药学学报

Name	Company	Treatment of diseases	Target	Delivery route	Status	Reference
	Endonuclease (RNase H)-mediated RNA degradation
Mipomersen	Ionis	Familial hypercholesterolemia	apo-B-100	Subcutaneous	Approved	[13]
Inotersen	Ionis	Familial amyloid polyneuropathy	Transthyretin	Subcutaneous	Approved	[14]
Volanesorsen	Ionis	Familial chylomicronemia syndrome	apoC3	Subcutaneous	Approved	[15]
QR-1123	ProQR	Retinitis pigmentosa (adRP)	Rhodopsin	Intravitreal	Phase Ⅱ	[17]
FB-LRX	Ionis	Age-related macular degeneration	CFB	Subcutaneous	Phase Ⅱ	[18]
	siRNA interference
Patisiran	Alnylam	Familial amyloid polyneuropathy	TTR	Intravenous	Approved	[20]
Givosiran	Alnylam	Hemophilia (AHP)	ALAS1	Subcutaneous	Approved	[21]
Lumasiran	Alnylam	Hyperoxaluria	GO	Subcutaneous	Approved	[22]
Inclisiran	Alnylam	Hypercholesterolemia	PCSK9	Subcutaneous	Approved	[23]
Nedosiran	Dicerna	Primary hyperoxaluria	LDHA	Subcutaneous	Phase Ⅲ	[55]
Cemdisiran	Alnylam	Paroxysmal hemoglobinuria	C5	Subcutaneous	Phase Ⅲ	[56]
QPI-1007	Quark	Optic nerve injuries	Caspase-2	N.a.	Phase Ⅲ	[57]
ARO-APOC3	Arrowhead	Hyperlipoproteinemia type I	APOCⅢ	Subcutaneous	Phase Ⅲ	[58]
Teprasiran	Quark	Acute kidney injury	P53	Intravenous	Phase Ⅲ	[59]
Fiturisan	Alnylam	Hemophilia	AT3	Subcutaneous	Phase Ⅲ	[60]
Tivanisiran	Sylentis	Xerophthalmia	TRPV1	Subcutaneous	Phase Ⅲ	[61]
ARO-ANG-3	Arrowhead	Familial hypercholesterolemia	ANGPTL3	N.a.	Phase Ⅱ	[62]
	Splicing regulatory mechanism
Drisapersen	Biomarin	Duchenne muscular dystrophy	Dystrophin	N.a.	Exit R & D	[46]
Eteplirsen	Sarepta	Duchenne muscular dystrophy	Dystrophin	Intravenous	Approved	[47]
Casimersen	Sarepta	Duchenne muscular dystrophy	Dystrophin	Intravenous	Approved	[49]
Golodirsen	Sarepta	Duchenne muscular dystrophy	Dystrophin	Intravenous	Approved	[48]
Spinraza	Ionis	Spinal muscular atrophy	SMN2	Intravenous	Approved	[63]
Viltolarsen	Shinyaku	Duchenne muscular dystrophy	Dystrophin	Intravenous	Approved	[64]
Sepofarsen	ProQR	Leber congenital amaurosis	CEP290	Intravenous	Phase Ⅲ	[54]
QR-421a	ProQR	Usher syndromes	USH2A	Intravenous	Phase Ⅲ	[53]

Name	Company	Treatment of diseases	Target	Delivery route	Status	Reference
	Endonuclease (RNase H)-mediated RNA degradation
Mipomersen	Ionis	Familial hypercholesterolemia	apo-B-100	Subcutaneous	Approved	[13]
Inotersen	Ionis	Familial amyloid polyneuropathy	Transthyretin	Subcutaneous	Approved	[14]
Volanesorsen	Ionis	Familial chylomicronemia syndrome	apoC3	Subcutaneous	Approved	[15]
QR-1123	ProQR	Retinitis pigmentosa (adRP)	Rhodopsin	Intravitreal	Phase Ⅱ	[17]
FB-LRX	Ionis	Age-related macular degeneration	CFB	Subcutaneous	Phase Ⅱ	[18]
	siRNA interference
Patisiran	Alnylam	Familial amyloid polyneuropathy	TTR	Intravenous	Approved	[20]
Givosiran	Alnylam	Hemophilia (AHP)	ALAS1	Subcutaneous	Approved	[21]
Lumasiran	Alnylam	Hyperoxaluria	GO	Subcutaneous	Approved	[22]
Inclisiran	Alnylam	Hypercholesterolemia	PCSK9	Subcutaneous	Approved	[23]
Nedosiran	Dicerna	Primary hyperoxaluria	LDHA	Subcutaneous	Phase Ⅲ	[55]
Cemdisiran	Alnylam	Paroxysmal hemoglobinuria	C5	Subcutaneous	Phase Ⅲ	[56]
QPI-1007	Quark	Optic nerve injuries	Caspase-2	N.a.	Phase Ⅲ	[57]
ARO-APOC3	Arrowhead	Hyperlipoproteinemia type I	APOCⅢ	Subcutaneous	Phase Ⅲ	[58]
Teprasiran	Quark	Acute kidney injury	P53	Intravenous	Phase Ⅲ	[59]
Fiturisan	Alnylam	Hemophilia	AT3	Subcutaneous	Phase Ⅲ	[60]
Tivanisiran	Sylentis	Xerophthalmia	TRPV1	Subcutaneous	Phase Ⅲ	[61]
ARO-ANG-3	Arrowhead	Familial hypercholesterolemia	ANGPTL3	N.a.	Phase Ⅱ	[62]
	Splicing regulatory mechanism
Drisapersen	Biomarin	Duchenne muscular dystrophy	Dystrophin	N.a.	Exit R & D	[46]
Eteplirsen	Sarepta	Duchenne muscular dystrophy	Dystrophin	Intravenous	Approved	[47]
Casimersen	Sarepta	Duchenne muscular dystrophy	Dystrophin	Intravenous	Approved	[49]
Golodirsen	Sarepta	Duchenne muscular dystrophy	Dystrophin	Intravenous	Approved	[48]
Spinraza	Ionis	Spinal muscular atrophy	SMN2	Intravenous	Approved	[63]
Viltolarsen	Shinyaku	Duchenne muscular dystrophy	Dystrophin	Intravenous	Approved	[64]
Sepofarsen	ProQR	Leber congenital amaurosis	CEP290	Intravenous	Phase Ⅲ	[54]
QR-421a	ProQR	Usher syndromes	USH2A	Intravenous	Phase Ⅲ	[53]

反义药物作用机制及化学修饰研究进展

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李磊 , 郑艺璇 , 马春英 , 冯文化 ^*

药学学报 | 综述 2022,57(9): 2709-2719

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药学学报 | 综述 2022, 57(9): 2709-2719

反义药物作用机制及化学修饰研究进展

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李磊, 郑艺璇, 马春英, 冯文化^*

作者信息

中国医学科学院、北京协和医学院药物研究所, 北京 100050

通讯作者:

*冯文化, E-mail: fwh@imm.ac.cn

Research progress in antisense drug mechanism and chemical modification strategies

Lei LI, Yi-xuan ZHENG, Chun-ying MA, Wen-hua FENG^*

Affiliations

Institute of Materia Medical, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

出版时间: 2022-09-12 doi: 10.16438/j.0513-4870.2022-0479

文章导航

摘要

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在过去的30年里, 越来越多的反义药物获批上市或进入临床, 反义技术凭借其在治疗疾病方面独特的优势和在临床上较强的发展潜力, 已成为新药研究领域的焦点。与传统的化药小分子及大分子蛋白生物药物存在较大差异, 是一种非常独立的药物形式, 起源于用于治疗单基因突变的疾病, 但近期逐渐开始用于常见病的治疗。因此, 基于遗传学的基础合理进行反义药物设计对于疾病的治疗至关重要。本文针对当前反义药物的作用机制、化学修饰、递送策略领域的最新进展进行了阐述, 并对目前存在的瓶颈问题进行了分析, 相信伴随着这些问题的解决, 反义药物的研究可以到达一个新的高度。

关键词

反义药物 / 反义药物设计 / 核酸靶点 / 化学修饰 / 药物递送

Abstract

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Over the past three decades, more and more antisense drugs have been approved for marketing or clinical trails. Antisense technology has become the focus of pharmaceutical research due to its unique advantages in treating diseases and strong clinical development potential. There is a big difference from traditional small molecule chemical drugs, and macromolecular protein biological drugs. Antisense drugs are a very independent drug form. Antisense drugs were initially used to treat diseases with single gene mutations, but recently they have gradually begun to be used for the treatment of common diseases. Rational antisense drug design is crucial for disease treatment based on genetics. This paper reviews the latest progress in the field of action mechanism, chemical modification and delivery strategy of antisense drugs, and analyzes the current intractable problems. It is believed that with the resolution of these problems, the research of antisense drugs can reach a new level.

