药学学报

No.	Drug name/commercial name	Type	Owner	Time-to-market/place of approval	Therapeutic indication	Retouching/delivery	Mode of administration
1	Formivirsen/Vitravene	ASO	Ionis/Novartis	1998, FDA (Delisted)	CMVR	PS	IVI
2	Mipomersen/Kynamro	ASO	Ionis/Kastle Therapeutics	2013, FDA (Delisted)	HOFH	PS, 2′-O-MOE	SC
3	Nusinersen/Spinraza	ASO	Ionis/Biogen	2016, FDA	SMA	PS, 2′-O-MOE	IT
4	Eteplirsen/Exondys 51	ASO	Sarepta Therapeutics	2016, FDA	DMD	PMO	IV
5	Inotersen/Tegsedi	ASO	Ionis	2018, FDA	hATTR	PS, 2′-O-MOE	SC
6	Golodirsen/Vyondys 53	ASO	Sarepta Therapeutics	2019, FDA	DMD	PMO	IV
7	Volanesorsen/Waylivra	ASO	Ionis	2019, EU	FCS	PS, 2′-O-MOE	SC
8	Viltolarsen/Viltepso	ASO	Nippon Shinyaku	2020, FDA	DMD	PMO	IV
9	Casimersen/Amondys 45	ASO	Sarepta Therapeutics	2021, FDA	DMD	PMO	IV
10	Patisiran/Onpattro	siRNA	Alnylam	2018, FDA	hATTR	PS, 2′-F, 2′-O-Me/ LNPs	IV
11	Givosiran/Givlaari	siRNA	Alnylam	2019, FDA	AHP	PS, 2′-F, 2′-O-Me/ GalNAc	SC
12	Lumasiran/Oxlumo	siRNA	Alnylam	2020, FDA	PH1	PS, 2′-F, 2′-O-Me/ GalNAc	SC
13	Inclisiran/Leqvio	siRNA	Alnylam/Novartis	2020, EU 2021, FDA	FP	PS, 2′-F, 2′-O-Me/ GalNAc	SC
14	Pegaptanib/Macugen	Aptamer	Pfizer/Eyetech,	2004, FDA (Delisted)	AMD	——	IVI
15	Vutrisiran/Amvuttra	siRNA	Alnylam	2022, FDA	hATTR	PS, 2′-F, 2′-O-Me/ GalNAc	SC
16	Eplontersen/Wainua	ASO	Ionis/Astra Zeneca	2023, FDA	hATTR	PS, 2′-O-Me/ GalNAc	SC
17	Tofersen/Qalsody	ASO	Ionis/Biogen	2023, FDA	ALS	PS, 2′-O-Me	IT
18	Nedosiran/Rivfloza	siRNA	Dicerna (Novo Nordisk)/ Alynlam	2023, FDA	PH1	PS, 2′-F, 2′-O-Me/ GalNAc	SC
19	Avacincaptad pegol/Lzervay	Aptamer	Lveric Bio/Archemix	2023, FDA	GA	——	IVI

No.	Drug name/commercial name	Type	Owner	Time-to-market/place of approval	Therapeutic indication	Retouching/delivery	Mode of administration
1	Formivirsen/Vitravene	ASO	Ionis/Novartis	1998, FDA (Delisted)	CMVR	PS	IVI
2	Mipomersen/Kynamro	ASO	Ionis/Kastle Therapeutics	2013, FDA (Delisted)	HOFH	PS, 2′-O-MOE	SC
3	Nusinersen/Spinraza	ASO	Ionis/Biogen	2016, FDA	SMA	PS, 2′-O-MOE	IT
4	Eteplirsen/Exondys 51	ASO	Sarepta Therapeutics	2016, FDA	DMD	PMO	IV
5	Inotersen/Tegsedi	ASO	Ionis	2018, FDA	hATTR	PS, 2′-O-MOE	SC
6	Golodirsen/Vyondys 53	ASO	Sarepta Therapeutics	2019, FDA	DMD	PMO	IV
7	Volanesorsen/Waylivra	ASO	Ionis	2019, EU	FCS	PS, 2′-O-MOE	SC
8	Viltolarsen/Viltepso	ASO	Nippon Shinyaku	2020, FDA	DMD	PMO	IV
9	Casimersen/Amondys 45	ASO	Sarepta Therapeutics	2021, FDA	DMD	PMO	IV
10	Patisiran/Onpattro	siRNA	Alnylam	2018, FDA	hATTR	PS, 2′-F, 2′-O-Me/ LNPs	IV
11	Givosiran/Givlaari	siRNA	Alnylam	2019, FDA	AHP	PS, 2′-F, 2′-O-Me/ GalNAc	SC
12	Lumasiran/Oxlumo	siRNA	Alnylam	2020, FDA	PH1	PS, 2′-F, 2′-O-Me/ GalNAc	SC
13	Inclisiran/Leqvio	siRNA	Alnylam/Novartis	2020, EU 2021, FDA	FP	PS, 2′-F, 2′-O-Me/ GalNAc	SC
14	Pegaptanib/Macugen	Aptamer	Pfizer/Eyetech,	2004, FDA (Delisted)	AMD	——	IVI
15	Vutrisiran/Amvuttra	siRNA	Alnylam	2022, FDA	hATTR	PS, 2′-F, 2′-O-Me/ GalNAc	SC
16	Eplontersen/Wainua	ASO	Ionis/Astra Zeneca	2023, FDA	hATTR	PS, 2′-O-Me/ GalNAc	SC
17	Tofersen/Qalsody	ASO	Ionis/Biogen	2023, FDA	ALS	PS, 2′-O-Me	IT
18	Nedosiran/Rivfloza	siRNA	Dicerna (Novo Nordisk)/ Alynlam	2023, FDA	PH1	PS, 2′-F, 2′-O-Me/ GalNAc	SC
19	Avacincaptad pegol/Lzervay	Aptamer	Lveric Bio/Archemix	2023, FDA	GA	——	IVI

No.	Strain	Target	Mechanism	Delivery carrier	Result	Ref.
1	E. coli, MRSA	acpP mRNA	Inhibition of fatty acid synthesis	GP-SiNPs	100% Kill human-derived resistant bacteria; the uptake efficiency (51.6%) surpassed that of CPPs. The MIC for E. coli and S. aureus was reduced to 0.8 μmol·L^-1	[4]
2	E. coli K12	The start codon region of acpP mRNA	Inhibition of fatty acid synthesis	CPPs	The MIC of anti-acpP peptide-PNAs was reduced to 0.2 μmol·L^-1	[5]
3	S. aureus	fmhB mRNA	Inhibition of peptidoglycan synthesis Inhibition of DNA replication	CPPs	The growth of S. aureus was effectively inhibited in a dose-dependent using PNAs	[6]
4	E. coli, S. aureus, MRSA	ftsZ genes	Inhibition of the ftsZ gene, thereby inhibiting bacterial division	ZIF-8	Synergistic, broad-spectrum antibacterial; the biofilm removal efficacy reached 88.2%, while the MIC exhibited a reduction to 16 μg·mL^-1	[7]
5	S. aureus	ftsZ genes	Inhibition of the ftsZ gene, thereby inhibiting bacterial division	6HB	ASO and Ag⁺ exhibit a synergistic effect in antimicrobial therapy, providing a broad-spectrum antimicrobial activity	[8]
6	S. aureus	yycF mRNA	Regulation negatively of yycF transcription by the two-component system, regulates formation of biofilm	GO	The onset of the log phase was delayed by a duration of 3h; there was an approximate twofold reduction in biofilm formation	[9, 10]
7	MRSA, Staphylococcus epidermidis	mecA genes	Inhibition of mecA gene expression	AuNPs, PEI, MLAuNPs	The uptake efficiency was observed to be 74%; when combined with oxacillin, a significant reduction of 29% in MRSA was achieved	[11]
8	MRSA	mecA genes	Inhibition of mecA gene expression	Anionic liposome	The expression of penicillin-binding protein 2a is inhibited, thereby enhancing the affinity of antibiotics; combined with oxacillin, the MIC was reduced to 0.13 μg·mL^-1	[12]
9	E. coli	β-Lactamase CTX-M-group 1	Inhibition of the expression of β-lactamase CTX-M-group 1	Tetrahedral framework	Reinstatement of cefotaxime susceptibility in strains exhibiting susceptible phenotypes. The MIC of cefotaxime (CTX 16 mg·L^-1) was reduced from 35 to 16 mg·L^-1	[13]

