药学学报

Name	Proper name/target	Indication	Vector	Organization	Stage
Gendicine^TM	Recombinant human p53 adenovirus particle	Head and neck cancer	Adenovirus type 5-p53	Shenzhen SiBiono GeneTech	Launched
Glybera^TM	Alipogene Tiparvovec	LPLD	rAAV1-LPL	UniQure	Withdrawn
Strimvelis^TM	Autologous CD34⁺ cells transduced to express ADA	ADA-SCID	Retroviral-ADA	Orchard Therapeutics	Launched
Luxturna^TM	Voretigene Neparvovec-rzl	IRD	rAAV2-PRE65	Spark Therapeutics	Launched
Zolgensma^TM	Onasemnogene Abeparvovec-xioi	SMA	rAAV9-SMN1	AveXis	Launched
Zynteglo^TM	Autologous CD34⁺ cells encoding β^A-T87Q-globin gene	Β-thalassemia	Lentiviral-β-globin	Bluebird Bio	Launched
Invossa^TM	TissueGene-C	OA	Retroviral-TGF-β1	Kolon Life Science	Launched
RZ-001	hTERT mRNA	HCC	Adenovirus	Rznomics	Phase Ⅰ/Ⅱ
PRT-mirl22aT	mTERT mRNA	HCC	Adenovirus	Dong-A University	Preclinical
Ad.CMV.Rz.p53	mTERT mRNA	HCC	Adenovirus	Gyeongsang National University	Biological testing
Ad5mTR	mTERT mRNA	Head and neck cancer	Adenovirus type 5	National Cancer Center of Korea	Preclinical
Ad5CMV.Rz.HSVtk.miR-145	mTERT mRNA	Brain cancer	Adenovirus type 5	National Cancer Center of Korea	Preclinical
Ad5CMV.mTR.sPD1	mTERT mRNA	Colorectal cancer, non-Hodgkin's lymphoma	Adenovirus type 5	National Cancer Center of Korea	Biological testing
pRib100AS-HSVtk	AIMP2-DX2 pre-mRNA	Lung cancer	DNA plasmids	Dankook University	Biological testing
Ad-3R1-TK/GCV	KRAS G12V pre-mRNA	Cancer	Adenovirus	Dankook University	Preclinical
AAV2/8-bRho-PTM20	RHO pre-mRNA	Autosomal dominant retinitis pigmentosa	AAV	INSERM	Preclinical
pA5c-9v1	DENV mRNA	Viral	pA5c	University of Notre Dame	Biological testing
pMU2-tsRNAHA	SMN pre-mRNA	SMA	rAAV	Boston University School of Medicine	Biological testing

Name	Proper name/target	Indication	Vector	Organization	Stage
Gendicine^TM	Recombinant human p53 adenovirus particle	Head and neck cancer	Adenovirus type 5-p53	Shenzhen SiBiono GeneTech	Launched
Glybera^TM	Alipogene Tiparvovec	LPLD	rAAV1-LPL	UniQure	Withdrawn
Strimvelis^TM	Autologous CD34⁺ cells transduced to express ADA	ADA-SCID	Retroviral-ADA	Orchard Therapeutics	Launched
Luxturna^TM	Voretigene Neparvovec-rzl	IRD	rAAV2-PRE65	Spark Therapeutics	Launched
Zolgensma^TM	Onasemnogene Abeparvovec-xioi	SMA	rAAV9-SMN1	AveXis	Launched
Zynteglo^TM	Autologous CD34⁺ cells encoding β^A-T87Q-globin gene	Β-thalassemia	Lentiviral-β-globin	Bluebird Bio	Launched
Invossa^TM	TissueGene-C	OA	Retroviral-TGF-β1	Kolon Life Science	Launched
RZ-001	hTERT mRNA	HCC	Adenovirus	Rznomics	Phase Ⅰ/Ⅱ
PRT-mirl22aT	mTERT mRNA	HCC	Adenovirus	Dong-A University	Preclinical
Ad.CMV.Rz.p53	mTERT mRNA	HCC	Adenovirus	Gyeongsang National University	Biological testing
Ad5mTR	mTERT mRNA	Head and neck cancer	Adenovirus type 5	National Cancer Center of Korea	Preclinical
Ad5CMV.Rz.HSVtk.miR-145	mTERT mRNA	Brain cancer	Adenovirus type 5	National Cancer Center of Korea	Preclinical
Ad5CMV.mTR.sPD1	mTERT mRNA	Colorectal cancer, non-Hodgkin's lymphoma	Adenovirus type 5	National Cancer Center of Korea	Biological testing
pRib100AS-HSVtk	AIMP2-DX2 pre-mRNA	Lung cancer	DNA plasmids	Dankook University	Biological testing
Ad-3R1-TK/GCV	KRAS G12V pre-mRNA	Cancer	Adenovirus	Dankook University	Preclinical
AAV2/8-bRho-PTM20	RHO pre-mRNA	Autosomal dominant retinitis pigmentosa	AAV	INSERM	Preclinical
pA5c-9v1	DENV mRNA	Viral	pA5c	University of Notre Dame	Biological testing
pMU2-tsRNAHA	SMN pre-mRNA	SMA	rAAV	Boston University School of Medicine	Biological testing

Name	Sequence (sense/anti-sense strand)/vector	Indication	Target	Organization	Stage
Patisiran (Onpattro^TM)	5'-GUAACCAAGAGUAUUCCAUTT-3'/ 5'-AUGGAAUACUCUUGGUUACTT-3'	FAP	TTR mRNA	Alnylam Pharma	Launched
Vutrisiran (Amvuttra^TM)	5'-UCUUGGUUACAUGUAACCAAGA-3'/ 5'-UGGGAUUUCAUGUAACCAAGA-3'	ATTR	TTR mRNA	Alnylam Pharma	Launched
Givosiran (Givlaari^TM)	5'-UGGUCUUUCUCACAGAGUAGAA-3'/ 5'-AUUCUACUCUCUGUGAGAAAGAC-3'	AHP	ALAS1 mRNA	Alnylam Pharma	Launched
Lumasiran (Oxlumo^TM)	GACUUUCAUCCUGGAAAUAUA/ ACCUGAAAGUAGGACCUUUAUAU	PH	HAO1 mRNA	Alnylam Pharma	Launched
Inclisiran (Leqvio^TM)	5'-CUAGACCUGUTUUGCUUUUGU-3' 5'-ACAAAAGCAAAACAGGUCUAGAA-3'	Atheroscle-rosis	PCSK9 mRNA	Novartis	Launched
Fitusiran	5'-GGUUAACACCAUUUACUUCAA-3'/ 5'-UUGAAGUAAAUGGUGUUAACCAG-3'	Hemophilia A/B	SERPINC1 mRNA	Sanofi Genzyme	Phase Ⅲ
Nusinersen (Spinraza^TM)	5'-TCACTTTCATAATGCTGG-3'	SMA	SMN2 Ex7	Ionis Pharma, Biogen	Launched
E1^MOv11	5'-CUAUAUAUAGUUAUUCAACA-3'	SMA	SMN2 E1	Shift Pharma	Preclinical
MO HSMN2-Ex7D	5'-GTAAGATTCACTTTCATAATGCTGG-3'	SMA	SMN2 Ex7	IRCCS Ospedale Maggiore Policlinico	Preclinical
E1.4	CTGAAAGgttagtggacagccatgc	FTDP-17	MAPT Ex1	University of Pennsylvania	Preclinical
E5.3	GCCAAGgtaagctgacgatgccacagg	FTDP-17	MAPT Ex5	University of Pennsylvania	Preclinical
7-26S	GTCGCAAACAGTACAATGGC	FD	IKBKAP Ex20	Cold Spring Harbor Laboratory	Preclinical
Eteplirsen (Exondys 51^TM)	5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3'	DMD	Dystrophin Ex51	Sarepta Therapeutics	Launched
Drisapersen	5'-UCAAGGAAGAUGGCAUUUCU-3'	DMD	Dystrophin Ex51	BioMarin Pharma Inc	Phase Ⅲ (discontinued)
Golodirsen (Vyondys 53^TM)	5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3'	DMD	Dystrophin Ex53	Sarepta Therapeutics	Launched
Viltolarsen (Viltepso^TM)	5'-CCUCCGGUUCUGAAGGUGUUC-3'	DMD	Dystrophin Ex53	National Center of Neurology and Psychiatry	Launched
Casimersen	5'-CAATGCCATCCTGGAGTTCCTG-3'	DMD	Dystrophin Ex45	Sarepta Therapeutics	Launched
DS-5141	5'-CCUACCGUAACCCGUCGC-3'	DMD	Dystrophin Ex45	Daiichi Sankyo Co Ltd	Phase Ⅰ/Ⅱ

