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Eighteen nucleic acid therapeutics have been approved for treatment of various diseases in the last 25 years. Their modes of action include antisense oligonucleotides (ASOs), splice-switching oligonucleotides (SSOs), RNA interference (RNAi) and an RNA aptamer against a protein. Among the diseases targeted by this new class of drugs are homozygous familial hypercholesterolemia, spinal muscular atrophy, Duchenne muscular dystrophy, hereditary transthyretin-mediated amyloidosis, familial chylomicronemia syndrome, acute hepatic porphyria, and primary hyperoxaluria. Chemical modification of DNA and RNA was central to making drugs out of oligonucleotides. Oligonucleotide therapeutics brought to market thus far contain just a handful of first- and second-generation modifications, among them 2′-fluoro-RNA, 2′-O-methyl RNA and the phosphorothioates that were introduced over 50 years ago. Two other privileged chemistries are 2′-O-(2-methoxyethyl)-RNA (MOE) and the phosphorodiamidate morpholinos (PMO). Given their importance in imparting oligonucleotides with high target affinity, metabolic stability and favorable pharmacokinetic and -dynamic properties, this article provides a review of these chemistries and their use in nucleic acid therapeutics. Breakthroughs in lipid formulation and GalNAc conjugation of modified oligonucleotides have paved the way to efficient delivery and robust, long-lasting silencing of genes. This review provides an account of the state-of-the-art of targeted oligo delivery to hepatocytes.
过去 25 年中，已经批准了 18 种核酸治疗剂来治疗各种疾病。它们的作用方式包括反义寡核苷酸（ASOs）、剪接调节寡核苷酸（SSOs）、RNA 干扰（RNAi）和针对蛋白质的 RNA 适配体。这种新型药物治疗的疾病包括同型家族高胆固醇血症、脊髓肌萎缩症、杜氏肌营养不良症、遗传性转甲状腺素介导的淀粉样变性、家族型脂肪乳症、急性肝性神经病和原发性肾小管中毒。化学修饰 DNA 和 RNA 是将寡核苷酸转化为药物的关键。到目前为止，已上市的寡核苷酸治疗药物中只包含了少数几种第一代和第二代修饰，其中 2'-氟内酰胺 RNA、2'-O-甲基 RNA 和 50 多年前引入的磷酸硫酸盐等化学修饰成为了中心。另外两种有优先选择的化学体系是 2'-O-(2-甲氧基乙基)-RNA（MOE）和以磷酸二酰胺为基础的形态恩酰基吗啡核酸（PMO）。鉴于它们在赋予寡核苷酸高靶向亲和力、代谢稳定性和有利的药物代动力学和动力学特性方面的重要性，本文综述了这些化学体系及其在核酸治疗药物中的应用。脂质配方和修饰寡核苷酸的甘氨酰胺酰联已为有效传递和稳健持久的基因沉默打开了道路。本文对靶向寡核苷酸输送到肝细胞的现代技术进行了介绍。
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Half a century may seem like an eternity but occasionally it feels like the years just flew by. Consider what has been achieved in roughly that time frame as we went from: the Wright brothers’ first flight (1903) to manned space flight (Gagarin/Sheppard, 1961), Turing's paper on computable numbers and the decision problem (Turing machine, 1936) to the release of the Cray-2 supercomputer (1985), and Sanger's sequencing of bovine insulin (1953) to the completion of the human genome project (2003). These amazing feats have in common that they happened within some short 50 years! Viewed from such a perspective, chemically modified oligonucleotide drugs getting US FDA approval, VITRAVENE® (fomivirsen)—1998, MACUGEN® (pegaptanib sodium)—2004, and KYNAMRO® (mipomersen)—2013, just half a century after the publication of the structure of DNA (1953), is no small achievement.
