Martin Egli, Muthiah Manoharan
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HEPLISAV-B is a recombinant vaccine that prevents hepatitis B liver infection caused by hepatitis B virus (HBV). It is composed of a 0.5 mL aqueous mixture of 20 μg of hepatitis B surface antigen (HBsAg) derived from Hansenula polymorpha with 3 mg of CpG 1018, an oligonucleotide adjuvant Toll-like receptor 9 (TLR9) agonist. The vaccine received US FDA approval in 2017 for use in adults ≥ 18 years. HEPLISAV-B is given by intramuscular injection in a 2-dose regimen spaced one month apart. CpG 1018 is a 22mer oligo-2′-deoxynucleotide phosphorothioate (PS-DNA) of sequence 5′-ps-d(TG AC TG TG AACGTTCG AG AT GA)-3′ with two CpG steps (boldface) and a palindromic section (underlined). The mechanism of action of this adjuvant oligo is quite different from those presented thus far and including ASOs, SSOs, siRNAs and aptamers (Figure 3).
乙肝疫苗 HEPLISAV-B®使用了一种寡核苷酸佐剂 CpG 1018，该疫苗是一种预防由乙型肝炎病毒（HBV）引起的乙型肝炎肝脏感染的重组疫苗。它由 20μg 来自 Hansenula polymorpha 的乙型肝炎表面抗原（HBsAg）与 3mg 的 CpG 1018 组成，后者是一种寡核苷酸佐剂，能激活 Toll 样受体 9（TLR9）。该疫苗于 2017 年获得美国 FDA 批准，适用于 18 岁及以上的成年人。HEPLISAV-B®通过肌肉注射，分两次注射，间隔一个月。CpG 1018 是一种 22 核苷酸磷酸硫酸酯修饰的寡聚-2'-脱氧核苷酸（PS-DNA），序列为 5'-ps-d(TG AC TG TG AACGTTCG AG AT GA)-3'，其中包含两个 CpG 位点（粗体标记）和一个回文结构（下划线标记）。这种佐剂寡聚体的作用机制与前述的 ASOs、SSOs、siRNAs 和适配体有很大的区别（见图 3）。
The presence of the CpG motif is central to its mode of action and underlies the adjuvant's role as a TLR9 agonist. In eukaryotic DNAs CpG dinucleotides are present at lower frequency than in the prokaryotic DNA sequences. Moreover, the frequency of methylation at CpG sites is higher in eukaryotic DNA than in the DNA of microbes. Differential CpG-methylation affords a molecular basis for TLR9 to distinguish self from non-self DNA in the host's defense immune response to microbial infections and thereby potential therapeutic applications of (unmethylated) agonistic CpG-containing oligonucleotides . Thus, CpG 1018 was used in combination immunotherapy with rituximab to treat patients with non-Hodgkin lymphoma. Conversely, ASOs have 5MeC rather than C, primarily to avoid any immune-stimulation when present along with G, but also to increase the binding affinity to the target RNA by 0.5°C/mod. The structural basis for a CpG-containing oligo to act as agonist (or antagonist in the methylated form) is depicted in Figure 24.
CpG 基序的存在对其作用方式至关重要，并构成了该佐剂作为 TLR9 激动剂的基础。在真核 DNA 中，CpG 二核苷酸的频率比原核 DNA 序列中低。此外，真核 DNA 中 CpG 位点的甲基化频率比微生物 DNA 高。不同的 CpG 甲基化为 TLR9 提供了在宿主的免疫防御应答中区分自身 DNA 和非自身 DNA 的分子基础，因此有潜在的治疗应用前景（甲基化）激动性含 CpG 寡核苷酸。因此，CpG 1018 与利妥昔单抗联合免疫疗法用于治疗非霍奇金淋巴瘤患者。相反，ASOs 中的 C 位点被 5MeC 取代，主要是为了避免在存在 G 时产生任何免疫刺激，同时也提高了与靶向 RNA 的结合亲和力（每个甲基引起 0.5°C 的亲和力提高）。CpG 寡核苷酸作为激动剂（或甲基化形式下的拮抗剂）的结构基础如图 24 所示。
图 24.
