siRNA 设计和 GalNAc 增强的肝靶向递送

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Abstract 摘要

Small interfering RNA (siRNA) is a clinically approved therapeutic modality, which has attracted widespread attention not only from basic research but also from pharmaceutical industry. As siRNA can theoretically modulate any disease-related gene’s expression, plenty of siRNA therapeutic pipelines have been established by tens of biotechnology companies. The drug performance of siRNA heavily depends on the sequence, the chemical modification, and the delivery of siRNA. Here, we describe the rational design protocol of siRNA, and provide some modification patterns that can enhance siRNA’s stability and reduce its off-target effect. Also, the delivery method based on N-acetylgalactosamine (GalNAc)-siRNA conjugate that is widely employed to develop therapeutic regimens for liver-related diseases is also recapitulated.

小干扰 RNA（siRNA）是一种已经获得临床批准的治疗方式，不仅在基础研究中广受关注，也吸引了制药行业的广泛兴趣。由于 siRNA 理论上能够调控任何与疾病相关的基因表达，许多生物技术公司已经建立了大量的 siRNA 治疗管线。siRNA 的药物性能在很大程度上取决于其序列、化学修饰以及递送方式。本文描述了 siRNA 的合理设计方案，并提供了一些能够增强 siRNA 稳定性和减少脱靶效应的修饰模式。此外，本文还概述了基于N-乙酰半乳糖胺（GalNAc）-siRNA 偶联物的递送方法，这种方法被广泛用于开发治疗肝脏相关疾病的方案。

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1 Introduction 介绍

RNA interference (RNAi) based on small interfering RNA (siRNA) is not only widely used to regulate gene expression in basic research areas but also represents a clinically approved therapeutic platform that can be investigated for treating diverse life-threaten diseases, such as metabolic disorders, genetic or orphan diseases, infectious diseases, and cancers. Mature siRNA can be generated by both chemical synthesis and processing from short hairpin RNA (shRNA) or long double-stranded RNA with Dicer (Fig. 1). Once the siRNA duplex is introduced into the cytoplasm of cells, it will be incorporated into the RNA-induced silencing complex (RISC). Then the sense (passenger) strand of siRNA is released from RISC, and the antisense (guide) strand of siRNA will recognize the target complementary mRNA in a complete match manner. Finally, the target mRNA will be cleaved by Argonaut protein in RISC. The cleaved mRNA fragments are further degraded by cellular 5′ and 3′ exonucleases (Fig. .1). In contrast to the recognition mode of “complete match with target mRNA,” microRNA (miRNA), another representative member of RNAi family, modulates gene’s expression in a “partial match” or “seed match” manner. In which way, only the seed region of the miRNA (position of 2–8 from 5′ end) needs to match with the targeted gene. Nucleic acid therapeutics based on both siRNA and miRNA are investigated for treating diseases currently, although siRNA is more potent and popular for drug development. siRNA and miRNA are usually considered to be generated exogenously and endogenously, respectively. Hence, the sequences of siRNA are artificially designed, while the sequences of miRNA are selected from naturally existing miRNA library. Actually, the activity of siRNA molecules is highly dependent on the sequence since not all double-stranded RNA molecules that completely matched with target mRNA can trigger efficient gene silence. Many factors contribute to the overall activity of siRNA, such as G and C content, the stability of the 3′ terminus of the sense strand (it will determine which strand of the duplex will be loaded into RISC), the nucleotide (base) type of the 5′ and 3′ end of antisense strand. Taken together, rational design of siRNA is essentially required.