Key words

antisense drug / antisense drug design / nucleic acid target / chemical modification / drug delivery

引用本文

李磊, 郑艺璇, 马春英, 冯文化. 反义药物作用机制及化学修饰研究进展. 药学学报, 2022 , 57 (9) : 2709 -2719 . DOI: 10.16438/j.0513-4870.2022-0479

Lei LI, Yi-xuan ZHENG, Chun-ying MA, Wen-hua FENG. Research progress in antisense drug mechanism and chemical modification strategies[J]. Acta Pharmaceutica Sinica, 2022 , 57 (9) : 2709 -2719 . DOI: 10.16438/j.0513-4870.2022-0479

正文

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基因表达过程为: 前体RNA (Pre-mRNA) 在细胞核中由DNA转录合成, 经过加工与修饰形成信使RNA (mRNA), 之后mRNA从细胞核运送到细胞质, 在细胞质中翻译出蛋白质并随后降解^[1]。然而蛋白质的异常表达或非功能蛋白质的产生会导致机体疾病的发生。过去几十年里新药研发的策略是利用小分子药物纠正异常蛋白质功能, 从而改变细胞发生的不正常生理活动^[2]。但小分子药物研发有一些难以避免的缺陷和不足, 难以发现具有明确作用机制的靶点, 即由靶点开发出安全有效的小分子药物越来越难。随着基因表达机制的深入研究, 科学家们开始将目光投注到遗传物质中, 其中之一是利用反义技术构建反义药物, 从而达到治疗疾病的目的。这是一种具有远大前景的药物研发方法。

反义技术是一种根据碱基互补配对原理开发的基因调控方法, 利用天然存在或人工合成的互补寡核苷酸片段, 与目的基因(单链、双链DNA) 或mRNA的特定序列相结合, 从基因复制、转录、剪接、翻译等水平上调节靶基因的表达, 干扰遗传信息从核酸向蛋白质的传递, 从而达到抑制、封闭或破坏靶基因的目的, 达到治疗效果。

反义药物是根据这一原理开发的药物, 通常是指反义寡核苷酸^[3], 一般设计为20~30个核苷酸组成的短链, 可干扰遗传信息的传递, 精准调控蛋白质的合成, 用于治疗由于基因突变引起的疾病^[4]。与以蛋白质作为靶点的药物相比, 反义技术更容易合理设计新药物。反义药物具有极大的临床应用价值和发展前景, 了解目前反义药物的作用机制、化学修饰及递送策略的研究进展是有必要的。

1 反义药物作用机制

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目前反义药物涉及的作用机制主要有3种: 核糖核酸酶H (RNase H) 介导RNA降解、RNA干扰(RNAi) 介导的转录本降解及剪接调节(splice-modulating) 机制。本文对以上作用机制及相关反义药物进行总结。

1.1 核糖核酸酶H (RNase H) 介导RNA降解机制

利用细胞内广泛表达的核糖核酸内切酶RNase H特异性地水解DNA-RNA杂合链中的RNA的机制^[5]。设计单链寡核苷酸(反义药物) 递送到体内, 与目标mRNA结合时, 结合物被细胞核和细胞质中存在的RNase H识别并降解, 阻断蛋白质的产生, 从而达到治疗疾病的目的。

根据这一原理, 选择疾病相关的特定mRNA序列, 根据碱基互补配对原理, 利用合成的特异互补寡核苷酸片段与目标核酸序列结合以此调整蛋白功能。这类反义药物的结构特征是两端通常为化学修饰的RNA碱基, 包裹住中央称为Gap的8~10个DNA碱基^[6]。两个主要的部分分别起到不同的作用, RNA碱基可增强对互补序列的亲和力, 与目标mRNA结合, 而DNA碱基则可作为核酸内切酶的识别底物。通过诱导RNase H1切割破坏目标mRNA, 从而发挥反义药物的治疗效果(图 1), 这也是治疗性寡核苷酸药物设计的关键特征^{[7, 8]}。

此类寡核苷酸结合RNA并激活RNase H的机制已经被充分研究, Furdon等^[9]发现含有甲基磷酸酯、硫代磷酸酯和磷酸二酯键的寡核苷酸结合RNA可被RNase H切割。例如反义药物inotersen: 分子两端5个核苷酸分别为2位氧-甲氧乙基糖修饰的核苷酸, 形成结构为5-10-5的间隔反义分子, 此外在序列5′端所有的嘧啶碱基均进行甲基化修饰以增强与互补mRNA的亲和力, inotersen包含的20个碱基对分别为5′UCUUGGTTACATGAAAUCCC3′^[10], 此药物可与靶标肝脏TTR mRNA结合, 随后激活RNase H1, 进而切割并降解mRNA, 使得产生的TTR蛋白减少, 形成较少的淀粉样蛋白原纤维, 达到治疗疾病遗传性转甲状腺素淀粉样变性的目的^[11]。

在过去几年中, 应用RNase H1机制开发的药物发展迅速, 其中, 已经有3种药物被批准用于临床治疗, 分别是: mipomersen、inotersen和volanesoren^[12], 全部由Ionis制药公司研发。

Mipomersen是一种靶向apo-B-100 mRNA的由20个碱基排列而成的寡核苷酸, 通过肌肉注射, 降低血浆胆固醇水平治疗家族性高胆固醇血症(FH)^[13]; inotersen被批准用于治疗致命性的遗传性转甲状腺素淀粉样变性(hATTR)^[14]。通过RNase H1降解转甲状腺素mRNA, 阻止转甲状腺素淀粉样蛋白沉积在周围神经系统中, 以此达到治疗效果; volanesorsen靶向apo C3 RNA, 用于治疗家族性乳糜微粒血症综合征(FCS)^[15]。

治疗肝、肺、眼部、肌肉、中枢神经系统及肿瘤细胞的反义药物正处于临床开发阶段^[16]。QR-1123^[17]目前正由ProQR公司进行临床试验, 通过RNase H1特异性识别并敲除突变等位基因以此抑制突变的视紫红质蛋白, 从而增加视网膜感光细胞中的野生型视紫红质功能蛋白, 治疗由视紫红质基因突变引起的常染色体显性视网膜色素变性(adRP); 另一种正在进行临床试验的寡核苷酸FB-L_RX^[18], 皮下注射后靶向肝脏中的补体因子B (CFB基因), 用于治疗年龄相关性黄斑变性(AMD)。

1.2 RNA干扰(RNAi) 介导的转录本降解机制

RNAi是由与目标基因互补的双链小干扰RNA (siRNA) 诱发, 从而引起同源mRNA高效特异性降解关闭其功能, 显著降低目标RNA和蛋白质水平^[19], 以此达到治疗目的。

小干扰RNA是一种双链RNA分子, 其中一条为引导链, 另一条为承载链, 引导链与目的基因序列的编码互补, 通过沃森克里克碱基对特异性地与目标RNA特定序列杂交。siRNA长度通常为21~23个核苷酸, 可与Argonaute蛋白等生物大分子形成RNA诱导沉默复合体(RISC), 由于Argonaute蛋白中部分结构域有RNase H切割活性, 当结合为复合物且siRNA的感知链(承载链) 被降解后, RISC的切割活性被激活, 利用siRNA引导链引导靶向切割靶mRNA, 致使基因沉默(图 1)。

由于siRNA需与目标RNA结合且siRNA具有高度特异性, 故siRNA引导链与目标mRNA特定序列完全互补, 即感知链与目标mRNA特定序列一致, 在设计小干扰RNA时, 根据mRNA靶标序列设计其序列结构, 并通过适当的化学修饰增强其在体内的稳定性。如反义药物patisiran是一种化学合成的双链寡核苷酸, 由21个核苷酸组成, 反义链结构为5′AUGGAAU ACUAUUGGUUACTT3′, 其中一些核苷酸进行了2位氧甲氧基修饰以提高稳定性。Patisiran与目标mRNA结合, 激活RISC切割靶RNA活性, 进而减少蛋白产生, 减少淀粉样蛋白原纤维的生成, 达到治疗疾病的目的。

根据此类作用机制开发药物是非常有前景的, 目前共有4种成功上市的siRNA药物, 均由美国的Alnylam制药公司研发, 分别是治疗转甲状腺素运载蛋白淀粉样变性(hATTR) 的patisiran^[20]、治疗急性肝卟啉的givosiran^[21]、治疗原发性高草酸尿Ⅰ型的iumasiran^[22]和治疗原发性高胆固醇血症的inclisiran^[23]。其中inclisiran药物使得反义药物研发从罕见病走向常见病。

Patisiran是第一个被批准的siRNA药物。除了编码链上的所有嘧啶和反义链上的两个尿苷甲基化外, patisiran大多未经修饰, 主要靶向野生型和突变型转录本的3′非翻译区域的一个保守序列, 以脂质纳米颗粒(LNPs) 的方式静脉给药, 可提高药物的生物利用度、促进细胞摄取和核内体逃逸^[24]。因此, patisiran不仅被认为是RNA治疗领域的一个关键里程碑, 同时也为快速开发Covid-19的mRNA疫苗铺平了道路^[25]。Givosiran是一种2位甲氧基和部分磷硫化(PS) 修饰的siRNA药物, 靶向肝脏中氨基乙酰丙酸合成酶1 (ALAS1) mRNA。Givosiran不是依赖LNPs来靶向肝细胞, 而是与一种被称为N-乙酰半乳糖胺(GalNAc) 的三触角偶连体结合。这种结合方式可以改善受体肝细胞中siRNA的递送效果^[26], 使药物在组织中缓慢释放, 并允许皮下给药。Lumasiran是一种靶向乙醇酸氧化酶mRNA的反义药物, 也通过皮下注射给药, 相似的还有inclisiran, 靶向PCSK9转录本。这两种siRNA药物的化学性质与givosiran相似^[27]。