半个世纪可能看起来像是永远，但偶尔也会感觉年岁飞逝。考虑到在大约这个时间范围内所取得的成就：我们从莱特兄弟的第一次飞行（1903）到有人驾驶的太空飞行（加加林/谢伯德，1961），图灵论文中的可计算数字和决策问题（图灵机，1936）到 Cray-2 超级计算机的发布（1985）（5），以及桑格测序牛胰岛素（1953）到完成人类基因组计划（2003）。这些惊人的成就有一个共同点，就是在短短的 50 年内就完成了！从这样的角度来看，化学修饰寡核苷酸药物获得美国 FDA 批准，VITRAVENE®（fomivirsen）-1998，MACUGEN®（pegaptanib sodium）-2004 和 KYNAMRO®（mipomersen）-2013，仅仅在发现 DNA 结构（1953）后的半个世纪，这是一项不小的成就。
A look back at the beginnings of molecular biology makes clear how little was known about concepts we now take for granted, concepts that foreshadowed a watershed of techniques and discoveries that can be traced back to early insights. The model of the DNA double helix sparked intense interest in the structural properties of RNA. It was unclear at the time how the ribose 2′-hydroxyl group would affect the conformation of RNA and whether RNA could form a duplex at all. In a landmark publication in 1956, Rich and Davies demonstrated the existence of an RNA duplex by mixing poly-A and poly-U. Remarkably, the same paper also put forth the concept of nucleic acid hybridization, i.e. the spontaneous pairing using specific interactions between two long nucleic acid strands. In 1960, Rich presented evidence that DNA and RNA can hybridize such that the resulting duplex is held together by hydrogen bonds (H-bonds) between purine and pyrimidine residues. This finding provided a mechanism for information transfer between DNA and RNA.
回顾分子生物学的起步，可以清楚地看到我们现在视为理所当然的概念所知甚少，这些概念预示着一系列技术和发现的分界点，可以追溯到早期的洞察力。DNA 双螺旋模型激起了人们对 RNA 结构性质的浓厚兴趣。当时还不清楚核糖 2'-羟基基团将如何影响 RNA 的构象，以及 RNA 是否能够形成双链。Rich 和 Davies 在 1956 年的一篇具有里程碑意义的文章中通过混合 Poly-A 和 Poly-U 证明了 RNA 双链的存在。值得注意的是，同一篇文章还提出了核酸杂交的概念，即利用两条长的核酸链之间的特定相互作用自发配对。1960 年，Rich 提出了 DNA 和 RNA 可以杂交的证据，从而使产生的双链被嘌呤和嘧啶残基之间的氢键（H-键）所支撑。这一发现提供了在 DNA 和 RNA 之间进行信息传递的机制。
Hybridization reactions are the backbone of modern molecular biology; approaches from the Southern blot to microarrays and the polymerase chain reaction (PCR), as well as genome sequencing and the forensic sciences, all depend at their core on nucleic acid hybridization. The same applies to the inhibition of biological information transfer, first successfully demonstrated by Zamecnik and Stephenson using the antisense and antigene approaches . In just a few years in the late 1950s and early 1960s, discoveries were made that laid the foundation for therapeutic intervention targeting RNA (e.g. hybridization via antisense, alternative splicing or RNA interference) and identified the RNA duplex as a key component of RNA structure (and, we now know, as an important actor in therapeutic intervention in the form of siRNA duplexes). Indeed, the earliest suggestion that RNA might have both a phenotype and a genotype, hinting at the existence of an RNA world, also dates back to the early 1960s.