Name	Sequence (sense/anti-sense strand)/vector	Indication	Target	Organization	Stage
Patisiran (Onpattro^TM)	5'-GUAACCAAGAGUAUUCCAUTT-3'/ 5'-AUGGAAUACUCUUGGUUACTT-3'	FAP	TTR mRNA	Alnylam Pharma	Launched
Vutrisiran (Amvuttra^TM)	5'-UCUUGGUUACAUGUAACCAAGA-3'/ 5'-UGGGAUUUCAUGUAACCAAGA-3'	ATTR	TTR mRNA	Alnylam Pharma	Launched
Givosiran (Givlaari^TM)	5'-UGGUCUUUCUCACAGAGUAGAA-3'/ 5'-AUUCUACUCUCUGUGAGAAAGAC-3'	AHP	ALAS1 mRNA	Alnylam Pharma	Launched
Lumasiran (Oxlumo^TM)	GACUUUCAUCCUGGAAAUAUA/ ACCUGAAAGUAGGACCUUUAUAU	PH	HAO1 mRNA	Alnylam Pharma	Launched
Inclisiran (Leqvio^TM)	5'-CUAGACCUGUTUUGCUUUUGU-3' 5'-ACAAAAGCAAAACAGGUCUAGAA-3'	Atheroscle-rosis	PCSK9 mRNA	Novartis	Launched
Fitusiran	5'-GGUUAACACCAUUUACUUCAA-3'/ 5'-UUGAAGUAAAUGGUGUUAACCAG-3'	Hemophilia A/B	SERPINC1 mRNA	Sanofi Genzyme	Phase Ⅲ
Nusinersen (Spinraza^TM)	5'-TCACTTTCATAATGCTGG-3'	SMA	SMN2 Ex7	Ionis Pharma, Biogen	Launched
E1^MOv11	5'-CUAUAUAUAGUUAUUCAACA-3'	SMA	SMN2 E1	Shift Pharma	Preclinical
MO HSMN2-Ex7D	5'-GTAAGATTCACTTTCATAATGCTGG-3'	SMA	SMN2 Ex7	IRCCS Ospedale Maggiore Policlinico	Preclinical
E1.4	CTGAAAGgttagtggacagccatgc	FTDP-17	MAPT Ex1	University of Pennsylvania	Preclinical
E5.3	GCCAAGgtaagctgacgatgccacagg	FTDP-17	MAPT Ex5	University of Pennsylvania	Preclinical
7-26S	GTCGCAAACAGTACAATGGC	FD	IKBKAP Ex20	Cold Spring Harbor Laboratory	Preclinical
Eteplirsen (Exondys 51^TM)	5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3'	DMD	Dystrophin Ex51	Sarepta Therapeutics	Launched
Drisapersen	5'-UCAAGGAAGAUGGCAUUUCU-3'	DMD	Dystrophin Ex51	BioMarin Pharma Inc	Phase Ⅲ (discontinued)
Golodirsen (Vyondys 53^TM)	5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3'	DMD	Dystrophin Ex53	Sarepta Therapeutics	Launched
Viltolarsen (Viltepso^TM)	5'-CCUCCGGUUCUGAAGGUGUUC-3'	DMD	Dystrophin Ex53	National Center of Neurology and Psychiatry	Launched
Casimersen	5'-CAATGCCATCCTGGAGTTCCTG-3'	DMD	Dystrophin Ex45	Sarepta Therapeutics	Launched
DS-5141	5'-CCUACCGUAACCCGUCGC-3'	DMD	Dystrophin Ex45	Daiichi Sankyo Co Ltd	Phase Ⅰ/Ⅱ

Name	Indication	Target	Organization	Stage
Risdiplam (Evrysdi^TM)	SMA	SMN2 Ex7	PTC Therapeutics, Roche, SMA Foundation	Approved
Branaplam	SMA/HD	SMN2 Ex7/HTT pre-mRNA	Novartis	Phase Ⅱ/Ⅲ (discontinued)
PK4C9	SMA	SMN2 TSL2	Roche	Preclinical
LDN-2014	SMA	SMN2 pre-mRNA	Brigham and Women's Hospital, Indiana University Research Technology, University of Massachusetts	Preclinical
Kinetin	FD	IKBKAP pre-mRNA	New York University	Phase Ⅰ (discontinued)
RECTAS	FD	IKBKAP Ex20	Kyoto University	Preclinical
BPN-15477	FD/FTDP-17	IKBKAP Ex20/MAPT Ex10	PTC Therapeutics	Preclinical

Name	Indication	Target	Organization	Stage
Risdiplam (Evrysdi^TM)	SMA	SMN2 Ex7	PTC Therapeutics, Roche, SMA Foundation	Approved
Branaplam	SMA/HD	SMN2 Ex7/HTT pre-mRNA	Novartis	Phase Ⅱ/Ⅲ (discontinued)
PK4C9	SMA	SMN2 TSL2	Roche	Preclinical
LDN-2014	SMA	SMN2 pre-mRNA	Brigham and Women's Hospital, Indiana University Research Technology, University of Massachusetts	Preclinical
Kinetin	FD	IKBKAP pre-mRNA	New York University	Phase Ⅰ (discontinued)
RECTAS	FD	IKBKAP Ex20	Kyoto University	Preclinical
BPN-15477	FD/FTDP-17	IKBKAP Ex20/MAPT Ex10	PTC Therapeutics	Preclinical

靶向pre-mRNA的选择性剪接过程的疾病治疗策略研究概述

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郭欣茹 , 张翔 ^*

药学学报 | 综述 2023,58(12): 3557-3571

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药学学报 | 综述 2023, 58(12): 3557-3571

靶向pre-mRNA的选择性剪接过程的疾病治疗策略研究概述

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郭欣茹, 张翔^*

作者信息

中国医学科学院、北京协和医学院药物研究所, 活性物质发现与适药化研究北京市重点实验室, 北京 100050

通讯作者:

*张翔, Tel: 86-10-63165248, E-mail: simon@imm.ac.cn

An overview of disease treatment strategies targeting the alternative splicing of pre-mRNA

Xin-ru GUO, Xiang ZHANG^*

Affiliations

Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

出版时间: 2023-12-12 doi: 10.16438/j.0513-4870.2023-0586

文章导航

摘要

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前体信使RNA (pre-mRNA) 的选择性剪接是人类转录组和蛋白质组多样性的关键机制。选择性剪接是复杂的基因调控过程, 全转录组分析表明95%的人外显子基因是选择性剪接的, 涉及多种顺式作用元件和反式作用因子。其中, 任一环节或组分发生改变都可能引起错误剪接事件, 进而导致多种相关疾病的发生。除直接改变剪接结果的基因替代治疗外, RNA剪接修饰有望成为一种新的治疗策略, 通过靶向并纠正异常pre-mRNA剪接来达到缓解或治疗疾病的目的。目前所开发的剪接修饰工具有RNA反式剪接、反义寡核苷酸、小干扰RNA和小分子药物等, 它们可通过不同的方式纠正异常剪接。本文综述了近年来对pre-mRNA选择性剪接的表观遗传调控研究进展, 探讨了选择性剪接的发生与调节、相关的剪接缺陷导致的疾病种类以及当前用于靶向和改变剪接的工具, 展望了剪接修饰策略在未来人类疾病治疗中的重要作用。

关键词

前体信使RNA / 选择性剪接 / 剪接修饰 / 遗传代谢疾病 / 癌症

Abstract

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Alternative splicing of pre-messenger RNA (pre-mRNA) is a crucial mechanism for the diversity of the human transcriptome and proteome. Alternative splicing is a complex gene regulation process. Whole-transcriptome analysis shows that 95% of human exonic genes are alternatively spliced, involving various cis-acting elements and trans-acting factors. Any changes in any component or step may cause erroneous splicing events and lead to the occurrence of various related diseases. In addition to gene replacement therapy that directly changes the splicing results, RNA splicing modification is expected to become a new therapeutic strategy to alleviate or treat diseases by targeting and correcting abnormal pre-mRNA splicing. Splicing modification tools currently developed including RNA trans-splicing, antisense oligonucleotides, small interfering RNA, and small molecule drugs can correct abnormal splicing through different ways. This article reviews the resent progress of epigenetic regulation of pre-mRNA alternative splicing in recent years, and discusses the occurrence and regulation of alternative splicing, the types of diseases caused by related splicing defects, and the current-used tools for targeting and altering splicing. The importance of splicing modification strategies in the future treatment of human diseases is envisioned.