基于小干扰 RNA (siRNA) 的 RNA 干扰 (RNAi) 技术不仅在基础研究领域广泛用于调控基因表达，而且还是一种经过临床验证的治疗平台，可用于治疗多种危及生命的疾病，例如代谢紊乱、遗传病或罕见病、传染病和癌症。成熟的 siRNA 可以通过化学合成以及由短发夹 RNA（shRNA）或长双链 RNA 经 Dicer 处理生成（见图 1）。siRNA 双链进入细胞质后，会被整合到 RNA 诱导沉默复合物 (RISC) 中。然后，siRNA 的正义（乘客）链将从 RISC 中释放，而反义（引导）链将以完全匹配的方式识别目标互补 mRNA。最终，目标 mRNA 将被 RISC 中的 Argonaut 蛋白切割，并被细胞的 5′和 3′外切酶进一步降解（见图 1）。与“完全匹配目标 mRNA”的识别模式不同，微小 RNA（miRNA）是 RNAi 家族的另一代表成员，它通过“部分匹配”或“种子匹配”的方式调控基因表达。在这种方式中，仅需要 miRNA 的种子区域（位于 5′末端的 2-8 位）与目标基因匹配。尽管 siRNA 在药物开发中更有效且更受欢迎，但目前基于 siRNA 和 miRNA 的核酸疗法都在用于治疗疾病的研究中。通常认为，siRNA 和 miRNA 分别是外源性和内源性生成的。因此，siRNA 的序列可以是人工设计的，而 miRNA 的序列是从自然存在的 miRNA 库中选择的。实际上，siRNA 分子的活性高度依赖于其序列，因为并非所有完全匹配目标 mRNA 的双链 RNA 分子都能引发有效的基因沉默。许多因素影响 siRNA 的整体活性，例如 G 和 C 的含量、正义链 3′末端的稳定性（这将决定双链中的哪条链被加载到 RISC 中）、以及反义链 5′和 3′末端的核苷酸（碱基）类型。综上所述，siRNA 的合理设计是必不可少的。

Fig. 1 Working mechanism of siRNA. Synthetic siRNA or Dicer-processed siRNA is incorporated into RNA-induced silencing complex (RISC). The sense (passenger) strand is removed and the antisense (guide) strand will recognize the full complementary mRNA. Then Argonaut protein in RISC cleaves the target mRNA, and the protein translation is blocked.siRNA 的工作机制。合成的 siRNA 或 Dicer 处理的 siRNA 被整合到 RNA 诱导沉默复合物 (RISC) 中。正义链 (乘客链) 被移除，反义链 (引导链) 将识别完整的互补 mRNA。然后 RISC 中的 Argonaut 蛋白切割目标 mRNA，并阻断蛋白质翻译

In addition, the issues of metabolic stability, specificity, and off-target effect are the key determinants of drug performances in vivo. Both the sequence and chemical modification may contribute to these issues. For instance, siRNA with UA/UA and/or CA/UG paring in the sequence is more sensitive to RNase attacking, and modifications in these bases can enhance its resistance to enzyme’s degradation. All the mismatch type (e.g., A-G, A-C, A-A, and G-U, G-A, G-G, etc.), mismatch position (from 5′-end to 3′-end of siRNA, and on coding region or untranslated region of mRNA), and mismatch number (one or two or more mismatches) could affect the activity and specificity of siRNA. Chemical modification may also change the strand selection of RISC, thus contributing to enhancing the specificity of siRNA. The off-target effect of siRNA may occur through several different mechanisms, including: (1) passenger strand of siRNA binds to undesired target mRNA; (2) the seed regions of passenger or guide strand of siRNA recognize certain mRNA sequence, especially at the 3′-untranslated region (UTR) region, and silence gene’s expression in a “miRNA-like” way; (3) passenger or guide strand of siRNA bind to toll-like receptors (TLRs, e.g., TLR3), stimulate immune response, and affect downstream gene’s expression. Various chemical modification strategies have been investigated to reduce the off-target effect of siRNA. For example, 2′-O-methyl (OMe) modification at position 14 of the siRNA passenger strand can be used to abolish the activity of the passenger strand. Unlocked nucleic acid (UNA) and glycol nucleic acid (GNA) at position 7 of the siRNA guide strand dramatically reduced the “miRNA-like” off-target effect. Actually, recognition of toll-like receptors and stimulation of immune response directly caused the termination of Phase III study of Bevasiranib, the first-ever clinically investigated siRNA therapeutic, in 2009. Disturbance of the expression of undesired target genes resulted from the seed regions of both strands of siRNA may also trigger significant hepatotoxicity in animals . Therefore, rational chemical modifications are extremely important for drug development.

