾链, 烷基尾链可通过影响LNP的pKa值、亲脂性、流动性和膜的融合性影响核酸的递送效力。1, 2-二油醇-3-二甲基氨基丙烷(1, 2-dioleyloxy-3-dimethylaminopropane, DODMA) 及其类似物1, 2-二油酰氧基-3-(二甲氨基)丙烷[1, 2-dioleoyloxy-3-(dimethylamino)propane, DODAP] 是第一类用于RNA递送的可离子化脂质^[68]。在此基础上, 研究者又陆续合成了DLin-DMA和DLin-MC3-DMA^[69], 后者已作为包封核酸的关键组分用于制备FDA批准的首款siRNA药物Onpattro^TM, 并已进一步被证明可用于mRNA的体内递送^[70]。

市售LNP处方中的常见辅助磷脂为二硬脂酰磷脂酰胆碱(1, 2-distearoyl-sn-glycero-3-phosphocholine, DSPC) 和二油酰磷脂酰乙醇胺(1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE)。辅助磷脂通常是半合成磷脂, 占LNP配方中总脂质的10%~20%^[67], 位于LNP外周。DSPC是由1个胆碱头基和2条饱和的十八碳烷基链形成的磷脂分子, 是Onpattro^TM和2019新型冠状病毒(sever acute respiratory syndrome coronavirus 2, SARS-CoV-2) mRNA疫苗的关键辅料^[57]。DOPE内吞后可通过形成六方晶相(HII) 扰动内体膜结构, 促进荷载RNA逃逸内体^[71]。采用天然存在的糖脂替代基于MC3处方中的DSPC可改善mRNA胞内转染效率并提高LNP稳定性^[72]。还有研究表明, 通过设计两性辅助磷脂可促进内体膜扰动和逃逸。这些辅助两性离子磷脂均由含胺基的头部、磷酸酯和短的疏水烷基链组成。经优化筛选得到的两性辅助离子磷脂9A1P9可高效递送核酸至肝脏和肺部^[73]。

除调节转染效率外, 辅助磷脂可影响LNP的体内分布。在LNP中加入带电磷脂可在不显著改变表面电荷的前提下实现选择性器官靶向。添加正电脂质(2, 3-二油酰基-丙基)-三甲基铵-氯盐(1, 2-dioleoyl-3-trimethylammonium-propane, DOTAP) 可优先转染肺, 添加负电脂质18∶1 PA将优先转染脾^[74]。此外, DOPE能促进C12-200 LNP的肝脏积累, 而DSPC在静脉给药后会导致LNP的脾脏蓄积^[75]。

胆固醇占LNP总脂质的20%~50%, 主要分布于LNP外壳中, 可影响LNP的脂质流动和稳定性, 调节细胞识别和摄取。以天然存在的植物甾醇(如β-谷甾醇)^[76]或胆固醇氧化衍生物^[77]代替胆固醇能显著改善mRNA体内递送, 这可能由于胆固醇可影响LNP结构和内吞途径, 并由此增强LNP中mRNA的内体逃逸。此外, 还有研究表明ApoE会导致胆固醇从LNP核心迁移至外层脂质, 因此胆固醇的结构修饰可能引起LNP表面性状变化^[78]。

聚乙二醇化脂质(PEG-脂质) 是LNP处方中的重要组分, 能延长制剂体内循环时间。由于PEG良好的亲水性及较大的空间体积, PEG-脂质在自组装时常位于LNP外壳, 形成外周水化层防止纳米粒在血液中聚集, 阻碍蛋白吸附和单核巨噬细胞系统的清除, 延长LNP的体内循环时间。也有研究将PEG链末端功能化, 使LNP共价键合配体或生物大分子, 实现LNP的表面功能化^[71]。

PEG-脂质的含量和烷基链性质可显著影响核酸体内递送效率, 即过量的PEG-脂质将抑制LNP的摄取和转染^[79]。一方面, PEG-脂质通过疏水烷基链锚定于LNP表面, 其PEG组分可在体内诱导产生抗PEG抗体, 加速LNP清除; 另一方面, 锚定于LNP表面的PEG-脂质在体循环时会脱落, 影响体内蛋白冠的形成, 改变体内分布和清除速率。通常认为PEG-脂质的锚定作用随着烷基链长度的增加而加强^[80], 改变烷基链长度可调控PEG-脂质的脱落, 影响体内LNP表面PEG-脂质的比例, 调控体内清除速率, 促进核酸的体内可控递送。

