已获批准的寡核苷酸疗法的化学、结构和功能(六)

文摘   科学   2023-06-09 07:00   美国  

Martin Egli, Muthiah Manoharan

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已获批准的寡核苷酸疗法的化学、结构和功能(一)
已获批准的寡核苷酸疗法的化学、结构和功能(二)
已获批准的寡核苷酸疗法的化学、结构和功能(三)
已获批准的寡核苷酸疗法的化学、结构和功能(四)
接上篇《已获批准的寡核苷酸疗法的化学、结构和功能(五)

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THIRD-GENERATION SUGAR MODIFICATION CHEMISTRIES 第三代糖化学修饰

We reviewed the concept of conformational preorganization in one of the above sections and discussed various effects on the equilibrium between the C2′-endo (DNA-like) and C3′-endo (RNA-like) pucker modes of the sugars in the backbones of DNA and RNA. Particularly modifications targeting the 2′-position such as 2′-F, 2′-O-Me, 2′-O-MOE and 2′-O-substitution in general favor the A-form backbone geometry. The sugar ring flips between the North and South regions of the pseudorotation phase cycle and the Eastern region is only sparsely populated, FANA being the prime example of a modification that prefers the East O4′-endo pucker . However, without further constraints the pentose retains its ability to fluctuate conformationally, which typically results in an entropically unfavorable contribution to pairing stability opposite an RNA target.

在上述的某个部分,我们回顾了构象预组织的概念,并讨论了对 DNA 和 RNA 骨架中糖的 C2′-endo(类似 DNA)和 C3′-endo(类似 RNA)脱氧核糖的平衡产生的各种影响。特别是针对 2′位点的修饰,如 2′-F、2′-O-Me、2′-O-MOE 和 2′-O的取代,通常有利于 A 型骨架几何结构。糖环在伪旋转相位周期中在北部和南部区域之间翻转,而东部区域只有稀疏的占据,FANA 是更倾向于东部的 O4′-endo脱氧核糖修饰的主要例子。然而,在没有进一步的约束条件下,脱氧核糖仍然保持着构象的波动性,这通常会导致与 RNA 靶标相对配对稳定性的熵不利贡献。

Chemical modifications that could freeze or lock the sugar moiety in the nucleic acid backbone would offer the ultimate control of conformational preorganization. Bicyclic frameworks achieve just that, and so-called locked nucleic acid (LNA) has arguably become the most well-known representative of this class of analogues since its inception in 1997/1998 (Figure 14) . Bridging ribose O2′ and C4′ with a methylene group effectively locks the sugar moiety in the C3′-endo conformation. The concept to bridge various atoms of 2′-deoxyribose, ribose and other sugar stereoisomers and using various linker chemistries has given rise to a wealth of bridged nucleic acids (BNAs, see Figure 14 for a small selection) . As expected, pairing between an oligonucleotide containing LNA and either complementary DNA or RNA results in a strongly stabilized duplex - up to 10°C per modified nucleotide compared to the parent duplex. In principle, this renders LNA a promising modification for antisense applications, although the analogue could only be used in the wings of ASOs so as not to abolish RNase H activity. Indeed, LNA oligos show 5- to 10-fold increased potencies relative to MOE-ASOs in animal studies. However, in rodents, non-human primates and humans, LNA-ASO were found to cause renal and hepatic toxicities and adverse injection site reactions. Clinical trials with a number of LNA-ASOs against various targets as well as with an LNA anti-miRNA oligo (antagomir) are continuing.

能够冻结或锁定核酸骨架中的糖基团的化学修饰将提供对构象预组织的终极控制。双环框架正是实现这一目标的方法,自 1997/1998 年以来,所谓的锁定核酸(LNA)可能已成为这类类似物中最为知名的代表(见图 14)。通过用亚甲基桥接核糖 O2′和 C4′,可以有效地将糖基团锁定在 C3′-endo构象中。通过在 2′-脱氧核糖、核糖和其他糖立体异构体的不同原子之间桥接,并使用不同的连接化学手段,产生了丰富的桥接核酸(BNAs,见图 14 中的一小部分)。如预期的那样,含有 LNA 的寡核苷酸与互补的 DNA 或 RNA 之间的配对形成了强烈稳定的双链结构,相对于未修饰的双链结构,每个修饰核苷酸可使稳定温度提高高达 10°C。原则上,这使得 LNA 成为一种有前景的反义应用修饰,尽管该类似物只能在 ASO 的翅膀部分使用,以免抑制 RNase H 的活性。实际上,在动物研究中,与 MOE-ASO 相比,LNA 寡核苷酸显示出 5 至 10 倍增强的活性。然而,在啮齿动物、非人灵长类动物和人类中,发现 LNA-ASO 会导致肾脏和肝脏毒性以及不良的注射部位反应。对多个靶点的 LNA-ASO 以及 LNA 抗-miRNA 寡核苷酸(antagomir)进行的临床试验仍在进行中。

图 14.

锁定核酸(LNA)、桥接核酸(BNAs)、双环-DNA(bcDNA)、三环-DNA(tc-DNA)以及北甲烷环核酸(N-MC)和南甲烷环核酸(S-MC)的结构如下图所示。RNA 和 DNA 类似物的优选糖环构型在结构式下方给出。

The LNA O2′-C4′ bridge allows for further chemical modification. The cEt BNA isomers feature methyl substituents at C4′ and the cMOE BNA isomers are the result of stitching together C4′ and the inner carbon of the ethyl moiety in the 2′-O-MOE substituent (Figure 14). Not only do these BNAs maintain the ability to stabilize duplexes relative to MOE-modified strands, but both cyclic analogs exhibit better resistance to 3′-exonuclease degradation than the parent MOE modification. Surprisingly, cEt BNA affords better protection than cMOE and crystal structures of modified duplexes and models of 3′-terminally modified strands bound to exonuclease point to a steric origin of the favorable resistance. Several clinical trials with cEt-modified oligonucleotides are underway. A carbocyclic analog of LNA (cLNA) with an exocyclic methylene group in place of the 2′-oxygen (methylene-cLNA) afforded similar pairing stability gains and mismatch discriminations as the parent LNA. The corresponding ASOs showed similar affinities but reduced toxicity relative to LNA. The methylene-cLNA modification was superior to Me-cLNA isomers, both in terms of affinity and ASO activity.

LNA O2′-C4′桥能够进行进一步的化学修饰。cEt BNA 异构体在 C4′位置具有甲基取代基,而 cMOE BNA 异构体是通过将 C4′和 2′-O-MOE 取代基中乙基部分的内部碳连接在一起形成的(见图 14)。这些 BNAs 不仅相对于 MOE 修饰链能够保持稳定双链的能力,而且这两种环状类似物相对于原始的 MOE 修饰具有更好的抵抗 3′-外切核酸酶降解的能力。令人惊讶的是,cEt BNA 提供了比 cMOE 更好的保护效果,修改双链的晶体结构和 3′端修饰链与外切核酸酶结合的模型表明有利的耐受性源于立体位阻效应。目前正在进行几项使用 cEt 修饰寡核苷酸的临床试验。LNA 的环状类似物(cLNA)中,以 2′-氧原子的位置具有外环亚甲基取代基(methylene-cLNA),其所提供的成对稳定性增益和错配区分能力与原始的 LNA 相似。相应的 ASOs 显示了与 LNA 相似的亲和力,但毒性降低。相对于 Me-cLNA 异构体,methylene-cLNA 修饰在亲和力和 ASO 活性方面表现出更优越的特性。

Bicyclic frameworks do not just allow one to freeze the sugar conformation in the C3′-endo mode, but judicious use of isomers and types of bridges has resulted in analogs that are limited to a DNA-like sugar pucker. Thus, α-L-LNA adopts solely the South-type C3′-exo pucker (Figure 14) , but nonetheless does not elicit RNase H when paired with RNA. The synthesis of bicyclo-DNA (bcDNA) represents a very early attempt to rigidify the DNA sugar-phosphate backbone. The analogue formed entropically favored duplexes and displayed a propensity for triplex formation, whereby the preferred sugar pucker was either C2′-endo or C1′-exo. Tricyclo-DNA (tcDNA) contains an additional cyclopropyl ring compared with bcDNA, but its sugar adopts a C3′-endo pucker instead. In the crystal structure of a DNA duplex with tcDNA residues, the cyclopropyl ring was positioned at the edge of the major groove. This observation helped rationalize the considerably increased nuclease resistance seen with tcDNA-modified oligos. Tricyclo-DNA was also extensively tested inside PS-modified ASO gapmers and found to exhibit robust antisense activity in the absence of hepatotoxicity.