此外，代谢稳定性、特异性和脱靶效应是体内药物性能的关键因素。siRNA 的序列和化学修饰都可能影响这些因素。例如，含有 UA/UA 和/或 CA/UG 配对的 siRNA 序列更易受到 RNase 攻击，对这些碱基进行修饰可以提高其抗酶降解能力。错配类型（如 A-G、A-C、A-A 和 G-U、G-A、G-G 等）、错配位置（从 siRNA 的 5′端到 3′端，或在 mRNA 的编码区或非翻译区）以及错配数量（一个、两个或更多）都可能影响 siRNA 的活性和特异性。化学修饰还可能改变 RISC 的链选择，从而提高 siRNA 的特异性。siRNA 的脱靶效应可能通过几种不同的机制发生，包括：（1）siRNA 的乘客链结合到不希望的目标 mRNA；（2）siRNA 的乘客链或引导链的种子区域识别某些 mRNA 序列，尤其是在 3′-非翻译区（UTR）区域，以类似 miRNA 的方式沉默基因表达；（3）siRNA 的乘客链或引导链结合到 Toll 样受体（TLRs，如 TLR3），刺激免疫反应，并影响下游基因表达。为了减少 siRNA 的脱靶效应，已经研究了各种化学修饰策略。例如，siRNA 乘客链第 14 位的 2′-O-甲基（OMe）修饰可以消除乘客链的活性。siRNA 引导链第 7 位的解锁核酸（UNA）和乙二醇核酸（GNA）显著减少了类似 miRNA 的脱靶效应。实际上，Toll 样受体的识别和免疫反应的刺激直接导致了 2009 年首个临床研究的 siRNA 疗法 Bevasiranib 的三期研究终止。siRNA 双链种子区域对不希望的目标基因表达的干扰也可能在动物中引发显著的肝毒性。因此，合理的化学修饰对药物开发极为重要。

Moreover, insufficient delivery of siRNA to the targeted tissue and cell significantly hampered siRNA therapeutic development for a long period. Because the cell membrane is primarily made up of zwitterionic and negatively charged phospholipids, and shows a negative potential (−40 to −80 mV) across the cell membrane, it is difficult for the negatively charged siRNA to cross cell membrane by itself. Hence, various delivery platforms, including chemically synthesized materials (such as liposomes, polymers, dendrimers, and inorganic nanoparticles), biological agents (such as peptides, antibodies, aptamers , CpG, 3-Way Junction, and exosomes), and physical approaches (such as electroporation, microneedle poking, hydrodynamic injection, and microfluidic extrusion), have been developed to deliver siRNA to desired tissues and cells (Fig. 2). Among them, N-Acetylgalactosamine (GalNAc)-siRNA conjugate exhibits excellent hepatocyte-targeted delivery efficiency and safety profile in vivo (Fig. 2d, e, g) . GalNAc can be recognized by asialoglycoprotein receptor (ASGPR) that is conserved across species and highly expressed by hepatocytes, and can mediate clathrin-involved endocytosis. It takes only 15 min for GalNAc from binding to ASGPR to being internalized into the cell and re-displaying on cell surface. After thoroughly exploring the effects of valence of oligosaccharides, distance among the targeting moieties, and size of the particle on the binding performance, researchers reached a consensus that triantennary GalNAc with a mutual distance of ~20 Å exhibits the highest affinity with ASGPR. By conjugating three GalNAc moieties to the siRNA with proper linker chemistry, siRNA can be efficiently delivered to hepatocytes via subcutaneous injection. Benefiting from the enhanced stabilization modification, GalNAc-siRNA conjugate can remain in the circulation system and cytoplasm for more than 100 days, thus permitting a long-term gene silencing and therapeutic effect in human beings. The reduction profile could be well maintained for more than 6 months or even 1 year, after a single dose of GalNAc-siRNA, as demonstrated by abundant clinical data.