The following are the principal actions of CpG 1018 in combination with HBsAg in the HEPLISAV-B vaccine against HBV infection: (i) Activation of plasmacytoid dendritic cells (pDCs) for secretion of interferons (IFNs) and cytokines. (ii) Conversion of pDCs into efficient antigen-presenting cells such that they present processed HBsAg peptides to CD4+ T cells. (iii) Promotion of T cell differentiation to functional helper T cells via pDC-derived IFNs and cytokines. Multiple signals to B cells specific for intact HBsAg are then provided by the antigen-specific helper T cells, resulting in the generation of antibody responses that afford protective immunity to HBV.
CpG 1018 与 HBsAg 在 HEPLISAV-B 疫苗中对抗 HBV 感染的主要作用包括：(i) 激活浆细胞样树突状细胞（pDCs），促使其分泌干扰素（IFNs）和细胞因子。(ii) 将 pDCs 转化为高效的抗原呈递细胞，从而向 CD4+ T 细胞呈递经过处理的 HBsAg 肽段。(iii) 通过 pDC 源性的 IFNs 和细胞因子促进 T 细胞分化为功能性辅助 T 细胞。然后，特异性抗原辅助 T 细胞向 HBsAg 特异性 B 细胞提供多重信号，从而产生对 HBV 的保护性免疫应答。
CpG 1018 exerts its biological actions locally at the injection site and draining lymph nodes. These stimulatory actions decline quite rapidly, such that biomarkers of 1018 activity return to baseline values within a week. At the doses that CpG 1018 is administered in HEPLISAV-B, the adjuvant is cleared from circulation within a couple of hours. Hence the oligonucleotide does not reach levels that would result in systemic TLR9-mediated immune stimulation. Therefore, it is the long-lived antibody responses and T and B cell memory that make up the durable effects of HEPLISAV-B immunization. Once these responses that are highly specific for the HBs antigen itself are generated, the continuing presence of either antigen or CpG 1018 adjuvant is no longer needed.
CpG 1018 在注射部位和引流淋巴结处发挥其生物作用。这些刺激性作用迅速下降，因此 1018 活性的生物标志物在一周内恢复到基线水平。在 HEPLISAV-B 中使用的 CpG 1018 剂量下，佐剂从循环中清除的时间只需几个小时。因此，寡核苷酸不会达到导致全身 TLR9 介导的免疫刺激的水平。因此，持久的 HEPLISAV-B 免疫效果是由长期存在的抗体应答和 T、B 细胞记忆所构成的。一旦产生了高度特异性的 HBs 抗原特异性应答，不再需要持续存在的抗原或 CpG 1018 佐剂。
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With many thousands of prescription drug products approved for marketing in the US alone, and small molecule compounds constituting the largest share among them, oligonucleotide therapeutics occupy niches. But SSOs and siRNAs have enabled breakthrough treatments for rare genetic disorders that have proven difficult targets for small molecules. In fact, both ASOs and siRNAs are in development or approved for at least several rare disorders, giving these patients options to participate in clinical research and access to treatment choices for the first time.
在仅美国市场上，已经批准上市的处方药品数量超过几千种，其中小分子化合物占据了最大份额，而寡核苷酸治疗药物则在特定领域占据了一席之地。然而，SSOs 和 siRNAs 为罕见的遗传性疾病提供了突破性的治疗方法，这些疾病对于小分子化合物而言往往是难以攻克的靶点。事实上，ASOs 和 siRNAs 已经在开发中或已经获得批准用于治疗至少数种罕见疾病，为这些患者提供了参与临床研究和获得治疗选择的机会，这也是他们首次获得这些选择。
The traditional competition between antibody- and oligo-based therapeutics looks to increasingly become a synergistic partnership if mRNA vaccines live up to their immense promise. Thus, the ever-expanding RNA therapeutic toolbox includes molecules that range from those controlling gene expression (ASO, SSO, siRNA), regulation (antagomirs), proinflammatory effects (ssRNA and dsRNA) and recognizing proteins and receptors (aptamers) to RNA immunotherapeutics, such as mRNA vaccines that encode antigens of interest as well as self-amplifying RNAs. Provided the proteins encoded by mRNA vaccines elicit a robust neutralizing antibody response, the traditional antibody vs. oligo therapeutic ‘100m dash’ is being transformed into a ‘400m relay’, with RNA doing the early legwork and antibodies carrying the baton over the finishing line. Thus, early evidence in the development of a SARS-CoV-2 mRNA vaccine looked promising and the program received fast track designation by the US FDA on 12 May 2020. As well a preliminary account of a phase 1, dose-escalation, open-label trial with mRNA-1273 that included 45 healthy adults reported vaccine-induced anti-SARS-CoV-2 immune responses in all participants and an absence of trial-limiting safety concerns. A timeline of COVID-19 vaccine news and updates from the early days to the FDA-authorized emergency use of the Moderna SPIKEVAX® and Pfizer-BioNTech COMIRNATY® COVID-19 vaccines in children down to 6 months of age can be found here: https://www.fda.gov/emergency-preparedness-and-response/coronavirus-disease-2019-covid-19/covid-19-vaccines#authorized-vaccines ( https://www.fda.gov/emergency-preparedness-and-response/coronavirus-disease-2019-covid-19/covid-19-vaccines#authorized-vaccines ).