No.	Strain	Target	Mechanism	Delivery carrier	Result	Ref.
1	E. coli, MRSA	acpP mRNA	Inhibition of fatty acid synthesis	GP-SiNPs	100% Kill human-derived resistant bacteria; the uptake efficiency (51.6%) surpassed that of CPPs. The MIC for E. coli and S. aureus was reduced to 0.8 μmol·L^-1	[4]
2	E. coli K12	The start codon region of acpP mRNA	Inhibition of fatty acid synthesis	CPPs	The MIC of anti-acpP peptide-PNAs was reduced to 0.2 μmol·L^-1	[5]
3	S. aureus	fmhB mRNA	Inhibition of peptidoglycan synthesis Inhibition of DNA replication	CPPs	The growth of S. aureus was effectively inhibited in a dose-dependent using PNAs	[6]
4	E. coli, S. aureus, MRSA	ftsZ genes	Inhibition of the ftsZ gene, thereby inhibiting bacterial division	ZIF-8	Synergistic, broad-spectrum antibacterial; the biofilm removal efficacy reached 88.2%, while the MIC exhibited a reduction to 16 μg·mL^-1	[7]
5	S. aureus	ftsZ genes	Inhibition of the ftsZ gene, thereby inhibiting bacterial division	6HB	ASO and Ag⁺ exhibit a synergistic effect in antimicrobial therapy, providing a broad-spectrum antimicrobial activity	[8]
6	S. aureus	yycF mRNA	Regulation negatively of yycF transcription by the two-component system, regulates formation of biofilm	GO	The onset of the log phase was delayed by a duration of 3h; there was an approximate twofold reduction in biofilm formation	[9, 10]
7	MRSA, Staphylococcus epidermidis	mecA genes	Inhibition of mecA gene expression	AuNPs, PEI, MLAuNPs	The uptake efficiency was observed to be 74%; when combined with oxacillin, a significant reduction of 29% in MRSA was achieved	[11]
8	MRSA	mecA genes	Inhibition of mecA gene expression	Anionic liposome	The expression of penicillin-binding protein 2a is inhibited, thereby enhancing the affinity of antibiotics; combined with oxacillin, the MIC was reduced to 0.13 μg·mL^-1	[12]
9	E. coli	β-Lactamase CTX-M-group 1	Inhibition of the expression of β-lactamase CTX-M-group 1	Tetrahedral framework	Reinstatement of cefotaxime susceptibility in strains exhibiting susceptible phenotypes. The MIC of cefotaxime (CTX 16 mg·L^-1) was reduced from 35 to 16 mg·L^-1	[13]

反义核酸在抗菌治疗领域的研究进展

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李佳 ¹ , 韩晓璐 ¹^,² , 宋世昱 ³ , 林锦涛 ¹ , 唐志强 ¹^,⁴ , 王增明 ¹ , 徐亮 ¹^,^* , 郑爱萍 ¹^,^*

药学学报 | 综述 2025,60(2): 337-347

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药学学报 | 综述 2025, 60(2): 337-347

反义核酸在抗菌治疗领域的研究进展

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李佳¹, 韩晓璐¹^,², 宋世昱³, 林锦涛¹, 唐志强¹^,⁴, 王增明¹, 徐亮¹^,^*, 郑爱萍¹^,^*

作者信息

1.军事医学研究院国家安全特需药品全国重点实验室, 北京 100850

2.中国人民解放军32104部队, 内蒙古阿拉善盟 735400

3.重庆医科大学, 重庆 400016

4.延边大学药学院, 吉林延吉 133002

通讯作者:

^*徐亮, Tel: 13366515305, E-mail: wj24998@163.com

郑爱萍, Tel: 86-10-66931694, E-mail: apzheng@163.com

Progress on antisense oligonucleotide in the field of antibacterial therapy

Jia LI¹, Xiao-lu HAN¹^,², Shi-yu SONG³, Jin-tao LIN¹, Zhi-qiang TANG¹^,⁴, Zeng-ming WANG¹, Liang XU¹^,^*, Ai-ping ZHENG¹^,^*

Affiliations

1. State Key Laboratory of National Security Specially Needed Medicines, Academy of Military Medical Sciences, Beijing 100850, China

2. Troops 32104 of People's Liberation Army of China, Alashanmeng 735400, China

3. Chongqing Medical University, Chongqing 400016, China

4. College of Pharmacy, Yanbian University, Yanji 133002, China

出版时间: 2025-02-12 doi: 10.16438/j.0513-4870.2024-0607

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摘要

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随着抗生素广泛使用, 耐药菌感染已经成为人类健康重要威胁, 寻找能有效控制耐药菌感染的新型抗菌策略已成为当务之急。区别于以细菌蛋白为靶点的小分子药物, 反义寡核苷酸可以靶向细菌耐药、致病、生长繁殖及生物膜形成等机制相关的基因, 通过调控相关基因表达来抑制或者杀灭细菌, 为抗菌药物的研发提供了全新的思路。为改变反义核酸细菌内递送难题, 已有多种递药系统应用于细菌递送领域, 主要包括细胞穿透肽、脂质纳米颗粒及无机纳米颗粒等, 为反义核酸在抗菌领域的发展提供了崭新动力。本综述围绕近些年小核酸药物的发展现状, 反义核酸抗菌机制、靶点及序列, 以及递送载体等方面进行了总结归纳, 为反义核酸在抗菌感染治疗领域的研究发展提供参考。

关键词

核酸药物 / 反义寡核苷酸 / 抗菌 / 药物递送 / 基因治疗

Abstract

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With the widespread use of antibiotics, drug-resistant bacterial infections have become a significant threat to human health. Finding new antibacterial strategies that can effectively control drug-resistant bacterial infections has become an urgent task. Unlike small molecule drugs that target bacterial proteins, antisense oligonucleotide (ASO) can target genes related to bacterial resistance, pathogenesis, growth, reproduction and biofilm formation. By regulating the expression of these genes, ASO can inhibit or kill bacteria, providing a novel approach for the development of antibacterial drugs. To overcome the challenge of delivering antisense oligonucleotide into bacterial cells, various drug delivery systems have been applied in this field, including cellpenetrating peptides, lipid nanoparticles and inorganic nanoparticles, which have injected new momentum into the development of antisense oligonucleotide in the antibacterial realm. This review summarizes the current development of small nucleic acid drugs, the antibacterial mechanisms, targets, sequences and delivery vectors of antisense oligonucleotide, providing a reference for the research and development of antisense oligonucleotide in the treatment of bacterial infections.