Key words

pre-mRNA / alternative splicing / splicing modification / genetic metabolic disease / cancer

引用本文

郭欣茹, 张翔. 靶向pre-mRNA的选择性剪接过程的疾病治疗策略研究概述. 药学学报, 2023 , 58 (12) : 3557 -3571 . DOI: 10.16438/j.0513-4870.2023-0586

Xin-ru GUO, Xiang ZHANG. An overview of disease treatment strategies targeting the alternative splicing of pre-mRNA[J]. Acta Pharmaceutica Sinica, 2023 , 58 (12) : 3557 -3571 . DOI: 10.16438/j.0513-4870.2023-0586

正文

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人类基因组从一个包含2万多个不同基因的遗传密码中产生了多达100万种不同的蛋白质^[1], 蛋白质组的多样性提示了遗传信息的流动不是传统上所认为的DNA转录成RNA并翻译成蛋白质的简单过程, 基因表达的控制网络是极为复杂的。随着对基因表达调控研究的深入, RNA的重要作用逐渐凸显, 对RNA加工的控制是基因调控的关键组成部分。

基因由内含子和外显子组成, 但只有外显子含有合成蛋白质所必需的信息, 蛋白质编码基因的初始转录产物, 即前体信使RNA (pre-mRNA), 需要通过加工去除内含子并连接外显子, 才能形成成熟的mRNA作为翻译模板, 这一过程称为剪接(splicing)。人类基因组学的研究显示人类基因平均含有8.8个外显子, 平均大小为145个核苷酸(nucleotide, nt)。内含子平均长度为3 365 nt, 5'和3'非翻译区分别为770 nt和300 nt。因此, 一个“标准基因”的长度约27千碱基对(kilobase, kb)。在pre-mRNA加工后, 输出到胞质溶胶中的mRNA平均由1 340 nt的编码序列、1 070 nt的非翻译区和poly (A) 尾巴组成^[2]。这表明pre-mRNA上超过90%的部分作为内含子被除去。

大量的研究发现, 一种pre-mRNA可在不同组合的剪接位点被识别, 以不同的剪接方式进行剪接, 准确地切除内含子序列, 将不同的外显子序列拼接到一起, 从而产生多种成熟mRNA, 编码结构和功能多样的蛋白质, 这一现象定义为选择性剪接(alternative splicing, AS)。选择性剪接是真核生物基因表达中的一种高效、精准的调控机制, 广泛存在于超过95%的人类基因表达过程中^{[3, 4]}, 在细胞内环境稳定、细胞分化和组织器官发育等多个生理过程中发挥关键作用^[5]。选择性剪接过程的任一环节、任一组分发生突变都有可能导致疾病的发生。本文概述了多种基因序列和蛋白因子参与选择性剪接的复杂过程, 分析了选择性剪接过程中导致疾病的相关突变, 旨在通过在RNA层面上对选择性剪接相关疾病的机制进行阐述, 探寻靶向RNA剪接的剪接修饰工具对选择性剪接所致疾病的积极影响, 展望了RNA剪接修饰策略在疾病治疗中的前景。

1 选择性剪接

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1.1 选择性剪接的发生

识别外显子和内含子边界, 以通过剪接机制正确去除内含子的过程需要pre-mRNA上4个核心剪接位点元件: 5'剪接位点(5' splice site, 5' SS)、分支点序列(branch point sequence, BPS)、多嘧啶束(polypyrimidine, Py) 和3'剪接位点(3' splice site, 3' SS)^[6]。核pre-mRNA内含子通过两个连续的酯交换反应去除(图 1A^[7])。首先, 内含子分支点腺苷的2' OH基团对5'外显子和内含子之间的磷酸基团进行亲核攻击, 形成2'-5'磷酸二酯键和套索结构。其次, 3'外显子和内含子间的磷酸基团被5'外显子的3' OH基团攻击, 随后5'和3'外显子连接形成mRNA, 并释放内含子套索^[8]。

1.1.1 剪接体

上述的剪接过程由称为剪接体的大型核糖核蛋白(ribonucleoprotein, RNP) 机器催化^[9], 该机器由5种富含尿苷的小核RNA (small nuclear RNA, snRNA; 包括U1、U2、U4、U5和U6) 和200种其他蛋白质组成^[10]。对pre-mRNA剪接位点的识别随着剪接体的组装和拆分高度动态化进行, 提高了剪接过程的效率和灵活性。体外分离出选择性剪接过程中的几种中间体, 按时间顺序分别为复合体E、A、B和B^*。如图 1B^[11-16]所示, 在小核核糖核蛋白(small nuclear ribonucleoprotein, snRNP) 和剪接因子结合之前, pre-mRNA被异质核糖核蛋白(heterogeneous ribonucleoprotein particle, hnRNP) 蛋白结合, 形成复合物H。U1 snRNA和pre-mRNA通过碱基互补配对而结合, U1 snRNP与5' SS (GU) 以非ATP依赖的相互作用启动剪接体组装, 此过程中, U2 snRNP也松散地结合在pre-mRNA上^[17], 剪接因子SF1/BBP (splicing factor 1/branchpoint binding protein) 和U2AF (U2 auxiliary factor) 协同识别BPS、Py和3' SS (AG) 以组装复合物E。在后续依赖ATP的步骤中, U2 snRNA通过碱基互补与BPS配对, 使U2 snRNP稳定结合并形成复合体A (也称为预剪接体)。在剪接体组装的过程中所产生的snRNA或其他蛋白质和pre-mRNA之间的相互作用对剪接位点的识别和选择具有重要意义。因此, 选择性剪接的pre-mRNA的调节通常是通过在复合体E和A形成过程中调节剪接体成分的结合来实现的。接下来, 预先装配好的U4/U6.U5三元-snRNP与复合体A结合, 复合体B形成^{[18, 19]}。复合体B经过重排, U1和U4 snRNP去稳定化或丢失, 活化剪接体复合物B^*形成, 它催化两个剪接步骤中的第一步。随后产生复合体C, 催化第二步^[20]。然后mRNA和切除的内含子被释放, 剪接体解离, 经过重塑后, 释放的snRNPs参与到另外的剪接过程中去^{[21, 22]}。

1.1.2 顺式作用元件和反式作用因子

剪接需要极高的精确度, 因为即使在外显子连接位点的单个核苷酸添加或缺失也会改变阅读框, 对蛋白质编码产生不利后果。剪接位点在体内的准确识别是各种调节机制共同作用的结果。剪接的分子基础研究揭示了外显子和内含子顺式作用调控序列的存在, 它们结合反式作用因子[通常是被称为剪接因子(splicing factor, SF) 的蛋白质] 共同影响剪接位点的选择。这些顺式作用元件相对较短, 通常为4~18 nt, 分为外显子剪接增强子(exonic splicing enhancer, ESE)、外显子剪接沉默子(exonic splicing silencer, ESS)、内含子剪接增强子(intronic splicing enhancer, ISE) 和内含子剪接沉默子(intronic splicing silencer, ISS)^[23]。反式作用因子, 即一些可以调节剪接的蛋白质, 可分为富含丝氨酸-精氨酸的蛋白[serine and arginine-rich (SR) proteins] 和hnRNP, SR蛋白在N端具有一个或两个RNA识别基序(RNA recognition motif, RRM), 在C端具有富含精氨酸和丝氨酸的结构域(RS结构域), 其介导多种蛋白质-蛋白质和蛋白质-RNA相互作用^{[24, 25]}。这些蛋白质通过其RRM结合pre-mRNA内的顺式调节元件、募集剪接体和相关蛋白质以及调节剪接位点选择来发挥作用。典型的hnRNP包含RRM结构域、RGG盒和通过促进多种蛋白质-蛋白质相互作用来促进功能多样性的附加结构域。SR蛋白通常结合剪接增强子促进外显子识别, 而hnRNP通常结合剪接沉默子并抑制外显子识别。然而最近的研究显示, 剪接因子作为激活剂或者阻遏剂的功能并不固定, 通常取决于它们结合pre-mRNA的位置^{[26, 27]}。剪接调节蛋白与这些剪接调节元件的特异性结合有助于将剪接体置于适当的剪接位点从而调控剪接过程。

1.2 选择性剪接的分类

选择性剪接主要存在5种模式(图 2^[28]): ①外显子跳跃(exon skipping, 又称盒式外显子), 指外显子可以与其侧翼内含子一起从转录本中剪接出来, 占人类和小鼠基因组保守选择性剪接事件的38%; ② 5' SS的选择(alternative 5' SS usage) 和③ 3' SS的选择(alternative 3' SS usage), 指外显子一端有两个或多个剪接位点可供识别, 是否识别该剪接位点取决于该位点的强弱, 分别占人类和小鼠基因组保守选择性剪接事件的18%和8%; ④内含子保留(intron retention), 指内含子可以保留在成熟mRNA分子中, 占人类和小鼠基因组保守选择性剪接事件的3%; ⑤互斥外显子(mutually exclusive exons), 指两个外显子可以交替包含或跳过^[29]。此外, 还有其他一些不太常见的复杂类型, 包括选择性转录起始位点和多聚腺苷酸化位点, 可产生选择性转录变体, 这些类型和互斥外显子占选择性剪接事件剩余的33%^[30]。上述这些重排可能发生在mRNA的编码区或非编码区, 当出现在编码区时, 可能导致剪接转录本产生具有广泛不同功能的蛋白质亚型, 包括亚细胞定位、蛋白质-蛋白质相互作用和翻译后修饰的变化; 出现在非编码区时, 不影响蛋白质序列, 但可调节mRNA的稳定性及蛋白质的表达。