3.3.2 聚合物类纳米粒

聚合物纳米粒是另一大类RNA递送载体。常用的聚合物主要有聚乳酸-羟基乙酸共聚物[poly(lactic-co-glycolic acid, PLGA], 聚乙烯亚胺(polyethyleneimine, PEI)、聚赖氨酸(polylysine, PLL)、聚β-氨基酯(poly-β-aminoester, PBAE) 及树枝状聚合物等^[58] (图 5b)。研究者可通过调整聚合物的电荷、可降解性和分子质量调控聚合物纳米粒的核酸递送。

PLGA在中性pH下带负电, 不易与同带负电荷的RNA结合, 可向PLGA中添加壳聚糖等阳离子组分实现siRNA的包载与递送^[81]。此外, 由于PLGA在酸性条件下带正电, 相较于不带电的聚苯乙烯颗粒具有更强的内体逃逸能力^[18]。PEI和PLL均为阳离子聚合物, 可通过静电与RNA结合, 借助质子海绵效应和内体膜扰动递送RNA^{[18, 82]}。然而未经修饰的PEI和PLL具有与分子质量正相关的细胞毒性^{[83, 84]}, 因此需通过结构改造提高其体内功效和安全性。如高稳定性、低细胞毒性的PEG-PEI纳米粒可将mRNA递送至肺中的免疫细胞进行免疫调节^[85]; 氧化铁-PLL纳米粒可将基因传递到小鼠的中枢神经系统, 并可在较大浓度范围内保持低细胞毒性^[86]。PBAE是胺单体与二丙烯酸酯共轭合成的阳离子聚合物, 具有可离子化叔胺及可生物降解的酯键, 生物相容性良好^[87]。基于PBAE的聚合物纳米粒可通过雾化给药, 将Cas13a的mRNA递送至小鼠的呼吸道用以防治SARS-CoV-2^[88]。另有研究发现在PBAE中添加脂质可进一步改善核酸的血清稳定性^[89], 故脂质-聚合物体系将有望实现高效的核酸递送。以聚酰胺(polyamidoamine, PAMAM) 和PLL为代表的树枝状聚合物也可用于RNA递送。树枝状聚合物由一定数量的核心分子发出的支链单体组成, 带正电的树枝状聚合物可与RNA通过静电作用络合, 内吞后扰动负电内体膜, 在脂质双分子层中产生瞬时孔提高内渗透压实现内体逃逸^[90]。通过合理优化和修饰树枝状大分子载体, 研究者得以调控核酸药物的体内分布和细胞摄取^[90], 并陆续开发了埃博拉病毒和H1N1病毒的mRNA疫苗^[91], 实现了RNA的中枢神经系统递送^[92]。

目前已有多种针对聚合物的化学修饰及偶联策略, 但高分子链末端或侧链上的靶向基团易被包裹屏蔽。此外也难以精确控制聚合物的分子质量与结构, 导致批间重复性低^[93]。聚合物纳米载体的粒径也可影响递送效率: 研究者比较了不同粒径的聚苯乙烯聚合物纳米粒的胞内转运模式, 发现大颗粒(43 nm) 主要在溶酶体中积累并降解, 而小颗粒(24 nm) 则能完全绕过内体通路, 在核周区域积累^[94]。