不仅如此, 正在进行临床研究阶段的siRNA药物不胜枚举。已处于临床Ⅲ期的药物有: Dicerna制药研发的治疗原发性高草酸尿症的药物nedosiran, Alnylam公司研发的治疗阵发性血红蛋白尿症的药物cemdisiran, Quark公司研发的治疗视神经损伤的药物QPI-1007, Arrowhead公司研发的治疗Ⅰ型高脂蛋白血症的药物ARO-APOC3, Quark公司研发的治疗急性肾损伤的药物teprasiran, Alnylam公司研发的治疗血友病的fitusiran和Sylentis公司研发的治疗干眼症的tivanisiran等; 处于临床Ⅱ期的siRNA药物则更多, 如Arrowhead公司研发的治疗纯合子家族性高胆固醇血症的ARO-ANG3等。

然而, 基于RNA干扰机制的反义药物上市前也需要解决很多难题。首先是siRNA的序列设计、选择和优化, 并遵循必要的规则以此产生更有效和更具靶向特异性的siRNA^{[28, 29]}; 其次, 由于对类似靶mRNA的非特异性杂交, 干扰过程可能会产生脱靶效应^[30]; 此外, 对于以任何反义机制为基础的治疗性药物设计, 都面临着一些共同的挑战, 如RNA本身容易受到体内核酸酶的降解、外源引入的RNA可能会诱导免疫应答等问题^[31]。由于这些问题的存在, 早期siRNA药物, 例如用于治疗湿性年龄相关性黄斑变性(wAMD) 的bevasiranib和AGN211745, 临床试验未能达到指标^[32]。

此外, 科学家注意到微小RNA (miRNA) 可以像siRNA一样抑制基因表达, 被认为是一种具有潜力的基因沉默疗法^[33]。但是与siRNA相比, miRNA与mRNA的结合机制更不完善, 且不能只靶向单个转录本, 因此目前该技术的发展非常缓慢^{[34, 35]}。

1.3 剪接调节(splice-modulating)机制

在前体mRNA剪接形成mRNA过程中, 非编码的内含子序列被移除, 编码片段(外显子) 被连接在一起的过程称为剪接, 发生在被称为剪接体(spliceosome) 的大型RNA-蛋白质复合物中^[36], 在该复合物中, hnRNA中的重要序列元素被识别, 这些序列元素的识别以高度协调的方式发生, 确保了剪接的准确和高效^[37], 形成一条成熟的mRNA链^[38]。但是在某些情况下, 如序列元素发生基因突变, 会产生不需要的亚型, 导致疾病发生^[39], 约10%的人类遗传疾病是由基因突变导致的hnRNA剪接缺陷引起。

研究表明化学修饰的小RNA寡聚体可以通过和剪接因子识别, 而后与前体mRNA结合来调节选择性剪接^{[40, 41]} (图 1), 从而修正剪接缺陷达到疾病治疗的目的^[42]。由于3′和5′剪接位点和分支位点存在于不同的内含子中, 且序列相对保守, 因此通常将位于内含子或外显子的剪接增强子作为理想靶点。

开发此类机制的反义药物首先需要找到特定mRNA序列, 此序列可通过剪接调节机制改变部分序列, 恢复蛋白质表达或者恢复部分功能。随后测试在外显子与内含子连接附近的互补序列寡核苷酸, 能否调节前体mRNA剪接; 随后将可调节前体mRNA剪接的序列进行化学修饰, 以增强其稳定性。例如反义药物eteplirsen是数量为30个核苷酸的磷酸二酯型吗啡啉低聚物, 其分子序列为5′CTCCAACATCAAGGAA GATGGCATTTCT3′, eteplirsen与DMD的51外显子杂交, 通过剪接因子剪接改变了阅读框, 跳过51外显子产生缩短的mRNA, 进而产生缩短但保留一定功能的抗肌萎缩蛋白, 达到治疗疾病的目的^[43]。这一作用机制最早由Dominski和Kole^[40]在1993年提出并成为反义药物治疗中的一个全新领域^[44]。

2009年, van Deutekom等^[45]成功将利用剪接调节原理的寡核苷酸的研究推向临床, 这是一种可以治疗杜氏肌萎缩症(Duchenne) 的反义药物, 后被称为drisapersen。其设计核心是跳过肌萎缩蛋白基因的第51外显子, 但由于Ⅲ期临床表现不佳而中断了研发^[46]。与此同时, Sarepta制药公司开发了一种具有相同作用模式的寡核苷酸(称为Exondys 51或eteplirsen), 同样靶向肌萎缩蛋白基因的第51个外显子。但eteplirsen使用了吗啉修饰主干, 与更常见磷硫化修饰不同, 这种修饰的主干呈现中性, 可以避免细胞RNA酶的降解。Eteplirsen最终被FDA批准用于治疗杜氏肌萎缩症^[47]。此外, 同样经过吗啉修饰的反义药物golodirsen^[48]和casimersen^[49], 最近被FDA批准分别用于治疗杜氏肌萎缩基因外显子53和45突变的患者。

另一种基于反义药物剪接调节机制治疗的疾病是脊髓性肌萎缩症(SMA), 这种疾病主要由SMN1基因突变或缺失产生非功能蛋白, 而后引起的神经肌肉遗传疾病。在人类群体中, 包括SMA患者, 都存在一种名为SMN2的基因, 这是一种与SMN1基因序列几乎一样的同源基因, 仅存在5个不同的核苷酸, 其中一个是外显子7。这会导致转录剪接SMN2时, 跳过第7外显子, 从而产生无功能且可被快速降解的SMN蛋白。罗氏药业成功开发了磷硫化修饰的寡核苷酸nusinersen, 在SMN2的hnRNA剪接过程中促进包含外显子7的表达, 以达到疾病治疗的目的^{[50, 51]}。这是RNA治疗领域取得的重要一步, 在仅仅10年的时间里, 便获得了FDA的批准。

除此之外, 两种针对视网膜疾病的剪接调节机制的寡核苷酸, 即靶向CEP290的sepofarsen^[52]和靶向USH2A^[53]的QR-421a, 分别用于治疗莱伯氏先天性黑蒙病和Ⅱ型Usher综合征, 在Ⅰ期和Ⅱ期临床研究中取得了良好的临床结果, 正在进入Ⅲ期临床试验^[54]。

反义药物涉及的作用机制及其相关药物临床研究进度见表 1^{[13-15, 17, 18, 20-23, 46-49, 53-64]}。

2 反义药物的化学修饰

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反义药物都需要进入细胞发挥作用, 由于体内具有广泛表达的核酸酶, 未经化学修饰的核酸在体内稳定性差, 非常容易被降解。因此化学修饰是反义药物从理论走向实践的物质基础^[65], 增加体内物理化学稳定性, 并能通过细胞膜或靶向到细胞, 同时化学修饰能够很好地保留或增强药物与靶标RNA碱基配对能力或调节mRNA功能蛋白的结合。

反义药物的化学修饰通常在其磷酸主链和糖环上。最常见的修饰方法是硫代主链, 磷硫化的寡核苷酸通常被认为是第一代的反义药物, 主要代表为硫代磷酸酯寡聚脱氧核苷酸。硫代磷酸脂寡核苷酸也是至今为止研究最多和应用最广泛的反义寡核苷酸, 其优点是改善了寡核苷酸在体内对酶的稳定性, 缺点是对靶RNA分子的亲和性较低及存在某些不良反应。

为了解决第一代反义药物的种种问题, 科学家们做出了许多尝试, 如: 2位烷基化、吗啉化、锁核酸(locked nucleic acid, LNA) 等多种方式对反义药物骨架进行了适当的修饰, 此类改进修饰的反义药物被称为第二代反义药物。

第三代反义药物主要在呋喃核糖、核糖磷酸键和核苷酸中进行大量修饰, 从而提高反义药物对核酸酶稳定性、靶标亲和力和药代动力学特征, 例如肽核酸^[66]和吗啡啉^[67]修饰。肽核酸耐核酸酶和多肽酶的降解, 在生物体内稳定, 与目标mRNA有强杂交亲和性; 吗啡啉修饰具有静电中性主干, 这减少了非特异性相互作用的可能性, 克服了体内药物递送的困难。由于化学修饰对寡核苷酸性质的影响, 该领域在过去几十年见证了重大的发展。现已经开发了多种核苷酸修饰的化学组合^{[68, 69]}, 不同的化学修饰赋予了寡核苷酸不同的性质。