Key words

nucleic acid drug / antisense oligonucleotide / antibacterial / drug delivery / gene therapy

引用本文

李佳, 韩晓璐, 宋世昱, 林锦涛, 唐志强, 王增明, 徐亮, 郑爱萍. 反义核酸在抗菌治疗领域的研究进展. 药学学报, 2025 , 60 (2) : 337 -347 . DOI: 10.16438/j.0513-4870.2024-0607

Jia LI, Xiao-lu HAN, Shi-yu SONG, Jin-tao LIN, Zhi-qiang TANG, Zeng-ming WANG, Liang XU, Ai-ping ZHENG. Progress on antisense oligonucleotide in the field of antibacterial therapy[J]. Acta Pharmaceutica Sinica, 2025 , 60 (2) : 337 -347 . DOI: 10.16438/j.0513-4870.2024-0607

正文

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随着临床耐药等问题频发, 传统小分子抗生素研发方法已不能应对日益严峻的耐药菌感染, 许多新抗菌策略, 如CRISPR-Cas疗法、噬菌体疗法、工程毒素及抗菌肽等相继被用于新型抗菌剂。然而, 这些新抗菌策略由于存在免疫原性、脱靶等潜在风险, 不同程度上限制了相应的临床转化。小核酸药物是以DNA或RNA为载体或靶点的药物, 与现有的以蛋白为靶点的小分子和抗体药物相比, 其靶点筛选快、研发成功率高、不易产生耐药, 在传统药物“不可成药”靶点问题上具有更好的优势, 成为目前国际上重点关注的一类新型生物技术药物。近些年, 越来越多原核生物研究中发现, 外源核酸通常在大多数细菌质粒及遗传基因中表达, 与目标RNA/DNA结合, 可以参与破坏DNA到信使核糖核酸(messenger RNA, mRNA) 再到蛋白质的信息流。随着化学修饰和递送载体的发展, 菌内反义核酸递药有望成为逆转细菌耐药性的一种新方法。本文围绕近些年小核酸药物发展现状, 反义核酸的抗菌机制及生物学靶点, 以及反义核酸递送载体等方面进行了总结归纳, 为反义寡核苷酸(antisense oligonucleotide, ASO) 在抗菌治疗领域的发展提供了新的认识。

1 小核酸药物

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小核酸药物是以DNA或RNA为载体或靶点的药物, 目前在研的小核酸药物种类很多, 包括ASO、小干扰核糖核酸(small interfering RNA, siRNA)、微小核糖核酸(microRNA, miRNA)、核酸适配体(aptamer) 等, 不同的小核酸药物通常具有不同的结构特点和作用机制。其中ASO、siRNA和miRNA通过碱基互补配对原则靶向mRNA或前体mRNA, 通过基因沉默抑制靶蛋白表达, 具有包括RNA干扰等介导的切割靶标降解、剪接调节、非编码RNA抑制、基因激活和程序性基因编辑的功能, 在治疗或控制多种疾病上展现了巨大潜力。截至2023年底, 共11款ASO药物、6款siRNA药物和2款aptamer药物获批上市, 表 1对已上市的小核酸的种类、上市公司、上市时间/批准地、适应症、修饰/递送及给药方式信息进行汇总^[1]。目前, 获批上市的小核酸药物的适应症基本为罕见遗传病, 其中有12款药物未使用递送系统, 2款为aptamer, 剩余10款均为ASO。ASO主要采用裸核酸进行化学修饰后进行给药, 这主要是由于ASO分子质量小, 进入细胞能力较强, 特别是ASO经过鞘内注射, 不需要递送系统就可以进入中枢神经系统。在7款使用递送系统的药物中, 6款使用GalNAc共轭连接递送系统, 其中5款为siRNA, 1款为ASO。1款药物使用脂质纳米颗粒(lipid nanoparticles, LNPs) 递送, 为siRNA药物^[1]。

综上所述, ASO的开发及应用占据了小核酸药物发展的半壁江山, 其应用领域最为广泛, 已在严重和罕见遗传疾病、心血管和代谢疾病取得了巨大进展, 逐渐向肿瘤、神经疾病及抗感染治疗等领域拓展。在早期研究阶段, ASO存在靶向性不强、生物活性不足、脱靶毒副作用等诸多短板, 而今, 一些经化学修饰ASO (核酸主链、核糖的糖部分和核苷碱基的修饰) 很好地解决了上述问题。随着对ASO作用机制的深入研究, 以及大规模更具针对性临床试验的开展, 制药行业逐渐掀起一股ASO研发热。在针对原核细胞的研究中发现, 大肠杆菌(Escherichia coli, E. coli)、金黄色葡萄球菌(Staphylococcus aureus, S. aureus)、枯草芽孢杆菌、结核分枝杆菌等菌株中均发现了反义RNA的存在, 这些小RNA分子具有重要的调节功能。此外, 在原核生物中也发现了类似于真核生物的RNAi现象, 即成簇的规律间隔的短回文重复序列及其相关系统介导的适应性免疫机制^[2]。此类研究成果的发现, 也推动了反义技术在原核生物中的研究日趋广泛, 使得反义核酸在抗菌、抗感染领域走入了新阶段。

2 反义核酸在抗菌领域的应用

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2.1 反义核酸的优势及抗菌机制

使用外源性核酸作为抗菌剂的概念于上世纪90年代初提出, 通过递送外源核酸与细菌遗传物质的碱基互补配对, 调控细菌靶基因表达, 可以参与到细菌基因编码的信息流中, 并影响几种代谢和毒力蛋白质的产生。1997年, White等通过设计反义序列抑制E. coli的marRAB操纵子, 恢复了E. coli对诺氟沙星的敏感性, 成功提高了诺氟沙星对E. coli的杀菌活性, 首次证明通过设计反义序列可以抑制耐药基因表达^[3]。

与传统抗生素相比, ASO具有很多优点: ①易于设计: 理论上, ASO可以靶向所有病原菌中所有与疾病相关的基因, 了解核苷酸序列就可以设计ASO, 从而达到抗菌目的; ②精准靶点: ASO可以靶向其互补的mRNA, 选择性地阻止密切相关基因中单个异构体的表达, 优于传统小分子药物; ③不易诱发耐药: ASO不像抗菌肽等其他抗菌药物那样容易诱导细菌耐药。目前, 传统抗生素发展跟不上生物变异和耐药速度, 一旦发现耐药基因, 反义技术可能是抗击超级细菌最后的快速手段。