此外，siRNA 在目标组织和细胞中的递送不足长期以来严重阻碍了 siRNA 疗法的发展。由于细胞膜主要由带双电荷和负电荷的磷脂组成，并且细胞膜显示负电位（-40 到-80 mV），导致带负电荷的 siRNA 难以自行穿过细胞膜。因此，开发了多种递送平台，包括化学合成材料（如脂质体、聚合物、树枝状分子和无机纳米颗粒）、生物制剂（如肽、抗体、适配体、CpG、三向结点和外泌体）以及物理方法（如电穿孔、微针刺、液体动力注射和微流体挤出）来将 siRNA 递送到目标组织和细胞（图 2）。其中，N-乙酰半乳糖胺（GalNAc）-siRNA 偶联物在体内表现出优异的肝细胞靶向递送效率和安全性（图 2d、e、g）。GalNAc 可以被去唾液酸糖蛋白受体（ASGPR）识别，这种受体在肝细胞表面高表达，并且在不同物种间保守。GalNAc 与 ASGPR 结合后，可以介导网格蛋白 (clathrin) 依赖性内吞作用，将 siRNA 送入肝细胞。从 GalNAc 与 ASGPR 结合到被细胞内化并重新返回到细胞表面仅需 15 分钟。经过深入研究寡糖的价数、靶向基团之间的距离和颗粒大小对结合性能的影响，研究人员一致认为，三天线 GalNAc 相距约 20 Å时与 ASGPR 具有最高亲和力。通过合适的化学连接子将三个 GalNAc 基团偶联到 siRNA 上，可以通过皮下注射有效地将 siRNA 递送到肝细胞。得益于增强的稳定性修饰，GalNAc-siRNA 偶联物可以在循环系统和细胞质中停留超过 100 天，从而在人体内实现长期的基因沉默和治疗效果。丰富的临床数据表明，单剂量的 GalNAc-siRNA 的减少效果可以保持超过 6 个月甚至 1 年。

Fig. 2

Schemes and structures of representative siRNA delivery platforms. Schemes of (a) lipid nanoparticles, (b) DPC2.0™ or EX-1™, (c) TRiM™, (d) GalNAc-siRNA conjugates, (e) GalXC™ and (f) LODER™, respectively. (g) Chemical structures of the key component(s) of the discussed delivery platforms, including Dlin-DMA, Dlin-MC3-DMA, C12-200, cKK-E12, MLP-based DPC2.0, PLGA-based LODER, and GalNAc-siRNA conjugates. DPC, Dynamic PolyConjugates; TRiM, Targeted RNAi Molecule; LODER, LOcal Drug EluteR; PLGA, poly(lactic-co-glycolic) acid; MLP, membrane-lytic peptide, with the amino sequence NH-LIGAILKVLATGLPTLISWIKNKRKQ-COOH; CDM, carboxylated dimethyl maleic acid; PEG, polyethylene glycol; NAG, N-acetylgalactosamine (GalNAc)代表性 siRNA 递送平台的方案和结构。分别为 (a) 脂质纳米粒子、(b) DPC2.0™ 或 EX-1™、(c) TRiM™、(d) GalNAc-siRNA 结合物、(e) GalXC™ 和 (f) LODER™ 的方案。(g) 所讨论的递送平台的关键组件的化学结构，包括 Dlin-DMA、Dlin-MC3-DMA、C12-200、cKK-E12、基于 MLP 的 DPC2.0、基于 PLGA 的 LODER 和 GalNAc-siRNA 结合物。DPC，动态多聚结合物；TRiM，靶向 RNAi 分子；LODER，局部药物洗脱剂；PLGA，聚（乳酸-乙醇酸）共聚物；MLP，膜溶解肽，氨基酸序列为 NH-LIGAILKVLATGLPTLISWIKNKRKQ-COOH；CDM，羧化二甲基马来酸；PEG，聚乙二醇；NAG，N-乙酰半乳糖胺 (GalNAc)

In this chapter, we will introduce the siRNA design method with online-accessible tools, discuss chemical modification strategies, and recapitulate the experimental design process of GalNAc-mediated liver-targeted siRNA delivery. It may provide a brief roadmap for researches from both academic institute and pharmacy industry.