核酸修饰方法很多, 目的也各自不同, 但是一个核酸药物的成功, 可使用不同的修饰方法来达到不同治疗效果。本文对目前几种常见的化学修饰方法进行总结。

2.1 反义药物核苷连接磷酸酯修饰

由于核酸酶对磷酸二酯键有较强的降解作用导致磷酸二酯键的固有不稳定性, 所以反义药物化学修饰的首要目标就是寡核苷酸的主链。修饰方式主要有磷硫化修饰、甲基磷酸修饰、硫代磷酸氨基酯修饰、吗啡啉修饰、肽核酸修饰、二硫代磷酸酯修饰等。

磷硫化修饰即磷酸酯的1个磷原子被1个硫原子取代, 这是目前应用广泛的一种主干化学修饰(图 2)。大部分单链反义寡核苷酸通常是完全磷硫化修饰的, 而通常双链siRNA在其末端修饰, 以防止其被核酸酶降解^[70]。磷硫化主干修饰显著增强了寡核苷酸对核酸酶降解的抗性^[71]。因此, 在全身给药后, 增强核酸与血浆和细胞表面蛋白的结合, 从而促进细胞摄取^[72]、组织吸收, 减少肾脏快速排泄, 有效提高了生物利用度, 显著改善了药代动力学性质。磷硫化的寡核苷酸通常被认为是第一代的反义药物, 它能通过核糖核酸酶H介导高效诱导靶RNA的裂解。

然而, 经硫磷化修饰的寡核苷酸, 无论2位是否修饰, 均会导致部分凝血活酶时间的短暂延长; 也可导致替代补体途径峰血浆浓度依赖性激活, 但此类问题目前存在于非灵长类动物研究中, 尚未在人类使用中观测到^[73]。

此外, 磷酸酯连接核苷的3位氧原子被氮原子取代称为硫代磷酸氨基酯修饰, 形成的硫磷酰胺和磷酰胺都对互补RNA表现出较高的亲和力和较强的核酸酶抗性^[74]; 除了磷硫化的主链修饰外, 还有利用等距体替换磷酸糖主链的设计, 例如磷酸吗啡啉寡核苷酸的设计, 此类修饰的寡核苷酸呈电中性, 与DNA的亲和力相似且对核酸酶稳定, 缺点是这种电中性的吗啡啉修饰与肽核酸修饰类似, 在血液中有较快的清除速率^{[75, 76]}。

2.2 糖和碱基修饰

至今为止, 糖基2位的化学修饰在增强反义药物性质方面具有极大价值, 通常将这种糖基2位化学修饰的寡核苷酸称为第二代反义药物。

糖基2位化学修饰的寡核苷酸虽然增加了与靶RNA的结合亲和力, 但是却极大地减少或者完全抑制了RNase H对靶RNA的切割。后来科学家开发出嵌合策略, 这种缺点得以被规避或最大程度减小。在这种策略中, 2位修饰的核苷酸区域位于寡核苷酸的中心DNA区域两侧, 将未修饰的DNA在中心嵌合^[6]。在此研究中发现, 糖基2位的电负性会影响与靶标mRNA的亲和力, 2位氟修饰可增加结合亲和力^[77]; 而2位烷氧基的修饰则对结合亲和力的作用较小, 但却能产生相当程度的核酸酶抗性, 采用此修饰的嵌合反义寡核苷酸已进入临床试验。当由2位甲氧基和2位氟原子修饰结合设计成siRNA时, 使得反义药物可以完全消除目标RNA, 效力相比于未修饰的siRNA提高500倍并可以增强双链稳定性^[78], 目前已经有许多使用2位氟原子修饰和2位甲氧基修饰的siRNA类似物进入临床试验^[79], 如givosiran等^[80]。目前进展较快的反义药物化学修饰还有2位氧-甲氧乙基修饰(图 2), 这种修饰增加了亲和力和核酸酶抗性^[77], 也能减少和某些非特异性蛋白结合, 从而降低毒性^[81]。如: mipomersen、volanesorsen、IONIS-FXI_Rx、ATL 1103等已经作为反义药物成功上市或进入临床。

此外, 糖基4位碳原子和2位氧原子连接的双环系统在靶RNA结合亲和力方面同样表现优异^[82], 该修饰也称为2, 4位桥连核酸锁核酸^[83]。锁核酸能够提高杂合特性, 显著改善核酶抗性。目前已出现大量的锁核酸修饰的类似物, 其中一些表现出优良的性质^{[84, 85]}。但完全锁核酸修饰的寡核苷酸也不支持内切酶机制, 所以同样需要采用嵌合策略^[86]。虽然采用了嵌合策略的锁核酸修饰的寡核苷酸更具优势, 但是此类修饰也增加了毒性^{[87, 88]}, 因此需要进行体内生物学评价。修饰策略有桥杂原子取代^[89]、立体异构体^[90]、烷基取代^[91]、更刚性的乙醇衍生体系取代双环^{[92, 93]}等, 其中含有S构型乙基修饰的寡核苷酸通常表现出良好的特性^{[94, 95]}, 并且目前很多已经进入临床试验, 这些研究进一步说明这类修饰的寡核苷酸具有良好的成药潜力。

然而, 2位修饰寡核苷酸的化学性质对药物不良反应类型、发生率以及严重程度有显著影响^[96], 因此, 目前药物相互作用以及临床安全性仍在考察中^[97]。

2.3 综合修饰

当单纯修饰不能完全满足于临床需求时, 就需要根据药物的性质考虑综合修饰的策略, 这也是核酸药物开发的关键。

已经上市的反义药物inotersen、mipomersen和volanosersen均采用了磷硫化、2位甲氧乙基嵌合修饰策略, 其中磷硫化骨架位于中央间隙区域, 甲氧乙基修饰核苷酸位于外侧^[98], 间隙区支持核酸内切酶介导的降解, 而甲氧乙基修饰的核苷酸通过外切酶介导的降解以增强RNA结合亲和力和稳定性^[99]; Spinraza是第一个全磷硫化、甲氧乙基修饰的反义寡核苷酸药物^[63], 甲氧乙基修饰增强了对靶互补RNA的亲和力, 磷硫化修饰提高了代谢稳定性, 促进了组织分布和细胞进入; eteplirsen、golidersen和viltorarsen是调节mRNA剪切的反义寡核苷酸, 并且使用了吗啡啉环修饰^{[100, 101]}。当呋喃糖环被吗啡啉环取代时, 吗啡啉环的电中性^[102]对核酸酶介导的降解表现出优良的稳定性。但是此种修饰的蛋白质结合性能较差, 并且能迅速被肾脏清除, 因此在临床上需要使用更高的剂量^{[103, 104]}。

3 反义药物的递送策略

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反义药物的递送一直是此领域面临的最大挑战, 在其有效进入细胞的目标位点前, 面临着诸多障碍, 如核酸内切酶介导的降解、内体吞噬(endosomal entrapment) 等, 目前已有多种递送策略帮助反义药物成功到达目标位点。

对于具有磷硫化骨架修饰的单链寡核苷酸药物的递送是可行的。大部分反义寡核苷酸(AON) 被肝脏和肾脏吸收, 其他组织可部分吸收, 但神经系统由于大多数AON化学物质无法穿过血脑屏障而基本不吸收。因此, 对于单链反义寡核苷酸的全身给药, 通常使用每月注射多次的频繁给药方案。

对于双链寡核苷酸药物的递送则更加具有挑战性。磷硫化修饰的骨架结构被双链结构所限制, 从而影响了细胞的摄取吸收。可采用脂质纳米颗粒(LNPs) 的方式包裹药物促进肝细胞的吸收。如对于siRNA, 临床上使用LNP的方式静脉给药, 可提高药物的生物利用度, 同时也为开发mRNA疫苗铺平了道路^[25]。

受体介导的反义药物偶联配体靶向递送方式可增强细胞和组织对药物的吸收。胆固醇是研究最多的一类偶联配体, 寡核苷酸偶联胆固醇可在增加药物对肝脏吸收的同时减少肾毒性^{[105, 106]}。寡核苷酸偶联胆固醇的策略已被用于多种反义机制, 包括RNase H机制、RNAi机制等。除了胆固醇外, 糖胺类、脂肪酸类和维生素E等也可改变反义药物在动物体内的分布, 增强靶点的表达效果^[107-109]。如使用三价乙酰半乳糖胺(GalNAc) 修饰介导的肝靶向递送策略, 使得受体介导的递送方式成为反义药物递送的重要手段(图 2)。通过对siRNA进行GalNAc修饰, 实现siRNA向肝脏的特异性递送, 并通过ASGPR介导的细胞内吞作用使siRNA进入细胞和发挥功能^[110]。此外, 2位甲氧乙基修饰的寡核苷酸和GalNAc偶联, 在人体内的作用比相同序列的亲本寡核苷酸强30倍^[111]。因此, 这种递送方式可降低治疗成本, 增强患者的依从性, 有望成为一种新型的临床治疗方式。但这种递送策略能否拓展到除肝脏以外的其他组织器官尚不清楚, 这也是未来寡核苷酸偶联药物研究的主要方向。