近几十年研究显示, ASO抗菌作用机制主要是反义核酸通过专有占位或者剪切依赖机制与细菌的mRNA进行互补配对, 干扰其正常翻译表达, 反义核酸抑菌抑制过程与机制如图 1所示。其中, 专有占位是指反义核酸与靶mRNA通过碱基互补配对原理结合后, 形成空间位阻效应, 影响核糖体多肽的延长与成熟, 阻断其翻译; 剪切依赖机制是指反义核酸与mRNA结合后激活内源性核糖核酸酶(ribonuclease, RNase) 等, 降解mRNA。其主要有3种方式, 即由双链RNA介导的mRNA的降解、由RNase H介导的RNA/DNA杂交双链中的RNA链降解, 以及通过RNase P介导的降解作用。

2.2 反义核酸抗菌的生物学靶点

反义核酸抗菌的优点已在生物医学研究领域得到广泛认可, 大量研究表明, 反义核酸在替代传统抗生素抗菌方面具有显著优势。近年来, 越来越多研究者采用反义技术在基因组内筛选致病菌必需基因作为靶点抗菌, 其抗菌、抗感染策略主要集中在细胞侵袭和细菌生长的抑制剂、生物膜形成的抑制剂、辅助递送抗菌剂、细菌毒素的抑制剂、抗生素增强剂及耐药相关酶的抑制剂开发等方面, 总结及分析反义抗菌靶点、递送载体及抗菌效果见表 2^[4-13]。

2.2.1 抑制细菌生长代谢的必需基因

丝状温度敏感蛋白Z (filamenting temperature-sensitive mutant Z, FtsZ) 是一种高度保守的细菌管蛋白同源物, 细菌细胞分裂由FtsZ介导, 形成一个收缩环, 将2个子细胞分开^[14]。ftsZ基因被认为是新抗菌剂的理想靶点^[15]。针对ftsZ基因的反义核酸已被证明对铜绿假单胞菌^[16]、肠球菌^[17]和E. coli^[18]等具有显著的抗菌作用。Meng等^[15]将靶向ftsZ mRNA的锁核酸(locked nucleic acid, LNA) 与细胞穿透肽(cell-penetrating peptides, CPPs) 偶联, 生成肽-LNA (peptide-LNA, PLNA)。在上皮细胞培养中, 合成的PLNA787抑制了细菌生长, 提高了感染小鼠生存率, 有较好的体内外抑制作用。

肽聚糖(peptidoglycan, PG) 是组成细菌细胞壁的重要组成成分, PG包裹在细菌外层, 与细菌菌体分裂、形态维持和小分子信号识别息息相关^[19]。自青霉素被发现后, 阻断PG的合成一直是小分子抗生素抗菌的常见靶点方向, 尤其是β-内酰胺类抗生素。在S. aureus中, PG特征性五甘氨酸间肽合成第一步的催化因子是由基因fmhB编码的, 基因fmhB可作为新型抗菌剂的潜在靶标, 用于控制新出现的多重耐药金黄色葡萄球菌感染威胁。ASO可阻断FmhB蛋白的合成, 消耗细菌中细胞壁组装所需的各种酶。研究发现, 对基因起始密码子具有特异性的PNA能通过耗尽细菌FmhB蛋白, 在一定浓度下完全抑制细菌体外生长。靶向革兰氏阴性菌(Gram-negative bacteria, GNB) PG合成机制的ASO已在E. coli^[18]和铜绿假单胞菌^[16]等细菌中进行了相关研究。

在抗生素开发策略中, 针对抑制脂质生物合成是小分子药物相对不常见的抗菌靶点方向^[20]。酰基载体蛋白(acyl carrier protein, ACP) 是细菌脂质延伸的支架^[21], 研究者证明基因acpP主要编码GNB鲍曼不动杆菌^[22]、E. coli^[18]、铜绿假单胞菌^[16]的ACP。Moustafa等^[23]证明了肽偶联PMO可以抑制铜绿假单胞菌中特定必需基因的互补mRNA的翻译, 使其停止生长从而达到抗菌目的, 为铜绿假单胞菌抗生素耐药性相关的问题提供了新方法。

2.2.2 抗生素抗性基因

细菌抵抗抗生素抑制或杀伤作用主要来源于耐药基因和靶点的表达, 这些基因可编码多种耐药机制, 包括抗生素修饰酶、药物外排系统、药物结合位点突变, 以及细菌生理变化(如生物膜形成和膜通透性改变)。开发治疗性佐剂、抑制导致抗生素有效性降低的抗性机制, 是新型抗生素替代方案。小分子抗生素佐剂已广泛用于恢复β-内酰胺抗生素活性, 常见的抗生素β-内酰胺酶抑制剂组合治疗包括阿莫西林-克拉维酸、头孢他啶-阿维巴坦和亚胺培南-曲马巴坦等。抗菌佐剂已经在反义核酸领域进行了探索, 即小分子抗生素的功效通过ASO的加入而使细菌恢复对原有抗生素敏感性, 协同增效。

mecA基因是编码产生青霉素结合蛋白2a的结构基因, 由转座子携带并整合至葡萄球菌染色体的mec部位。mec片段是葡萄球菌染色体上获得的外源片段, 为大约30~50 kb的染色体DNA, mecA基因对几乎所有β-14内酰胺类抗生素产生耐药^[24], 其在葡萄球菌物种中高度保守, 是理想的反义抑制靶点。Meng等^[12]在mecA mRNA编码区发现了一个对反义抑制特别敏感的靶序列(854~871 nt), 随后, 他们通过反义核酸抗生素抑制mecA mRNA的表达, 使MRSA耐药性降低, 为对抗MRSA感染提供了一种有效的抗生素佐剂。

2.2.3 调控细菌的生物膜及细菌毒力

生物膜耐药性主要与细菌生长缓慢和抗菌剂难以扩散有关。YycFG双组分系统普遍存在于细菌中, 以适应外部环境刺激和营养变化, 是细菌应对外界环境的重要信息传递系统^[9]。其中, 表达反应调节子YycG的是一种组氨酸激酶, 在一定刺激下可以磷酸化, 并将磷酸化基转移到同源反应调节因子YycF^[25]。当YycFG缺失时, S. aureus的生存能力被基本抑制。反义核酸可实现S. aureus中YycFG的调控, 在毒力和生物膜代谢表达调控中发挥重要作用。Wu等^{[9, 10]}利用氧化石墨烯(graphene oxide, GO) 作为反义yycF RNA的递送载体, 使反义yycF RNA通过转录后调节和物理切割效应触发强抗菌活性, 加速感染的糖尿病伤口的愈合, 抗菌效果显著。