3.3.3 外泌体

外泌体属于细胞外囊泡(extracellular vesicles, EV), 主要由被称为多囊泡体的特定内涵体与质膜融合后分泌到细胞外基质, 是一种参与调控细胞间通讯的内源性囊泡(30~200 nm) (图 5c), 具有较好的生物相容性和靶向性^[59]。目前, 由外泌体递送的核酸以小RNA为主, 例如由小鼠成纤维细胞L929细胞衍生的外泌体可递送转化生长因子-β1 (transforming growth factor-β1, TGF-β1) siRNA至肿瘤细胞, 显著降低TGF-β1的表达, 抑制肿瘤细胞的活性和转移, 促进细胞凋亡^[95]。外泌体的RNA装载方式可分为内源性与外源性。内源性装载利用外泌体生成的内源性途径, 通过促进生产细胞系中目标RNA的表达和外泌体分泌, 间接提高目标外泌体产量; 外源性加载是通过电穿孔、超声、递送载体等方式将外源性核酸直接导入已获得的外泌体^[96]。与脂质体及聚合物纳米粒类似, 外泌体也可通过表面修饰调控其体内靶向递送。目前表面修饰策略主要包括化学修饰和基因工程改造。化学修饰主要通过化学反应在脂质或膜结合蛋白上偶联功能成分^[97]。如PSMA的多肽配体修饰的外泌体可在体内将siRNA递送至异种移植瘤并诱导肿瘤消退。基因工程改造是另一类外泌体修饰方法。研究者通过基因工程技术将编码目的蛋白或多肽的基因序列与特定外泌体膜蛋白的基因序列融合, 达到借助外泌体膜蛋白递送目标蛋白的效果。该方法对目的蛋白/多肽和外泌体膜蛋白的结构和大小均有要求, 在实际应用中需重点考量^[96]。在COVID-19疫苗研发背景下, 有研究发现外泌体能高效(~90%) 封装编码刺突蛋白和核衣壳蛋白的mRNA, 介导其体内翻译诱导产生免疫应答及记忆。相较于mRNA的LNP, mRNA的外泌体制剂特异性和安全性更好, 有望实现更加高效安全的体内递送^[98]。

3.4 其他递送策略

除已介绍的递送策略, 还有其他多种方案可实现RNA递送(表 1^[60-64]), 如能穿透多种生物屏障的球形核酸颗粒(spherical nucleic acid, SNA)、结构设计灵活精准的DNA纳米结构、以金属有机框架材料(metal-organic frameworks, MOFs) 为代表的多孔纳米材料和静电作用介导的多肽-核酸自组装纳米粒等, 其结构形态各异(图 5d~g), RNA荷载方式也多种多样, 各具鲜明优势, 受到了广泛关注。

4 RNA现有递送策略的不足

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虽然目前已有许多RNA药物取得了较好的临床前结果, 但最终通过FDA授权用于临床治疗的RNA药物仍非常有限。缺乏合适的递送系统依然是RNA药物临床转化的关键制约因素。首先, 临床可用的体内RNA递送系统必须要具备良好的生物安全性。最早用于体内核酸药物递送的病毒载体已被证实具有一定的毒副作用^[59], 目前多局限于临床前的机制研究, 极少成为临床可接受的RNA递送方式。化学合成的载体系统如阳离子脂质^[66]、表面具有高正电荷的PLL/PEI^{[83, 84]}和大部分无机纳米材料^{[62, 63]}等已被证实可在生物体内引发细胞凋亡及炎症反应。此外, 目前RNA药物临床前研究中广泛采用的小鼠模型并非严格的RNA毒性评价模型, 基于小鼠模型得到的RNA量-效关系并不能直接运用于人体。其次, 所合成的RNA体内递送系统必须具备可靠的化学及物理稳定性。递送系统化学稳定性在于既要在RNA药物入胞前保护其免受酶降解, 又要控制RNA在入胞后快速脱离递送系统进入细胞质保证疗效。已有相关文献^[99]报道, 部分与RNA偶联的配体或带正电的阳离子脂质及聚合物载体因与RNA药物结合过于紧密, 导致RNA因释放困难而效果减弱。物理稳定性主要为静电稳定性, 即RNA递送系统须在人体体液正常的生理盐浓度下, 克服因静电效应导致的颗粒团聚及由此产生的细胞内吞障碍。最后, RNA递送系统要实现大规模的临床应用还须保证其易于生产、质控和运输。然而, 在基于聚合物的RNA递送系统中, 高分子链末端或侧链上的靶向基团易被聚合物分子包裹屏蔽, 分子质量与结构难以精确控制, 批间重复性低^[93]; 而外泌体则由于内容物高度复杂, 不易质控; 以LNP为代表的RNA递送系统则会在低温冷冻条件下因结晶而稳定性降低^[100]。