2 选择性剪接和人类疾病

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随着对人类遗传学和全基因组研究的深入, 越来越多的研究揭示了选择性剪接与诸多疾病的密切关系, 特别是神经退行性疾病、神经肌肉疾病和癌症。选择性剪接的失调通常发生在开放阅读框内, 可导致异常蛋白质亚型的产生。这些异常功能蛋白影响神经肌肉的正常功能, 从而导致相关疾病。例如3R/4R tau蛋白(microtubule-associated protein tau) 亚型比例异常在阿尔茨海默症(Alzheimer's disease, AD) 中普遍存在; 运动神经元生存基因2 (survival motor neuron gene 2, SMN2) 突变导致约90%的无功能蛋白产生从而引起脊髓性肌萎缩(spinal muscular atrophy, SMA)^[31]。此外, 这些异常亚型还与癌症的进展和转移密切相关。随着细胞获得增殖能力, 并在上皮-间充质和间充质-上皮移动的致癌过程, 许多基因的特定亚型的剪接模式被改变, 使其获得血管生成、侵入性、抗细胞凋亡和存活特性, 摆脱生长因子依赖和生长抑制, 改变自身新陈代谢以应对缺氧, 从而获得免疫逃逸机制。每个“癌症标志”都与选择性剪接有关, 向更具侵袭性的癌症表型转变。

相关疾病机制的阐明揭示了剪接在机体生理病理状态中的关键作用。据估计, 破坏正常剪接的突变约占所有致病突变的1/3^[32]。选择性剪接主要在3个方面影响多种病理过程: ①通过降低剪接位点选择的特异性、保真度或激活不常使用的隐秘剪接位点来影响顺式作用元件序列的突变或遗传变异, 这些改变会影响单个基因; ②反式作用剪接因子的功能改变, 包括核心剪接体成分和调节因子, 这种变化可能会改变多个RNA靶标的表达; ③剪接因子在核苷酸重复扩增序列中的调节失衡, 这种抑制机制广泛影响基因表达^[28]。

2.1 顺式作用元件突变: 改变剪接模板

剪接位点和调控序列等顺式作用元件对剪接的控制是至关重要的。其中, 最常见的突变是5' SS或3' SS的单核苷酸替换, 可导致外显子跳跃、隐蔽剪接位点激活或较小程度的内含子保留。类似地, 内含子和外显子突变(如错义、无义或其他沉默突变) 通常可以通过增强子/沉默子的获得或缺失来影响剪接。

外显子跳跃是常见的致病突变, 特定外显子是否包含在成熟mRNA中主要取决于剪接机制对侧翼剪接位点的识别和使用, 研究显示这可能由ESE/ISE/ESS/ISS序列控制^[33]。如SMA是一种常染色体隐性神经肌肉疾病, SMA患者中染色体5q13位点上的运动神经元生存基因1 (survival motor neuron gene 1, SMN1) 的缺失或突变, 导致其编码的SMN蛋白水平降低, 引起α神经元的缺失和进行性肌肉萎缩。人类还同时存在SMN2基因, 该基因除外显子7第6位发生C→T的替换外与SMN1序列完全相同, 但该突变导致剪接过程中外显子7的5'端与U1 snRNP的结合变弱, 从而引起外显子7的跳跃, 使得90%的SMN2基因产生的mRNA转录本编码产生缺乏外显子7的蛋白, 此蛋白是无功能的, 并被迅速降解。此外, 有研究显示C→T的替换导致了SR蛋白SRSF1识别的ESE的损失以及hnRNPA1识别的ESS的增益^{[34, 35]}。

隐蔽剪接位点的激活可能导致如Hutchinson-Gilford早衰症(Hutchinson-Gilford progeria syndrome, HGPS) 的发生。HGPS是一种表现在儿童早期的引起过早死亡的加速衰老障碍, 临床表现为出生后生长迟缓、面中部发育不全、小颌畸形、过早动脉粥样硬化、皮下脂肪缺失和全身性骨发育不良等多种早衰特征。HGPS的突变发生在核纤层蛋白A (lamin A, LMNA) 基因中。核纤层蛋白分布在整个核质中, 并参与包括DNA复制、转录、染色质组织、核定位和形状, 以及细胞分裂期间核的组装/拆卸在内的许多功能。大多数典型的Hutchinson-Gilford早衰患者是由于LMNA mRNA外显子11中携带单个C→T沉默突变。该突变激活隐蔽的5' SS, 导致mRNA编码显性失活的LMNA, 导致核基因组不稳定^[36]。

信号传导及转录激活蛋白2 (signal transducer and activator of transcription 2, STAT2) 是一种转录因子, 是Janus活化激酶(Janus kinase, JAK) /STAT信号通路的主要成分^[37]。在干扰素(interferon, IFN) 刺激下, STAT2与STAT1形成异二聚体进入细胞核并激活IFN应答基因的转录。通过该途径, IFN诱导癌细胞凋亡^[37]。IFN已被证明可用于治疗多种癌症, 对血液系统恶性肿瘤最有效^[38]。但是, 癌细胞经常表现出对IFN的耐药性限制了其使用。Du等^[39]发现IFN抵抗细胞产生含有内含子19的STAT 2剪接变体。内含子19的保留导致在Src同源2结构域(Src homology 2 domain, SH2) 之前引入终止密码子, 使得STAT无法二聚化, 破坏了细胞间的信号传递, 从而导致IFN诱导癌细胞凋亡失调。

2.2 反式作用因子突变: 改变剪接模式

2.2.1 突变影响snRNP

肌萎缩侧索硬化症(amyotrophic lateral sclerosis, ALS) 是一种大脑和脊髓运动神经元的缺失引起的神经退行性疾病。肉瘤融合蛋白(fused in sarcoma protein, FUS) 基因突变和TAR-DNA结合蛋白43 (TAR-DNA binding protein 43, TDP-43) 免疫阳性包涵体缺失与ALS有关。FUS是一种hnRNP样蛋白, 与U1和U2 snRNA相互作用^[40-42]。FUS突变蛋白也与这些snRNA结合并留在细胞质中, 导致细胞核中的U1和U2 snRNP减少。TDP-43与FUS相互作用, 其失调也影响snRNA的丰度。最近的结果表明, TDP-43抑制了隐秘外显子的剪接, 并且在TPD-43缺陷的胚胎干细胞中激活这些外显子诱导细胞死亡^[43]。

2.2.2 核心剪接蛋白突变

骨髓增生异常综合征(myelodysplastic syndromes, MDS) 是一组异质性血液系统癌症。2011年, Yoshida及其同事^[44]报告了MDS中RNA剪接机制编码基因的复发性体细胞突变, 其中, 编码剪接因子SF3B1 (splicing factor 3b subunit 1)、U2AF1 (U2 small nuclear RNA auxiliary factor 1)、SRSF2 (serine/arginine rich splicing factor 2) 和ZRSR2 (zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) 的基因突变最频繁。SF3B1突变频率最高, 其作为一种剪接因子在剪接位点识别中起着关键作用, 它与U2AF和pre-mRNA的相互作用有助于U2 snRNP募集, 其磷酸化与剪接催化相结合影响剪接结果。在对SF3B1的基因测序中发现, 20%的MSD患者中存在SF3B1突变。其中, 对于难治性贫血伴环形铁粒幼细胞亚型和难治性血细胞减少伴多系异型增生和环形铁粒幼细胞亚型, 分别有68%和57%的患者对SF3B1突变呈阳性。SF3B1突变在其他骨髓增生异常综合征亚型中发生率较低, 一般不超过10%。此外, SF3B1突变也见于其他髓系癌症, 包括急性髓系白血病(5%)、原发性骨髓纤维化(4%)、原发性血小板增多症(3%) 和慢性粒单核细胞白血病(5%)。SF3B1突变还见于1%~5%的其他类型的肿瘤患者: 乳腺癌(1%)、肾癌(3%)、慢性淋巴细胞白血病(5%)、多发性骨髓瘤(3%) 和腺样囊性癌(4%)^[45]。

2.2.3 剪接调节因子突变

Ron是巨噬细胞刺激蛋白(macrophage stimulating protein, MSP) 的酪氨酸激酶受体, 可以促进细胞生长和抑制细胞凋亡, 还参与控制细胞解离、运动和细胞外基质的侵袭。ΔRon是外显子11跳跃产生的突变亚型, 外显子11的跳跃导致具有影响蛋白水解成熟功能的胞外结构域缺失, 并增加癌症侵袭性^[46]。ΔRon mRNA的异常积累发生在乳腺和结肠肿瘤中。外显子12中的增强子和沉默子在外显子11的剪接程序中起着关键作用, 其活性与外显子跳跃和外显子包合物之间的比率相似。剪接增强子的活性关键取决于剪接因子SF2/ASF (也称SRSF1, 最常见的SR蛋白之一) 直接结合的序列基序。因此, 高水平的SF2/ASF增加增强子的活性, 引起外显子11的跳跃, 并促进ΔRon亚型的产生。SF2/ASF已被证明是一种原癌基因^[47], 能够调节剪接与细胞运动之间的直接联系, 影响胚胎发生、组织形成和肿瘤转移^[33]。