3 反义核酸抗菌递送的研究进展

收起

虽然, 反义核酸可用于抗菌治疗具有巨大前景, 但由于寡核苷酸分子质量高、高亲水性和负电荷等特性, 导致皮肤、组织渗透率及细胞摄取低下, 特别是在具有药物外排机制的多重耐药性(multi-drug resistance, MDR) 细菌中, 细菌对核酸的摄取更加有限。一般来说, 由于细菌细胞壁结构特殊, 尤其是GNB的外膜, 会进一步加大反义核酸进入菌内的难度^[26]。小分子化合物(如抗生素) 通过外膜狭窄的β-桶状蛋白质进入细菌。相关研究表明, 只有含有胺的小分子具有两亲性、刚性和球状度低等特性, 才能穿过外膜在GNB中聚集。而对于反义核酸来说, 不同取代的反义核酸主链都是极性和亲水的, 往往分布在细胞外液。此外, 除PNA和PMO外, 所有ASO药物均带负电, 不易与带负电的细菌膜发生相互作用。因此, 未经修饰或包裹的核酸无法跨膜运输, 改进ASO递送最常用的策略包括化学修饰改善“类药性”、共价或非共价到靶向或穿透细胞的纳米颗粒上。反义核酸在细菌中的表达与在真核细胞中有很大不同, 并且用于哺乳动物细胞的常规载体, 如脂质体和阳离子聚合物, 对细菌细胞的递送效率低。CPPs是目前用于反义核酸递送的主要方式之一, 其递送效率和载药量也受到了限制。如何在整个抗菌过程中准确追踪或靶向致病菌并实现高效递送, 仍然是抗菌反义核酸开发的一大难题。截至目前, 各种递送载体如图 2所示, 除CPPs^[5]、LNPs^[27]外, 一些DNA纳米结构^[13]、无机纳米结构和新型递送载体^[28]等均在解决抗菌反义核酸的递送上展开了相关探索。

3.1 细胞穿透肽

CPPs也称为蛋白质转导域、“特洛伊木马”肽或转导肽等, 是一大类由10~30个氨基酸组成的短肽。到目前为止发现的CPPs主要包括转录反式激活蛋白、信号转导肽和富含精氨酸序列肽等。在生理pH条件下, 精氨酸含有的胍基可与细胞表面膜上带负电荷的磷酸基团和硫酸基团结合形成氢键, 从而产生细胞内化作用^[29]。作为纳米级转运载体, 大多数CPPs是无毒的, 这使其成为药物治疗、诊断, 以及蛋白质、核酸等大分子递送研究的新工具^[30]。从目前研究报道可以看出, 反义核酸与CPPs共价结合是目前应用最广泛的抗菌治疗方案。

Good等^[5]首次将反义PNA与CPPs偶联, 得到了一种新的PNA-CPPs偶联物。PNA-CPPs或PMO-CPPs的偶联复合物合成路径上较为容易, 且PNA和PMO的电中性, 与带负电荷的ASO相比, 更容易与CPPs偶联。CPPs中最有效的序列是KFFKFFKFFK和RXRRXRRXRXB (X是6-氨基己酸, B是β-丙氨酸)。已有研究表明, KFFKFFKFFK成功介导PMO和PNA进入E. coli、鼠伤寒沙门氏菌(Salmonella typhimurium, S.typhimurium)、MRSA等。在过去的几十年中, ASO-CPPs偶联物已被证明具有良好的体外抗菌效果, 但其疗效(体外10~40 μmol·L^-1) 低于传统抗生素。ASO-CPPs偶联物的进一步开发和应用还需要克服一些问题, 如CPPs耐药诱导, CPPs在体液和血浆中的降解影响ASO-CPPs偶联物的稳定性, ASO-CPPs介导的递送系统缺乏细菌特异性, 以及对它们在不同细菌细胞中的摄取机制仍未探明^[31]。

3.2 脂质纳米颗粒

LNPs通常是由4种脂质成分(可电离的阳离子磷脂、中性辅助磷脂、胆固醇及聚乙二醇修饰的磷脂) 混合形成的直径为50~150 nm的均匀球体, 可载荷各种小核酸及mRNA, 在体内发挥基因沉默作用, 或者翻译成纳米抗体、抗体和抗原发挥治疗作用。其中, 阳离子脂质是LNPs逃离内体的关键成分, 并可高效结合荷负电的核酸^{[32, 33]}, 形成具有极高封装率且中性的LNPs, 并保护其不被降解。Nature杂志在其官方发布的“2022年值得关注的七项技术”中为LNPs保留了位置, 与基因编辑和量子计算等前沿技术同列。尤其是当下, LNPs在有效保护mRNA并将其运输到细胞当中发挥着举足轻重的作用, 不仅是抗击新冠疫情的重要武器—mRNA疫苗的关键成分, 也应用到了医学影像、营养品、农业、化妆品及纳米反应器等其他领域。新一代LNPs, 包括固体脂质纳米粒、纳米结构脂质载体和阳离子脂质-核酸复合物^{[34, 35]}, 表现出更复杂的内部结构和更优秀的物理稳定性^[36]。LNPs能够控制体内药物递送的靶向位置和时间, 可为多种疾病提供治疗方案^{[37, 38]}。

LNPs由于递送系统的成熟性和先进性, 作为反义核酸的载体, 已被应用于对抗MDR细菌、提高抗生素敏感性的相关研究^[39]。Xue等^[31]制备了一种新型的阴离子脂质体, 其介导抗青霉素结合蛋白2a基因可使MRSA在含苯唑西林(6 μg·mL^-1) 的Mueller-Hinton琼脂培养基中的mecA表达降低45%~93%, 并抑制了MRSA的生长, 恢复了耐氟喹诺酮E. coli的抗生素敏感性。此外, 商业化的Lipofectamine^TM 2000也具有包封ASO且抑制细菌繁殖的潜力, 但对哺乳动物细胞显示出明显的抗菌活性的同时, 细胞毒性较大。因此, 人们还需开发能够将ASO高效递送到细菌中且低细胞毒性的LNPs。

3.3 DNA纳米结构

随着DNA纳米技术的快速发展, 一些具有三维稳定纳米结构的DNA纳米材料可作为药物载体, 用于递送多种药物和进行基因治疗^[40]。功能核酸是一类功能超出核酸常规遗传作用的核酸, 主要包括天然的核酶(RNAzymes)、核糖开关, 以及体外通过指数富集配体系统进化技术筛选的aptamers、RNAzymes和脱氧核酶等^[41]。通过Watson-Crick碱基配对规则, 成功地设计和合成了一系列具有明确尺寸、形状、表面化学和功能的自组装DNA纳米结构^[42]。这类通过互补序列的杂交精确地组装成各种DNA多面体线框笼, 例如四面体(tetrahedral DNA nanostructure, TDN)、立方体和二十面体等, 被用作各种领域的研究工具, 并且几种不同形状的DNA纳米结构在递送siRNA和ASO到哺乳动物细胞方面已经有了广泛的研究^[43]。

TDN由于其修饰简单、稳定性高和生物相容性佳, 可作为化疗药物、核酸药物和成像探针的载体^[44], 其连接反义核酸在治疗细菌感染中有了新发展。Readman等^[13]将可抑制E. coli中抗CTX基因表达的PNA加于TDN中使其穿透E. coli的细胞壁, 证明在TDN携带PNA4 (40 μmol·L^-1) 的条件下, CTX对E. coli的MIC从35 mg·L^-1降至16 mg·L^-1。相反, 仅存在PNA时未观察到MIC的降低。在Setyawati等^[45]的另一项研究中, 将放线菌素D插于TDN中传递杀灭细菌, 在这些纳米结构进入细菌细胞后才观察到足够的细菌死亡, 带有放线菌素D的TDN杀灭传染性细菌效果优于游离放线菌素D。这两项研究可以证明TDN可以有效穿透细菌细胞壁, 作为细菌内递送反义核酸的载体。