综合而言, RNA药物及相关系统的开发, 须以全面的研究思路, 结合复杂的人体内环境条件和递送屏障, 对载体介导的递送过程进行逐步的量化分析, 以高度可控且可重复的方式实现RNA药物的高效递送, 并解决其在生产及运输等过程中可能面临的挑战。而RNA药物具体的给药途径、药物载量、给药频率及人体反应等参数, 则需基于充分开展的人体实验来确定, 以保证治疗效力安全性。

5 总结与展望

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RNA药物相较于小分子或重组蛋白等药物, 不受靶点种类限制, 可在核酸水平对组织细胞的生理活动进行精准干预, 目前已被用于多种疾病的防治。RNA药物具有广阔的应用前景, 但当前其临床应用仍须进一步提高自身的稳定性、半衰期、免疫原性、组织靶向性、细胞摄取及内涵体逃逸效率。为实现上述目标, 研究者开展了核苷酸化学修饰、配体或抗体偶联、纳米递送载体在内的许多研究。通过优化核苷酸的磷酸骨架、核糖、碱基及核酸链整体结构, 可大幅提高RNA体内稳定性, 降低免疫原性, 有效地促进沉默或翻译等生物学功能。通过共价缀合脂质、半乳糖、蛋白/多肽或适配体等配体, 则可延长RNA体循环时间, 增强对特定组织或细胞的递送。本文重点介绍了包含LNP、聚合物类纳米粒和外泌体在内的RNA的纳米递送载体。当前LNP已在临床大规模应用, 通过精细调控LNP组分可获得较好的体内安全性及递送效率。聚合物纳米粒通过精细控制聚合物结构, 调控质子海绵效应及内体膜扰动实现核酸的内体逃逸。外泌体生物相容性好, 具备体内靶向性, 作为核酸载体具有极大潜力。此外, 本文也对球形核酸颗粒、DNA纳米结构、多孔纳米材料和阳离子多肽-核酸复合物等进行了介绍。随着RNA药物递送相关研究的深入, 其瓶颈正被逐一攻克。目前其临床化推广和应用尚存在一定不足, 仍需深入开发。待RNA药物的临床能力得到综合性地提高, 有望开启继小分子、抗体药物之后的第三次新药创制浪潮。

作者贡献: 李丹负责撰写; 黄宇坤和高小玲负责设计与修改。

利益冲突: 所有作者均声明不存在利益冲突。

基金

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国家自然科学基金资助项目(81722043)
国家自然科学基金资助项目(81973272)
国家自然科学基金资助项目(92068111)

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2023年第58卷第3期

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doi: 10.16438/j.0513-4870.2022-0500

接收时间：2022-04-27
首发时间：2025-11-21
出版时间：2023-03-12

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收稿日期：2022-04-27
修回日期：2022-05-20

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国家自然科学基金资助项目(81722043)

国家自然科学基金资助项目(81973272)

国家自然科学基金资助项目(92068111)