双环框架不仅可以将糖的构象冻结在 C3′-endo模式下,而且通过适当选择异构体和桥接类型,可以得到限制在 DNA 样式糖构象的类似物。因此,α-L-LNA 仅采用南型 C3′-exo糖构象(见图 14),但与 RNA 配对时并不引发 RNase H 反应。双环-DNA(bcDNA)的合成代表了刚性化 DNA 糖磷酸骨架的非常早期尝试。该类似物形成了熵上有利的双链结构,并具有三链结构形成的倾向,其中首选的糖构象为 C2′-endo或 C1′-exo。与 bcDNA 相比,三环-DNA(tcDNA)多了一个环丙基环,但其糖构象取代为 C3′-endo。在含有 tcDNA 残基的 DNA 双链结构中,环丙基环位于主沟边缘。这一观察结果有助于解释 tcDNA 修饰寡核苷酸显示出显著增强的核酸酶抗性。三环-DNA 也被广泛用于修饰了 PS 的 ASO 间隙探针内进行了大量测试,并且在没有肝毒性的情况下表现出强大的反义活性。

Finally, the aforementioned methanocarbacyclic N-MC and S-MC sugar modifications also feature a cyclopropyl moiety, between C4′ and C7′ (replaces O4′) and C7′ and C1′, respectively (Figure 14). This rather simple measure locks the pucker in the North C2′-exo (N-MC) or South C3′-exo (S-MC) mode. The N-MC analogue was combined with the 2′-fluoro modification (2′-F-NMC), initially only at the level of thymidine. More recently all four nucleoside phosphoramidites have been prepared and incorporated into siRNAs to evaluate the activity of this modification in the context of RNAi.

最后,前述的甲基环丙基 N-MC 和 S-MC 糖修饰也具有环丙基基团,分别位于 C4′ 和 C7′(取代 O4′)以及 C7′ 和 C1′ 之间(见图 14)。这种相对简单的措施将糖构象锁定在北向 C2′-exo(N-MC)或南向 C3′-exo(S-MC)模式。N-MC 类似物与 2′-氟修饰(2′-F-NMC)结合,最初仅在胸腺嘧啶水平上进行。最近,已制备了所有四种核苷酸磷酰胺酯,并将其合并到 siRNA 中,以评估该修饰在 RNAi 环境中的活性。

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RNAi THERAPEUTICS: DOUBLE-STRANDED MECHANISM OF ACTION RNAi 治疗:双链作用机制


   

A natural defense and regulatory system 一种天然的防御和调控系统

The most obvious difference between ASOs and small interfering RNAs (siRNAs) as embodied by current oligonucleotide therapeutics is that the former are single-stranded and the latter double-stranded (Figure 4). The origin of the double-stranded nature of siRNA is not entirely clear. It may be rooted in the archaeal ancestry of the eukaryotic Argonaute (eAgo) proteins or a consequence of a subsequently acquired mode of action during the assembly of the eukaryotic RNA interference (RNAi) machinery from prokaryotic sources. Thus, all cell-based life forms rely on defense systems that provide protection against viruses or transposable elements. The actual mechanism involves a DNA- or RNA-based guide that directs a nuclease (slicer) to destroy the target, whereby specificity is conferred by base complementarity between guide and target.

当前核酸药物中的短寡核苷酸(ASO)和小干扰 RNA(siRNA)的最明显区别在于前者是单链结构,而后者是双链结构(图 4)。siRNA 的双链性质的起源尚不完全清楚。它可能源于真核生物阿尔卡纳酶(eAgo)蛋白的古菌祖先,或者是在真核生物 RNA 干扰(RNAi)机制的组装过程中从原核源头后续获得的一种作用模式。因此,所有细胞生命形式都依赖于提供对抗病毒或可转座元件的保护的防御系统。实际的机制涉及 DNA 或 RNA 为基础的导向物,将核酸酶(切割酶)定向破坏目标,其中导向物和目标之间的碱基互补性赋予了特异性。

A ribozyme would represent the primordial version of this defense mechanism in that the catalytic RNA combines guide and nuclease. Because host and parasite have co-evolved since the earliest stages of cellular life, defense mechanisms must have emerged simultaneously as part of the diversification of host cells and given rise to the host-parasite arms race that is a central element in the evolution of life. The prokaryotic Ago system (pAgo) is the direct predecessor of eAgo and constitutes the core of proteins and domains that underlie DNA- or RNA-guided innate immunity. Proteins containing MID, PIWI and/or PAZ domains (Figures 8 and 15A) and potentially associated with other nucleases, helicases and nucleic acid-binding proteins served RNA guide processing, maturation and amplification and the subsequent recognition and destruction of targets.

在这种防御机制中,核酸酶可以被视为原始版本,因为催化 RNA 结合了导向物和核酸酶的功能。由于宿主和寄生体在细胞生命的早期阶段就共同进化,防御机制必须同时出现作为宿主细胞多样化的一部分,并导致了宿主-寄生体的军备竞赛,这是生命进化的一个核心元素。原核生物中的阿尔卡纳酶系统(pAgo)是真核生物阿尔卡纳酶(eAgo)的直接前身,它构成了在 DNA-或 RNA 引导下的固有免疫中起核心作用的蛋白质和结构域。包含 MID、PIWI 和/或 PAZ 结构域的蛋白质(图 8 和图 15A)以及潜在与其他核酸酶、解旋酶和核酸结合蛋白相关的蛋白质,用于 RNA 导向物的处理、成熟和扩增,以及随后对目标的识别和破坏。

图 15.

(A) 人类 Ago2 与引导(红色)-乘客(蓝色)链 siRNA 双链复合物的晶体结构,分辨率为 2.5 Å(PDB ID 4w5t)。各个酶结构域以不同的颜色进行标记;N 末端和连接区域以不同的灰色阴影进行标记。MID 结构域含有引导链 5'-磷酸基团的结合位点,以黑色表示,在球棒模式下突出显示,并用红色 P 标记。PIWI RNA 内切酶结构域(“切割酶”)在引导 siRNA 相对于靶向 mRNA 的位置进行切割。PAZ 结构域结合于引导链的 3'-末端突出部分。虚线表示蛋白质或 RNA 的缺失部分,例如引导链的核苷酸 15 至 19。箭头指向由 AS6 和 AS7 引起的碱基对之间的急剧弯曲。
(B) Drosophila Dicer-2:R2D2 异二聚体与引导(红色)-乘客(蓝色)链 siRNA 双链和一段双链 RNA(dsRNA)的低温电镜结构,分辨率为 3.3 Å(PDB ID 7v6c)。Dicer-2 和 R2D2 各自的结构域以不同的颜色进行标记(R2D2 RBD,RNA 结合域和 CTD,C 末端域)。Dicer-2 中螺旋酶、RNase III 和平台-PAZ 结构域之外的区域以浅灰色进行标记,虚线表示蛋白链中的缺失部分。

The eAgo system features additional components such as Dicer (Figure 15B) with a helicase domain of archaeal origin and RNase III domains of bacterial origin as well as RNA-dependent RNA polymerase (RdRp) of phage origin that emerged from a predecessor DdRp and is concerned with siRNA amplification in eukaryotes. Dicer is the endoribonuclease that processes double-stranded RNA (dsRNA) into siRNA (hence the double-stranded nature of fragments), and pre-microRNA into miRNA. In eukaryotic cells, the RNAi machinery sets up the innate immunity response and the piwiRNA branch constitutes the distinct type of adaptive immunity. In the evolution of the eukaryotic RNAi machinery, the prokaryotic CRISPR/Cas system that represents adaptive immunity in the lower organisms was thus discarded. Not only is the eAgo system inherently more complex than its pAgo predecessor, but it exclusively uses RNA guides and chiefly targets RNA, a change that was presumably triggered by the transition from the prokaryotic world of viruses mainly governed by DNA to a eukaryotic virosphere that is dominated by RNA viruses.

eAgo 系统具有额外的组分,例如具有古菌起源的螺旋酶结构域和具有细菌起源的 RNase III 结构域的 Dicer(图 15B),以及具有噬菌体起源的 RNA 依赖性 RNA 聚合酶(RdRp),它起源于前体 DdRp,并参与真核生物中的 siRNA 扩增。Dicer 是内切核酸酶,可将双链 RNA(dsRNA)处理成 siRNA(因此片段具有双链性),并将前体微小 RNA 处理成微小 RNA。在真核细胞中,RNAi 机制建立了先天免疫响应,而 piwiRNA 分支构成了独特类型的适应性免疫。在真核 RNAi 机制的演化过程中,代表低级生物中适应性免疫的原核 CRISPR/Cas 系统被舍弃。eAgo 系统不仅固有比其 pAgo 前体更复杂,而且仅使用 RNA 引导,并主要靶向 RNA,这种变化可能是由于从主要由 DNA 主导的原核病毒世界过渡到以 RNA 病毒为主导的真核生物病毒界。

The discovery of RNAi as the agent of gene silencing in C. elegans and later the demonstration that short dsRNA fragments could be used to silence genes in mammalian cells without triggering an immune response was greeted with excitement, as it offered a path to a new therapeutic approach by hitchhiking on a natural defense and regulatory machine. Andrew Z. Fire and Craig C. Mellow were jointly awarded the 2006 Nobel Prize in Physiology or Medicine for their discovery of RNA interference - gene silencing by double-stranded RNA. However, the challenges encountered with early clinical trials echoed those with ASOs a decade before and included toxicity related to immune stimulation, problems with delivery and insufficient therapeutic efficacy .