杂交反应是现代分子生物学的支柱；从 Southern blot、微阵列和聚合酶链反应（PCR）的方法，到基因组测序和法医科学，所有这些方法的核心都依赖于核酸杂交。同样适用于抑制生物信息传递，这是由 Zamecnik 和 Stephenson 通过反义和抗基因方法首次成功实现的。在 20 世纪 50 年代末和 60 年代初的短短几年内的发现为干预 RNA（如通过反义、选择性剪接或 RNA 干扰进行杂交）铺平了基础，并确定了 RNA 双链作为 RNA 结构的关键组成部分（而我们现在知道，它还是 siRNA 双链治疗干预的重要因素）。实际上，最早提出 RNA 可能具有表型和基因型（暗示着 RNA 世界的存在）的建议也可以追溯到 20 世纪 60 年代初。
Beginning in 1955, nucleotide and oligonucleotide syntheses started in the laboratories of Todd, Khorana, Letsinger and Eckstein . Following these initial discoveries, DNA and RNA oligonucleotide syntheses and methods for the same were developed in the laboratories of Caruthers and Ogilvie, respectively. Phosphorothioate oligonucleotide synthesis was developed by Stec. These synthetic revolutions led to the discovery of the genetic code, and paved the way to molecular biology, chemical biology, and nucleic acid-based medicine (see also
https://www.trilinkbiotech.com/a-short-history-of-oligonucleotide-synthesis).
从 1955 年开始，核苷酸和寡核苷酸合成在 Todd、Khorana、Letsinger 和 Eckstein 的实验室中开始。在这些最初的发现之后，Caruthers 和 Ogilvie 的实验室分别开发了 DNA 和 RNA 寡核苷酸合成和相应的合成方法。磷酸硫代酯寡核苷酸合成是由 Stec 开发的。这些合成革命导致了遗传密码的发现，并铺平了分子生物学、化学生物学和基于核酸的医学的道路（也可参见
https://www.trilinkbiotech.com/a-short-history-of-oligonucleotide-synthesis）。
Figure 1 depicts a brief history of key discoveries, nucleic acid structure analysis, and breakthroughs in DNA and RNA synthesis as well as modification strategies on the way to FDA-approved oligonucleotide therapeutics. Chemical modification efforts at the levels of nucleosides and nucleotides got underway >50 years ago and the syntheses of 2′-fluorothymidine, 2′-fluoro-2′-deoxyuridine (2′-F-U), nucleoside phosphorothioates (PSOs) , and 2′-O-methylated (2′-O-Me) polyadenylic acidwere all reported in the 1960s (Figure 2A).
图 1 描述了关键发现、核酸结构分析以及在制备 FDA 批准的寡核苷酸治疗药物上进行 DNA 和 RNA 合成和修饰策略的突破性历程。五十多年前，核苷和核苷酸水平的化学修饰工作已开始，2'-氟胸腺嘧啶、2'-氟-2'-脱氧尿嘧啶（2'-FU）（46）、核苷酸磷硫酸酯（PSOs）和 2'-O-甲基聚腺苷酸（2'-O-Me）在 1960 年代均已报道（图 2A）。
图 1.
化学修饰（洋红色）、寡核苷酸合成方法（黑色）、生物信息传递干扰（绿色）、DNA 和 RNA 结构（蓝色）以及 FDA 批准的寡核苷酸疗法（橙色）的发展时间表。
图 2.
(A) 迄今为止在治疗学中获得批准的天然 DNA、RNA 和化学修饰。 (B) 不同的传递系统； LNP = 脂质纳米颗粒。
The 2′-F and 2′-O-Me modifications figure prominently in MACUGEN, an anti-VEGF aptamer for the treatment of wet age-related macular degeneration, and GIVLAARI® (givosiran), an siRNA for the treatment of acute hepatic porphyria (Figure 3). The 2′-O-Me modification is also present in ONPATTRO® (patisiran), the first FDA-approved RNAi therapeutic (2018), indicated for the treatment of the polyneuropathy of hereditary transthyretin-mediated amyloidosis (hATTR) . The delivery system for ONPATTRO is a lipid nanoparticle (LNP, Figure 2B). VITRAVENE, an antisense drug indicated for cytomegalovirus retinitis and the very first oligonucleotide therapeutic to receive clinical approval was a fully PS-modified 21mer oligo-2′-deoxynucleotide. These first-generation chemistries are still commonly encountered in the backbones of oligonucleotide therapeutic candidates that are currently in phase I to III clinical trials in the US. This is especially true for the phosphorothioates (PS-DNA/-RNA) but 2′-O-Me/2′-F modified oligonucleotides are also represented and include LEQVIO® (inclisiran, Figure 3 Table 1), an siRNA against hypercholesterolemia that was approved by regulators in the European Union in 2021 and the US FDA in 2022.