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上海交通大学医学院, 上海 200025

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*高小玲， Tel: 86-21-63846590-776580, E-mail: shellygao1@sjtu.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Strategy	Principle	Advantage	Challenge
Spherical nucleic acid (SNA)^[60]	Consist of hydrophobic core nanoparticles (including gold, silica, or other materials) linked by thiol bonds to hydrophilic oligonucleotides (e.g., ASO, siRNA)	They can transfect a variety of tissues and cell types non-toxic with a mild immune response, and can penetrate various biological barriers (such as skin, blood-brain barrier, and blood-tumor barrier)	① The biodistribution in vivo is greatly affected by its composition; ② The targeted delivery efficiency is low; ③ The oligonucleotides may be degraded prematurely during the delivery process
DNA nanostructures^[61]	Formed by self-assembly of complementary bases. Modular design of RNA drugs (e.g. siRNA or immunostimulatory oligonucleotides) and targeting ligands (e.g. aptamers)	The structure can be precisely and flexibly designed to tune its physicochemical properties; the small size (about 20 nm) of the designable structure may enable extrahepatic delivery	Unfunctionalized DNA nanostructures are smaller in size, and have higher renal clearance. The overall bioavailability is reduced
Metal organic frameworks (MOFs)^{[62, 63]}	Encapsulation of RNA drugs into MOFs by pore encapsulation or in situ synthesis, and targeted delivery in vivo by surface modification of MOFs (such as covering the membrane of naturally derived platelets)	MOFs have high specific surface area and porosity, enabling high-load delivery of drugs. According to the size of the RNA drug, the pore size of MOFs can be precisely designed and the physical and chemical properties can be adjusted to achieve stable and effective delivery of the RNA drug	Research on the drug loading and release kinetics of MOFs is still limited, and their preclinical evaluation performance should be optimized through systematic in vivo studies on their stability, degradation mechanism, and side effects on normal organs
Peptide-nucleic acid complex^[64]	Positively charged lysine or arginine residues can bind to nucleic acids through electrostatic interactions at physiological pH; hydrophobic amino acid residues or hydrophobic components (such as alkyl chains) facilitate their self-assembly to form nanoparticles or fibers; histidine residues enhance endosomal escape via the proton sponge effect	The secondary structure of peptides in aqueous solution and the conformational changes following peptide-nucleic acid interactions are tuned by modulating the distribution of cationic amino acids (arginine, lysine, or histidine) and hydrophobic amino acids. The complexation strength and membrane penetration ability of peptides and nucleic acids can be flexibly adjusted to improve delivery efficiency. It has good biocompatibility and low immunogenicity	There is insufficient information on the biodistribution, clearance, immunocompatibility, and specific organ toxicity of such polypeptide-nucleic acid complexes. It is necessary to develop polypeptide gene carriers with higher delivery efficiency through further optimized design and more structure-activity relationship studies to meet the needs of clinical applications

Strategy

Principle

Advantage

Challenge

Spherical nucleic acid (SNA)^[60]

Consist of hydrophobic core nanoparticles (including gold, silica, or other materials) linked by thiol bonds to hydrophilic oligonucleotides (e.g., ASO, siRNA)

They can transfect a variety of tissues and cell types non-toxic with a mild immune response, and can penetrate various biological barriers (such as skin, blood-brain barrier, and blood-tumor barrier)

① The biodistribution in vivo is greatly affected by its composition; ② The targeted delivery efficiency is low; ③ The oligonucleotides may be degraded prematurely during the delivery process

DNA nanostructures^[61]

Formed by self-assembly of complementary bases. Modular design of RNA drugs (e.g. siRNA or immunostimulatory oligonucleotides) and targeting ligands (e.g. aptamers)

The structure can be precisely and flexibly designed to tune its physicochemical properties; the small size (about 20 nm) of the designable structure may enable extrahepatic delivery

Unfunctionalized DNA nanostructures are smaller in size, and have higher renal clearance. The overall bioavailability is reduced

Metal organic frameworks (MOFs)^{[62, 63]}

Encapsulation of RNA drugs into MOFs by pore encapsulation or in situ synthesis, and targeted delivery in vivo by surface modification of MOFs (such as covering the membrane of naturally derived platelets)

MOFs have high specific surface area and porosity, enabling high-load delivery of drugs. According to the size of the RNA drug, the pore size of MOFs can be precisely designed and the physical and chemical properties can be adjusted to achieve stable and effective delivery of the RNA drug

Research on the drug loading and release kinetics of MOFs is still limited, and their preclinical evaluation performance should be optimized through systematic in vivo studies on their stability, degradation mechanism, and side effects on normal organs

Peptide-nucleic acid complex^[64]

Positively charged lysine or arginine residues can bind to nucleic acids through electrostatic interactions at physiological pH; hydrophobic amino acid residues or hydrophobic components (such as alkyl chains) facilitate their self-assembly to form nanoparticles or fibers; histidine residues enhance endosomal escape via the proton sponge effect

The secondary structure of peptides in aqueous solution and the conformational changes following peptide-nucleic acid interactions are tuned by modulating the distribution of cationic amino acids (arginine, lysine, or histidine) and hydrophobic amino acids. The complexation strength and membrane penetration ability of peptides and nucleic acids can be flexibly adjusted to improve delivery efficiency. It has good biocompatibility and low immunogenicity

There is insufficient information on the biodistribution, clearance, immunocompatibility, and specific organ toxicity of such polypeptide-nucleic acid complexes. It is necessary to develop polypeptide gene carriers with higher delivery efficiency through further optimized design and more structure-activity relationship studies to meet the needs of clinical applications