在线虫 C. elegans 中发现 RNAi 作为基因沉默的机制,随后证明可以使用短的双链 RNA 片段在哺乳动物细胞中沉默基因而不引发免疫反应,这一发现令人兴奋,因为它为一种新的治疗方法提供了可能,通过利用天然的防御和调控机制。安德鲁·Z·费尔(Andrew Z. Fire)和克雷格·C·梅洛(Craig C. Mellow)因其发现 RNA 干扰-双链 RNA 引起的基因沉默而共同获得了 2006 年诺贝尔生理学或医学奖。然而,早期临床试验中遇到的挑战与 10 年前 ASO 的问题相似,包括与免疫刺激相关的毒性、递送问题和治疗效果不足。

However, RNAi established itself very quickly as a powerful tool in cell-based assays, using either transfection or vector-based delivery in combination with high-throughput screens to knockdown gene expression to ferret out their function and for target validation. As well, existing ASO modification chemistries could be applied immediately to siRNAs and tested. Although effective for downregulating genes in vitro, unmodified siRNAs are quickly degraded in vivo and protective steps need to be taken to prevent degradation by nucleases. Unlike natural noncoding RNAs that are composed mostly of native ribonucleotides and sparsely modified, RNAi therapeutics require extensive chemical modification.

然而,RNAi 迅速成为细胞基因分析的有力工具,在转染或基于载体的递送方式结合高通量筛选中使用,以降低基因表达并揭示其功能和进行靶点验证。此外,现有的 ASO 修饰化学方法可以立即应用于 siRNA 并进行测试。虽然在体外能有效下调基因表达,但未修饰的 siRNA 在体内很快被核酸酶降解,因此需要采取保护措施来防止降解。与主要由天然核苷酸组成且稀疏修饰的自然非编码 RNA 不同,RNAi 治疗需要进行广泛的化学修饰。

Shared features between ASOs and siRNAs contrast with clear differences. As pointed out above, siRNAs are 21mer duplexes that consist of the paired guide (antisense, AS) and passenger (sense, S) strands (Figure 4). Although activity based on the guide strand alone (ss-siRNA) has been demonstrated, the use of duplex constructs remains much more common. The nuclease in RNAi-mediated cleavage of target mRNA is Ago2, whereby the PIWI domain that adopts an RNase H-like fold harbors the endonuclease (Figure 8). Deviating architectures of RNase H and Ago2 create fundamentally different protein interactions with ASO and guide RNA that direct degradation of their respective targets. Among the Ago family of proteins, Ago2 is the only one that possesses endonuclease activity. Other family members are involved in miRNA-mediated pathways of post-transcriptional regulation.

ASO 和 siRNA 之间存在一些共同特征,但也存在明显的差异。正如上面指出的那样,siRNA 是由成对的导向链(反义链,AS)和乘客链(正义链,S)组成的 21 碱基双链结构(图 4)。虽然单独使用导向链(ss-siRNA)已经被证明具有活性,但双链结构的使用更为普遍。RNAi 介导的靶 mRNA 裂解的核酸酶是 Ago2,其中采用 RNase H 样折叠的 PIWI 结构域具有内切酶活性(图 8)。RNase H 和 Ago2 的不同结构决定了它们与 ASO 和导向 RNA 的蛋白质相互作用的根本差异,从而指导了它们各自目标的降解。在 Ago 蛋白家族中,Ago2 是唯一具有内切酶活性的成员,其他成员参与 miRNA 介导的转录后调控途径。

The various steps of RNA interference using an exogenous siRNA are depicted in Figure 16. Following delivery and cellular uptake, (i) the siRNA duplex is loaded into RISC Ago2, (ii) the passenger strand is peeled off the guide strand and discarded, (iii) after which the target mRNA is accommodated opposite the guide siRNA, (iv) prompting cleavage of the former by Ago2 and (v) removal of the sliced target and initiation of the next round of endo-nucleolytic cleavage. The illustration in Figure 16 highlights another difference between guide siRNA and ASO, namely a 5′-phosphate group in the former that is important for recognition and stable binding of the guide as well as discriminating between guide and passenger strands (Figure 15A).

图 16 展示了使用外源 siRNA 进行 RNA 干扰的各个步骤。在递送和细胞摄取之后,(i)siRNA 双链结构被加载到 RISC Ago2 中,(ii)乘客链从导向链上剥离并丢弃,(iii)之后,目标 mRNA 与导向 siRNA 相互配对,(iv)促使 Ago2 剪切目标 mRNA,(v)移除剪切的目标 mRNA 并启动下一轮内切剪切。图 16 中的插图突出了导向 siRNA 和 ASO 之间的另一个差异,即导向 siRNA 具有重要的 5'-磷酸基团,该基团对于识别和稳定结合导向链以及区分导向链和乘客链起着重要作用(图 15A)。

图 16.

(A) 外源 siRNA 引导的 RNA 干扰的各个步骤:(1) RISC 加载,(2) 乘客链剥离,(3) 与 mRNA 目标结合,(4) 目标剪切,(5) 释放剪切产物并与下一个目标配对,开始新一轮剪切。导向 siRNA、乘客 siRNA 和 mRNA 目标分别以红色、蓝色和黑色表示。Ago2 MID 结构域以绿色和红色突出显示,灰色圆点标记 5'-端磷酸基团和 3'-端 TT 末端突出。

The presence of two strands and the lack of proper discrimination between them can trigger an off-target effect (OTE). An OTE can arise from a passenger strand directing cleavage of a complementary target, i.e. sense-mediated silencing. Alternatively, downregulation of an mRNA target is based on partial complementarity between siRNA and mRNA target, also referred to as seed-mediated silencing (the seed region entails nucleotides 2–8 of the guide strand). Other OTEs are the induction of type I interferons and inflammatory cytokines (immune stimulation) and upregulation of miRNA-controlled genes as a result of a saturation of the RNAi machinery . Shortened passenger strands as well as blunt-end siRNA designs were introduced to counter the likelihood of OTEs.

存在两条链和缺乏正确鉴别它们之间的区别可能会引发离靶效应(OTE)。OTE 可以源自乘客链引导对互补靶点的剪切,即正义介导的沉默。另外,mRNA 靶点的下调基于 siRNA 与 mRNA 靶点之间的部分互补性,也称为种子介导的沉默(种子区域包括导向链的核苷酸 2-8)。其他 OTE 包括诱导 I 型干扰素和炎性细胞因子(免疫刺激)以及 miRNA 调控基因的上调,这是由于 RNAi 机制的饱和所致。为了减少 OTE 的可能性,人们引入了缩短的乘客链以及带有钝端的 siRNA 设计。

Compared to ASOs, the double-stranded nature of siRNAs also creates unique challenges for delivery that need to be addressed with tailored approaches for formulation and new modification/conjugation chemistries. Not unexpectedly, single-stranded ASO and siRNA duplexes exhibit divergent pharmacokinetic and pharmacodynamic profiles. To make siRNAs into drugs, chemical modification strategies need to consider both the guide and the passenger strand, thus precluding a uniform modification design seen, for example, with SSO therapeutics such as SPINRAZA or EXONDYS 51.

将 siRNA 与 ASO 相比,siRNA 双链结构的特性也为传递提出了独特的挑战,需要针对性地进行配方和新的修饰/共轭化化学策略。预料之中的是,单链 ASO 和 siRNA 双链在药代动力学和药效动力学特性上表现出差异。为将 siRNA 转化为药物,化学修饰策略需要同时考虑引导链和载客链,这使得无法像 SPINRAZA 或 EXONDYS 51 等 SSO 药物一样采用统一的修饰设计。


   

Initial RNA modifications: PS, 2′-O-Me and 2′-F 初始 RNA 修饰:PS、2'-O-Me 和 2'-F

We discussed in detail the benefits of the phosphorothioate and 2′-O-modifications for ASOs and SSOs. They include nuclease protection, conformational preorganization and enhanced pairing strength (2′-O-mods.), low toxicity and favorable pharmacokinetic and pharmacodynamic behavior (PS, MOE). In a study dating back almost a decade, we compared the in vitro and in vivo efficacies of various 2′-modifications in siRNA against Factor VII, an established endogenous target system. In both the guide (AS) and passenger (S) strands every pyrimidine ribonucleotide was replaced with the 2′-F, 2′-O-Me, 2′-O-MOE or LNA analogues. Relative levels of Factor VII protein were measured in mice on day 2 and up to three weeks after administering 2, 3 or 5 mg/kg of F/F, OMe/OMe, MOE/MOE, LNA/LNA, OMe/F, MOE/F or LNA/F (S/AS strands) siRNA in a lipid formulation. Factor VII amounts resulting from treatment with the F/F combination were >3-fold lower than those seen with the second ranked siRNA, LNA/F and ca. 10-fold reduced relative to the levels measured for OMe/OMe, MOE/MOE and LNA/LNA.