传统上，抗体类和寡核苷酸类治疗药物之间存在竞争关系，但随着 mRNA 疫苗兑现其巨大的潜力，二者之间的关系将越来越趋向于合作伙伴关系。因此，不断扩大的 RNA 治疗工具箱包括了一系列分子，涵盖了基因表达调控（ASO，SSO，siRNA），调节（antagomirs），促炎作用（ssRNA 和 dsRNA），以及识别蛋白质和受体（aptamers）的分子，还包括编码感兴趣抗原和自放大 RNA 等 RNA 免疫治疗药物。如果 mRNA 疫苗所编码的蛋白质能引发强效的中和抗体反应，传统的抗体与寡核苷酸治疗的“百米冲刺”正在转变为“400 米接力赛”，其中 RNA 负责早期工作，而抗体在终点线上接力完成。因此，在 SARS-CoV-2 mRNA 疫苗的开发早期，有迹象表明该计划前景看好，并于 2020 年 5 月 12 日获得美国 FDA 的快速通道认可。此外，一项针对 mRNA-1273 的Ⅰ期剂量递增、开放标签试验的初步报告显示，在包括 45 名健康成年人的试验参与者中，疫苗诱导了针对 SARS-CoV-2 的免疫反应，并且没有出现限制试验的安全问题。关于 COVID-19 疫苗新闻和更新的时间线，从早期到 FDA 授权紧急使用 Moderna SPIKEVAX®和 Pfizer-BioNTech COMIRNATY® COVID-19 疫苗适用于 6 个月以上儿童的情况，请参阅此处的链接：https://www.fda.gov/emergency-preparedness-and-response/coronavirus-disease-2019-covid-19/covid-19-vaccines#authorized-vaccines ( https://www.fda.gov/emergency-preparedness-and-response/coronavirus-disease-2019-covid-19/covid-19-vaccines#authorized-vaccines )。
The two FDA-approved mRNA COVID-19 vaccines, Moderna's SPIKEVAX and Pfizer-BioNTech's COMIRNATY are generally effective and safe. Chemical modification, i.e. the use of N1-methyl-pseudouridine in place of U (Figure 2A), reduces the intrinsic immune stimulation potential of exogenous mRNAs. Moreover, refined delivery strategies, LNP technology in the case of both the Moderna and Pfizer-BioNTech vaccines (Figure 21B), are crucial and indispensable instruments in the development and clinical implementation of mRNA vaccines. Stay tuned – the future of RNA therapeutics is finally here!
两种经 FDA 批准的 mRNA COVID-19 疫苗，Moderna 的 SPIKEVAX 和 Pfizer-BioNTech 的 COMIRNATY，一般来说是有效和安全的。化学修饰，即在 U 的位置使用 N1-甲基伪尿苷（见图 2A），降低了外源 mRNA 的固有免疫刺激潜力。此外，在现代和 Pfizer-BioNTech 疫苗中采用了 LNP 技术（见图 21B）的改进递送策略，在 mRNA 疫苗的开发和临床应用中是至关重要且不可或缺的工具。敬请关注- RNA 治疗的未来终于来临了！
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This article was written to celebrate a milestone in the 30-odd years nucleic acid chemistry, modification and drug discovery saga, the approval of the first 18 nucleic acid-based therapeutics (Figure 3, Table 1). Those following the evolution of the blueprint to turn oligos into drugs from the very first hours will remember the ups and downs the field experienced over the years and countless occasions that led doubters to predict the premature end of the pursuit to create ASO therapeutics. Enter RNAi and more apparent challenges to protect double-stranded siRNA against nuclease attacks and facilitate uptake and tissue-specific delivery. Chemical modification played a crucial role in overcoming these, and just five backbone chemistries make up the oligo drugs that were brought to market since 1998. That three of them - 2′-F, 2′-O-Me and PS - were the first nucleoside and nucleotide modifications explored by chemists in the 1960s is quite astonishing.
