siRNA 药物在肝病治疗中的应用（一）

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Abstract 摘要

Small interfering RNA (siRNA)-based therapeutics holds the promise to treat a wide range of human diseases that are currently incurable using conventional therapies. Most siRNA therapeutic efforts to date have focused on the treatment of liver diseases due to major breakthroughs in the development of efficient strategies for delivering siRNA drugs to the liver. Indeed, the development of lipid nanoparticle-formulated and GalNAc-conjugated siRNA therapeutics has resulted in recent FDA approvals of the first siRNA-based drugs, patisiran for the treatment of hereditary transthyretin amyloidosis and givosiran for the treatment of acute hepatic porphyria, respectively. Here, we describe the current strategies for delivering siRNA drugs to the liver and summarize recent advances in clinical development of siRNA therapeutics for the treatment of liver diseases.

基于小干扰 RNA（siRNA）的治疗方法有望治疗许多目前无法通过传统疗法治愈的人类疾病。目前，大多数 siRNA 治疗的研究都集中在肝脏疾病上，这是由于在高效输送 siRNA 药物至肝脏方面取得了重大突破。实际上，脂质纳米粒制剂和 GalNAc-缀合 siRNA 疗法的发展，促使首批 siRNA 药物获得了 FDA 批准，即用于治疗遗传性转甲状腺素蛋白淀粉样变性症的 patisiran 和用于治疗急性肝卟啉病的 givosiran。本文描述了当前 siRNA 药物输送到肝脏的策略，并总结了在肝脏疾病治疗中 siRNA 疗法临床开发的最新进展。

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1 Introduction 简介

The discovery of RNA interference (RNAi) and the use of synthetic 21–23 nucleotide (nt) double-stranded RNAs, named siRNAs, to trigger post-transcriptional gene silencing in mammalian cells in a sequence-specific manner have revolutionized functional genomic studies and biomedical research . Furthermore, marked improvements in the design, chemical stabilization, and delivery of siRNA molecules have greatly facilitated their therapeutic utility resulting in a diverse pipeline of siRNA therapeutics currently in clinical trials. In 2018, almost 20 years since the discovery of the RNAi pathway, the first siRNA-based drug was approved by the US Food and Drug Administration (FDA) for the treatment of polyneuropathy caused by hereditary transthyretin-mediated amyloidosis . In this review, we will describe the different chemical modifications used in siRNA drugs, summarize the currently deployed strategies for delivering siRNA therapeutics to the liver, and highlight recent advances in the development of siRNA therapeutics for the treatment of liver diseases.

RNA 干扰（RNAi）的发现以及使用合成的 21-23 核苷酸（nt）双链 RNA（siRNA）在哺乳动物细胞中以序列特异性方式触发转录后基因沉默的应用，彻底改变了功能基因组学研究和生物医学研究。此外，siRNA 分子的设计、化学稳定性和递送方面的显著改进，大大提高了其治疗效用，使得目前有多种 siRNA 治疗药物正在进行临床试验。2018 年，距离 RNAi 途径发现近 20 年后，第一个基于 siRNA 的药物被美国食品药品监督管理局（FDA）批准用于治疗由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。在这篇综述中，我们将介绍 siRNA 药物中使用的不同化学修饰，概述当前用于将 siRNA 药物递送到肝脏的策略，并强调在肝脏疾病治疗中 siRNA 药物开发的最新进展。

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2 Chemical Modifications in siRNA Drugs siRNA 药物中的化学修饰