本章将介绍 siRNA 设计方法及在线工具，讨论化学修饰策略，并重述 GalNAc 介导的肝靶向 siRNA 递送的实验设计过程。它可为学术机构和制药行业的研究提供简要的路线图。

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2 Materials 材料

2.1 Software, Database, and Equipment 软件、数据库与设备

1. DNAMAN 8, and/or online alignment tool (http://molbiol-tools.ca/Alignments.htm).

   DNAMAN 8，和/或在线比对工具（http://molbiol-tools.ca/Alignments.htm)

2. “LALIGN,” use following website: https://embnet.vital-it.ch/software/LALIGN_form.html(see Note 1). LALIGN”，使用以下网站：https://embnet.vital-it.ch/software/LALIGN_form.html（见 注释 1）

3. Online siRNA design tools (see Note 2).

   在线 siRNA 设计工具（见 注释 2）

4. NCBI database, including the sub-databases of “Gene” (https://www.ncbi.nlm.nih.gov/gene/) and “Nucleotide” (https://www.ncbi.nlm.nih.gov/nucleotide/).

   NCBI 数据库，包括“基因”子数据库（https://www.ncbi.nlm.nih.gov/gene/）和“核苷酸”子数据库（https://www.ncbi.nlm.nih.gov/nucleotide/）

5. NCBI tool of Blastn: https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome

   Blastn 的 NCBI 工具：https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome

6. Extended Nucleic Acid Sequence Massager: https://www.cmbn.no/tonjum/seqMassager-saf.htm

   扩展核酸序列“按摩器”：https://www.cmbn.no/tonjum/seqMassager-saf.htm

7. A personal computer with Microsoft office and access to the World Wide Web.

   一台装有 Microsoft Office 并能访问万维网的个人电脑

8. Microvolume Spectrophotometers.

   微量分光光度计

9. Real-time PCR machine.

   实时荧光定量 PCR 仪

10. Multi-Detection Microplate Reader.

    多重检测酶标仪

11. Luminex 100 system.

    Luminex 100 系统

2.2 Reagents 试剂

1. siRNA, including GalNAc-siRNA conjugate.

   siRNA，包括 GalNAc-siRNA 偶联物。

2. Lipofectamine 2000 (Thermo Fisher Scientific, USA).

3. DMEM medium: Dulbecco’s Modified Eagle’s Medium (DMEM) without supplement.

   DMEM 培养基：杜氏改良伊格尔培养基（DMEM），无添加。

4. HEK293A medium: DMEM medium supplemented with 1% penicillin-streptomycin, and 10% fetal bovine serum. HEK293A 培养基：添加 1%青霉素-链霉素和 10%胎牛血清的 DMEM 培养基。

5. Perfusion solution 1: Hank’s Balanced Salt Solution (HBSS) supplemented with 0.5 mM EDTA, pH = 8.

   灌注液 1：添加 0.5 mM EDTA 的 Hank 平衡盐溶液（HBSS），pH=8。

6. Perfusion solution 2: DMEM medium supplemented with 0.8 mg/mL collagenase type I.

   灌注液 2：添加 0.8 mg/mL I 型胶原酶的 DMEM 培养基

7. Primary mouse hepatocyte medium: DMEM medium supplemented with 10% fetal bovine serum, but without adding antibiotics.

   小鼠原代肝细胞培养基：添加 10%胎牛血清的 DMEM 培养基，但不添加抗生素

8. Opti-MEM medium (Thermo Fisher Scientific, USA) with no supplement.

   Opti-MEM 培养基（美国赛默飞世尔科技公司），无添加

9. Trypsin.

   胰蛋白酶

10. Trizol.

11. Cell culture plates.

    细胞培养板

12. Serological pipettes.

    血清移液器

13. Reverse transcription kit.

    逆转录试剂盒

14. Real-time PCR kit (UltraSYBR Mixture).

    实时 PCR 试剂盒（UltraSYBR Mixture）

15. RNAlater.

16. Dual luciferase reporter system: the primary plasmid vectors of siQuant and psiCheck.

    双荧光素酶报告系统：siQuant 和 psiCheck 的初级质粒载体

17. Luciferase detection kit.

    荧光素酶检测试剂盒

18. Plasmid isolation kit.

    质粒分离试剂盒

19. Anesthetic: 4% Chloral hydrate solution (w/v).

    麻醉剂：4% 水合氯醛溶液（w/v）

2.3 Cells and Animals 细胞与动物

1. HEK293A originally derived from human embryonic kidney cells . Culture HEK293A cells in HEK293A medium. HEK293A 最初来源于人胚胎肾细胞。在 HEK293A 培养基中培养 HEK293A 细胞。