DNA纳米结构还可以在递送反义核酸的同时, 共递送其他抗菌成分, 实现协同抗菌。Long等^[8]的研究中, 首次使用6HB纳米结构来共递送ASO和Ag⁺。Ag⁺-6HB偶联物对E.coli和S. aureus都表现出优异的抗菌效果, 但ASO-6HB偶联物可以选择性抑制S. aureus, Ag⁺-6HB-ASO共偶联物对S. aureus的抗菌效果增强。从机制而言, Ag⁺-6HB偶联物通过破坏细胞壁而延缓细菌生长, 而靶向S. aureus的ftsZ基因的ASO与6HB偶联后, 则通过抑制ftsZ基因的表达而有效地抑制细菌在对数生长期的生长。进一步说明Ag⁺在S. aureus的生长初期具有延迟作用, ASO在对数期抑制生长。此外, 共递送载体在低浓度下表现出优异的抗菌活性, 对正常哺乳动物细胞没有有害影响, 研究人员推断其选择性可能是由于细菌细胞壁的组成不同引起的。这种由DNA纳米结构共同递送的系统为抗菌治疗提供了一个有前景的平台, 不同形态的DNA纳米结构被不同细菌细胞摄取的效率及穿透细菌细胞壁的机制尚不清楚, 有待进一步研究。

3.4 无机纳米颗粒

3.4.1 氧化石墨烯

GO作为从氧化石墨上剥离下来的单层材料, 由于在表面及边缘上引入大量含氧基团, 可在水溶液及极性溶剂中稳定存在^[46], 因其具有较大表面积和多功能的化学和生物相容性, 可以有效保护复杂活性材料免受酶降解和生物干扰, 用于靶向递送、基因调控、细胞内检测和成像, 具有高摄取效率、高细胞内稳定性和极低毒性^[47]。GO本身具有抗菌功能, 其独特的二维蜂窝状疏水平面结构和其边缘上的羧基和羟基的亲水基团, 可以促进与脂质、蛋白质、DNA等的生物分子的相互作用, 并诱导细菌死亡而无需细胞内过程^[48]。

Wu等^[10]构建了一种新型抗菌、促进愈合的甲基丙烯酰化透明质酸(hyaluronic acid methacryloyl, HAMA)/羧甲基壳聚糖(carboxymethyl chitosan, CMCS) 核壳微针贴片, 以GO为载体包载反义yycF基因, 通过转录后调节和物理切割效应触发强抗菌活性。该微针刺穿生物膜后, 其微针聚合物将部分降解并破坏带负电的细菌细胞壁, GO和反义yycF基因会立即分散到生物膜中, 由GO的截断效应和反义yycF基因的转录后调节共同杀死细菌。微针在S. aureu中释放反义yycF-GO, 控制蛋白yycF的表达和透明质酸酶的分泌, 促使HAMA微针加速降解。同时, GO本身在一定浓度下具有杀菌效果且不伤害宿主细胞, 这使基因转化效率更高, 抗菌效果增强。

3.4.2 金纳米颗粒(Au nanoparticles, AuNPs)

AuNPs是通过Au (I) 或Au (III) 还原成Au (0) 而制备的纳米尺寸颗粒, 因其较大的比表面积来运输药物, 表现出良好的生物相容性^[49]。在过去的几十年中, 具有独特性质的AuNPs已被证明是高效的药物递送载体, 不同ASO与AuNPs的结合已经广泛用于医学研究中。AuNPs能够不断释放金离子, 这些金离子可以黏附在细胞壁和细胞质膜上, 膜蛋白是金离子的重要靶标, 黏附的离子可以改善细胞质膜通透性并导致细菌包膜的破坏^[50]。经共价或非共价缀合与核酸选择性协同修饰后, 核酸链通过硫醇基团共价连接到AuNPs核心上, 递送到其靶标, 可以通过生物内部刺激或外部光激活控制核酸药物的释放产生疗效^[51]。Beha等^[11]使用多层AuNPs作为载体, 包载针对MRSA耐药基因的ASO, 并成功地将ASO递送到MDR细菌中, 诱导抗性基因沉默, 致使高达74%的mecA基因以高选择性、剂量依赖性方式不表达, 从而恢复其对抗生素的敏感性来抑制细菌生长。研究还发现AuNPs本身的物理化学特性, 允许其更容易穿透细菌细胞壁, 与相对被动的真核细胞内吞过程相比更快。也进一步证明AuNPs本身也具有抗菌能力, 可影响细菌和人细胞的代谢功能和生长, 可以在细胞中积累并破坏细胞膜, 引起低的抗微生物活性^[52]。

3.5 以“特洛伊木马”策略作为思路的递送载体

早在20世纪80年代, 科学家就提出应对耐药抗生素的“特洛伊木马”策略, 该策略可简单地认为GNB和革兰氏阳性菌(Gram-positive bacteria, GPB) 都可以强烈且有选择性地“食用”它们的“假冒食品”。利用该特性可搭便车地将具有抗菌活性物质与铁载体^[53]、维生素B₁₂^[54]、特异性糖原^[55]等通过细菌特异的转运系统将抗菌物质转运到细菌细胞内, 从内部将细菌杀死。铁霉素是一种新型抗生素, 其母体抗生素与铁载体相连接, 并通过铁运输途径主动转运至细菌内。Wang等^[56]提出一种双重“特洛伊木马”策略, 儿茶酚可以作为铁载体被铜绿假单胞菌识别并主动摄取, 使金属-铁霉素复合物同时通过铁载体受体转运进细胞内, 以此达到协同抗菌效果, 提高铁霉素抗菌活性。在传递抗菌活性分子方面, “特洛伊木马”递送策略能够将药物分子传递到特定药物或类药物分子的靶定位点, 展示出了一定前景。

维生素B₁₂是一种天然的有机金属分子, 是哺乳动物代谢中的必需营养辅助因子^[57]。维生素B₁₂不能在人体内合成, 因此必须包含在饮食中, 靠体内细菌摄取吸收。在微生物学研究中发现, 大多数需氧细菌需要维生素B₁₂才能生长, 在细菌中存在完善的维生素B₁₂摄取机制, 这使得维生素B₁₂成为一种易于开发的药物载体候选物^[48]。维生素B₁₂已被用作哺乳动物细胞中的递送载体, 为了利用维生素B₁₂天然吸收特性, 有研究将该分子修饰并缀合至PNA寡聚物上, 在某些位置的缀合显著改变了哺乳动物细胞中维生素B₁₂的结合特性^[58]。所以, 如何在PNA转运中成功利用维生素B₁₂的摄取途径, 且不影响PNA与其靶标的相互作用是研究的重点。Równicki等^[28]采用铜催化的1, 3-偶极环加成反应合成PNA-维生素B₁₂偶联物, 作用于E. coli和S.typhimurium。研究发现维生素B₁₂可以将PNA转运到E. coli内, 且比广泛使用的CPPs更有效, 在E. coli和S.typhimurium中的反义抑制效力不同, 这主要是由于细胞壁结构和维生素B₁₂摄取机制存在差异。