The endogenous RNAi machinery can be engaged by synthetic siRNA molecules to specifically silence the expression of a given target gene for therapeutics. However, unmodified siRNAs are large, polyanionic, and susceptible to degradation by nucleases, which make their clinical use difficult due to poor cellular uptake and rapid clearance in vivo. Furthermore, gene silencing by RNAi-based drugs requires optimization of the siRNA triggers for target specificity with minimized off-target effects and immunogenic reactions due to sensing of double-stranded RNA molecules by Toll-like receptor (TLR) 3/7/8 and protein kinase R (PKR), and for improved nuclease resistance. This can be achieved using a variety of chemical modifications of the sugar, the nucleobase, or the internucleotide linkages (Fig. 1). Commonly used 2′ sugar modifications in siRNA molecules include 2′-O-methyl (2′-O-Me) and 2′-fluoro (2′-F), which enhance serum stability and reduce immune activation of siRNA drugs (Fig. 1). In addition, structurally constrained bicyclic locked nucleic acids (LNA) and the flexible acyclic unlocked nucleic acids (UNA) have been deployed to increase or decrease, respectively, the binding affinity of siRNAs to their cognate target RNA. Moreover, substitution of phosphodiester linkages with phosphorothioate (PS) linkages, in which a sulfur atom replaces one of the non-bridging oxygen atoms in the phosphate group also improve nuclease resistance (Fig. 1). More recently, additional chemistries have been applied to enhance siRNA potency and safety. For example, incorporation of a 5′(E)-vinyl-phosphonate in the guide (antisense) strand improves the potency and duration of RNAi activity in vivo, while incorporation of a (S)-glycol nucleic acid modification in the seed region of the siRNA guide strand decreases hepatotoxicity and further improves potency. In addition to the chemical modifications, selection of the siRNA sequence has a major impact on both on-target potency and off-targets activities. For instance, ensuring exclusive selection and loading of the guide strand into the RISC complex is crucial to minimize off-target activities. This can be reinforced by decreasing the thermodynamic stability of the 5′ end of the guide strand by selecting sequences with AU-rich motifs. Asymmetric siRNAs with a 2-nt 3′ overhang in one end and a blunt end in the other have also been reported to favor guide strand incorporation into the RISC complex. Finally, using LNAs allows the design of small internally segmented inferring RNAs (sisiRNAs), which are characterized by an intact guide strand and a segmented passenger (sense) strand modified with LNAs preventing loading into the RISC complex, thereby minimizing passenger-strand induced off-target effects.

内源性 RNA 干扰（RNAi）机制可以通过合成的 siRNA 分子来启动，特异性地沉默特定靶基因的表达以用于治疗。然而，未修饰的 siRNA 分子较大且带有多阴离子，容易被核酸酶降解，这使得其在临床应用中面临细胞摄取效率低和体内快速清除的挑战。此外，RNAi 药物的基因沉默效果需要对 siRNA 进行优化，以确保其靶向特异性，同时尽量减少脱靶效应和因双链 RNA 被 Toll 样受体（TLR）3/7/8 和蛋白激酶 R（PKR）感应而引起的免疫反应，并提高对核酸酶的抵抗力。通过对糖、碱基或核苷酸间连接进行各种化学修饰可以实现这一点（图 1）。常用的 siRNA 分子中的 2′位糖修饰包括 2′-O-甲基（2′-O-Me）和 2′-氟（2′-F），它们可以增强血清稳定性并减少 siRNA 药物的免疫激活（图 1）。此外，结构受限的双环锁核酸（LNA）和灵活的无环解锁核酸（UNA）分别用于增加或减少 siRNA 与其靶 RNA 的结合亲和力。此外，用磷硫酰（PS）连接取代磷酸二酯连接，其中硫原子取代磷酸基团中的一个非桥氧原子，也能提高对核酸酶的抵抗力（图 1）。最近，更多的化学修饰被应用于增强 siRNA 的效力和安全性。例如，在引导链（反义链）中加入 5′(E)-乙烯基磷酸酯，可以提高 RNAi 活性在体内的效力和持续时间，而在 siRNA 引导链的种子区加入(S)-乙二醇核酸修饰则可以减少肝毒性并进一步提高效力。除了化学修饰，siRNA 序列的选择对靶向效力和脱靶效应也有重大影响。例如，确保引导链被选择并独特地加载到 RISC 复合体中对于最小化脱靶效应至关重要。选择含有 AU 富集基序的序列可以降低引导链 5′末端的热力学稳定性，从而加强这一点。据报道，具有一端 2 个核苷酸 3′悬垂末端和另一端平末端的非对称 siRNA 也有助于引导链加载到 RISC 复合体中。最后，使用 LNA 可以设计小型内部分段干扰 RNA（sisiRNA），其特点是完整的引导链和经过 LNA 修饰的分段乘客链（正义链），防止其加载到 RISC 复合体中，从而最小化乘客链引起的脱靶效应。