2'-F 和 2'-O-Me 修饰在马库根（MACUGEN）和 GIVLAARI®（givosiran）等寡核苷酸治疗药物中具有重要作用，用于治疗湿性年龄相关性黄斑变性和急性肝性卟啉症的 siRNA（图 3）。2'-O-Me 修饰物还存在于 ONPATTRO®（patisiran）中，这是首个 FDA 批准的 RNAi 治疗药物（2018 年），用于治疗遗传性转甲状腺素介导的淀粉样变性（hATTR）的多神经病。ONPATTRO 的传递系统是脂质纳米粒（LNP，图 2B）。反义药物 VITRAVENE 用于巨细胞病毒性视网膜炎，是第一个获得临床批准的全思环磷酸酯修饰的 21 肽联脲基-2'-脱氧核糖核酸寡核苷酸，这些第一代化学物质仍然普遍存在于当前在美国 I 至 III 期临床试验中的寡核苷酸治疗候选药物骨干中，尤其是磷酸硫酸酯（PS-DNA/-RNA），但 2'-O-Me/2'-F 修饰的寡核苷酸也有代表，并包括 LEQVIO®（inclisiran）（图 3，表 1），这是一种用于治疗高胆固醇血症的 siRNA，已被欧洲联盟监管机构于 2021 年批准，并于 2022 年获得美国 FDA 批准。
图 3.
目前批准的寡核苷酸药物和 mRNA 疫苗、批准年份（1998 年至 2022 年）和适应症。 从左上到右下：VITRAVENE®、MACUGEN®、KYNAMRO®、EXONDYS 51®、VYONDYS 53®、SPINRAZA®、HEPLISAV-B®、ONPATTRO®、TEGSEDI®、WAYLIVRA®、GIVLAARI®、OXLUMO®、VILTEPSO®、 AMONDYS 45®、LEQVIO®、AMVUTTRA®、COMIRNATY® 和 SPIKEVAX®。 作用机制是：反义寡核苷酸（ASO）、适体、剪接转换寡核苷酸（SSO）、小干扰 RNA（siRNA）和 mRNA（疫苗）。
表 1.
批准的基于寡核苷酸的疗法的总结
Among second-generation antisense modifications, 2′-O-(2-methoxyethyl)-RNA (MOE-RNA) has emerged as the most successful chemistry (Figure 2A). The synthesis of MOE-modified building blocks and oligonucleotides was first reported in 1995 and MOE chemistry subsequently moved from bench to bedside within about 20 years (图 1). The first of three MOE-based therapeutics approved by the FDA was KYNAMRO for the treatment of homozygous familial hypercholesterolemia (图 3, 表 1). The 20mer antisense oligonucleotide is an all-PS modified gapmer with a central DNA decamer gap and pentameric MOE flanks. The gapmer approach is needed to elicit RNase H, the endonuclease that cleaves the RNA target opposite the DNA window.
在第二代反义修饰中，2'-O-(2-甲氧基乙基)-RNA（MOE-RNA）已成为最成功的化学修饰体系（图 2A）。MOE 修饰的构建块和寡核苷酸的合成于 1995 年首次报道，约 20 年后，MOE 化学从实验室转移到临床（图 1）。被 FDA 批准的三种基于 MOE 的治疗药物中，第一种是 KYNAMRO，用于治疗家族性高胆固醇血症（图 3，表 1）。20mer 反义寡核苷酸是一种全 PS 修饰的 Gapmer，具有中央 DNA 十聚体间隙和五聚体 MOE 侧翼。Gapmer 方法需要引发 RNase H，即裂解 RNA 靶标的内切酶，以发挥作用。
SPINRAZA® (nusinersen) that was approved three years later acts via an entirely different mechanism (Figures 1and 3). The all-PS/MOE-modified RNA 18mer is used in the treatment of spinal muscular atrophy and modulates alternate splicing of the survival motor neuron 2 (SMN2) gene. The third therapeutic is TEGSEDI® (inotersen) that resembles KYNAMRO in that it is also a PS-modified 5-10-5 gapmer with MOE flanks and a central DNA gap. The drug was approved in 2018 for the treatment of the polyneuropathy in hereditary transthyretin-mediated amyloidosis. Lastly, another MOE based 5-10-5 gapmer, WAYLIVRA® (volanesorsen), was approved by the European Commission as an adjunct therapy to diet for treatment of a rare lipid disorder, familial chylomicronemia syndrome.