我们详细讨论了磷酸硫酯和 2'-O-修饰对 ASO 和 SSO 的益处。这些益处包括核酸酶保护、构象预先组织和增强配对强度(2'-O-修饰)、低毒性以及良好的药代动力学和药效动力学特性(PS、MOE)。在将近十年前的一项研究中,我们比较了针对凝血因子 VII 的 siRNA 中不同 2'-修饰在体外和体内的疗效。在引导链(AS)和载客链(S)上,每个嘧啶核苷酸被 2'-F、2'-O-Me、2'-O-MOE 或 LNA 类似物所取代。在小鼠体内,给予 2、3 或 5 mg/kg F/F、OMe/OMe、MOE/MOE、LNA/LNA、OMe/F、MOE/F 或 LNA/F(S/AS 链)的 siRNA 脂质配方后,测量了第 2 天和至少三周内因子 VII 蛋白的相对水平。F/F 组合处理后的因子 VII 水平比第二高的 LNA/F siRNA 低 3 倍以上,并且相对于 OMe/OMe、MOE/MOE 和 LNA/LNA 的水平降低了约 10 倍。

The overall ranking was as follows: F/F < LNA/F < OMe/F < MOE/F ≈ MOE/MOE ≈ OMe/OMe ≈ LNA/LNA. The F/F siRNA also outperformed the parent duplex (OH/OH) in terms of silencing and was non-immunostimulatory, unlike OH/OH that stimulated IFN-α and TNF-α. The melting temperature (Tm) of the F/F duplex was about 20°C above that of the parent duplex, but LNA/LNA and MOE/MOE as well as LNA/F and MOE/F duplexes were all more stable than F/F. The increased stability of the F/F duplex is chiefly enthalpy-based which points to improved stacking and/or H-bonding interactions because of fluorine modification. Indeed, we subsequently demonstrated using solution NMR and thermodynamic assays with RNA hairpins that featured 2′-F-modified overhangs at the 3′-end that the fluorine modification strengthens both Watson-Crick H-bonds and base stacking.

整体排序如下:F/F < LNA/F < OMe/F < MOE/F ≈ MOE/MOE ≈ OMe/OMe ≈ LNA/LNA。与 OH/OH 相比,F/F siRNA 在基因沉默方面表现出色,并且不具有免疫刺激性,而 OH/OH 会刺激 IFN-α和 TNF-α的产生。F/F 双链的熔化温度(Tm)比母体双链高约 20°C,但 LNA/LNA、MOE/MOE 以及 LNA/F 和 MOE/F 双链都比 F/F 更稳定。F/F 双链的增加稳定性主要基于焓,这表明氟修饰改善了叠合和/或氢键作用。事实上,我们随后通过使用溶液核磁共振和热力学测定,针对具有 2'-F 修饰的 RNA 发夹结构,特别是在 3'-端具有氟修饰的突出部分,证明了氟修饰强化了 Watson-Crick 氢键和碱基叠合。

Fluorine is smaller than the 2′-hydroxyl group, hydrophobic and typically lacks the ability to participate in H-bonds. This is definitely the case in the RNA minor groove as we showed using crystallographic data and osmotic stressing assays (Figure 17). However, organic fluorine leads somewhat of a double life and there have been observations where it does act as an H-bond acceptor. The poor hydration in the minor groove precludes the need for de-solvation of 2′-F-modified siRNA upon transport and uptake and may have an impact on cellular trafficking and endosomal release. One limitation of the 2′-F modification is the limited protection against nuclease degradation it affords compared to other 2′-modifications such as 2′-O-Me and 2′-O-MOE. Moreover, at high concentrations 2′-F nucleotides are recognized, although somewhat poorly, by human RNA polymerases.

氟原子比 2'-羟基基团要小,具有疏水性,并且通常缺乏参与氢键的能力。我们使用晶体学数据和渗透压应激实验(图 17)证明了这一点,尤其是在 RNA 的小凹槽中。然而,有时有观察到有机氟会作为氢键受体发挥作用。小凹槽中的贫水化情况消除了在转运和摄取过程中需要对 2'-F 修饰的 siRNA 进行去溶剂化的需求,并可能对细胞转运和内体释放产生影响。2'-F 修饰的局限性之一是相对于其他 2'-修饰(如 2'-O-Me 和 2'-O-MOE),它对核酸酶降解的保护作用有限。此外,在高浓度下,2'-F 核苷酸会被人类 RNA 聚合酶识别,尽管识别效果不佳。

图 17.

化学修饰对 RNA 周围水结构的影响。(A) RNA 和(B) 2'-F-RNA 中的小凹槽贫水化。RNA 中的 2'-羟基基团作为小凹槽中串联水桥的桥头。氟原子的引入(绿色高亮显示)破坏了 2'-取代基与水之间的氢键,通过距离的改变和水分子的偏移可观察到。水分子用灰色球体表示,氢键用细实线表示,选择的距离用虚线表示。

A design principle that emerged as a solution to these shortcomings is to combine 2′-F and 2′-O-Me modified nucleotides in fully modified siRNAs. An example of the successful application of this approach is GIVLAARI (givosiran), an siRNA approved by the US FDA in 2019 for treatment of acute hepatic porphyria (Figures 1 and 18). GIVLAARI also contains pairs of PS modifications at the 5′- and 3′-ends of the guide siRNA as well as PS modifications on the first and second phosphates in the passenger siRNA as well as the triantennary GalNAc linker at its 3′-end to facilitate uptake (vide infra).

将 2'-F 和 2'-O-Me 修饰的核苷酸结合在完全修饰的 siRNA 中成为解决这些缺点的设计原则。成功应用该方法的一个例子是 GIVLAARI(givosiran),这是一种在 2019 年获得美国 FDA 批准用于治疗急性肝性卟啉病的 siRNA(图 1 和图 18)。GIVLAARI 还包含在引导 siRNA 的 5'-和 3'-末端的 PS 修饰对以及旅客 siRNA 中第一个和第二个磷酸基的 PS 修饰,以及三糖基 GalNAc 连接剂位于其 3'-末端以促进摄取(见下文)。

图 18.

迄今为止获得市场批准的五种 RNAi 治疗药物的引导链和旅客链的序列和修饰。(A) ONPATTRO, (B) GIVLAARI, (C) OXLUMO, (D) LEQVIO 和(E) AMVUTTRA。核糖核苷酸(Ribo-)、2'-脱氧核糖核苷酸(2'-deoxyribo-)、2'-*O*-Me 核苷酸和 2'-F 核苷酸分别以红色、蓝色、黑色和绿色的圆圈表示。GIVLAARI、OXLUMO、LEQVIO 和 AMVUTTRA 在旅客链的 3'-末端具有 PS 修饰(橙色条)和三糖基 GalNAc 偶联物。

As mentioned above, MOE modification of siRNA strands did not result in favorable in vitro or in vivo efficacies against the Factor VII targe. The modification is sterically quite demanding in the minor groove and a model of a modified RNA duplex bound to Piwi protein revealed clashes between protein side chains and MOE substituents at the protein-siRNA interface. Conversely the small fluorine substituent is well tolerated and mimics 2′-hydroxyl groups in the complex with the parent duplex. Thus, it is the only 2′-modification that is tolerated at the second position of the guide strand (AS2), where Ago2 forces the RNA to make a tight turn. We also used the crystal structure of human Ago2 in complex with miR-20a to build a model with 2′-O-MOE substituents attached to all residues of the guide strand seed region (AS2–AS8). The model exposed clashes between all MOE moieties and Ago2 side chains, indicating that the MOE modification is likely too bulky to be accommodated at many sites in the siRNA guide and passenger strands. An MOE-modified residue was introduced at the first position of the guide strand (AS1), but the crystal structure of the complex with Ago2 did not reveal an interaction between the 2′-O-substituent and MID domain residues.

如上所述,MOE 修饰的 siRNA 链对于 Factor VII 靶点的体外或体内疗效不佳。这种修饰在次要沟槽中的空间要求较高,在与 Piwi 蛋白结合的修改 RNA 双链模型中,蛋白质侧链与 MOE 取代基在蛋白质-siRNA 界面存在冲突。相反,较小的氟取代基能够很好地容忍,并在与母体双链的复合物中模拟 2'-羟基基团。因此,它是唯一能够容忍在引导链第二位置(AS2)的 2'-修饰,Ago2 迫使 RNA 产生紧密转向。我们还利用人类 Ago2 与 miR-20a 复合物的晶体结构,构建了一个模型,其中引导链种子区(AS2-AS8)的所有残基都附有 2'-O-MOE 取代基。该模型显示所有 MOE 基团与 Ago2 侧链之间存在冲突,表明 MOE 修饰可能过于庞大,无法适应 siRNA 引导链和旅客链中的许多位点。在引导链的第一个位置(AS1)引入了一个 MOE 修饰残基,但与 Ago2 的结晶结构并未显示 2'-O-取代基与 MID 结构域残基之间的相互作用。

Another modification at that site, 2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), in combination with a 5′-(E)-vinylphosphonate ((E)-VP) moiety, a metabolically stable phosphate mimic (Figure 19), improved RISC loading and showed robust siRNA-mediated gene silencing. A structural model of 5′-terminal guide strand residues with 5′-(E)-VP/2′-O-NMA-U at AS1 bound to Ago2 was consistent with an H-bond between the NMA substituent and the side chain of Gln-548 that together with Asn-551 stabilizes the tight turn in the guide strand at that site.