2. Male C57BL/6 mice (age 6–8 weeks).

   雄性 C57BL/6 小鼠（6-8 周龄）

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3 Methods 方法

RNAi therapeutic companies may establish their own siRNA design algorithm and develop their confidential software. Here we just introduce how to design siRNAs with online accessible tools. Firstly, design siRNAs with online tools that are accessible for public (Table 1). Then evaluate the activity, stability, and off-target effect in vitro. After narrowing down the number of siRNA candidates, apply chemical modifications to the siRNAs, and evaluate the performance of siRNA again. Then conjugate three or four (typically three) GalNAc moieties to the siRNA with proper linker chemistry. Finally, inject the GalNAc-siRNA conjugate into the mice subcutaneously and assess the in vivo gene silence and toxicity.

RNAi 治疗公司可能会建立自己的 siRNA 设计算法并开发保密软件。在这里，我们仅介绍如何使用在线可访问工具设计 siRNA。首先，使用对公众开放的在线工具设计 siRNA（表 1）。然后在体外评估其活性、稳定性和脱靶效应。在缩小 siRNA 候选范围后，对 siRNA 进行化学修饰，并再次评估其性能。然后使用适当的连接化学将三个或四个（通常为三个）GalNAc 基团偶联到 siRNA 上。最后，将 GalNAc-siRNA 偶联物皮下注射到小鼠体内，并评估其体内基因沉默效果和毒性。

Table 1 Selected online siRNA design tools and their features 可选择的在线 siRNA 设计工具及其功能

Ref.(s) reference(s), NA not available

3.1 Primary Mouse Hepatocyte Isolation 小鼠原代肝细胞分离

Isolate primary mouse hepatocytes from C57BL/6 mice according to the protocol reported previously and described as following.

根据之前报道的方案从 C57BL/6 小鼠中分离小鼠原代肝细胞，如下所述。

1. Anesthetize the animals by intraperitoneally injecting a saline solution containing 80 mg/kg Ketamine and 5 mg/kg Xylazine.

   通过腹膜内注射含有 80 mg/kg 氯胺酮和 5 mg/kg 赛拉嗪的盐水溶液对动物进行麻醉。

2. Prepare the animal for surgery (clean/sanitize).

   准备动物进行手术（清洁/消毒）

3. Immobilize the animal with tape. Dissect the abdominal cavity, move the gastric system to the right, and expose liver.

   用胶带固定动物。解剖腹腔，将胃系统移到右侧，暴露肝脏

4. To start perfusion, insert the catheter into the vena cava through the right atrium. The flow rate should be 5 mL/min.

   将导管通过右心房插入下腔静脉，流速设为 5 mL/min，开始灌注。

5. Create back pressure by blocking the visceral vena cava with a finger when the blood is drained out. Perfuse for 5–7 min.

   当血液流尽时，用手指阻塞内脏下腔静脉以产生背压。灌注 5-7 分钟。

6. Position a lamp a few inches above the animal to maintain the cavity temperature of 37 °C throughout the procedure.

   将灯放置在动物上方几英寸处，以在整个过程中保持腔内温度为 37 °C

7. Switch the tubing from perfusion solution 1 to perfusion solution 2, and continue perfusion for 7–8 min (flow rate 5 mL/min).

   将管从灌注溶液 1 切换到灌注溶液 2，并继续灌注 7-8 分钟（流速 5 mL/min）

8. Collect the liver into perfusion solution 2, and cut the liver sac to release the hepatocytes.

   将肝脏收集到灌注溶液 2 中，切开肝囊以释放肝细胞

9. Add DMEM medium and pass the liver suspension through a cell strainer.

   加入 DMEM 培养基，并通过细胞滤器过滤肝细胞悬液

10. Spin cells at 800 rpm for 5 min. Wash the pellet twice with DMEM medium.

    将细胞以 800 rpm 离心 5 分钟。用 DMEM 培养基清洗沉淀物两次

11. Count cells with a hemocytometer and culture in primary mouse hepatocyte medium.

    使用血细胞计数板计数细胞，并用原代小鼠肝细胞培养基进行培养