Fig. 1 Structures of chemical modifications used in siRNA drugs. Commonly used sugar modifications in siRNA molecules include 2′-O-methyl (2′-O-Me), 2′-fluoro (2′-F), locked nucleic acid (LNA), and unlocked nucleic acid (UNA). Replacement of phosphate backbone linkages with phosphorothioate (PS) linkages increases nuclease resistance of siRNAsiRNA 药物中使用的化学修饰结构。siRNA 分子中常用的糖修饰包括 2′-O-甲基 (2′-O-Me)、2′-氟 (2′-F)、锁定核酸 (LNA) 和解锁核酸 (UNA)。用硫代磷酸酯 (PS) 键替换磷酸主链键可提高 siRNA 的核酸酶抗性

The importance of chemical modifications in siRNA drugs is underscored by the results from the ENDEAVOUR Phase III clinical trial (Clinical Trial Number: NCT02319005). The study showed that revusiran, Alnylam’s first GalNAc-conjugated siRNA drug targeting transthyretin (TTR), had an imbalance in mortality with 18 deaths in the revusiran arm compared with only 2 deaths in the placebo group, as well as increased peripheral neuropathy. The design of revusiran is based on Alnylam’s Standard Template Chemistry (STC), in which the siRNA sequence is fully modified with 2′-OMe and 2′-F sugars and has two terminal PS backbone linkages at the 3′-end of the guide strand. To improve this siRNA drug design, Alnylam developed enhanced stability chemistry (ESC), which includes modifications improving metabolic stability, potency, and duration of action, resulting in decreased dose level and toxicity. The ESC modifications include among others PS backbone adjustments at the 5′-ends of both strands, structural motifs that extend the guide strand to 23 nt, and an augmented number of sequential 2′-O-Me modifications. Recently, Alnylam reported on the development of the ESC+ system that further improves the specificity and therapeutic index . All siRNA drugs from Alnylam entering clinical studies are based on the ESC or ESC+ design.

在 siRNA 药物中进行化学修饰的重要性在 ENDEAVOUR 第三期临床试验（临床试验编号：NCT02319005l）的结果中得到了充分体现。该研究表明，Alnylam 公司的第一个 GalNAc-偶联 siRNA 药物 revusiran，在治疗转甲状腺素（TTR）相关疾病时，revusiran 组有 18 人死亡，而安慰剂组只有 2 人死亡，同时还观察到周围神经病变增加。revusiran 的设计基于 Alnylam 的标准模板化学（STC），即 siRNA 序列完全由 2′-OMe 和 2′-F 糖修饰，并在引导链的 3′端有两个末端磷硫酰（PS）骨架连接。为了改进这种设计，Alnylam 开发了增强稳定性化学（ESC），包括提高代谢稳定性、效力和作用持续时间的修饰，从而降低了剂量水平和毒性。ESC 修饰包括对两条链的 5′端进行 PS 骨架调整，将引导链延长至 23 个核苷酸的结构模体，以及增加 2′-OMe 修饰的数量。最近，Alnylam 公司报道了 ESC+系统的开发，进一步提高了特异性和治疗指数。所有进入临床研究的 Alnylam 公司的 siRNA 药物都基于 ESC 或 ESC+设计。

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3 Strategies for Delivering siRNA Drugs to the Liver siRNA 药物肝脏递送策略