在该位置上的另一种修饰是 2'-O-[2-(甲基氨基)-2-氧代乙基](2'-O-NMA),结合具有代谢稳定性的磷酸盐模拟物 5'-(E)-乙烯基膦酸酯((E)-VP)(见图 19),改善了 RISC 的装载并展示了强大的 siRNA 介导的基因沉默作用。将 5'-(E)-VP/2'-O-NMA-U 与 AS1 处的 5'-端引导链残基结合于 Ago2 的结构模型与 NMA 取代基和 Gln-548 的侧链之间的 H 键相一致,Gln-548 与 Asn-551 一起稳定了引导链在该位点的紧密转向。

图 19.

磷酸盐模拟物E-乙烯基膦酸酯(E-VP,左图),(S)-GNA(中图),和酰胺骨架连接(AM1,右图)的结构。


   

Conformational flexibility and reduced off-target effects: GNA

We touched upon off-target effects (OTEs) in the siRNA introductory section and discussed the various origins and types of OTEs, including silencing of genes due to passenger strand activity and RNAi-mediated silencing caused by matching sequences between guide-strand seed region and non-targeted RNAs. Careful selection of target sequence and region, avoidance of non-Ago2 mediated silencing involving guide-strand seed pairing opposite non-targeted sequences (activation of miRNA pathway of silencing) and reduced dosage combined with chemical modification to modulate seed pairing can all reduce OTEs. The latter idea was inspired by a kink between nucleotides AS6 and AS7 in the siRNA seed region in the structures of Ago2 complexes bound either to miRNA or guide/passenger strand seed duplexes (Figure 15A). The wrinkle in the RNA chain causes local unstacking and appears to be promoted by an isoleucine side chain that inserts itself between AS6 and AS7 nucleobases. Moreover, the RNA backbone at the site of the kink is characterized by closely spaced phosphate groups, whereby potential Coulombic repulsion is relieved by adjacent arginine residues.

我们在 siRNA 的介绍部分提到了非特异性作用效应(OTEs),并讨论了各种 OTEs 的起源和类型,包括由于乘客链活性引起的基因沉默,以及由导引链种子区与非靶向 RNA 之间匹配序列导致的 RNAi 介导的沉默。通过仔细选择目标序列和区域、避免非 Ago2 介导的沉默、减少剂量以及化学修饰来调节种子区的配对,都可以减少 OTEs。后一种思路受到了 siRNA 种子区中 AS6 和 AS7 核苷酸之间的弯曲现象的启发,该现象在结合 miRNA 或导引链/乘客链种子双链结构的 Ago2 复合物中观察到(图 15A)。RNA 链的弯曲导致局部非叠加,并且似乎由一支异亮氨酸侧链插入 AS6 和 AS7 核苷酸之间所促进。此外,弯曲部位的 RNA 磷酸骨架具有密集排列的磷酸基团,邻近的精氨酸残基可以缓解潜在的库仑斥力。

Incorporation of an (S)-glycol nucleic acid (GNA, Figure 19) residue or GNA base pair at the site of the kink resulted in favorable in vitro and in vivo silencing activity of the modified siRNA relative to the parent duplex (ca. 2-fold gain in potency). GNA is the simplest phosphate-based nucleic acid, lacks a cyclic sugar moiety and its backbone is shortened by a bond compared to DNA and RNA. GNA is chiral and the pairing behavior is quite unusual. GNA self-pairing is of the Watson-Crick type, but base pairs are inverted—like in left-handed Z-DNA—inside the (S)-GNA right-handed duplex with a backbone geometry that resembles the A-form. Paired opposite RNA—only (S)-GNA pairs with RNA but not (R)-GNA—GNA does not alter its inverted base orientation. Thanks to its shorter backbone (S)-GNA mimics the kinked RNA conformation between AS6 and AS7, thereby mitigating OTEs. First, GNA conformationally preorganizes the RNA backbone for the kinked conformation that is sculpted by Ago2 after loading. Second, the conformationally flexible GNA residue incorporated into RNA lowers the thermodynamic stability of modified duplexes and depresses the affinity of pairing between guide siRNA seed region and matching sequences of non-targeted RNAs. This work demonstrated the benefits of structure-based and regiospecific chemical modification of siRNAs.

在弯曲点的位置引入(S)-甘醇核酸(GNA,见图 19)残基或 GNA 碱基对相对于母体双链的修饰 siRNA 表现出有利的体外和体内沉默活性(效力增加约 2 倍)。GNA 是最简单的基于磷酸的核酸,不含有环糖基团,其骨架相对于 DNA 和 RNA 而言缩短了一条化学键。GNA 是手性的,其碱基配对行为非常不寻常。GNA 自身的配对是属于 Watson-Crick 型,但碱基对是倒置的——就像左手 Z-DNA 一样——在(S)-GNA 右手双链内,其骨架几何形状类似 A 型。与 RNA 相配对时,只有(S)-GNA 与 RNA 配对,而不是(R)-GNA。GNA 不改变其倒置的碱基取向。由于其较短的骨架,(S)-GNA 模拟了 AS6 和 AS7 之间弯曲的 RNA 构象,从而减轻了 OTEs。首先,GNA 在构象上预组织了 RNA 骨架,使其形成由 Ago2 负载后塑造的弯曲构象。其次,结构具有构象灵活性的 GNA 残基降低了修饰双链的热力学稳定性,并降低了导引 siRNA 种子区与非靶向 RNA 匹配序列之间的配对亲和力。这项研究展示了基于结构和区域特异性的化学修饰对 siRNA 的益处。

There are many other design strategies to combat RNAi OTEs and improve siRNA potency. A recent example is the blockage of 5′-phosphorylation of the passenger strand by a 5′-morpholino modification. This modification results in improved antisense strand selection and RNAi activity. When the morpholino moiety is placed at the 5′-end of the guide strand it triggers complete loss of activity. Morpholino modification makes the sense strand siRNA a more effective passenger and can be combined with the (E)-VP moiety instead of phosphate at the 5′-end of the guide strand (Figure 19). The interaction between the 5′-phosphate and a strongly positively polarized binding pocket on the MID domain is probably key to loading and discrimination between guide and passenger strand. The (E)-VP but not the (Z)-VP modification mimics the 5′-phosphate bound to MID and affords enhanced potency by precluding dephosphorylation and repeated phosphorylation in cellular environments. The enhanced stability and activity afforded by the (E)-VP phosphate analog is particularly important for ss-siRNA efficacy.

有许多其他的设计策略可以应对 RNAi 的 OTEs 并提高 siRNA 的效力。最近的一个例子是通过 5'-morpholino 修饰阻断载体链的 5'-磷酸化。这种修饰导致改善了反义链的选择和 RNAi 活性。当 morpholino 基团位于导引链的 5'-末端时,会导致完全失去活性。Morpholino 修饰使正义链的 siRNA 成为更有效的载体,并且可以与(E)-VP 基团结合在导引链的 5'-末端,取代磷酸酯(见图 19)。5'-磷酸与 MID 结构域上强正极化的结合口袋之间的相互作用可能对导引链和载体链的加载和区分起关键作用。(E)-VP 但不是(Z)-VP 修饰模拟了结合到 MID 的 5'-磷酸酯,并通过阻止细胞环境中的脱磷酸化和重复磷酸化来提供增强的效力。(E)-VP 磷酸酯类似物提供的增强稳定性和活性对于 ss-siRNA 的疗效尤为重要。

Finally, yet another way for reducing potential OTEs was demonstrated with a neutral amide modification (Figure 19) that replaces the RNA phosphate group. Amides are well tolerated at many sites in the backbone of guide and passenger strands and were found to emulate interactions between the negatively charged phosphate group and protein side and main chain atoms . However, the amide moiety is not a suitable mimic of the phosphate at the site of a sharp turn between the AS1 and AS2 residues of guide siRNA bound to Ago2. Thus, an amide linkage incorporated at the corresponding site in passenger siRNA, between S1 and S2, can prevent off-target activity by the sense strand.