2.3 核苷酸重复扩增

微卫星重复扩增(microsatellite expansion) 是指染色质基因组内小片段DNA重复序列异常扩增, 这些扩增的重复序列经常折叠成发夹结构, 干扰正常的RNA加工。据研究, 这类突变与30多种人类疾病相关, 其中绝大多数是神经退行性疾病和神经肌肉疾病, 包括亨廷顿舞蹈病(Huntington's disease, HD)、ALS、脆性X染色体相关震颤/共济失调综合征(fragile X-associated tremor/ataxia syndrome, FXTAS) 及1型和2型杜氏肌营养不良(Duchenne muscular dystrophy, DMD)。如DMD是一种常染色体显性遗传的多系统疾病, 主要特征为肌强直和进行性肌无力。大约60%~70%的患者存在DMD基因内一个或多个外显子的缺失。其他不太常见的突变包括基因内核苷酸重复扩增、单核苷酸变异、剪接位点改变及少数核苷酸的缺失和插入, 以下仅以核苷酸重复扩增引起DMD为例。两个位点中的不同微卫星扩增导致具有相似特征的不同形式的疾病: DM1型(DM1) 是由位于染色体19q13.3位点上的营养不良肌紧张蛋白激酶(DM1 protein kinase, DMPK) 基因3'-非翻译区(3'-untranslated regions, 3'-UTR) 的三(CTG) 核苷酸扩增引起的, 疾病严重程度和发病年龄与重复长度相关, 重复长度范围为80至数千个重复, 而未感染个体的重复数少于40; DM2型(DM2) 是由染色体3q21位上锌指蛋白9 (zinc finger protein 9, ZNF9) 基因内含子1内的四(CCTG) 核苷酸扩增引起的。这些基因的pre-mRNA中扩展的CUG和CCUG重复序列可以自身折叠, 形成相对较长的双链RNA, 通过与RNA结合蛋白的相互作用引起疾病。对与CUG重复序列结合的能力进行鉴定, 筛选出两种重要的蛋白质: 肌盲样蛋白1 (muscle blind-like protein 1, MBNL1) 和CUG结合蛋白1 (CUG binding protein 1, CUGBP1)。MBNL1和CUGBP1都是重要的选择性剪接调节器。剪接过程中, MBNL1被隔离到由CUG重复序列形成的双链发夹结构中, 将其从核质中耗尽。另一方面, CUG重复通过PKC活化诱导CUGBP1过度磷酸化和稳定。两种蛋白在选择性剪接调控中出现拮抗反应^[45], 这说明降低MBNL1活性和增加CUGBP1活性的综合作用共同调节选择性剪接的变化。

3 靶向AS的治疗工具

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设计安全有效的治疗策略来克服异常剪接事件所导致的疾病以达到精准治疗的目的仍然是一项巨大挑战。目前已经开发了如下几种治疗工具来影响选择性剪接过程从而改变剪接结果, 长期研究显示这些可以通过纠正或诱导RNA异常剪接、靶向外显子连接突变、靶向增强子和沉默子等作用于选择性剪接多个环节的剪接工具具有强大的治疗潜力, 为新的靶点治疗方法开发指明了方向。

3.1 基因替代疗法

基因替代疗法为治疗遗传性的神经肌肉疾病提供了新的途径, 有可能实现一次性治愈性修复或改变个体受影响的基因, 最大限度地减少甚至消除患者生命周期的疾病症状^[48]。对于由基因表达缺失或减少引起的单基因疾病, 如SMA和DMD, 该基因疗法使用基因替代策略将被破坏基因的完整拷贝(称为转基因) 传递给细胞, 使它们能够表达正常的功能性蛋白来缓解疾病。基因替代治疗能够直接纠正疾病相关的剪接变异而不影响基因组。

Onasemnogene abeparvovec是一种复制、重组、自我互补、基于腺相关病毒(adeno-associated virus, AAV) 载体的用于治疗SMA的新型基因替代疗法。它使用腺相关病毒血清型9 (adeno-associated virus serotype 9, AAV9), 在巨细胞病毒增强子/鸡-β-肌动蛋白杂交启动子的控制下, 将功能性SMN1基因递送到运动神经元^[49]。AAV可以穿过血脑屏障, 并通过神经元启动子驱动其在运动神经元中的活动。2019年1月, Onasemnogene abeparvovec获得FDA批准, 其已被证明可改善重度Ⅰ型SMA婴儿的运动功能。

Mendell等^[50]在2010年首次将AAV介导的微DMD基因转移至人体中进行试验, 结果显示, 该疗法具有较好的安全性, 但并未显著增加微肌营养不良蛋白的表达。随后, 为了确保转导效率和组织靶向, 该团队选用腺相关病毒血清型74 (adeno-associated virus serotype 74, AAVrh74) 载体和杂合α-肌球蛋白重链增强子/MCK增强子(MHCK7) 启动子对4名DMD患儿进行全身性微肌营养不良蛋白的开放标签Ⅰ/Ⅱa期试验, 结果显示该疗法能够将AAVrh74-MHCK7-微肌营养不良蛋白成功地送至骨骼肌, 增加微肌营养不良蛋白表达并促进肌营养不良蛋白相关蛋白复合物的恢复和重建^[51]。

基因替代疗法还应用在ALS^[52]、X连锁肌管性肌病(X-linked myotubular myopathy, XLMTM) ^[53-55]、包括AD^{[56, 57]}、帕金森病(Parkinson's disease, PD)^{[58, 59]}、卡纳万病(Canavan disease, CD)^[57]等在内的中枢神经系统疾病和包括血友病^[60-62]、溶酶体贮积症(lysosomal storage diseases, LSD)^[63-65]等在内的出血性疾病等(表 1列举了部分基因替代疗法药物)。

基因疗法相较于其他治疗策略, 其免疫原性、稳定性较差、所递送的基因在人体内的可控性难以预测及载体的安全性和转移效率低等问题更加突出, 并且这些问题一旦出现对患者所造成的影响是致命性的。此外, 基因治疗的价格极其昂贵, 这也是限制其临床应用最主要的原因之一。但是基因替代治疗可能带来的独特的一次可治愈性仍使其成为极具吸引力的治疗手段。

3.2 剪接体介导的反式剪接(spliceosome-mediated RNA trans-splicing, SMaRT)

剪接体介导的RNA反式剪接是基于反式剪接过程的基因重编码技术, 其通过设计用于替换靶剪接位点上游或下游的整个编码序列来校正异常mRNA, 从而实现相关疾病的治疗。该方法涉及3个不同的组成部分: 靶mRNA、剪接体机器和前反式剪接分子(pre-trans-splicing molecule, PTM)。前两种成分存在于细胞中, 而第3种由外源提供引入靶细胞内, 诱导外源RNA和内源pre-mRNA之间的反式剪接, 产生具有野生型序列(无突变) 的嵌合RNA^[16]。经典的PTM由以下部分组成: ①能够通过碱基配对识别内源性pre-mRNA上靶内含子的结合结构域; ②催化剪接反应的人工内含子; ③包含取代编码序列的cDNA^[66]。根据pre-mRNA的靶向区域, SMaRT可分为① 5'-反式剪接, 其靶向5'部分; ② 3'-反式剪接, 其靶向3'部分; ③内部外显子置换(IER), 其靶向pre-mRNA的内部。

SMaRT方法已经用于多种疾病治疗。Coady等^[67]开发了一种靶向SMN2治疗SMA的反式剪接因子, 该因子包括包含大约130 nt的SMN内含子6退火序列、优化的异源剪接位点、SMN1外显子7序列以及SMN外显子7下游血凝素(hemagglutinin, HA) 基序的两个串联拷贝, 以AAV为载体输送至SMA患者成纤维细胞内。研究结果显示, SMN外显子7适合反式剪接替换策略, 内源性SMN2转录本可以作为标靶进行反式剪接, 从而使SMN2基因产生全长SMN。此外, 反式剪接不仅可以增加来自内源性SMN2基因的SMN蛋白水平, 而且经反式剪接产生的蛋白质可以恢复患者成纤维细胞中的关键SMN功能。

SMaRT策略还用于其他疾病的治疗, 例如囊性纤维化(cystic fibrosis, CF)^[68-70]、DMD^{[71, 72]}、血友病A (hemophilia A)^[73]、SMA^{[67, 74]}、色素性视网膜炎(retinitis pigmentosa, RP)^[75]、X连锁高IgM综合征^[76]、额颞叶痴呆伴帕金森综合征-17 (frontotemporal dementia and parkinsonism linked to chromosome 17, FTDP-17)^{[77, 78]}、严重联合免疫缺陷(severe combined immunodeficiency disease, SCID)^[79]和大疱性表皮松解症(epidermolysis bullosa, EB)^[80-82]等疾病(表 1列举了部分SMaRT治疗药物)。

虽然PTM合成和效率评估技术繁琐, 并且由于病毒表达和递送的限制, 反式剪接的特异性和效率可能偏离预期。但是对于5' SS或3' SS突变, 特别是突变位于内含子的第一个或最后一个核苷酸中时, 反式剪接是纠正该类突变最简单有效的方法。在反式剪接过程中, PTM仅取代基因的一部分, 由内源性启动子控制pre-mRNA的转录^[83], 因此靶基因pre-mRNA的表达仍处于内源性控制之下。并且反式剪接分子只能与现有的pre-mRNA相互作用, 因此基因的组织特异性、时间特异性和数量特异性表达不会改变。此外, 一个PTM分子在单个反应中同时具有减少突变蛋白合成和促进正常蛋白质合成的双重能力^[66]。