本文旨在庆祝核酸化学、修饰和药物发现领域 30 多年的里程碑，即首批 18 种核酸药物的获批（见图 3、表 1）。那些一直关注将寡核苷酸转化为药物的发展蓝图的人会记得这个领域多年来经历的起伏，以及无数次让怀疑者预言 ASO 治疗的过早终结的情况。随着 RNAi 的出现，保护双链 siRNA 免受核酸酶攻击、促进其摄取和组织特异性递送变得更加困难。化学修饰在克服这些问题中起到了至关重要的作用，自 1998 年以来，市场上推出的寡核苷酸药物仅使用了五种骨架化学结构。其中，2′-F、2′-O-Me 和 PS 这三种修饰是化学家在上世纪 60 年代首次探索的核苷酸和核苷修饰，这一事实相当令人惊讶。
The current success and positive outlook for oligonucleotide therapeutics with dozens of candidates in late-stage clinical trials should not prevent one from looking beyond the apparent magic of the 2′-F, 2′-O-Me, 2′-O-MOE, PS and PMO modifications. Although it seems unlikely that a silver bullet has been missed among the expanding universe of modifications that have been synthesized to date, many need to be looked at more closely and may pave the road to more potent therapeutics. In this review we discussed many modifications beyond the clinically successful ones pointed out above. For example, LNA and cEt-BNA modifications are being evaluated in various modalities and results will be forthcoming. It is also important to point out the commercial and business considerations from biotechnology firms which are evaluating such chemistries and limiting their evaluation to selected few for various proprietary and economic reasons. Yet another mentioned chemistry, GNA, has entered clinical trials in an RNAi therapeutic as an efficient off-target mitigator.
目前，寡核苷酸药物在临床试验的晚期阶段有数十个候选药物取得了成功并展现了积极的前景，但这并不应阻止我们超越 2′-F、2′-O-Me、2′-O-MOE、PS 和 PMO 等修饰的显而易见的成功。尽管在迄今为止合成的各种修饰物中，很难说是否有一种灵丹妙药被忽视了，但许多修饰物需要更仔细地研究，可能为更强效的治疗药物铺平道路。在本综述中，我们讨论了许多超越上述临床成功修饰的修饰物。例如，LNA 和 cEt-BNA 修饰正在不同的疗法中进行评估，结果将会陆续公布。同时，还要指出生物技术公司在商业和业务考虑方面对这些化学修饰进行评估，并基于各种专有和经济原因将其限制在少数几种修饰上。另外提到的 GNA 修饰已作为高效的副靶位缓解剂进入了 RNAi 治疗的临床试验阶段。
Probably the most important breakthrough in the successful launch of two RNAi therapeutics concerns delivery using LNPs or conjugation chemistry for receptor-mediated uptake and tissue-specific targeting. LNPs validated the RNAi platform (ONPATTRO) and a new delivery platform. Further, the latter validated the mRNA vaccine platform. Regarding the conjugates, GalNAc-containing LEQVIO that lowers LDL-cholesterol (LDL-C) by targeting PCSK9 mRNA for the treatment of HeFH or atherosclerotic cardiovascular disease (ASCVD) and received regulatory approval in 2021/22, represents a game changer. ASCVD is the greatest health burden worldwide with an estimated 22 million deaths/year expected by 2040. In clinical studies, inclisiran siRNA provided durable, potent and consistent LDL-C lowering over 18 months with a safety profile similar to placebo. With maintenance dosing every 6 months, LEQVIO offers a potentially more convenient option compared to approved monoclonal antibody (mAb) options (REPATHA and PRALUENT) that are dosed every 2–4 weeks. Thus, RNAi treatment has become more similar to a ‘flu shot’ taken a couple of times per year, but in this case to prevent development or progression of ASCVD. Successful hepatic delivery by targeting ASGPR with GalNAc conjugation chemistry as applied with GIVLAARI, OXLUMO, LEQVIO and AMVUTTRA points the way to using other receptor/ligand pairs in order to enable future delivery of oligo therapeutics to various tissues.