ABC转运蛋白(ATP-binding cassette transporter, ABC transporter) 是一类依靠ATP水解提供能量跨膜转运底物的蛋白质家族, 使细菌吸收和代谢α(1-4)-糖苷键连接的葡萄糖聚合物^[59]。目前, 研究者们已经利用细菌特异性ABC转运蛋白成功地将修饰有葡萄糖聚合物(glucose polymer, GP) 的纳米颗粒转运到细菌中。Liu等^[4]将反义PNA偶联在GP包裹的荧光硅纳米颗粒上, 该纳米颗粒利用细菌特异性ABC转运蛋白途径被多种细菌选择性吞噬, 递送示意图如图 3所示。研究还进一步证明, ABC糖转运体突变的细菌很难内化该纳米颗粒。ASO进入细菌后, 会与靶点序列结合, 选择性抑制细菌中的基因表达, 有效地杀死细菌。其中, 反义PNA由两个连续块组成: Ec108 acpP和Sau101 fmhB。Ec108 acpP内化到E. coli细胞内后, 与acpP转录形成的mRNA的起始密码区相结合, 激活内源性RNA酶降解mEc108 acpP, 抑制脂肪酸的合成。Sau101 fmhB内化到S. aureus细胞内后, 会与fmhB转录形成的mRNA的起始密码区相结合, 激活内源性RNA酶降解Sau101 fmhB, 抑制PG的合成。

4 展望与挑战

收起

细菌耐药性问题是治疗细菌感染的主要障碍, 寻找疗效好、毒副作用小的抗菌治疗策略, 从而达到解决细菌耐药性的目的是当前研究热点。反义核酸突破以细菌蛋白为靶点的经典药物研究思路, 以细菌耐药、致病、生长繁殖及生物膜形成等基因为作用靶点, 通过阻断相关基因表达来抑制或者杀灭细菌, 具有特异性强、研发成本低的优点。通过近几十年研究, ASO对致病菌的必需基因、耐药基因等基因表达的抑制作用已被不同研究广泛证实, 各种递送载体, 如CPPs、LNPs、DNA纳米结构、无机纳米结构等, 均在解决抗菌反义核酸的递送上展开了相关探索。本综述围绕近些年小核酸药物的发展现状, 反义抗菌的抗菌机制、靶点及序列, 以及在反义核酸抗菌递送载体等方面进行了总结归纳。

总体来看, 部分策略在有效抗菌、逆转细菌耐药性等方面取得了可喜的成果, 但仍缺乏相关摄取机制的探索及临床试验数据的支撑。在未来工作中, 深入探讨反义靶点与抗菌机制之间的联系, 利用分子生物学等技术探究不同细菌对不同反义核酸载体的摄取机制, 仍有广泛的研究空间。然而, 基于ASO的抗菌药物的研究与开发仍然存在很多问题, 如递送系统的优化及耐药性评估等。因此, 仍然需要不断推进反义核酸抗菌的临床研究, 为反义抗菌在临床应用提供充分理论依据和实验数据。

作者贡献: 李佳和韩晓璐具体完成文章撰写; 宋世昱完成文献检索和筛选; 林锦涛和唐志强完成文章的修改; 王增明和徐亮确定文章方向及内容组成; 郑爱萍对文章撰写质量进行把关和审校。

利益冲突: 文章内容不涉及相关利益冲突, 未涉及任何厂家及相关经济组织直接或间接的经济或利益的赞助。

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军队后勤科研重点项目(BWS21T015)

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2025年第60卷第2期

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doi: 10.16438/j.0513-4870.2024-0607

接收时间：2024-07-01
首发时间：2025-11-07
出版时间：2025-02-12

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收稿日期：2024-07-01
修回日期：2024-11-20

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军队后勤科研重点项目(BWS21T015)

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1.军事医学研究院国家安全特需药品全国重点实验室, 北京 100850

2.中国人民解放军32104部队, 内蒙古阿拉善盟 735400

3.重庆医科大学, 重庆 400016

4.延边大学药学院, 吉林延吉 133002

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^*徐亮, Tel: 13366515305, E-mail: wj24998@163.com

郑爱萍, Tel: 86-10-66931694, E-mail: apzheng@163.com

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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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No.	Drug name/commercial name	Type	Owner	Time-to-market/place of approval	Therapeutic indication	Retouching/delivery	Mode of administration
1	Formivirsen/Vitravene	ASO	Ionis/Novartis	1998, FDA (Delisted)	CMVR	PS	IVI
2	Mipomersen/Kynamro	ASO	Ionis/Kastle Therapeutics	2013, FDA (Delisted)	HOFH	PS, 2′-O-MOE	SC
3	Nusinersen/Spinraza	ASO	Ionis/Biogen	2016, FDA	SMA	PS, 2′-O-MOE	IT
4	Eteplirsen/Exondys 51	ASO	Sarepta Therapeutics	2016, FDA	DMD	PMO	IV
5	Inotersen/Tegsedi	ASO	Ionis	2018, FDA	hATTR	PS, 2′-O-MOE	SC
6	Golodirsen/Vyondys 53	ASO	Sarepta Therapeutics	2019, FDA	DMD	PMO	IV
7	Volanesorsen/Waylivra	ASO	Ionis	2019, EU	FCS	PS, 2′-O-MOE	SC
8	Viltolarsen/Viltepso	ASO	Nippon Shinyaku	2020, FDA	DMD	PMO	IV
9	Casimersen/Amondys 45	ASO	Sarepta Therapeutics	2021, FDA	DMD	PMO	IV
10	Patisiran/Onpattro	siRNA	Alnylam	2018, FDA	hATTR	PS, 2′-F, 2′-O-Me/ LNPs	IV
11	Givosiran/Givlaari	siRNA	Alnylam	2019, FDA	AHP	PS, 2′-F, 2′-O-Me/ GalNAc	SC
12	Lumasiran/Oxlumo	siRNA	Alnylam	2020, FDA	PH1	PS, 2′-F, 2′-O-Me/ GalNAc	SC
13	Inclisiran/Leqvio	siRNA	Alnylam/Novartis	2020, EU 2021, FDA	FP	PS, 2′-F, 2′-O-Me/ GalNAc	SC
14	Pegaptanib/Macugen	Aptamer	Pfizer/Eyetech,	2004, FDA (Delisted)	AMD	——	IVI
15	Vutrisiran/Amvuttra	siRNA	Alnylam	2022, FDA	hATTR	PS, 2′-F, 2′-O-Me/ GalNAc	SC
16	Eplontersen/Wainua	ASO	Ionis/Astra Zeneca	2023, FDA	hATTR	PS, 2′-O-Me/ GalNAc	SC
17	Tofersen/Qalsody	ASO	Ionis/Biogen	2023, FDA	ALS	PS, 2′-O-Me	IT
18	Nedosiran/Rivfloza	siRNA	Dicerna (Novo Nordisk)/ Alynlam	2023, FDA	PH1	PS, 2′-F, 2′-O-Me/ GalNAc	SC
19	Avacincaptad pegol/Lzervay	Aptamer	Lveric Bio/Archemix	2023, FDA	GA	——	IVI

No.