Development of siRNA therapeutics holds great promise for the treatment of a wide array of human diseases. However, delivery of siRNA drugs is hampered by their high molecular weight (approximately 13–15 kDa) and hydrophilic and polyanionic properties, which prevent naked siRNA molecules to cross the cell membrane. Liver is an attractive organ for the development of siRNA therapeutics since it is amenable to both passive and targeted delivery of siRNAs drugs. Passive delivery exploits the inherent tendency of liposomes and lipid nanoparticles to accumulate in filtering organs of the reticuloendothelial system (RES), primarily the liver, but also lymph nodes, kidney, and spleen. Targeted delivery of siRNAs takes advantage of the asialoglycoprotein receptor (ASGPR), which is expressed at high density on the surface of hepatocytes. Thus, a key focus of RNAi therapeutics has been to develop siRNA drugs for treating liver diseases. Below, we will discuss some representative delivery systems used in the development of siRNA therapeutics for the treatment of liver diseases (Fig. 2).

siRNA 治疗的发展在治疗各种人类疾病方面展现了巨大潜力。然而，由于 siRNA 药物的高分子量（大约 13–15 kDa）及其亲水性和多阴离子特性，使得裸露的 siRNA 分子难以穿越细胞膜，从而阻碍了其递送。肝脏是 siRNA 治疗开发的理想靶器官，因为它适合于 siRNA 药物的被动和靶向递送。被动递送利用脂质体和脂质纳米颗粒在网状内皮系统（RES）过滤器官中积累的固有趋势，主要是肝脏，也包括淋巴结、肾脏和脾脏。siRNA 的靶向递送则利用了在肝细胞表面高密度表达的去唾液酸糖蛋白受体（ASGPR）。因此，RNA 干扰疗法的一个关键重点是开发用于治疗肝脏疾病的 siRNA 药物。以下，我们将讨论一些用于开发肝病治疗 siRNA 药物的典型递送系统（图 2）。

Fig. 2 Schematic illustration showing receptor-mediated and passive delivery of siRNAs drugs to the liver. Conjugation of the triantennary GalNAc ligand to the passenger strand of an siRNA enhances delivery to the liver. The siRNA molecule enters the hepatocyte by receptor-mediated endocytosis, where GalNAc binds to the asialoglycoprotein receptor (ASGPR). Upon internalization, the siRNA molecule escapes from the endosome and the guide strand (antisense) is incorporated into the RNA-induced silencing complex (RISC). Lipid nanoparticles (LNPs) facilitate siRNA delivery into the cytoplasm of target cells (hepatocytes) following intravenous (i.v.) administration. Passive delivery benefits from the propensity of LNPs to accumulate in filtering organs of the reticuloendothelial system, including the liver. The LNP encapsulated-siRNA system exploits different lipid components (e.g., PEG-lipid, DSPC, ionizable cationic lipid, and cholesterol) to increase uptake and thereby gene silencing through the RNAi pathway示意图显示了受体介导和被动递送 siRNA 药物至肝脏的过程。三天线 GalNAc 配体与 siRNA 的过客链结合可增强向肝脏的递送。siRNA 分子通过受体介导的内吞作用进入肝细胞，其中 GalNAc 与去唾液酸糖蛋白受体 (ASGPR) 结合。内化后，siRNA 分子从内体中逃逸，引导链 (反义) 被整合到 RNA 诱导的沉默复合物 (RISC) 中。脂质纳米颗粒 (LNP) 有助于在静脉 (i.v.) 给药后将 siRNA 递送到靶细胞 (肝细胞) 的细胞质中。被动递送受益于 LNP 倾向于在网状内皮系统的过滤器官（包括肝脏）中积累。LNP 封装的 siRNA 系统利用不同的脂质成分（例如 PEG 脂质、DSPC、可电离阳离子脂质和胆固醇）来增加吸收，从而通过 RNAi 途径实现基因沉默

3.1 Lipid Nanoparticles 脂质纳米粒

Lipid nanoparticles (LPNs) have been successfully deployed to deliver siRNA drugs to the liver. Many pharmaceutical companies have developed LNP-based delivery systems typically consisting of 20–200 nm regular LNP structures with a siRNA encapsulation efficiency of over 90%. Using helper lipids such as distearoylphosphatidylcholine (DSPC), DMG-PEG2000, and cholesterol, robust gene silencing in the liver is achievable via the enhanced permeability and retention effect, and penetrate leaky or more permeable vasculature. Consequently, most of the pharmaceutical companies have focused on developing siRNA drugs targeting hepatic genes (Table 1).