3.3 反义寡核苷酸(antisense oligonucleotides, ASOs)

ASOs通常为15~25 nt的短DNA或RNA序列, 以Watson-Crick碱基配对与特定pre-mRNA序列直接结合, 通过RNase H介导的靶RNA降解^[84]、在空间上干扰转录本RNA-RNA和/或RNA-蛋白质相互作用^{[85, 86]}或者改变剪接调节转录本的稳定性^[87]等途径来改变剪接过程。ASOs可被设计成靶向① 5' SS或3' SS, 空间上阻断剪接因子进入剪接位点, 将剪接重定向到相邻位点来促进剪接; ②增强子或沉默子元件, 防止反式作用调节剪接因子在靶位点的结合以阻断或促进剪接; ③ RNA茎环, 稳定或去稳定调节其结构并修饰剪接结果^{[16, 83]}。

未经修饰的寡核苷酸对循环核酸酶高度敏感, 可使其降解并通过肾脏排泄, 因此ASOs的化学修饰是非常关键的, 它们可以在体内稳定ASOs防止细胞降解并改善其细胞摄取、释放, 还能调节其与靶RNA序列的结合亲和力^[88]。常见的化学修饰包括寡核苷酸的磷酸骨架和/或糖组分的改变。如①第一代ASOs使用硫代磷酸(phosphorothioate, PS) 骨架, 是通过用硫原子替换主链中的一个非桥接氧原子来实现的。这种修饰的ASOs更稳定并且能够激活RNase H以下调靶RNA水平, 但也破坏了双链的稳定性, 导致熔化温度降低, 高浓度的PS还会产生细胞毒性; ②第二代ASOs对核糖糖环2' 位置进行修饰, 与PS骨架结合能更好地发挥作用。2'-O-甲基(2-OMe) 和2'-O-甲氧基乙基(2'-MOE) 被认为是最成功的糖基修饰, 与未经修饰的硫代磷酸盐相比, 这些修饰增加了其与靶RNA的结合力, 并且能减少PS主链产生的序列无关毒性, 但是这种方式修饰的ASOs不能诱导RNase H介导的靶RNA下调; ③其他ASOs有用聚酰胺键取代整个糖磷酸骨架而产生的肽核酸(PNA); 具有吗啉环而不是核糖环, 并且具有磷酰胺亚基间连接的二氨基磷酸吗啉基低聚物(PMO) 等。由于可能存在细胞摄取不良、水不溶性、在体内被迅速清除或在高剂量下毒性严重等问题应用受到限制^[89]。

2005年, Scaffidi等^[36]报道了一种25聚体的吗啉代寡核苷酸, 用于纠正HGPS中LMNA基因的异常剪接。该ASO能与pre-mRNA外显子11中产生HGPS突变的区域互补, 在空间上阻断隐蔽剪接位点的激活, 从而防止剪接机制进入异常的剪接位点, 有效阻断了内源性LMNA mRNA的异常剪接, 增加了功能性LMNA的水平。

Nusinersen于2016年12月由FDA批准上市, 是首个获批用于治疗SMA的药物^[90]。Nusinersen是一种2-OMe硫代磷酸酯ASO, 靶向位于外显子7的5' SS下游15 nt的抑制性RNA调控元件ISS-N1, 阻断hnRNP A1/A2与该区域两个基序的结合, 并破坏促进外显子7跳跃的抑制性二级结构末端茎环2 (terminal stem-loop 2, TSL2), 隔离外显子7的5' SS, 诱导外显子7包含并产生全长的功能性SMN蛋白^[91]。值得注意的是, 由于ASOs通常不会穿过血脑屏障, 因此Nusinersen需要重复鞘内给药。

ASOs靶向异常剪接的治疗策略不仅被用于CF^[92]、营养不良性大疱性表皮松解症(dystrophic epidermolysis bullosa, DEB)^[93]、DMD^{[94, 95]}、FTDP-17^[96]等遗传代谢性疾病, 而且也适用于许多癌症的治疗。如Dewaele等^[97]使用ASOs介导的外显子跳跃来降低MDM4的表达, MDM4是癌细胞中产生的剪接亚型。类似地, Hong等^[98]临床前和临床评价了一种化学修饰的ASO, 称为AZD9150, 其靶向STAT3编码基因, STAT3是JAK-STAT信号通路的转录激活因子和致癌介导物。Ross等^[99]使用一种含乙基的ASO (AZD4785) 下调了KRAS mRNA, 其在大约20%的人类癌症中发生突变(表 2列举了部分ASO药物)。

ASOs的序列特异性使它们能够精确地与内源性RNA结合, 并且其高保真度允许靶向不同的RNA亚型。虽然ASOs存在摄取较差、无法透过血脑屏障^[100], 具有一定的免疫原性以及通常采用周期性注射给药、患者依从性差等问题^[101]。但是易于递送的特点、良好的毒性特征和持久的效果正使其成为理想的疾病治疗工具^{[102, 103]}。

3.4 小干扰RNA (small interfering RNAs, siRNAs)

1998年, 随着siRNA通路的发现出现了RNA干扰(RNA interference, RNAi) 技术, 它使用21~25 nt的双链(ds) RNA通过RNA诱导沉默复合物(RISC) 降解靶pre-mRNA, 从而抑制内源和异源基因的表达^[104]。siRNA是RNAi的一种模式, 靶向外显子、内含子和外显子/内含子连接序列的siRNAs可以诱导选择性剪接和异常mRNA的降解, 而不影响正常mRNA的表达。

这种靶向方法已经用于如Ullrich先天性肌营养不良(Ullrich congenital muscular dystrophy, UCMD)、生长激素缺乏症(growth hormone deficiency, GHD) Ⅱ型和几种癌症的治疗。Bolduc等^[105]设计了不同的siRNAs, 靶向导致COL6A3基因外显子16跳跃的最常见突变, 并在UCMD衍生的真皮成纤维细胞中体外检测了siRNAs。这些siRNAs可以作为有效的等位基因特异性探针, 导致突变等位基因特异性敲除, 从而增加胶原Ⅵ基质产生的丰度和质量。类似地, Ryther等^[106]使用siRNA策略, 以外显子2~4为靶标, 特异性降解Ⅱ型GHD中外显子3跳过的转录物。结果显示, 即使将少量siRNAs递送到垂体也可能恢复Ⅱ型GHD患者GH的正常分泌。Hayes等^[107]已经表明, siRNA介导的SR蛋白激酶1 (serine/arginine-rich splicing factor protein kinase-1, SRPK1) 的下调(其在胰腺、乳腺和结肠肿瘤中显著上调) 导致细胞增殖剂量依赖性降低和凋亡潜力增加。此外, SRPK1表达的破坏导致肿瘤细胞对化疗剂如吉西他滨和顺铂的敏感性增加。

基于siRNA策略还开发了家族性淀粉样变性多发性神经病(familial amyloid polyneuropathy, FAP) 治疗药物patisiran^[108], 成人急性肝卟啉病(acute hepatic porphyria, AHP) 治疗药物givosiran^{[109, 110]}和高胆固醇血症治疗药物inclisiran^[111]等(表 2列举了部分siRNA药物)。

从理论上讲, 任何目的基因都可以被siRNA靶向, 这一优势使siRNA具有比小分子或抗体药物更短的研发时间和更广泛的治疗领域。尽管siRNA在药物开发中具有广阔的前景, 但稳定性不理想、药代动力学差、可能出现脱靶效应等仍是该技术未来发展中需要重点关注的问题。

3.5 小分子药物

小分子化合物也可用于调节RNA表达, 其可在剪接位点或调控序列(ESE、ESS、ISE、ISS) 通过直接干扰三级RNA结构或阻碍蛋白质-RNA相互作用来抑制或激活某个剪接位点的使用, 从而促进或抑制选择性剪接。

一些小分子剪接调节剂已经在治疗遗传代谢性疾病和癌症的临床试验中进行了评估。如普拉地内酯B的抗肿瘤活性已在体内和体外以及在源自胃癌、宫颈癌及红白血病的多种癌细胞系中得到证实^{[112, 113]}。普拉地内酯B可影响参与细胞凋亡的基因如p73, 通过增加和减少促细胞凋亡和抗细胞凋亡的异构体Tap73和DNp73来纠正异常剪接^[114]。此外, 普拉地内酯B已被证明可导致细胞周期停滞^[115]。普拉地内酯类似物E7107是靶向剪接体的新型抗癌剂的第一个化合物, 其靶向U2 snRNP复合体的SF3B, 与SF3B的亚基1相互作用以阻断癌基因的正常剪接。尽管E7107在临床前研究中展现出良好的效力, 但由于Ⅰ期临床试验中患者出现腹泻、呕吐、脱水和心肌梗塞等不良反应而导致试验停止^[116]。Herboxidiene、剪接抑素A、美亚霉素B和舒地霉素D6/K也已显示出通过靶向剪接体的SF3B亚基而表现出体内抗肿瘤活性^[117]。