Drug name/commercial name

Type

Owner

Time-to-market/place of approval

Therapeutic indication

Retouching/delivery

Mode of administration

Formivirsen/Vitravene

ASO

Ionis/Novartis

1998, FDA (Delisted)

CMVR

IVI

Mipomersen/Kynamro

ASO

Ionis/Kastle Therapeutics

2013, FDA (Delisted)

HOFH

PS, 2′-O-MOE

Nusinersen/Spinraza

ASO

Ionis/Biogen

2016, FDA

SMA

PS, 2′-O-MOE

Eteplirsen/Exondys 51

ASO

Sarepta Therapeutics

2016, FDA

DMD

PMO

Inotersen/Tegsedi

ASO

Ionis

2018, FDA

hATTR

PS, 2′-O-MOE

Golodirsen/Vyondys 53

ASO

Sarepta Therapeutics

2019, FDA

DMD

PMO

Volanesorsen/Waylivra

ASO

Ionis

2019, EU

FCS

PS, 2′-O-MOE

Viltolarsen/Viltepso

ASO

Nippon Shinyaku

2020, FDA

DMD

PMO

Casimersen/Amondys 45

ASO

Sarepta Therapeutics

2021, FDA

DMD

PMO

Patisiran/Onpattro

siRNA

Alnylam

2018, FDA

hATTR

PS, 2′-F, 2′-O-Me/
LNPs

Givosiran/Givlaari

siRNA

Alnylam

2019, FDA

AHP

PS, 2′-F, 2′-O-Me/
GalNAc

Lumasiran/Oxlumo

siRNA

Alnylam

2020, FDA

PH1

PS, 2′-F, 2′-O-Me/
GalNAc

Inclisiran/Leqvio

siRNA

Alnylam/Novartis

2020, EU
2021, FDA

PS, 2′-F, 2′-O-Me/
GalNAc

Pegaptanib/Macugen

Aptamer

Pfizer/Eyetech,

2004, FDA (Delisted)

AMD

——

IVI

Vutrisiran/Amvuttra

siRNA

Alnylam

2022, FDA

hATTR

PS, 2′-F, 2′-O-Me/
GalNAc

Eplontersen/Wainua

ASO

Ionis/Astra Zeneca

2023, FDA

hATTR

PS, 2′-O-Me/
GalNAc

Tofersen/Qalsody

ASO

Ionis/Biogen

2023, FDA

ALS

PS, 2′-O-Me

Nedosiran/Rivfloza

siRNA

Dicerna (Novo Nordisk)/
Alynlam

2023, FDA

PH1

PS, 2′-F, 2′-O-Me/
GalNAc

Avacincaptad pegol/Lzervay

Aptamer

Lveric Bio/Archemix

2023, FDA

——

IVI

No.	Strain	Target	Mechanism	Delivery carrier	Result	Ref.
1	E. coli, MRSA	acpP mRNA	Inhibition of fatty acid synthesis	GP-SiNPs	100% Kill human-derived resistant bacteria; the uptake efficiency (51.6%) surpassed that of CPPs. The MIC for E. coli and S. aureus was reduced to 0.8 μmol·L^-1	[4]
2	E. coli K12	The start codon region of acpP mRNA	Inhibition of fatty acid synthesis	CPPs	The MIC of anti-acpP peptide-PNAs was reduced to 0.2 μmol·L^-1	[5]
3	S. aureus	fmhB mRNA	Inhibition of peptidoglycan synthesis Inhibition of DNA replication	CPPs	The growth of S. aureus was effectively inhibited in a dose-dependent using PNAs	[6]
4	E. coli, S. aureus, MRSA	ftsZ genes	Inhibition of the ftsZ gene, thereby inhibiting bacterial division	ZIF-8	Synergistic, broad-spectrum antibacterial; the biofilm removal efficacy reached 88.2%, while the MIC exhibited a reduction to 16 μg·mL^-1	[7]
5	S. aureus	ftsZ genes	Inhibition of the ftsZ gene, thereby inhibiting bacterial division	6HB	ASO and Ag⁺ exhibit a synergistic effect in antimicrobial therapy, providing a broad-spectrum antimicrobial activity	[8]
6	S. aureus	yycF mRNA	Regulation negatively of yycF transcription by the two-component system, regulates formation of biofilm	GO	The onset of the log phase was delayed by a duration of 3h; there was an approximate twofold reduction in biofilm formation	[9, 10]
7	MRSA, Staphylococcus epidermidis	mecA genes	Inhibition of mecA gene expression	AuNPs, PEI, MLAuNPs	The uptake efficiency was observed to be 74%; when combined with oxacillin, a significant reduction of 29% in MRSA was achieved	[11]
8	MRSA	mecA genes	Inhibition of mecA gene expression	Anionic liposome	The expression of penicillin-binding protein 2a is inhibited, thereby enhancing the affinity of antibiotics; combined with oxacillin, the MIC was reduced to 0.13 μg·mL^-1	[12]
9	E. coli	β-Lactamase CTX-M-group 1	Inhibition of the expression of β-lactamase CTX-M-group 1	Tetrahedral framework	Reinstatement of cefotaxime susceptibility in strains exhibiting susceptible phenotypes. The MIC of cefotaxime (CTX 16 mg·L^-1) was reduced from 35 to 16 mg·L^-1	[13]

No.

Strain

Target

Mechanism

Delivery carrier

Result

Ref.

E. coli, MRSA

acpP mRNA

Inhibition of fatty acid synthesis

GP-SiNPs

100% Kill human-derived resistant bacteria; the uptake efficiency (51.6%) surpassed that of CPPs. The MIC for E. coli and S. aureus was reduced to 0.8 μmol·L^-1

[4]

E. coli K12

The start codon region of acpP mRNA

Inhibition of fatty acid synthesis

CPPs

The MIC of anti-acpP peptide-PNAs was reduced to 0.2 μmol·L^-1

[5]

S. aureus

fmhB mRNA

Inhibition of peptidoglycan synthesis
Inhibition of DNA replication

CPPs

The growth of S. aureus was effectively inhibited in a dose-dependent using PNAs

[6]

E. coli,
S. aureus, MRSA

ftsZ genes

Inhibition of the ftsZ gene, thereby inhibiting bacterial division

ZIF-8

Synergistic, broad-spectrum antibacterial; the biofilm removal efficacy reached 88.2%, while the MIC exhibited a reduction to 16 μg·mL^-1

[7]

S. aureus

ftsZ genes

Inhibition of the ftsZ gene, thereby inhibiting bacterial division

6HB

ASO and Ag⁺ exhibit a synergistic effect in antimicrobial therapy, providing a broad-spectrum antimicrobial activity

[8]

S. aureus

yycF mRNA

Regulation negatively of yycF transcription by the two-component system, regulates formation of biofilm

The onset of the log phase was delayed by a duration of 3h; there was an approximate twofold reduction in biofilm formation

[9, 10]

MRSA, Staphylococcus epidermidis

mecA genes

Inhibition of mecA gene expression

AuNPs, PEI, MLAuNPs

The uptake efficiency was observed to be 74%; when combined with oxacillin, a significant reduction of 29% in MRSA was achieved

[11]

MRSA

mecA genes

Inhibition of mecA gene expression

Anionic liposome

The expression of penicillin-binding protein 2a is inhibited, thereby enhancing the affinity of antibiotics; combined with oxacillin, the MIC was reduced to 0.13 μg·mL^-1

[12]

E. coli

β-Lactamase CTX-M-group 1

Inhibition of the expression of β-lactamase CTX-M-group 1

Tetrahedral framework

Reinstatement of cefotaxime susceptibility in strains exhibiting susceptible phenotypes. The MIC of cefotaxime (CTX 16 mg·L^-1) was reduced from 35 to 16 mg·L^-1

[13]