脂质纳米颗粒（LNPs）已成功应用于将 siRNA 药物递送至肝脏。许多制药公司开发了基于 LNP 的递送系统，通常由 20-200 纳米的规则 LNP 结构组成，siRNA 包封效率超过 90%。通过使用如二硬脂酰磷脂酰胆碱（DSPC）、DMG-PEG2000 和胆固醇等辅助脂质，通过增强的渗透性和滞留效应，可以在肝脏中实现强有力的基因沉默，并穿透渗漏或更具渗透性的血管。因此，大多数制药公司都专注于开发针对肝脏基因的 siRNA 药物（表 1）。

Table 1 siRNA drugs in clinical development for treatment of liver diseases

LNPs are divided into two major groups based on the different properties of the key lipids used: (1) cationic LNPs and (2) ionizable cationic LNPs. The positively charged cationic LNPs interact by electrostatic interactions with the polyanionic siRNA, leading to formation of siRNA lipocomplexes with enhanced cellular uptake and endosomal escape. Ionizable cationic LNPs were developed to achieve a system, where efficient encapsulation of negatively charged siRNAs could be accomplished at low pH, but which also exhibited a relatively uncharged surface at pH 7.4. Interestingly, apolipoprotein E (ApoE) was reported to function as a targeting ligand in an LDL-receptor-dependent manner for ionizable cationic LNPs , thereby enhancing hepatic delivery (Fig. 2) .

脂质纳米颗粒（LNPs）根据所使用的关键脂质的不同性质分为两大类：（1）阳离子 LNPs 和（2）可离子化阳离子 LNPs。带正电的阳离子 LNPs 通过静电相互作用与聚阴离子 siRNA 相互作用，形成具有增强细胞摄取和内吞体逃逸能力的 siRNA 脂质复合物。可离子化阳离子 LNPs 的开发旨在实现一种系统，在低 pH 值下能够有效封装带负电的 siRNA，但在 pH 7.4 时表现出相对无电荷的表面。有趣的是，有报道指出载脂蛋白 E（ApoE）作为靶向配体通过低密度脂蛋白受体（LDL 受体）依赖的方式作用于可离子化阳离子 LNPs，从而增强肝脏递送（图 2）

3.2 Dynamic Polyconjugates 动态多聚缀合物

The first dynamic polyconjugates (DPC) for targeted in vivo delivery of siRNA triggers to the liver were developed by Rozema and coworkers. DPCs comprise four important parts: a delivery platform that can reversibly conjugate a polymer (or polypeptide) with the siRNA, a hepatocyte targeting ligand (N-acetylgalactosamine (GalNAc)), a shielding agent (PEG fragment) to elongate blood retention, and a hydrophobic lipid for enhanced interaction with the cell membrane.

Rozsema 及其同事开发了用于 siRNA 触发物靶向肝脏内递送的首个动态聚合物缀合物 (DPC)。DPC 由四部分组成：一是递送平台，能够可逆地将聚合物 (或多肽) 与 siRNA 连接起来；二是肝细胞靶向配体 N-乙酰氨基葡萄糖 (GalNAc)，负责特异识别肝细胞；三是聚乙二醇 (PEG) 片段，作为屏蔽剂延长药物在血液中的停留时间；四是疏水脂质，能增强药物与细胞膜的相互作用，促进细胞摄取。

The second generation of Dynamic PolyConjugate technology (DPC 2.0) developed by Arrowhead Pharmaceuticals relies on cholesterol modifications to facilitate membrane penetration. In this system, a cholesterol-modified siRNA (chol-siRNA) is co-injected with a GalNAc-masked polypeptide due to the ease of manufacturing of the two components separately instead of a single molecule. However, frequent injections with simultaneous delivery of the siRNA trigger and the helper molecules to the target cells involve separate and sequential entry of the components into the blood. Moreover, safety issues, especially immunogenicity, might be a concern due to recognition by TLR 3/7/8 and/or PKR.