总之, 反义药物的递送策略是为了增加其在组织中的吸收分布, 靶向递送至细胞的目标位点, 从而提高药物的治疗效果。

4 结论与展望

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反义药物多年来一直是药学研究和临床试验的重点之一, 但至今依然存在许多瓶颈: 如何高效性、特异性地用药^[112]; 如何减少或规避脱靶问题^[113]; 如何减少不良反应的发生; 如何解决反义药物研发的高成本问题及能否针对普通疾病进行开发等^[114]。广大科研人员立足于多种已经阐明的反义药物机制, 同时通过化学修饰或偶联等手段, 在此领域已经产生了诸多喜人的成果, 目前已有多种反义药物获批, 在临床上有较强的发展潜力。

本文对反义药物的作用机制、化学修饰及递送策略的研究进展进行了汇总, 并对相关反义药物进行了梳理分析, 希望对未来反义药物的进一步研发提供参考, 相信在不久的将来, 反义药物的研究能够成功突破壁垒, 成为治疗疾病的主流药物, 为广大患者尤其是目前无药可用的患者带来福音。

作者贡献: 李磊、郑艺璇、马春英、冯文化共同参与了文章的撰写及修改。

利益冲突: 本文无任何利益冲突。

基金

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国家自然科学基金资助项目(81903432)

参考文献

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[1]

Jordan P, Goncalves V, Fernandes S, et al. Networks of mRNA processing and alternative splicing regulation in health and disease[J]. Adv Exp Med Biol, 2019, 1157: 1-27.

[2]

Robert A, Benoit-Vical F, Liu Y, et al. Small molecules: the past or the future in drug innovation?[J]. Met Ions Life Sci, 2019. DOI: 10.1515/9783110527872-008.

https://doi.org/10.1515/9783110527872-008

[3]

Crooke ST. Molecular mechanisms of action of antisense drugs[J]. Biochim Biophy Acta, 1999, 1489: 31-44.

https://doi.org/10.1016/S0167-4781(99)00148-7

[4]

Crooke ST. Antisense strategies[J]. Curr Mol Med, 2004, 4: 465-487.

https://doi.org/10.2174/1566524043360375

[5]

Lockhart A, Pires VB, Bento F, et al. RNase H1 and H2 are differentially regulated to process RNA-DNA hybrids[J]. Cell Rep, 2019, 29: 2890-2900.

https://doi.org/10.1016/j.celrep.2019.10.108

[6]

Monia BP, Lesnik EA, Gonzalez C, et al. Evaluation of 2'-modified oligonucleotides containing 2'-deoxy gaps as antisense inhibitors of gene-expression[J]. J Biol Chem, 1993, 268: 14514-14522.

https://doi.org/10.1016/S0021-9258(19)85268-7

[7]

Nakamura H, Oda Y, Iwai S, et al. How does RNase H recognize a DNA-RNA hybrid?[J]. Proc Natl Acad Sci U S A, 1991, 88: 11535-11539.

https://doi.org/10.1073/pnas.88.24.11535

[8]

Minshull J, Hunt T. The use of single-stranded DNA and RNase H to promote quantitative 'hybrid arrest of translation' of mRNA/DNA hybrids in reticulocyte lysate cell-free translations[J]. Nucleic Acids Res, 1986, 14: 6433-6451.

https://doi.org/10.1093/nar/14.16.6433

[9]

Furdon PJ, Dominski Z, Kole R. RNase H cleavage of RNA hybridized to oligonucleotides containing methylphosphonate, phosphorothioate and phosphodiester bonds[J]. Nucleic Acids Res, 1989, 17: 9193-9204.

https://doi.org/10.1093/nar/17.22.9193

[10]

Al Shaer D, Al Musaimi O, Albericio F, et al. 2018 FDA tides harvest[J]. Pharmaceuticals (Basel), 2019, 12: 52.

https://doi.org/10.3390/ph12020052

[11]

Mathew V, Wang AK. Inotersen: new promise for the treatment of hereditary transthyretin amyloidosis[J]. Drug Des Devel Ther, 2019, 13: 1515-1525.

https://doi.org/10.2147/DDDT.S162913

[12]

Chan L, Yokota T. Development and clinical applications of antisense oligonucleotide gapmers[J]. Methods Mol Biol, 2020, 2176: 21-47.

[13]

Parham JS, Goldberg AC. Mipomersen and its use in familial hypercholesterolemia[J]. Expert Opin Pharmacother, 2019, 20: 127-131.

https://doi.org/10.1080/14656566.2018.1550071

[14]

Mahfouz M, Maruyama R, Yokota T. Inotersen for the treatment of hereditary transthyretin amyloidosis[J]. Methods Mol Biol, 2020, 2176: 87-98.

[15]

Esan O, Wierzbicki AS. Volanesorsen in the treatment of familial chylomicronemia syndrome or hypertriglyceridaemia: design, development and place in therapy[J]. Drug Des Devel Ther, 2020, 14: 2623-2636.

https://doi.org/10.2147/DDDT.S224771

[16]

Scharner J, Aznarez I. Clinical applications of single-stranded oligonucleotides: current landscape of approved and in-development therapeutics[J]. Mol Ther, 2021, 29: 540-554.

https://doi.org/10.1016/j.ymthe.2020.12.022

[17]

Murray SF, Jazayeri A, Matthes MT, et al. Allele-specific inhibition of rhodopsin with an antisense oligonucleotide slows photoreceptor cell degeneration[J]. Invest Ophthalmol Vis Sci, 2015, 56: 6362-6375.

https://doi.org/10.1167/iovs.15-16400

[18]

Jaffe GJ, Sahni J, Fauser S, et al. Development of IONIS-FB-L-Rx to treat geographic atrophy associated with AMD[J]. Invest Ophthalmol Vis Sci, 2020, 61: 4305.

[19]

Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans[J]. Nature, 1998, 391: 806-811.

https://doi.org/10.1038/35888

[20]

Mullard A. FDA approves landmark RNAi drug[J]. Nat Rev Drug Discov, 2018, 17: 613.

[21]

Scott LJ. Givosiran: first approval[J]. Drugs, 2020, 80: 335-339.

https://doi.org/10.1007/s40265-020-01269-0

[22]

Scott LJ, Keam SJ. Lumasiran: first approval[J]. Drugs, 2021, 81: 277-282.

https://doi.org/10.1007/s40265-020-01463-0

[23]

Lamb YN. Inclisiran: first approval[J]. Drugs, 2021, 81: 389-395.

https://doi.org/10.1007/s40265-021-01473-6

[24]

Yonezawa S, Koide H, Asai T. Recent advances in siRNA delivery mediated by lipid-based nanoparticles[J]. Adv Drug Deliv Rev, 2020, 154-155: 64-78.

https://doi.org/10.1016/j.addr.2020.07.022

[25]

Buschmann MD, Carrasco MJ, Alishetty S, et al. Nanomaterial delivery systems for mRNA vaccines[J]. Vaccines (Basel), 2021, 9: 65.

https://doi.org/10.3390/vaccines9010065

[26]

Wang Y, Yu RZ, Henry S, et al. Pharmacokinetics and clinical pharmacology considerations of galnac3-conjugated antisense oligonucleotides[J]. Expert Opin Drug Metab Toxicol, 2019, 15: 475-485.

https://doi.org/10.1080/17425255.2019.1621838

[27]

Hu B, Zhong L, Weng Y, et al. Therapeutic siRNA: state of the art[J]. Signal Transduct Target Ther, 2020, 5: 101.

https://doi.org/10.1038/s41392-020-0207-x

[28]

Margus H, Padari K, Pooga M. Cell-penetrating peptides as versatile vehicles for oligonucleotide delivery[J]. Mol Ther, 2012, 20: 525-533.

https://doi.org/10.1038/mt.2011.284

[29]

Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference[J]. Nat Biotechnol, 2004, 22: 326-330.

https://doi.org/10.1038/nbt936

[30]

Jackson AL, Linsley PS. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application[J]. Nat Rev Drug Discov, 2010, 9: 57-67.

https://doi.org/10.1038/nrd3010

[31]

Alexopoulou L, Holt AC, Medzhitov R, et al. Recognition of double-stranded RNA and activation of NF-kappab by toll-like receptor 3[J]. Nature, 2001, 413: 732-738.

https://doi.org/10.1038/35099560

[32]

Weng Y, Xiao H, Zhang J, et al. RNAi therapeutic and its innovative biotechnological evolution[J]. Biotechnol Adv, 2019, 37: 801-825.

https://doi.org/10.1016/j.biotechadv.2019.04.012

[33]

Bajan S, Hutvagner G. RNA-based therapeutics: from antisense oligonucleotides to miRNAs[J]. Cells, 2020, 9: 137.

https://doi.org/10.3390/cells9010137

[34]

Zhang S, Cheng Z, Wang Y, et al. The risks of miRNA therapeutics: in a drug target perspective[J]. Drug Des Devel Ther, 2021, 15: 721-733.

https://doi.org/10.2147/DDDT.S288859

[35]

Hanna J, Hossain GS, Kocerha J. The potential for microRNA therapeutics and clinical research[J]. Front Genet, 2019, 10: 478.

https://doi.org/10.3389/fgene.2019.00478

[36]

Crick F. Split genes and RNA splicing[J]. Science, 1979, 204: 264-271.

https://doi.org/10.1126/science.373120

[37]

Kitamura K, Nimura K. Regulation of RNA splicing: aberrant splicing regulation and therapeutic targets in cancer[J]. Cells, 2021, 10: 923.

https://doi.org/10.3390/cells10040923

[38]

Warf MB, Berglund JA. Role of RNA structure in regulating pre-mRNA splicing[J]. Trends Biochem Sci, 2010, 35: 169-178.

https://doi.org/10.1016/j.tibs.2009.10.004

[39]

Urbanski LM, Leclair N, Anczukow O. Alternative-splicing defects in cancer: splicing regulators and their downstream targets, guiding the way to novel cancer therapeutics[J]. Wiley Interdiscip Rev RNA, 2018, 9: e1476.