Risdiplam是诺华公司开发的一种治疗SMA的脑渗透性口服液。目前, 该药物被批准用于所有年龄范围的1型、2型和3型SMA患者。在SMN2 pre-mRNA的剪接过程中, 外显子7的5' SS的特殊替换导致其与U1 snRNP的结合减弱, 直接导致剪接跳过。Risdiplam作为一种剪接修饰剂能够特异性地稳定由5' SS和U1 snRNP复合物组成的瞬时双链RNA结构, 将SMN2外显子7的弱5' SS转化为强5' SS, 有利于剪接的进行, 从而增加SMN2 mRNA中外显子7的含量和整个生物体中功能性SMN蛋白的水平。同时, 它还可以选择性地与SMN2外显子7中的外显子剪接增强子ESE2结合, 以增强靶向SMN2的特异性。

其他小分子药物包括恢复CF中囊性纤维化跨膜电导调节剂(cystic fibrosis transmembrane conductance regulator, CFTR) 基因突变、挽救功能性CFTR表达的DYRK/CLK双重激酶抑制剂CaNDY^[118], 在剪接失调的非小细胞肺癌(non-small cell lung carcinoma, NSCLC) 细胞中表现出抗增殖作用的PRMT5抑制剂PF06939999^[119], 促进CRL4-DCAF15泛素连接酶复合物对剪接因子RBM3的识别和降解而具有抗癌活性的indisulam^[120]以及诱导HTT pre-mRNA的内含子49中包含含有终止密码子的假外显子, 从而降低致病性蛋白质水平的HTT-C1和HTT-D1^[121]等(表 3列举了部分小分子药物)。

与上述治疗手段相比, 小分子化合物发挥作用的机制通常不够明确, 并且靶向特异性相对较低, 可能出现更多的非特异性和脱靶效应。但是小分子药物合成可及性较好、更容易递送至靶位点、药效良好并且通常毒性较低, 若通过口服给药, 还可以增加患者的依从性和便利性。具有小分子固有优势的化学药物的开发为操纵剪接提供了更为简便的治疗工具, 具有广阔的发展前景。

4 总结与展望

收起

Pre-mRNA的选择性剪接是基因表达调控的重要组成部分, 不同模式替代外显子的组合显著扩展了蛋白质组的多样性, 并对细胞、组织、器官的分化、发育和多样性具有重要影响。选择性剪接是复杂的生理过程, 由多种顺式作用元件和反式作用因子共同参与, 通过多样的RNA-蛋白质相互作用驱动, 调节机体的各项生理活动。越来越多的研究关注于选择性剪接与疾病之间的联系, 近年来, 与人类疾病相关的分子机制的研究为靶向剪接过程的治疗提供了可能性。正如文章中所概述的, 研究者们已经设计了多种工具来改变剪接反应的结果, 以治疗由剪接异常引起的疾病。该领域的广泛研究和一系列药物的上市已经证实了这种剪接修饰的策略对疾病治疗产生的现实可行性。然而, 这些工具在不同程度上以不同方式靶向体内的基因序列或蛋白结构, 因此其免疫原性、脱靶效应等所体现出的安全性问题是其走向临床必须深入细致研究的重要因素。此外, 剪接效率、靶向特异性和作用的持久性同样也是开发此类工具必须考虑的方面。因此, 更有效更安全的RNA剪接修饰的工具的开发仍任重道远, 也必将是该领域研究的热点和重点。

作者贡献: 郭欣茹检索文献、撰写文章、修改文章; 张翔指导撰写思路和文章修改。

利益冲突: 本文作者无利益冲突。

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国家自然科学基金青年科学基金资助项目(82103979)
中国医学科学院医学与健康科技创新工程项目(2021-I2M-1-028)

参考文献

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2023年第58卷第12期

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doi: 10.16438/j.0513-4870.2023-0586

接收时间：2023-05-08
首发时间：2025-11-21
出版时间：2023-12-12

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收稿日期：2023-05-08
修回日期：2023-06-02

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国家自然科学基金青年科学基金资助项目(82103979)

中国医学科学院医学与健康科技创新工程项目(2021-I2M-1-028)

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中国医学科学院、北京协和医学院药物研究所, 活性物质发现与适药化研究北京市重点实验室, 北京 100050

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*张翔, Tel: 86-10-63165248, E-mail: simon@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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EndNote
RefWorks
TxT

Name	Proper name/target	Indication	Vector	Organization	Stage
Gendicine^TM	Recombinant human p53 adenovirus particle	Head and neck cancer	Adenovirus type 5-p53	Shenzhen SiBiono GeneTech	Launched
Glybera^TM	Alipogene Tiparvovec	LPLD	rAAV1-LPL	UniQure	Withdrawn
Strimvelis^TM	Autologous CD34⁺ cells transduced to express ADA	ADA-SCID	Retroviral-ADA	Orchard Therapeutics	Launched
Luxturna^TM	Voretigene Neparvovec-rzl	IRD	rAAV2-PRE65	Spark Therapeutics	Launched
Zolgensma^TM	Onasemnogene Abeparvovec-xioi	SMA	rAAV9-SMN1	AveXis	Launched
Zynteglo^TM	Autologous CD34⁺ cells encoding β^A-T87Q-globin gene	Β-thalassemia	Lentiviral-β-globin	Bluebird Bio	Launched
Invossa^TM	TissueGene-C	OA	Retroviral-TGF-β1	Kolon Life Science	Launched
RZ-001	hTERT mRNA	HCC	Adenovirus	Rznomics	Phase Ⅰ/Ⅱ
PRT-mirl22aT	mTERT mRNA	HCC	Adenovirus	Dong-A University	Preclinical
Ad.CMV.Rz.p53	mTERT mRNA	HCC	Adenovirus	Gyeongsang National University	Biological testing
Ad5mTR	mTERT mRNA	Head and neck cancer	Adenovirus type 5	National Cancer Center of Korea	Preclinical
Ad5CMV.Rz.HSVtk.miR-145	mTERT mRNA	Brain cancer	Adenovirus type 5	National Cancer Center of Korea	Preclinical
Ad5CMV.mTR.sPD1	mTERT mRNA	Colorectal cancer, non-Hodgkin's lymphoma	Adenovirus type 5	National Cancer Center of Korea	Biological testing
pRib100AS-HSVtk	AIMP2-DX2 pre-mRNA	Lung cancer	DNA plasmids	Dankook University	Biological testing
Ad-3R1-TK/GCV	KRAS G12V pre-mRNA	Cancer	Adenovirus	Dankook University	Preclinical
AAV2/8-bRho-PTM20	RHO pre-mRNA	Autosomal dominant retinitis pigmentosa	AAV	INSERM	Preclinical
pA5c-9v1	DENV mRNA	Viral	pA5c	University of Notre Dame	Biological testing
pMU2-tsRNAHA	SMN pre-mRNA	SMA	rAAV	Boston University School of Medicine	Biological testing