Arrowhead Pharmaceuticals 公司研发的第二代动态聚合物缀合物技术 (DPC 2.0) 利用胆固醇修饰 siRNA (chol-siRNA) 来增强细胞膜的穿透力，简化了生产工艺。该系统采用单独注射胆固醇修饰的 siRNA 和 GalNAc 修饰的多肽，分别靶向 siRNA 和肝细胞，但这种方式需要频繁注射，siRNA 触发物和辅助分子需要分两次进入血液，过程复杂。另外，由于这种方法可能会被 TLR 3/7/8 和/或 PKR 等免疫识别系统识别，导致免疫原性，因此安全性也存在潜在的隐患。

3.3 GalNAc-siRNA Conjugates GalNAc-siRNA 缀合物

A major breakthrough in RNAi medicine was the discovery of targeted delivery of siRNA drugs to hepatocytes via ASGPR in the liver. An oligosaccharide termed GalNAc has high affinity toward the ASGPR receptor allowing selective delivery of GalNAc-siRNA conjugates to hepatocytes. The use of GalNAc-conjugated siRNAs for liver-targeted delivery is based on binding of the GalNAc moiety to the ASGPR on hepatocytes, which contain ∼500,000 receptors per cell, resulting in rapid endocytosis. ASGPR was discovered in 1965 by Ashwell and Morell, and shown to be predominantly expressed in hepatocytes, but not in other liver cells, such as Kupffer cells. Furthermore, GalNAc, which is an amino sugar derivative of galactose, was shown to possess high affinity to ASGPR. Binding of GalNAc to ASGPR occurs at the sinusoidal surface of the hepatocyte, initially on diffuse monomeric ASGPR receptors, followed by rapid local aggregation of the GalNAc-ASGPR complexes, and larger scale aggregation in clathrin-coated pits, resulting in endocytosis. Early studies noted that the half-life of ASGPR was much longer than the bound asialoglycoproteins. Subsequent studies showed that acidification during endosomal maturation leads to dissociation of the GalNAc ligand from ASGPR followed by degradation of GalNAc in the lysosome and rapid recycling of ASGPR to the cell surface. Several studies have demonstrated that the GalNAc moiety is able to facilitate efficient cellular uptake of GalNAc-siRNA conjugates by hepatocytes via ASGPR-mediated and clathrin-involved endocytosis, resulting in effective gene silencing with minimal adverse effects (Fig. 2) .

RNAi 药物领域的一大进步是利用 ASGPR 靶向将 siRNA 药物递送至肝细胞。一种叫做 GalNAc 的寡糖分子可以特异性地结合肝细胞表面的 ASGPR 受体，从而让携带 GalNAc 的 siRNA 复合物 (GalNAc-siRNA) 优先进入肝细胞。GalNAc 辅助的肝脏靶向递送方法依赖于 GalNAc 与肝细胞上丰富的 ASGPR 受体 (每个肝细胞约有 50 万个) 的结合，从而促进 siRNA 的快速细胞内吞作用。ASGPR 受体于 1965 年由 Ashwell 和 Morell 发现，主要表达于肝细胞，在其他肝脏细胞 (例如 Kupffer 细胞) 中几乎没有表达。GalNAc 是半乳糖的一种衍生物，也被称为氨基糖，研究表明它与 ASGPR 受体具有很高的亲和力。GalNAc 与 ASGPR 的结合发生在肝细胞面向血液的窦状面上，最初结合分散的单体 ASGPR 受体，然后迅速聚集形成局部复合物，并进一步聚集于包含网格蛋白的凹陷处，最终通过内吞作用进入细胞。早期研究发现，结合了 asialoglycoprotein 的 ASGPR 受体比游离受体具有更长的半衰期。后续研究表明，内吞体成熟过程中酸化会导致 GalNAc 配体从 ASGPR 上解离，GalNAc 在溶酶体中降解，而 ASGPR 则被迅速循环回细胞表面。有多项研究表明，GalNAc 可以促进 GalNAc-siRNA 复合物通过 ASGPR 介导的网格蛋白依赖性内吞作用有效进入肝细胞，从而实现基因沉默并具有良好的安全性 (见图 2)。