[40]

Dominski Z, Kole R. Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotides[J]. Proc Natl Acad Sci U S A, 1993, 90: 8673-8677.

https://doi.org/10.1073/pnas.90.18.8673

[41]

Havens MA, Hastings ML. Splice-switching antisense oligonucleotides as therapeutic drugs[J]. Nucleic Acids Res, 2016, 44: 6549-6563.

https://doi.org/10.1093/nar/gkw533

[42]

Maruyama R, Yokota T. Tips to design effective splice-switching antisense oligonucleotides for exon skipping and exon inclusion[J]. Methods Mol Biol, 2018, 1828: 79-90.

[43]

Lim KRQ, Maruyama R, Yokota T. Eteplirsen in the treatment of duchenne muscular dystrophy[J]. Drug Des Devel Ther, 2017, 11: 533-545.

https://doi.org/10.2147/DDDT.S97635

[44]

Lim KRQ, Yokota T. Invention and early history of exon skipping and splice modulation[J]. Methods Mol Biol, 2018, 1828: 3-30.

[45]

van Deutekom JC, Janson AA, Ginjaar IB, et al. Local dystrophin restoration with antisense oligonucleotide pro051[J]. N Engl J Med, 2007, 357: 2677-2686.

https://doi.org/10.1056/NEJMoa073108

[46]

Goemans N, Mercuri E, Belousova E, et al. A randomized placebo-controlled phase 3 trial of an antisense oligonucleotide, drisapersen, in duchenne muscular dystrophy[J]. Neuromuscul Disord, 2018, 28: 4-15.

https://doi.org/10.1016/j.nmd.2017.10.004

[47]

Aartsma-Rus A, Goemans N. A sequel to the eteplirsen saga: eteplirsen is approved in the united states but was not approved in Europe[J]. Nucleic Acid Ther, 2019, 29: 13-15.

https://doi.org/10.1089/nat.2018.0756

[48]

Anwar S, Yokota T. Golodirsen for duchenne muscular dystrophy[J]. Drugs Today (Barc), 2020, 56: 491-504.

https://doi.org/10.1358/dot.2020.56.8.3159186

[49]

Shirley M. Casimersen: first approval[J]. Drugs, 2021, 81: 875-879.

https://doi.org/10.1007/s40265-021-01512-2

[50]

Hua Y, Vickers TA, Baker BF, et al. Enhancement of SMN2 exon 7 inclusion by antisense oligonucleotides targeting the exon[J]. PLoS Biol, 2007, 5: e73.

https://doi.org/10.1371/journal.pbio.0050073

[51]

Hua Y, Krainer AR. Antisense-mediated exon inclusion[J]. Methods Mol Biol, 2012, 867: 307-323.

[52]

Cideciyan AV, Jacobson SG, Drack AV, et al. Effect of an intravitreal antisense oligonucleotide on vision in leber congenital amaurosis due to a photoreceptor cilium defect[J]. Nat Med, 2019, 25: 225-228.

https://doi.org/10.1038/s41591-018-0295-0

[53]

Dulla K, Slijkerman R, van Diepen HC, et al. Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH₂A exon 13 mutations[J]. Mol Ther, 2021, 29: 2441-2455.

https://doi.org/10.1016/j.ymthe.2021.04.024

[54]

Cideciyan AV, Jacobson SG, Ho AC, et al. Durable vision improvement after a single treatment with antisense oligonucleotide sepofarsen: a case report[J]. Nat Med, 2021, 27: 785-789.

https://doi.org/10.1038/s41591-021-01297-7

[55]

Ariceta G, Barrios K, Brown BD, et al. Hepatic lactate dehydrogenase A: an RNA interference target for the treatment of all known types of primary hyperoxaluria[J]. Kidney Int Rep, 2021, 6: 1088-1098.

https://doi.org/10.1016/j.ekir.2021.01.029

[56]

Badri P, Jiang X, Borodovsky A, et al. Pharmacokinetic and pharmacodynamic properties of cemdisiran, an RNAi therapeutic targeting complement component 5, in healthy subjects and patients with paroxysmal nocturnal hemoglobinuria[J]. Clin Pharmacokinet, 2021, 60: 365-378.

https://doi.org/10.1007/s40262-020-00940-9

[57]

Jiang J, Zhang X, Tang Y, et al. Progress on ocular sirna gene-silencing therapy and drug delivery systems[J]. Fundam Clin Pharmacol, 2021, 35: 4-24.

https://doi.org/10.1111/fcp.12561

[58]

Olkkonen VM, Sinisalo J, Jauhiainen M. New medications targeting triglyceride-rich lipoproteins: can inhibition of ANGPTL3 or apoC-Ⅲ reduce the residual cardiovascular risk?[J]. Atherosclerosis, 2018, 272: 27-32.

https://doi.org/10.1016/j.atherosclerosis.2018.03.019

[59]

Zhang MM, Bahal R, Rasmussen TP, et al. The growth of siRNA-based therapeutics: updated clinical studies[J]. Biochem Pharmacol, 2021, 189: 114432.

https://doi.org/10.1016/j.bcp.2021.114432

[60]

Sehgal A, Barros S, Ivanciu L, et al. An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia[J]. Nat Med, 2015, 21: 492-497.

https://doi.org/10.1038/nm.3847

[61]

Moreno-Montanes J, Bleau AM, Jimenez AI. Tivanisiran, a novel siRNA for the treatment of dry eye disease[J]. Expert Opin Investig Drugs, 2018, 27: 421-426.

https://doi.org/10.1080/13543784.2018.1457647

[62]

Tikka A, Jauhiainen M. The role of ANGPTL3 in controlling lipoprotein metabolism[J]. Endocrine, 2016, 52: 187-193.

https://doi.org/10.1007/s12020-015-0838-9

[63]

Finkel RS, Mercuri E, Darras BT, et al. Nusinersen versus sham control in infantile-onset spinal muscular atrophy[J]. N Engl J Med, 2017, 377: 1723-1732.

https://doi.org/10.1056/NEJMoa1702752

[64]

Dhillon S. Viltolarsen: first approval[J]. Drugs, 2020, 80: 1027-1031.

https://doi.org/10.1007/s40265-020-01339-3

[65]

Egli M, Manoharan M. Re-engineering RNA molecules into therapeutic agents[J]. Acc Chem Res, 2019, 52: 1036-1047.

https://doi.org/10.1021/acs.accounts.8b00650

[66]

Ray A, Norden B. Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future[J]. FASEB J, 2000, 14: 1041-1060.

https://doi.org/10.1096/fasebj.14.9.1041

[67]

Zhang N, Tan CY, Cai PQ, et al. Synthesis and properties of morpholino chimeric oligonucleotides[J]. Tetrahedron Lett, 2008, 49: 3570-3573.

https://doi.org/10.1016/j.tetlet.2008.04.035

[68]

Egli M, Minasov G, Tereshko V, et al. Probing the influence of stereoelectronic effects on the biophysical properties of oligonucleotides: comprehensive analysis of the RNA affinity, nuclease resistance, and crystal structure of ten 2'-O-ribonucleic acid modifications[J]. Biochemistry, 2005, 44: 9045-9057.

https://doi.org/10.1021/bi050574m

[69]

Patra A, Paolillo M, Charisse K, et al. 2'-Fluoro RNA shows increased watson-crick H-bonding strength and stacking relative to RNA: evidence from NMR and thermodynamic data[J]. Angew Chem Int Ed Engl, 2012, 51: 11863-11866.

https://doi.org/10.1002/anie.201204946

[70]

Nair JK, Attarwala H, Sehgal A, et al. Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAC-siRNA conjugates[J]. Nucleic Acids Res, 2017, 45: 10969-10977.

https://doi.org/10.1093/nar/gkx818

[71]

Stein CA, Subasinghe C, Shinozuka K, et al. Physicochemical properties of phosphorothioate oligodeoxynucleotides[J]. Nucleic Acids Res, 1988, 16: 3209-3221.

https://doi.org/10.1093/nar/16.8.3209

[72]

Crooke ST, Vickers TA, Liang XH. Phosphorothioate modified oligonucleotide-protein interactions[J]. Nucleic Acids Res, 2020, 48: 5235-5253.

https://doi.org/10.1093/nar/gkaa299

[73]