SPINRAZA®（努西尼生）是三年后获批的一种通过完全不同机制起作用的药物（图 1 和 3）。这种全 PS/MOE 修饰的 RNA 18mer 用于治疗脊髓性肌肉萎缩，并调节生存运动神经元 2（SMN2）基因的可变剪接。第三种治疗药物是 TEGSEDI®（依诺替生），它与 KYNAMRO 相似，也是一种 PS 修饰的 5-10-5 Gapmer，在 MOE 侧翼和中央 DNA 间隙部分。该药物于 2018 年获批用于治疗遗传性甲状腺素介导的多神经病。最后，另一种基于 MOE 的 5-10-5 Gapmer WAYLIVRA®（沃拉诺生）已获得欧洲委员会的批准，作为饮食辅助疗法用于治疗罕见的脂质紊乱症，家族性乳糜微粒增多症。
An antisense-like approach similar to the one exemplified by SPINRAZA and using a splice-switching oligonucleotide that targets exon 51 of the dystrophin mRNA was pursued for treatment of certain forms of Duchenne muscular dystrophy (DMD). EXONDYS 51® (eteplirsen), a phosphorodiamidate morpholino (PMO)30mer with an uncharged backbone was approved in 2016 (Figures1, 2A and 3). Another PMO oligonucleotide, VYONDYS 53 (golodirsen), that targets exon 53 of the dystrophin transcript (about 8% of the DMD community) was approved by the FDA in 2019.
对于治疗某些类型的杜氏肌营养不良症（Duchenne muscular dystrophy，DMD），采用与 SPINRAZA 类似的反义链方法，并使用针对 dystrophin mRNA 的外显子 51 的剪接切换寡核苷酸（splice-switching oligonucleotide）进行研究。EXONDYS 51®（埃特普利生）是一种磷酸二胺基甲酰吗啉（ phosphorodiamidate morpholino，PMO）30mer，具有无电荷的骨架结构，于 2016 年获得批准（图 1，2A 和 3）。另一种针对 dystrophin 转录本外显子 53（约占 DMD 患者 8%）的 PMO 寡核苷酸 VYONDYS 53（戈洛地生）于 2019 年获得 FDA 批准。
Over the course of the last 25 years, countless new nucleic acid modifications were introduced besides the first- and second-generation chemistries discussed above, and their activities have been evaluated at least in vitro. From the outset, it was clear that chemical modification of DNA and RNA was key to the successful therapeutic deployment of the antisense strategy, whereby increasing the binding affinity of the antisense oligonucleotide (ASO) was a particular concern. But there was an early recognition in the field that, besides affinity, multiple other hurdles needed to be overcome on the path to successful oligonucleotide drugs. These include chemical stability, resistance to degradation by a host of cellular exo- and endonucleases, i.e. metabolic stability, target site accessibility, delivery and biodistribution, i.e. binding to proteins and receptors, pharmacokinetics and pharmacodynamics as well as toxicity and complement activation.