最后,还有一种减少潜在 OTEs 的方法是通过中性酰胺修饰(见图 19)取代 RNA 的磷酸酯基团。酰胺在导引链和载体链的骨架中的许多位置上都能很好地耐受,并且发现它们模拟了磷酸酯基团与蛋白质侧链和主链原子之间的相互作用。然而,酰胺基团不适合模拟导引 siRNA 与 Ago2 结合时 AS1 和 AS2 残基之间的锐角转折点处的磷酸酯。因此,在载体 siRNA 的相应位置上引入酰胺连接,即 S1 和 S2 之间,可以阻止正义链的非特异性活性。


   

The first RNAi therapeutic: ONPATTRO 第一个 RNAi 治疗药物:ONPATTRO

In 2018, ONPATTRO® (patisiran) received approval in the US and Europe for the treatment of the hereditary disease transthyretin-mediated amyloidosis in adults. (hATTR) (Figures 1and 3, Table 1). As pointed out above, the PS/MOE-modified ASO gapmer TEGSEDI (inotersen) was granted approval by the EC and subsequently by the FDA in the same year. Both therapeutics target the 3′-UTR of TTR mRNA but are administered via different routes, namely intravenously and subcutaneously, respectively .

2018 年,ONPATTRO®(patisiran)在美国和欧洲获得批准,用于成人遗传性转甲状腺激素介导的淀粉样变性病(hATTR)的治疗(图 1 和图 3,表 1)。正如上文所指出的,经过 PS/MOE 修饰的 ASO gapmer TEGSEDI(inotersen)在同一年获得欧洲委员会的批准,并随后获得了美国食品药品监督管理局的批准。这两种治疗药物的靶点都是TTR mRNA 的 3'-非翻译区(3'-UTR),但给药途径有所不同,分别为静脉注射和皮下注射。

ONPATTRO features 21mer guide and passenger strands with deviating modification patterns (Figure 18A). They share the use of 2′-deoxythymidines at the 3′-ends and 2′-O-Me modification of selected pyrimidine nucleotides. However, in the guide only two uridines are 2′-modified as compared to five uridines and four cytidines in the passenger strand. 2′-O-Me modification affords increased nuclease resistance at sites that were found to be prone to strand cleavage. TEGSEDI and ONPATTRO exhibited somewhat different safety profiles in phase 3 clinical trials, whereby thrombocytopenia, renal dysfunction or elevated levels of liver enzymes were not observed with administration of ONPATTRO. ONPATTRO is delivered in a lipid nanoparticle (LNP) formulation and in order to minimize potential proinflammatory effects, patients are pretreated with H1 and H2 antihistamines, an analgesic and a glucocorticoid. LNP formulation is not unique to ONPATTRO, as 10 other LNP drugs have been given the green light by regulatory agencies over the last 30 years. However, ONPATTRO is the only oligonucleotide therapeutic so far that employs LNPs for delivery (Figures 2B and 20).

ONPATTRO 具有 21 碱基的引导链和过客链,并具有不同的修饰模式(图 18A)。它们共享在 3'-末端使用 2'-脱氧胸苷和选择性嘧啶核苷酸的 2'-O-Me 修饰。然而,在引导链中,只有两个尿嘧啶核苷酸进行 2'-修饰,而在旅行链中有五个尿嘧啶核苷酸和四个胞嘧啶核苷酸进行 2'-修饰。2'-O-Me 修饰可增强易受链断裂的位点的核酸酶抗性。在 3 期临床试验中,TEGSEDI 和 ONPATTRO 显示出略有不同的安全性特征,其中在使用 ONPATTRO 时未观察到血小板减少、肾功能障碍或肝酶水平升高。ONPATTRO 采用脂质纳米粒(LNP)制剂进行传递,并且为了最小化潜在的促炎作用,患者在治疗前接受 H1 和 H2 抗组胺药物、镇痛药和糖皮质激素预处理。LNP 制剂并不是 ONPATTRO 独有的,过去 30 年中已经有其他 10 种 LNP 药物获得了监管机构的批准。然而,ONPATTRO 是迄今为止唯一一种使用 LNP 进行传递的寡核苷酸治疗药物(图 2B 和 20)。

图 20.

用于将功能性 siRNA 传递到肝脏的平台:LNP 与 GalNAc-siRNA 偶联物。

The success with LNP formulation built on the initial breakthroughs with systemic delivery of modified siRNA by i.v. in mice and systemic delivery in a liposomal formulation to non-human primates. Although LNP formulation had been developed for delivery of small molecule drugs, those concepts did not yield viable formulations for clinical use of the much larger and negatively charged siRNAs. Ultimately, it took several breakthroughs to optimize LNPs with the ability to encapsulate the siRNA duplex and deliver it to the cytoplasm of hepatocytes in vivo. One of these was the identification of ionizable cationic lipids that are positively charged at acidic pH but neutral at physiological pH to prevent toxic effects due to immune stimulation triggered by positively charged systems. Another was the use of PEG lipids with C14 acyl chains for incorporation into the surface of LNPs to generate diameters in the 20–100 nm range and minimize interactions between PEG and target cells that could hamper delivery. Lastly, a large number of ionizable lipids was screened and evaluated initially in rodents and subsequently in non-human primates in the search for a chemistry that would afford optimal potency (Figure 21A). And so ONPATTRO became the first RNAi therapeutic to be brought to market, 20 years after the discovery of RNA interference.

在利用 LNP(脂质纳米粒子)制剂进行全身性递送修饰 siRNA 的初始突破之后,LNP 制剂在小鼠中经静脉注射和非人灵长类动物中的脂质体制剂进行全身性递送方面取得了成功。尽管 LNP 制剂已经被用于小分子药物的递送,但这些概念并没有产生可用于临床应用的体积更大且带有负电荷的 siRNA 的制剂。最终,需要进行多次突破来优化 LNPs,使其能够封装 siRNA 双链并将其递送到体内肝细胞的细胞质中。其中之一是识别出具有在酸性 pH 下呈正电荷但在生理 pH 下呈中性的离子化阳离子脂质,以防止由正电荷系统引发的免疫刺激导致的毒性作用。另一个突破是使用具有 C14 酰基链的 PEG 脂质,用于将其纳入 LNPs 表面,以生成直径在 20-100 nm 范围内,并最大限度减少 PEG 与目标细胞之间的相互作用,以防止递送受阻。最后,对大量离子化脂质进行了筛选和评估,最初在啮齿动物中,随后在非人灵长类动物中,以寻找能够提供最佳效力的化学成分(图 21A)。因此,ONPATTRO 成为第一个 RNAi 治疗药物,距离 RNA 干扰的发现已经有 20 年的时间。

图 21.

(A) LNP 多组分制剂,用于有效递送 RNAi 治疗药物 ONPATTRO 至肝细胞。
(B) LNP 多组分制剂,用于 COMIRNATY 和 SPIKEVAX mRNA 疫苗的肌肉注射给药。


   

GIVLAARI and acute hepatic porphyria GIVLAARI 与急性肝卟啉症

GIVLAARI® (givosiran) is used in the treatment of acute hepatic porphyria (AHP) in adults and was the second RNAi therapeutic to receive approval by the FDA. AHP are rare inherited, metabolic disorders of heme biosynthesis that lead to build-up of porphyrins which negatively affects skin or the nervous system, with some patients experiencing serious and potentially life-threatening attacks. Symptoms of an attack include chest and abdominal pain, vomiting, constipation, and high blood pressure and heart rate. Treatment options depend on the type of porphyria and a patient's symptoms. Avoiding sunlight helps with porphyria of the skin and the treatment of an attack may involve intravenous application of heme or glucose solution. The disease can be inherited from one parent or both and be of the autosomal dominant, autosomal recessive or X-linked dominant types.

GIVLAARI®(givosiran)用于治疗成年人的急性肝卟啉症(AHP),是第二个获得 FDA 批准的 RNAi 治疗药物。AHP 是罕见的遗传代谢性疾病,影响血红素生物合成,导致卟啉物质的积累,进而对皮肤或神经系统产生负面影响,部分患者可能出现严重且潜在危及生命的发作。发作症状包括胸痛、腹痛、呕吐、便秘、高血压和心率增快。治疗选择取决于卟啉症的类型和患者的症状。对于皮肤型卟啉症,避免阳光暴晒有助于缓解症状,而发作的治疗可能包括静脉注射血红素或葡萄糖溶液。该疾病可以从一个或两个父母遗传,并可属于常染色体显性遗传、常染色体隐性遗传或 X 连锁显性遗传类型。

AHP arises from an autosomal dominant loss-of-function of one of three enzymes in the heme biosynthesis pathways, namely the third (leading to acute intermittent porphyria), sixth (leading to hereditary coproporphyria) or seventh enzyme (leading to variegate porphyria). A fourth rarer type stems from an autosomal recessive inheritance. As a consequence of these disorders, precursors such as δ-aminolevulinic acid and porphobilinogen are being accumulated, eventually triggering AHP attacks. Notable individuals who were speculated to have suffered from porphyria include King George III and Vincent van Gogh, although in the case of the former this was debunked. A definitive diagnosis of porphyria in the case of the artist remains elusive, and various origins of his symptoms and mental health state have been put forth, including schizoaffective disorder and lead poisoning.