Crooke ST, Baker BF, Kwoh TJ, et al. Integrated safety assessment of 2'-O-methoxyethyl chimeric antisense oligonucleotides in nonhuman primates and healthy human volunteers[J]. Mol Ther, 2016, 24: 1771-1782.

https://doi.org/10.1038/mt.2016.136

[74]

Gryaznov S, Skorski T, Cucco C, et al. Oligonucleotide N3'- > P5' phosphoramidates as antisense agents[J]. Nucleic Acids Res, 1996, 24: 1508-1514.

https://doi.org/10.1093/nar/24.8.1508

[75]

McMahon BM, Mays D, Lipsky J, et al. Pharmacokinetics and tissue distribution of a peptide nucleic acid after intravenous administration[J]. Antisense Nucleic Acid Drug Dev, 2002, 12: 65-70.

https://doi.org/10.1089/108729002760070803

[76]

Solano EC, Kornbrust DJ, Beaudry A, et al. Toxicological and pharmacokinetic properties of QPI-1007, a chemically modified synthetic siRNA targeting caspase 2 mRNA, following intravitreal injection[J]. Nucleic Acid Ther, 2014, 24: 258-266.

https://doi.org/10.1089/nat.2014.0489

[77]

Freier SM, Altmann KH. The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA: RNA duplexes[J]. Nucleic Acids Res, 1997, 25: 4429-4443.

https://doi.org/10.1093/nar/25.22.4429

[78]

Allerson CR, Sioufi N, Jarres R, et al. Fully 2'-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA[J]. J Med Chem, 2005, 48: 901-904.

https://doi.org/10.1021/jm049167j

[79]

Nair JK, Willoughby JL, Chan A, et al. Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing[J]. J Am Chem Soc, 2014, 136: 16958-16961.

https://doi.org/10.1021/ja505986a

[80]

Agarwal S, Simon AR, Goel V, et al. Pharmacokinetics and pharmacodynamics of the small interfering ribonucleic acid, givosiran, in patients with acute hepatic porphyria[J]. Clin Pharmacol Ther, 2020, 108: 63-72.

https://doi.org/10.1002/cpt.1802

[81]

Henry S, Stecker K, Brooks D, et al. Chemically modified oligonucleotides exhibit decreased immune stimulation in mice[J]. J Pharmacol Exp Ther, 2000, 292: 468-479.

[82]

Obika S, Nanbu D, Hari Y, et al. Synthesis of 2'-O, 4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C-3, -endo sugar puckering[J]. Tetrahedron Lett, 1997, 38: 8735-8738.

https://doi.org/10.1016/S0040-4039(97)10322-7

[83]

Wengel J. Synthesis of 3'-C- and 4'-C-branched oligodeoxynucleotides and the development of locked nucleic acid (LNA)[J]. Acc Chem Res, 1999, 32: 301-310.

https://doi.org/10.1021/ar980051p

[84]

Fluiter K, Frieden M, Vreijling J, et al. On the in vitro and in vivo properties of four locked nucleic acid nucleotides incorporated into an anti-H-Ras antisense oligonucleotide[J]. Chembiochem, 2005, 6: 1104-1109.

https://doi.org/10.1002/cbic.200400419

[85]

Morita K, Hasegawa C, Kaneko M, et al. 2'-O, 4'-C-Ethylene-bridged nucleic acids (ENA): highly nuclease-resistant and thermodynamically stable oligonucleotides for antisense drug[J]. Bioorg Med Chem Lett, 2002, 12: 73-76.

https://doi.org/10.1016/S0960-894X(01)00683-7

[86]

Kurreck J, Wyszko E, Gillen C, et al. Design of antisense oligonucleotides stabilized by locked nucleic acids[J]. Nucleic Acids Res, 2002, 30: 1911-1918.

https://doi.org/10.1093/nar/30.9.1911

[87]

Burdick AD, Sciabola S, Mantena SR, et al. Sequence motifs associated with hepatotoxicity of locked nucleic acid-modified antisense oligonucleotides[J]. Nucleic Acids Res, 2014, 42: 4882-4891.

https://doi.org/10.1093/nar/gku142

[88]

Swayze EE, Siwkowski AM, Wancewicz EV, et al. Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals[J]. Nucleic Acids Res, 2007, 35: 687-700.

https://doi.org/10.1093/nar/gkl1071

[89]

Prakash TP, Siwkowski A, Allerson CR, et al. Antisense oligonucleotides containing conformationally constrained 2', 4'-(N-methoxy)aminomethylene and 2', 4'-aminooxymethylene and 2'-O, 4'-C-aminomethylene bridged nucleoside analogues show improved potency in animal models[J]. J Med Chem, 2010, 53: 1636-1650.

https://doi.org/10.1021/jm9013295

[90]

Seth PP, Jazayeri A, Yu J, et al. Structure activity relationships of alpha-L-LNA modified phosphorothioate gapmer antisense oligonucleotides in animals[J]. Mol Ther Nucleic Acids, 2012, 1: e47.

https://doi.org/10.1038/mtna.2012.34

[91]

Pallan PS, Allerson CR, Berdeja A, et al. Structure and nuclease resistance of 2', 4'-constrained 2'-O-methoxyethyl (cMOE) and 2'-O-ethyl (cEt) modified DNAs[J]. Chem Commun (Camb), 2012, 48: 8195-8197.

https://doi.org/10.1039/c2cc32286b

[92]

Egli M, Pallan PS, Allerson CR, et al. Synthesis, improved antisense activity and structural rationale for the divergent RNA affinities of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides[J]. J Am Chem Soc, 2011, 133: 16642-16649.

https://doi.org/10.1021/ja207086x

[93]

Seth PP, Allerson CR, Siwkowski A, et al. Configuration of the 5'-methyl group modulates the biophysical and biological properties of locked nucleic acid (INA) oligonucleotides[J]. J Med Chem, 2010, 53: 8309-8318.

https://doi.org/10.1021/jm101207e

[94]

Burel SA, Han SR, Lee HS, et al. Preclinical evaluation of the toxicological effects of a novel constrained ethyl modified antisense compound targeting signal transducer and activator of transcription 3 in mice and cynomolgus monkeys[J]. Nucleic Acid Ther, 2013, 23: 213-227.

https://doi.org/10.1089/nat.2013.0422

[95]

Pandey SK, Wheeler TM, Justice SL, et al. Identification and characterization of modified antisense oligonucleotides targeting DMPK in mice and nonhuman primates for the treatment of myotonic dystrophy type 1[J]. J Pharmacol Exp Ther, 2015, 355: 329-340.

https://doi.org/10.1124/jpet.115.226969

[96]

Crooke ST, Baker BF, Witztum JL, et al. The effects of 2'-O-methoxyethyl containing antisense oligonucleotides on platelets in human clinical trials[J]. Nucleic Acid Ther, 2017, 27: 121-129.

https://doi.org/10.1089/nat.2016.0650

[97]

Crooke ST, Baker BF, Pham NC, et al. The effects of 2-O-methoxyethyl oligonucleotides on renal function in humans[J]. Nucleic Acid Ther, 2018, 28: 10-22.

https://doi.org/10.1089/nat.2017.0693

[98]

Crooke ST, Witztum JL, Bennett CF, et al. RNA-targeted therapeutics[J]. Cell Metab, 2018, 27: 714-739.

https://doi.org/10.1016/j.cmet.2018.03.004

[99]

Altmann KH, Dean NM, Fabbro D, et al. Second generation of antisense oligonucleotides: from nuclease resistance to biological efficacy in animals[J]. Chimia, 1996, 50: 168-176.

https://doi.org/10.2533/chimia.1996.168

[100]

Kesselheim AS, Avorn J. FDA approval of eteplirsen for muscular dystrophy-reply[J]. JAMA, 2017, 317: 1481-1482.

https://doi.org/10.1001/jama.2017.2605

[101]

Frank DE, Schnell FJ, Akana C, et al. Increased dystrophin production with golodirsen in patients with duchenne muscular dystrophy[J]. Neurology, 2020, 94: e2270-e2282.

https://doi.org/10.1212/WNL.0000000000009233

[102]

Summerton J, Weller D. Morpholino antisense oligomers: design, preparation, and properties[J]. Antisense Nucleic Acid Drug Dev, 1997, 7: 187-195.

https://doi.org/10.1089/oli.1.1997.7.187

[103]

Sazani P, Magee T, Charleston JS, et al. Consistent safety and pharmacokinetic profiles of eteplirsen, SRP-4045, and SRP-4053, three phosphorodiamidate morpholino oligomers (PMOs) for treatment of patients with duchenne muscular dystrophy (DMD) suggest PMOs are a well-tolerated therapeutic class[J]. Ann Neurol, 2014, 76: S237-S238.