在两种 RNAi 治疗药物成功上市方面，可能最重要的突破涉及使用脂质体纳米粒（LNPs）或结合化学反应进行受体介导摄取和组织特异性靶向。LNPs 验证了 RNAi 平台（ONPATTRO）和一种新的递送平台。后者进一步验证了 mRNA 疫苗平台。至于共轭物，通过靶向PCSK9 mRNA 降低低密度脂蛋白胆固醇（LDL-C）的 GalNAc-LEQVIO 药物在 2021/22 年获得了监管批准，代表了一种革命性的突破。动脉粥样硬化性心血管疾病（ASCVD）是全球最大的健康负担，预计到 2040 年将有约 2200 万人死于该病。在临床研究中，inclisiran siRNA 在 18 个月内提供了持久、强效和一致的 LDL-C 降低效果，安全性与安慰剂相似。每 6 个月维持剂量的 LEQVIO 相比每 2-4 周剂量的已批准单克隆抗体（mAb）选项（REPATHA 和 PRALUENT），提供了一种更方便的选择。因此，RNAi 治疗变得更类似于每年接种几次流感疫苗，但这种情况是为了预防或延缓 ASCVD 的发展。通过使用 GalNAc 共轭化学反应以靶向 ASGPR 成功进行肝脏递送，如 GIVLAARI、OXLUMO、LEQVIO 和 AMVUTTRA，为利用其他受体/配体对以实现将寡核苷酸治疗药物递送到各种组织指明了方向。
Beyond the LNP and GalNAc platforms, new approaches are being assessed for delivery to extrahepatic tissues. A lipid-conjugated siRNA for CNS applications and an antibody-conjugated siRNA for muscle delivery have entered clinical studies. Additional ligands and delivery platforms are on the horizon. Future challenges and available opportunities for nucleic acid-based medicines have been recently addressed.
除了 LNP 和 GalNAc 平台，还在评估用于向肝脏之外组织递送的新方法。一种用于中枢神经系统应用的脂质共轭 siRNA 和一种用于肌肉递送的抗体共轭 siRNA 已进入临床研究。其他配体和递送平台也在不断涌现。关于核酸药物的未来挑战和可用机会最近已得到探讨。
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We thank our colleague Dhrubajyoti Datta for help with figures and references. We thank our colleagues Cindy Courtney, Jen Backo, Tim Long and Kevin Fitzgerald for their valuable comments. We are grateful to all our colleagues across the various disciplines who tirelessly contributed over the past several decades to the creation of the oligonucleotide-based medicines described herein. We especially acknowledge the vision, courage, and tenacity of Stanley T. Crooke (Isis-Ionis Pharmaceuticals) and John Maraganore (Alnylam Pharmaceuticals). Finally, we dedicate this review and perspective article to pioneers in the nucleic acid structure, chemistry and modification fields: Rosalind Franklin (100th birth anniversary on 25 July 2020), Robert L. Letsinger (100th birth anniversary on 31 July 2021), Har Gobind Khorana (100th birth anniversary on 9 January 2022), and Fritz Eckstein (on the occasion of his 90th birthday on 7 September 2022).
我们感谢我们的同事 Dhrubajyoti Datta 对图表和参考文献的帮助。我们感谢我们的同事 Cindy Courtney、Jen Backo、Tim Long 和 Kevin Fitzgerald 对本文的宝贵评论。我们衷心感谢各个领域的同事们在过去几十年中对本文所描述的基于寡核苷酸的药物的不懈贡献。特别感谢 Stanley T. Crooke（Isis-Ionis Pharmaceuticals）和 John Maraganore（Alnylam Pharmaceuticals）的远见、勇气和韧性。最后，我们将这篇综述和展望文章献给核酸结构、化学和修饰领域的先驱者：罗莎琳德·弗兰克林（2020 年 7 月 25 日诞辰 100 周年）、罗伯特·L·莱辛格（2021 年 7 月 31 日诞辰 100 周年）、哈·戈宾德·科拉纳（2022 年 1 月 9 日诞辰 100 周年）以及弗里茨·埃克斯坦（2022 年 9 月 7 日 90 岁生日）。
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Alnylam Pharmaceuticals. The open access publication charge for this paper has been waived by Oxford University Press – NAR Editorial Board members are entitled to one free paper per year in recognition of their work on behalf of the journal.
Conflict of interest statement. M.M. is an employee of Alnylam Pharmaceuticals.
Alnylam 制药公司。牛津大学出版社已免除本文的开放获取出版费——NAR 编辑委员会成员每年有权获得一篇免费论文，以表彰他们代表期刊所做的工作。
利益冲突声明。M.M.是 Alnylam Pharmaceuticals 的员工。