Name

Proper name/target

Indication

Vector

Organization

Stage

Gendicine^TM

Recombinant human p53 adenovirus particle

Head and neck cancer

Adenovirus type 5-p53

Shenzhen SiBiono GeneTech

Launched

Glybera^TM

Alipogene Tiparvovec

LPLD

rAAV1-LPL

UniQure

Withdrawn

Strimvelis^TM

Autologous CD34⁺ cells transduced to express ADA

ADA-SCID

Retroviral-ADA

Orchard Therapeutics

Launched

Luxturna^TM

Voretigene Neparvovec-rzl

IRD

rAAV2-PRE65

Spark Therapeutics

Launched

Zolgensma^TM

Onasemnogene Abeparvovec-xioi

SMA

rAAV9-SMN1

AveXis

Launched

Zynteglo^TM

Autologous CD34⁺ cells encoding β^A-T87Q-globin gene

Β-thalassemia

Lentiviral-β-globin

Bluebird Bio

Launched

Invossa^TM

TissueGene-C

Retroviral-TGF-β1

Kolon Life Science

Launched

RZ-001

hTERT mRNA

HCC

Adenovirus

Rznomics

Phase Ⅰ/Ⅱ

PRT-mirl22aT

mTERT mRNA

HCC

Adenovirus

Dong-A University

Preclinical

Ad.CMV.Rz.p53

mTERT mRNA

HCC

Adenovirus

Gyeongsang National University

Biological testing

Ad5mTR

mTERT mRNA

Head and neck cancer

Adenovirus type 5

National Cancer Center of Korea

Preclinical

Ad5CMV.Rz.HSVtk.miR-145

mTERT mRNA

Brain cancer

Adenovirus type 5

National Cancer Center of Korea

Preclinical

Ad5CMV.mTR.sPD1

mTERT mRNA

Colorectal cancer, non-Hodgkin's lymphoma

Adenovirus type 5

National Cancer Center of Korea

Biological testing

pRib100AS-HSVtk

AIMP2-DX2 pre-mRNA

Lung cancer

DNA plasmids

Dankook University

Biological testing

Ad-3R1-TK/GCV

KRAS G12V pre-mRNA

Cancer

Adenovirus

Dankook University

Preclinical

AAV2/8-bRho-PTM20

RHO pre-mRNA

Autosomal dominant retinitis pigmentosa

AAV

INSERM

Preclinical

pA5c-9v1

DENV mRNA

Viral

pA5c

University of Notre Dame

Biological testing

pMU2-tsRNAHA

SMN pre-mRNA

SMA

rAAV

Boston University School of Medicine

Biological testing

Name	Sequence (sense/anti-sense strand)/vector	Indication	Target	Organization	Stage
Patisiran (Onpattro^TM)	5'-GUAACCAAGAGUAUUCCAUTT-3'/ 5'-AUGGAAUACUCUUGGUUACTT-3'	FAP	TTR mRNA	Alnylam Pharma	Launched
Vutrisiran (Amvuttra^TM)	5'-UCUUGGUUACAUGUAACCAAGA-3'/ 5'-UGGGAUUUCAUGUAACCAAGA-3'	ATTR	TTR mRNA	Alnylam Pharma	Launched
Givosiran (Givlaari^TM)	5'-UGGUCUUUCUCACAGAGUAGAA-3'/ 5'-AUUCUACUCUCUGUGAGAAAGAC-3'	AHP	ALAS1 mRNA	Alnylam Pharma	Launched
Lumasiran (Oxlumo^TM)	GACUUUCAUCCUGGAAAUAUA/ ACCUGAAAGUAGGACCUUUAUAU	PH	HAO1 mRNA	Alnylam Pharma	Launched
Inclisiran (Leqvio^TM)	5'-CUAGACCUGUTUUGCUUUUGU-3' 5'-ACAAAAGCAAAACAGGUCUAGAA-3'	Atheroscle-rosis	PCSK9 mRNA	Novartis	Launched
Fitusiran	5'-GGUUAACACCAUUUACUUCAA-3'/ 5'-UUGAAGUAAAUGGUGUUAACCAG-3'	Hemophilia A/B	SERPINC1 mRNA	Sanofi Genzyme	Phase Ⅲ
Nusinersen (Spinraza^TM)	5'-TCACTTTCATAATGCTGG-3'	SMA	SMN2 Ex7	Ionis Pharma, Biogen	Launched
E1^MOv11	5'-CUAUAUAUAGUUAUUCAACA-3'	SMA	SMN2 E1	Shift Pharma	Preclinical
MO HSMN2-Ex7D	5'-GTAAGATTCACTTTCATAATGCTGG-3'	SMA	SMN2 Ex7	IRCCS Ospedale Maggiore Policlinico	Preclinical
E1.4	CTGAAAGgttagtggacagccatgc	FTDP-17	MAPT Ex1	University of Pennsylvania	Preclinical
E5.3	GCCAAGgtaagctgacgatgccacagg	FTDP-17	MAPT Ex5	University of Pennsylvania	Preclinical
7-26S	GTCGCAAACAGTACAATGGC	FD	IKBKAP Ex20	Cold Spring Harbor Laboratory	Preclinical
Eteplirsen (Exondys 51^TM)	5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3'	DMD	Dystrophin Ex51	Sarepta Therapeutics	Launched
Drisapersen	5'-UCAAGGAAGAUGGCAUUUCU-3'	DMD	Dystrophin Ex51	BioMarin Pharma Inc	Phase Ⅲ (discontinued)
Golodirsen (Vyondys 53^TM)	5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3'	DMD	Dystrophin Ex53	Sarepta Therapeutics	Launched
Viltolarsen (Viltepso^TM)	5'-CCUCCGGUUCUGAAGGUGUUC-3'	DMD	Dystrophin Ex53	National Center of Neurology and Psychiatry	Launched
Casimersen	5'-CAATGCCATCCTGGAGTTCCTG-3'	DMD	Dystrophin Ex45	Sarepta Therapeutics	Launched
DS-5141	5'-CCUACCGUAACCCGUCGC-3'	DMD	Dystrophin Ex45	Daiichi Sankyo Co Ltd	Phase Ⅰ/Ⅱ

Name

Sequence (sense/anti-sense strand)/vector

Indication

Target

Organization

Stage

Patisiran (Onpattro^TM)

5'-GUAACCAAGAGUAUUCCAUTT-3'/
5'-AUGGAAUACUCUUGGUUACTT-3'

FAP

TTR mRNA

Alnylam Pharma

Launched

Vutrisiran (Amvuttra^TM)

5'-UCUUGGUUACAUGUAACCAAGA-3'/
5'-UGGGAUUUCAUGUAACCAAGA-3'

ATTR

TTR mRNA

Alnylam Pharma

Launched

Givosiran (Givlaari^TM)

5'-UGGUCUUUCUCACAGAGUAGAA-3'/
5'-AUUCUACUCUCUGUGAGAAAGAC-3'

AHP

ALAS1 mRNA

Alnylam Pharma

Launched

Lumasiran (Oxlumo^TM)

GACUUUCAUCCUGGAAAUAUA/
ACCUGAAAGUAGGACCUUUAUAU

HAO1 mRNA

Alnylam Pharma

Launched

Inclisiran (Leqvio^TM)

5'-CUAGACCUGUTUUGCUUUUGU-3'
5'-ACAAAAGCAAAACAGGUCUAGAA-3'

Atheroscle-rosis

PCSK9 mRNA

Novartis

Launched

Fitusiran

5'-GGUUAACACCAUUUACUUCAA-3'/
5'-UUGAAGUAAAUGGUGUUAACCAG-3'

Hemophilia A/B

SERPINC1 mRNA

Sanofi Genzyme

Phase Ⅲ

Nusinersen (Spinraza^TM)

5'-TCACTTTCATAATGCTGG-3'

SMA

SMN2 Ex7

Ionis Pharma,
Biogen

Launched

E1^MOv11

5'-CUAUAUAUAGUUAUUCAACA-3'

SMA

SMN2 E1

Shift Pharma

Preclinical

MO HSMN2-Ex7D

5'-GTAAGATTCACTTTCATAATGCTGG-3'

SMA

SMN2 Ex7

IRCCS Ospedale Maggiore Policlinico

Preclinical

E1.4

CTGAAAGgttagtggacagccatgc

FTDP-17

MAPT Ex1

University of Pennsylvania

Preclinical

E5.3

GCCAAGgtaagctgacgatgccacagg

FTDP-17

MAPT Ex5

University of Pennsylvania

Preclinical

7-26S

GTCGCAAACAGTACAATGGC

IKBKAP Ex20

Cold Spring Harbor Laboratory

Preclinical

Eteplirsen (Exondys 51^TM)

5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3'

DMD

Dystrophin Ex51

Sarepta Therapeutics

Launched

Drisapersen

5'-UCAAGGAAGAUGGCAUUUCU-3'

DMD

Dystrophin Ex51

BioMarin Pharma Inc

Phase Ⅲ
(discontinued)

Golodirsen (Vyondys 53^TM)

5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3'

DMD

Dystrophin Ex53

Sarepta Therapeutics

Launched

Viltolarsen (Viltepso^TM)

5'-CCUCCGGUUCUGAAGGUGUUC-3'

DMD

Dystrophin Ex53

National Center of Neurology and Psychiatry

Launched

Casimersen

5'-CAATGCCATCCTGGAGTTCCTG-3'

DMD

Dystrophin Ex45

Sarepta Therapeutics

Launched

DS-5141

5'-CCUACCGUAACCCGUCGC-3'

DMD

Dystrophin Ex45

Daiichi Sankyo Co Ltd

Phase Ⅰ/Ⅱ

Name	Indication	Target	Organization	Stage
Risdiplam (Evrysdi^TM)	SMA	SMN2 Ex7	PTC Therapeutics, Roche, SMA Foundation	Approved
Branaplam	SMA/HD	SMN2 Ex7/HTT pre-mRNA	Novartis	Phase Ⅱ/Ⅲ (discontinued)
PK4C9	SMA	SMN2 TSL2	Roche	Preclinical
LDN-2014	SMA	SMN2 pre-mRNA	Brigham and Women's Hospital, Indiana University Research Technology, University of Massachusetts	Preclinical
Kinetin	FD	IKBKAP pre-mRNA	New York University	Phase Ⅰ (discontinued)
RECTAS	FD	IKBKAP Ex20	Kyoto University	Preclinical
BPN-15477	FD/FTDP-17	IKBKAP Ex20/MAPT Ex10	PTC Therapeutics	Preclinical

Name

Structure

Indication

Target

Organization

Stage

Risdiplam (Evrysdi^TM)

SMA

SMN2 Ex7

PTC Therapeutics, Roche, SMA Foundation

Approved

Branaplam

SMA/HD

SMN2 Ex7/HTT
pre-mRNA

Novartis

Phase Ⅱ/Ⅲ (discontinued)

PK4C9

SMA

SMN2 TSL2

Roche

Preclinical

LDN-2014

SMA

SMN2 pre-mRNA

Brigham and Women's Hospital, Indiana University Research Technology, University of Massachusetts

Preclinical

Kinetin

IKBKAP pre-mRNA

New York University

Phase Ⅰ (discontinued)

RECTAS

IKBKAP Ex20

Kyoto University

Preclinical

BPN-15477

FD/FTDP-17

IKBKAP Ex20/MAPT Ex10

PTC Therapeutics

Preclinical