[104]

Mendell JR, Rodino-Klapac LR, Sahenk Z, et al. Eteplirsen for the treatment of Duchenne muscular dystrophy[J]. Ann Neurol, 2013, 74: 637-647.

https://doi.org/10.1002/ana.23982

[105]

Soutschek J, Akinc A, Bramlage B, et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs[J]. Nature, 2004, 432: 173-178.

https://doi.org/10.1038/nature03121

[106]

Krutzfeldt J, Rajewsky N, Braich R, et al. Silencing of microRNAs in vivo with 'antagomirs'[J]. Nature, 2005, 438: 685-689.

https://doi.org/10.1038/nature04303

[107]

Herbert BS, Gellert GC, Hochreiter A, et al. Lipid modification of grn163, an N3'- > P5' thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition[J]. Oncogene, 2005, 24: 5262-5268.

https://doi.org/10.1038/sj.onc.1208760

[108]

Nishina K, Unno T, Uno Y, et al. Efficient in vivo delivery of siRNA to the liver by conjugation of alpha-tocopherol[J]. Mol Ther, 2008, 16: 734-740.

https://doi.org/10.1038/mt.2008.14

[109]

Nishina K, Piao W, Yoshida-Tanaka K, et al. DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing[J]. Nat Commun, 2015, 6: 7969.

https://doi.org/10.1038/ncomms8969

[110]

Prakash TP, Graham MJ, Yu J, et al. Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice[J]. Nucleic Acids Res, 2014, 42: 8796-8807.

https://doi.org/10.1093/nar/gku531

[111]

Prakash TP, Yu J, Migawa MT, et al. Comprehensive structure-activity relationship of triantennary N-acetylgalactosamine conjugated antisense oligonucleotides for targeted delivery to hepatocytes[J]. J Med Chem, 2016, 59: 2718-2733.

https://doi.org/10.1021/acs.jmedchem.5b01948

[112]

Gagliardi M, Ashizawa AT. The challenges and strategies of antisense oligonucleotide drug delivery[J]. Biomedicines, 2021, 9: 433.

https://doi.org/10.3390/biomedicines9040433

[113]

Winkler J, Stessl M, Amartey J, et al. Off-target effects related to the phosphorothioate modification of nucleic acids[J]. ChemMedChem, 2010, 5: 1344-1352.

https://doi.org/10.1002/cmdc.201000156

[114]

Austin CP, Cutillo CM, Lau LPL, et al. Future of rare diseases research 2017-2027: an irdirc perspective[J]. Clin Transl Sci, 2018, 11: 21-27.

https://doi.org/10.1111/cts.12500

2022年第57卷第9期

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doi: 10.16438/j.0513-4870.2022-0479

接收时间：2022-04-24
首发时间：2025-12-24
出版时间：2022-09-12

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收稿日期：2022-04-24
修回日期：2022-06-10

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国家自然科学基金资助项目(81903432)

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中国医学科学院、北京协和医学院药物研究所, 北京 100050

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*冯文化, E-mail: fwh@imm.ac.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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TxT

Name	Company	Treatment of diseases	Target	Delivery route	Status	Reference
	Endonuclease (RNase H)-mediated RNA degradation
Mipomersen	Ionis	Familial hypercholesterolemia	apo-B-100	Subcutaneous	Approved	[13]
Inotersen	Ionis	Familial amyloid polyneuropathy	Transthyretin	Subcutaneous	Approved	[14]
Volanesorsen	Ionis	Familial chylomicronemia syndrome	apoC3	Subcutaneous	Approved	[15]
QR-1123	ProQR	Retinitis pigmentosa (adRP)	Rhodopsin	Intravitreal	Phase Ⅱ	[17]
FB-LRX	Ionis	Age-related macular degeneration	CFB	Subcutaneous	Phase Ⅱ	[18]
	siRNA interference
Patisiran	Alnylam	Familial amyloid polyneuropathy	TTR	Intravenous	Approved	[20]
Givosiran	Alnylam	Hemophilia (AHP)	ALAS1	Subcutaneous	Approved	[21]
Lumasiran	Alnylam	Hyperoxaluria	GO	Subcutaneous	Approved	[22]
Inclisiran	Alnylam	Hypercholesterolemia	PCSK9	Subcutaneous	Approved	[23]
Nedosiran	Dicerna	Primary hyperoxaluria	LDHA	Subcutaneous	Phase Ⅲ	[55]
Cemdisiran	Alnylam	Paroxysmal hemoglobinuria	C5	Subcutaneous	Phase Ⅲ	[56]
QPI-1007	Quark	Optic nerve injuries	Caspase-2	N.a.	Phase Ⅲ	[57]
ARO-APOC3	Arrowhead	Hyperlipoproteinemia type I	APOCⅢ	Subcutaneous	Phase Ⅲ	[58]
Teprasiran	Quark	Acute kidney injury	P53	Intravenous	Phase Ⅲ	[59]
Fiturisan	Alnylam	Hemophilia	AT3	Subcutaneous	Phase Ⅲ	[60]
Tivanisiran	Sylentis	Xerophthalmia	TRPV1	Subcutaneous	Phase Ⅲ	[61]
ARO-ANG-3	Arrowhead	Familial hypercholesterolemia	ANGPTL3	N.a.	Phase Ⅱ	[62]
	Splicing regulatory mechanism
Drisapersen	Biomarin	Duchenne muscular dystrophy	Dystrophin	N.a.	Exit R & D	[46]
Eteplirsen	Sarepta	Duchenne muscular dystrophy	Dystrophin	Intravenous	Approved	[47]
Casimersen	Sarepta	Duchenne muscular dystrophy	Dystrophin	Intravenous	Approved	[49]
Golodirsen	Sarepta	Duchenne muscular dystrophy	Dystrophin	Intravenous	Approved	[48]
Spinraza	Ionis	Spinal muscular atrophy	SMN2	Intravenous	Approved	[63]
Viltolarsen	Shinyaku	Duchenne muscular dystrophy	Dystrophin	Intravenous	Approved	[64]
Sepofarsen	ProQR	Leber congenital amaurosis	CEP290	Intravenous	Phase Ⅲ	[54]
QR-421a	ProQR	Usher syndromes	USH2A	Intravenous	Phase Ⅲ	[53]

Name

Company

Treatment of diseases

Target

Delivery route

Status

Reference

Endonuclease (RNase H)-mediated RNA degradation

Mipomersen

Ionis

Familial hypercholesterolemia

apo-B-100

Subcutaneous

Approved

[13]

Inotersen

Ionis

Familial amyloid polyneuropathy

Transthyretin

Subcutaneous

Approved

[14]

Volanesorsen

Ionis

Familial chylomicronemia syndrome

apoC3

Subcutaneous

Approved

[15]

QR-1123

ProQR

Retinitis pigmentosa (adRP)

Rhodopsin

Intravitreal

Phase Ⅱ

[17]

FB-LRX

Ionis

Age-related macular degeneration

CFB

Subcutaneous

Phase Ⅱ

[18]

siRNA interference

Patisiran

Alnylam

Familial amyloid polyneuropathy

TTR

Intravenous

Approved

[20]

Givosiran

Alnylam

Hemophilia (AHP)

ALAS1

Subcutaneous

Approved

[21]

Lumasiran

Alnylam

Hyperoxaluria

Subcutaneous

Approved

[22]

Inclisiran

Alnylam

Hypercholesterolemia

PCSK9

Subcutaneous

Approved

[23]

Nedosiran

Dicerna

Primary hyperoxaluria

LDHA

Subcutaneous

Phase Ⅲ

[55]

Cemdisiran

Alnylam

Paroxysmal hemoglobinuria

Subcutaneous

Phase Ⅲ

[56]

QPI-1007

Quark

Optic nerve injuries

Caspase-2

N.a.

Phase Ⅲ

[57]

ARO-APOC3

Arrowhead

Hyperlipoproteinemia type I

APOCⅢ

Subcutaneous

Phase Ⅲ

[58]

Teprasiran

Quark

Acute kidney injury

P53

Intravenous

Phase Ⅲ

[59]

Fiturisan

Alnylam

Hemophilia

AT3

Subcutaneous

Phase Ⅲ

[60]

Tivanisiran

Sylentis

Xerophthalmia

TRPV1

Subcutaneous

Phase Ⅲ

[61]

ARO-ANG-3

Arrowhead

Familial hypercholesterolemia

ANGPTL3

N.a.

Phase Ⅱ

[62]

Splicing regulatory mechanism

Drisapersen

Biomarin

Duchenne muscular dystrophy

Dystrophin

N.a.

Exit R & D

[46]

Eteplirsen

Sarepta

Duchenne muscular dystrophy

Dystrophin

Intravenous

Approved

[47]

Casimersen

Sarepta

Duchenne muscular dystrophy

Dystrophin

Intravenous

Approved

[49]

Golodirsen

Sarepta

Duchenne muscular dystrophy

Dystrophin

Intravenous

Approved

[48]

Spinraza

Ionis

Spinal muscular atrophy

SMN2

Intravenous

Approved

[63]

Viltolarsen

Shinyaku

Duchenne muscular dystrophy

Dystrophin

Intravenous

Approved

[64]

Sepofarsen

ProQR

Leber congenital amaurosis

CEP290

Intravenous

Phase Ⅲ

[54]

QR-421a

ProQR

Usher syndromes

USH2A

Intravenous

Phase Ⅲ

[53]