AHP 起因于血红素生物合成途径中三个酶之一的常染色体显性失活,即第三酶(导致急性间歇性卟啉症),第六酶(导致遗传性卟胆卟啉症)或第七酶(导致杂色卟啉症)。第四种更罕见的类型源于常染色体隐性遗传。由于这些疾病,δ-氨基乙酸和卟胆原等前体物质会积累,最终触发 AHP 发作。被猜测曾经患有卟啉症的知名人物包括乔治三世国王和文森特·梵高,尽管对于前者的猜测已经被证实是错误的。关于艺术家患有卟啉症的明确诊断至今仍然难以确定,人们提出了各种关于他症状和心理健康状态起源的理论,包括情感性精神障碍和铅中毒等。

GIVLAARI targets aminolevulinate synthase 1 (ALAS1) mRNA and is composed of fully chemically modified 23mer and 21mer siRNA guide and passenger strands, respectively. The 3′-end of the passenger siRNA is linked to a triantennary N-acetylgalactosamine- (GalNAc-) containing ligand (Figure 18B). Except for the passenger terminus that is conjugated to the GalNAc ligand, the remaining termini are PS-modified at the last two bridging phosphates. The siRNA duplex contains 28 2′-O-Me modified nucleotides and 16 2′-F modified nucleotides, such that the modification chemistries in the guide siRNA are nearly balanced (12 2′-O-Me and 11 2′-F residues). The two analogs alternate along nearly the entire length of the guide; in the passenger strand the alternating mode is limited to the central 11mer (Figure 18B).

GIVLAARI 靶向δ-氨基乙酸合成酶 1(ALAS1)mRNA,由完全化学修饰的 23 碱基和 21 碱基的 siRNA 引导链和过客链组成。过客链的 3'-末端连接着一个三支化的乙酰半乳糖胺(GalNAc)配体(图 18B)。除了被动链末端与 GalNAc 配体结合外,其余的末端在最后两个桥联磷酸酯处都被 PS 修饰。siRNA 双链包含 28 个 2'-O-Me 修饰的核苷酸和 16 个 2'-F 修饰的核苷酸,因此在引导链的修饰化学结构中几乎达到平衡(12 个 2'-O-Me 和 11 个 2'-F 残基)。这两种模拟物几乎沿着引导链的整个长度交替出现;而在过客链中,交替模式仅限于中央的 11 碱基(图 18B)。

GalNAc conjugation chemistry requires no formulation and offers a delivery concept that is entirely different from i.v. administration of LNP-encapsulated siRNA (Figure 20). GalNAc-siRNA conjugates allow targeted delivery to liver thanks to GalNAc sugars being recognized by the asialoglycoprotein receptor carbohydrate recognition domain (ASGPR-CRD). ASGPR is predominantly expressed on the surface of hepatocytes but only exists in small quantities on extra-hepatic cells. This enables specific, receptor-mediated hepatocyte delivery of therapeutics for the treatment of hepatic afflictions while reducing risk of off-target effects. Galactose and GalNAc are sugars that ASGPR binds with high affinity, whereby the particular isomeric form, branching and galactose linkers, among others, influence ligand-receptor binding. Once docked to ASGPR, the receptor facilitates internalization of the conjugated siRNA by clathrin-mediated endocytosis.

GalNAc 共轭化学不需要制剂,并提供了一种与 i.v.注射 LNP 封装 siRNA 完全不同的传递概念(图 20)。GalNAc-siRNA 共轭物能够通过 GalNAc 糖与无糖型糖蛋白受体碳水化合物识别结构域(ASGPR-CRD)结合,实现靶向肝脏的传递。ASGPR 主要表达在肝细胞表面,但在肝外细胞中只存在少量。这使得治疗剂可以通过特异性的受体介导的肝细胞传递,用于治疗肝脏疾病,同时降低非靶效应的风险。半乳糖和 GalNAc 是 ASGPR 高亲和力结合的糖分子,其中特定的异构形式、支链和半乳糖连接部分等因素影响配体与受体的结合。一旦与 ASGPR 结合,受体通过衬膜蛋白介导的内吞作用促进共轭 siRNA 的内化。

GIVLAARI is administered subcutaneously once a month and exhibited a half-life of six hours, whereby metabolites were primarily eliminated in urine. Adverse events during clinical trials included nausea, injection site reactions, rash, increase in serum creatinine, and transaminase elevations. An immunogenic effect was observed in one patient (of 111) during placebo-controlled and open-label clinical studies.

GIVLAARI 以皮下注射的方式每月一次给药,并表现出半衰期为六小时,代谢产物主要通过尿液排泄。临床试验期间的不良事件包括恶心、注射部位反应、皮疹、血清肌酐增高和转氨酶升高。在安慰剂对照和开放标签的临床研究中,有一个患者(111 名患者中)观察到免疫原性效应。


   

OXLUMO and primary hyperoxaluria type 1 OXLUMO 与一型原发性高草尿酸症

Primary hyperoxaluria 1 (PH1) is a rare genetic disorder that results in increased hepatic oxalate production. Debilitating and life-threatening clinical manifestations include kidney stones, kidney failure and systemic oxalosis. Novel therapeutic approaches aimed at lowering oxalate levels and minimizing renal damage and focused on different targets range from cell-, gene- and protein-based therapies to small molecule inhibitors and oxalate-degrading enzymes and bacteria. Substrate reduction therapy is a potentially successful strategy to treat inborn metabolic diseases. Thus, inhibiting the biosynthetic pathway to production of the main precursor of oxalate should in principle be therapeutically beneficial to PH1 patients.

一型原发性高草酸尿症(PH1)是一种罕见的遗传性疾病,导致肝脏产生过多的草酸。该疾病会出现严重且危及生命的临床表现,包括肾结石、肾衰竭和全身性草酸沉积症。针对降低草酸水平、减少肾脏损伤的新型治疗方法涉及不同的靶点,包括细胞、基因和蛋白质治疗、小分子抑制剂以及降解草酸的酶和细菌。底物减少疗法是治疗先天代谢病的一种潜在成功策略。因此,原则上抑制草酸的主要前体物质的生物合成途径对于 PH1 患者在治疗上应该具有益处。

RNAi offers an attractive approach for addressing an unmet need to treat PH1 with high efficacy and sufficient durability. Lumasiran is an siRNA that targets the mRNA of the hydroxyacid oxidase 1 (HAO1) gene that encodes glycolate oxidase (GO), thereby precluding the conversion of glycolate to glyoxylate, the main precursor of oxalate. This siRNA was approved by the US FDA in 2020 as OXLUMO for treatment of PH1, to lower urinary and plasma oxalate levels in pediatric and adult patients. GO lies upstream from alanine-glyoxylate aminotransferase (AGT) that normally metabolizes glyoxylate to glycine in liver peroxisomes. AGT deficiency due to genetic mutations in PH1 patients means that glyoxylate is not efficiently converted to glycine. Rather, glycine is oxidized to oxalate by cytosolic lactate dehydrogenase (LDH), causing urinary oxalate excretion to spike. As a consequence of the poor solubility of oxalate, it can crystallize in the form of calcium oxalate and aggregate into kidney stones or cause inflammation and nephrocalcinosis. With OXLUMO silencing HAO1 mRNA, glyoxylate buildup is minimized and glycolate instead excreted in urine without resulting in kidney damage or toxic effects.

RNAi 为治疗一型原发性高草酸尿症(PH1)提供了一种具有高效和持久性的有吸引力的方法。Lumasiran 是一种针对羟基酸氧化酶 1(HAO1)基因的 mRNA 的 siRNA,该基因编码甘酸氧化酶(GO),从而阻断了甘酸转化为草酸的过程,草酸是草酸的主要前体物质。这种 siRNA 在 2020 年获得美国 FDA 批准,以 OXLUMO 的名义用于治疗 PH1,降低儿童和成人患者的尿液和血浆草酸水平。GO 位于正常情况下在肝脏过氧化物体中将草酸转化为甘氨酸的丙氨酰-草酰基转氨酶(AGT)之上。PH1 患者由于遗传突变导致 AGT 缺乏,使得草酰基不能有效地转化为甘氨酸。相反,甘氨酸会被细胞质中的乳酸脱氢酶(LDH)氧化为草酸,导致尿液中草酸的排泄增加。由于草酸溶解度较差,它会以草酸钙的形式结晶并聚集成肾结石,或引起炎症和肾脏钙化。通过沉默HAO1 mRNA,OXLUMO 能够最小化草酰基的积累,使甘酸代替草酰基在尿液中排泄,而不会导致肾脏损伤或毒性效应。

Like GIVLAARI, OXLUMO features fully modified 23mer and 21mer siRNA guide and passenger strands, respectively (Figure 18C). The 3′-end of the latter is linked to the triantennary GalNAc-containing ligand. Also, except for that passenger terminus, the three remaining termini are PS-modified at the last two bridging phosphates. In a double-blind phase 3 lumasiran trial, 39 PH1 patients aged 6 and older were randomly assigned in a 2:1 ratio to receive either the drug or placebo subcutaneously for a period of six months; afterwards all patients would receive lumasiran for a period of up to 54 months. It was found that the reduction in the 24-hour urinary oxalate excretion was 53.5% greater for patients on lumasiran compared to those receiving placebo during the six-month trial. Patients on lumasiran had normal or near-normal urinary oxalate levels at six months. Lumasiran treatment also resulted in substantially reduced plasma oxalate levels, thereby providing support for its mechanism of action. Patients in the lumasiran group showed elevated levels of plasma and urinary glycolate which is expected on the basis of the inhibition of GO enzyme production by the drug. In a separate case study of a teenage PH1 patient, lumasiran was found to consistently and rapidly reduce plasma oxalate and urinary excretion to normal or almost normal levels for 18 months. This patient did not develop new kidney stones and required no further urological interventions.