在过去的 25 年中，除了上述讨论的第一代和第二代化学修饰方法之外，还引入了无数种新的核酸修饰方法，并且它们的活性至少在体外得到了评估。从一开始就清楚，对 DNA 和 RNA 进行化学修饰对于成功应用反义链策略的治疗至关重要，其中提高反义链寡核苷酸（antisense oligonucleotide，ASO）的结合亲和力是一个特别关注的问题。但早期在这个领域就意识到，除了亲和力之外，在成功开发寡核苷酸药物的过程中还需要克服多个其他障碍。这些障碍包括化学稳定性，对多种细胞外核酸酶和内切酶的降解抵抗能力，即代谢稳定性，靶点位点的可接近性，输送和生物分布，即与蛋白质和受体的结合，药代动力学和药效学以及毒性和补体激活等。
Looking back, it is quite extraordinary that the breakthroughs achieved in bringing 18 oligonucleotide (nucleic acids) drugs to market (Figure 3, Table 1) are chiefly based on just a handful of sugar and backbone modifications, namely the very early 2′-F, 2′-O-Me, and PS chemistries as well as the 2′-O-MOE RNA and neutral PMO backbone analogs (Figure 2A). We included two mRNA vaccines in Figure 3 although these are technically not oligonucleotides. However, these mRNAs also feature chemical modification (N-Me-pseudouridine, and 2′-O-Me in the cap, Figure 2A, and, like ONPATTRO, their delivery entails LNPs (Figure2B).
回顾过去，令人难以置信的是，将 18 种寡核苷酸（核酸）药物推向市场的突破主要基于仅有的几种糖和骨架修饰，即早期的 2'-F、2'-O-Me 和 PS 化学修饰，以及 2'-O-MOE RNA 和中性 PMO 骨架模拟物（图 2A）。尽管这两种 mRNA 疫苗在技术上并不是寡核苷酸，但我们在图 3 中包括了它们。然而，这些 mRNA 也具有化学修饰（N-Me-假尿苷和 2'-O-Me 在帽子上，图 2A），并且像 ONPATTRO 一样，它们的传递涉及脂质纳米粒（LNPs）（图 2B）。
In this critical review and perspective article, we review the chemistries of FDA-approved antisense (ASO), splice-switching (SSO), small interfering RNAs (siRNAs) and aptamer oligonucleotides, and discuss properties of the 2′-F, 2′-O-Me, 2′-O-MOE, PS and PMO analogs that cemented their status as privileged modifications in oligonucleotide therapeutics. Particularly as far as the phosphorothioates are concerned, recent research has uncovered the origins of altered protein binding affinities and interaction modes as a consequence of replacing non-bridging phosphate oxygens by sulfur and cast a light on the benefits of stereopure PS modification. Another focus of this review concerns GalNAc conjugation chemistry for optimized delivery of oligonucleotides to liver (Figure 2B). GIVLAARI, LEQVIO, OXLUMO, and AMVUTTRA all employ GalNAc conjugation chemistry. For other summary articles, specifically regarding advances and challenges in the delivery of nucleic acid therapeutics, please see excellent recent reviews. Throughout this article we will refer to approved nucleic acid therapeutics with their US trade names.
在这篇批判性评论和前瞻性文章中，我们回顾了 FDA 批准的反义寡核苷酸（ASO）、剪接转换寡核苷酸（SSO）、小干扰 RNA（siRNA）和适配体寡核苷酸的化学特性，并讨论了 2'-F、2'-O-Me、2'-O-MOE、PS 和 PMO 模拟物的性质，这些模拟物巩固了它们作为寡核苷酸治疗中的特权修饰的地位。特别是对于磷酸硫酯化合物，最近的研究揭示了通过将非桥接磷酸氧原子替换为硫而导致的蛋白结合亲和性和相互作用模式变化的起源，并突显了立体纯的 PS 修饰的优点。本文的另一个重点是 GalNAc 连接化学在优化寡核苷酸递送到肝脏方面的应用（图 2B）。GIVLAARI、LEQVIO、OXLUMO 和 AMVUTTRA 都采用了 GalNAc 连接化学。关于核酸治疗递送方面的其他综述文章，请参阅出色的最新综述。在本文中，我们将使用美国的商标名称来指代已批准的核酸治疗药物。