与 GIVLAARI 类似,OXLUMO 的构成包括完全修饰的 23mer 和 21mer 的 siRNA 导向链和旅客链(图 18C)。后者的 3'端与三支 GalNAc 含有的配体连接。另外,除了旅客链末端外,其余三个末端的最后两个桥接磷酸根都被 PS 修饰。在一个双盲的 III 期 lumasiran 试验中,随机选取了 39 名年龄在 6 岁及以上的 PH1 患者,以 2:1 的比例分配到接受药物或安慰剂的皮下注射治疗组,在为期六个月的时间内进行治疗;之后,所有患者将接受长达 54 个月的 lumasiran 治疗。研究发现,在为期六个月的试验期间,与安慰剂组相比,接受 lumasiran 治疗的患者的 24 小时尿草酸排泄量减少了 53.5%。在六个月时,接受 lumasiran 治疗的患者的尿草酸水平正常或接近正常水平。lumasiran 治疗还显著降低了血浆草酸水平,从而支持其作用机制。lumasiran 组的患者显示出血浆和尿液中甘酸水平升高,这是由于药物抑制了 GO 酶的产生。在一项关于一名青少年 PH1 患者的单独病例研究中,发现 lumasiran 能够持续快速地将血浆草酸和尿液排泄物降至正常或接近正常水平,持续时间达 18 个月。该患者未出现新的肾结石,并且不需要进一步的泌尿学干预措施。


   

LEQVIO: the first ‘millions of people’ global RNAi therapeutic? LEQVIO:首个"百万人"级全球 RNAi 治疗药物?

Inclisiran is an siRNA targeting proprotein convertase subtilisin-kexin type 9 (PCSK9) mRNA for the treatment of heterozygous familial hypercholesterolemia (HeFH) or clinical atherosclerotic cardiovascular disease (ASCVD) to lower low-density lipoprotein cholesterol (LDL-C). Hypercholesterolemia is characterized by elevated levels of LDL-C that carries a risk of premature ASCVD. In two phase 3 trials, inclisiran was found to significantly lower LDL-C in adults compared to those who received placebo with an acceptable safety profile. Inclisiran (300 mg dose) or matching placebo were given by subcutaneous injection on days 1, 90, 270 and 450, whereby the two primary endpoints were the % change from baseline in the LDL-C level on day 510 and the % change from baseline in the LDL-C level between days 90 and 540. The minor common side effects include injection site reactions such as pain, redness and swelling. The twice-yearly dosing regimen constitutes a major advantage of inclisiran compared to a monoclonal antibody against PCSK9 protein that also reduces LDL-C but needs to be administered every two to four weeks. Inclisiran was given approval under the brand name LEQVIO in the EU and the US in 2021 and 2022, respectively (Figure 3).

Inclisiran 是一种靶向前蛋白转化酶枯草杆菌蛋白酶 9(PCSK9)mRNA 的 siRNA,用于治疗杂合性家族性高胆固醇血症(HeFH)或临床动脉粥样硬化性心血管疾病(ASCVD),以降低低密度脂蛋白胆固醇(LDL-C)水平。高胆固醇血症以升高的 LDL-C 水平为特征,存在患早发动脉粥样硬化性心血管疾病的风险。在两个 3 期试验中,与接受安慰剂的成年人相比,Inclisiran 显著降低了 LDL-C 水平,并具有可接受的安全性。Inclisiran(300 毫克剂量)或相应的安慰剂于第 1 天、第 90 天、第 270 天和第 450 天通过皮下注射给予,其中两个主要终点是第 510 天时与基线相比的 LDL-C 水平变化百分比,以及第 90 天至第 540 天期间与基线相比的 LDL-C 水平变化百分比。常见的轻微副作用包括注射部位反应,如疼痛、红肿等。与针对 PCSK9 蛋白的单克隆抗体相比,Inclisiran 每半年一次的给药方案具有重要优势,后者也可以降低 LDL-C,但需要每两到四周进行一次给药。Inclisiran 在 2021 年和 2022 年分别在欧盟和美国以商标名 LEQVIO 获得批准(图 3)。

Like GIVLAARI, LEQVIO features 2′-F and 2′-O-Me modification chemistry with pairs of PS moieties at the 5′- and 3′-ends of the 23mer guide strand and at the 5′-end of the 21mer passenger strand. The latter consists mainly of 2′-O-Me nucleotides (just two 2′-F nucleotides in the 5′-half and a central 2′-deoxynucleotide; Figure 18D). The 3′-end of the passenger is tethered to the triantennary GalNAc conjugate. Phase 3 trials of the drug were completed by The Medicines Company and Novartis subsequently acquired The Medicines Company and inclisiran in 2019. Compared to the majority of currently approved nucleic acid therapeutics that are used in the treatment of rare genetic disorders, LEQVIO targets both elevated LDL-C (in patients with the disease HeFH) and ASCVD due to elevated LDL-C, a condition that affects millions of patients worldwide. Thus, the drug will likely become the first ‘millions-of-people’ global RNAi therapeutic.

与 GIVLAARI 类似,LEQVIO 采用了 2'-F 和 2'-O-Me 修饰化学结构,其中 23 个核苷酸的导引链的 5'-端和 3'-端以及 21 个核苷酸的旅行链的 5'-端均配对有磷酸甘露糖胺(PS)基团。后者主要由 2'-O-Me 核苷酸组成(在 5'-半部分只有两个 2'-F 核苷酸和一个中央的 2'-去氧核苷酸;图 18D)。旅行链的 3'-端与三支 GalNAc 共轭物相连。该药物的 3 期临床试验由 The Medicines Company 完成,随后 Novartis 于 2019 年收购了 The Medicines Company 和 inclisiran。与目前大多数用于治疗罕见遗传疾病的核酸治疗药物相比,LEQVIO 针对的是同时存在高 LDL-C(HeFH 患者)和由高 LDL-C 引起的 ASCVD 的情况,这是一种影响全球数百万患者的疾病。因此,该药物有望成为首个面向“百万人群体”的全球 RNAi 治疗药物。


   

AMVUTTRA, a second-generation siRNA therapeutic for hATTR-mediated amyloidosis AMVUTTRA,第二代 siRNA hATTR 淀粉样变性病治疗药物

AMVUTTRA® (vutrisiran) is a transthyretin-directed siRNA indicated for the treatment of the polyneuropathy of hereditary transthyretin-mediated amyloidosis in adults. AMVUTTRA has the same sequence as the failed investigational drug revusiran but with different chemistry to provide extended metabolic stability of the drug (revusiran clinical development was voluntarily stopped in 2016). AMVUTTRA uses GalNAc-conjugation for efficient delivery into hepatocytes and further follows the design of GIVLAARI and OXLUMO for having enhanced metabolic stability (ESC chemistry with additional terminal PS modification, Figure 18E). AMVUTTRA received FDA approval in June of 2022. It is the fifth siRNA-based drug reaching approval status in five years (Figure 3). Compared to the first-generation ONPATTRO, it offers the benefit of infrequent dosing, i.e. 25 mg administered by subcutaneous injection once every three months, without the need for IV premedications to reduce infusion-related reactions.

AMVUTTRA®(vutrisiran)是一种转甲状腺素定向 siRNA 药物,用于治疗成年人遗传性转甲状腺素介导的淀粉样变性引起的多发性神经病变。AMVUTTRA 与失败的研究药物 revusiran 具有相同的序列,但具有不同的化学结构,以提供药物的长期代谢稳定性(revusiran 的临床开发于 2016 年自愿停止)。AMVUTTRA 利用 GalNAc 共轭技术实现高效递送至肝细胞,并采用 GIVLAARI 和 OXLUMO 的设计思路以提高药物的代谢稳定性(采用增强型代谢稳定性(ESC)化学结构,并额外进行了末端 PS 修饰,图 18E)。AMVUTTRA 于 2022 年 6 月获得 FDA 批准。它是在五年内获得批准的第五种 siRNA 药物(图 3)。与一代药物 ONPATTRO 相比,AMVUTTRA 具有不经常给药的优势,即每三个月一次皮下注射 25 毫克,无需进行静脉前用药以减少输注相关反应。


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