siRNA 设计和 GalNAc 增强的肝靶向递送（二）

3.2 siRNA Design and Screening siRNA 的设计与筛选

3.2.1 mRNA (cDNA) Selection mRNA（cDNA）选择

1. Identify the target gene that we intend to silence.

   确定需要沉默的靶基因

2. Determine the accession number(s) of the mRNA sequence(s) of the target gene, and/or download the mRNA (cDNA) sequence(s) in a FASTA format.

   确定靶基因 mRNA 序列的登录号，和/或以 FASTA 格式下载 mRNA (cDNA) 序列。

3. Analyze mRNA (cDNA) sequence(s) and determine the sequence(s) that will be used to design siRNA according to the workflow shown in Fig. 3(see Note 3).

   根据图 3 所示的工作流程分析 mRNA (cDNA) 序列并确定将用于设计 siRNA 的序列（参见 注释 3）。

Fig. 3 Workflow of mRNA (cDNA) sequence selection mRNA(cDNA)序列选择流程图

3.2.2 Generation of siRNA Sequence List 生成 siRNA 序列列表

1. Submit mRNA (cDNA) sequence of interest, or the accession number of the target gene, to the online siRNA design tools. The available online tools and related information are summarized in Tables 1 and 2.

   将目标基因的 mRNA (cDNA) 序列或基因序列编号提交到在线 siRNA 设计工具。常用的在线工具及其相关信息总结于表格 1 和 2 中

2. Establish a series of siRNA design algorithms by analyzing hundreds and thousands of siRNAs. The silencing activity, selectivity, off-target effect, as well as the mRNA secondary structure, alternative splicing, or the motif sequences may potentially trigger immune response when they were taken into accounts. One or more design tools can be used when submitting mRNA (cDNA) sequence.

   （siRNA 设计工具）通过分析数百乃至数千条 siRNA 序列，建立一系列设计算法。沉默活性、选择性、脱靶效应，以及 mRNA 二级结构、可变剪接或可能引发免疫反应的基序序列都需要考虑在内。在提交 mRNA (cDNA) 序列时，可以选择一个或多个设计工具进行 siRNA 设计。

3. siRNA list exported by the selected tool is automatically generated. Typically, 3–5 siRNA candidates that rank on the top of the list can be selected for further evaluation. High-ranking siRNAs means their scores are relatively high because the features of these sequences match better with the algorithm than other sequences (see Notes 4 and 5).

   选定的在线工具会自动生成 siRNA 列表。一般会选择列表中排名靠前的 3-5 个 siRNA 序列进行后续实验验证。排名越靠前的 siRNA 得分通常越高，这表示这些序列的特征更符合设计算法的要求 (参见注释 4 和 5)。

4. Synthesize the siRNAs without chemical modification (see Note 6).

   合成没有化学修饰的 siRNA（参见注释 6)

Table 2 Representative siRNA designing algorithms and their attributes

Ref. reference, NA not available

3.2.3 Activity Evaluation 活性评估

1. Evaluate the activities of siRNA candidates via either luciferase reporter system or reverser transcription quantitative PCR (RT-qPCR), or both (see Note 7).

   通过荧光素酶报告系统或逆转录定量 PCR（RT-qPCR）或两者结合评估 siRNA 候选物的活性（参见注释 7）

2. For luciferase reporter assay, insert the target sequences into the siQuant reporter vector according to standard molecular clone procedures (see Note 8).

   对于荧光素酶报告实验，根据标准分子克隆程序将目标序列插入 siQuant 报告载体中（参见注释 8）

3. Transfect siRNAs into the Hek293A cells with Lipofectamine 2000 at various transfection concentrations (at least three concentrations, e.g., 10 nM, 1 nM, and 0.1 nM; or more than seven concentration gradients that enable building a concentration-activity curve and calculating the IC₅₀), together with the reporter vectors (see Note 9).

   将 siRNA 与 Lipofectamine 2000 一起转染入 Hek293A 细胞，并使用不同的转染浓度（至少三个浓度，例如 10 nM、1 nM 和 0.1 nM；或超过七个浓度梯度以构建浓度-活性曲线并计算 IC₅₀），同时转染报告载体（参见注释 9）

4. 24 h later, harvest the cells and prepare cell lysates. Then, transfer 10μL cell lysates to the reader plate, and add the substrates of Firefly and Renilla luciferases into the samples sequentially. Detect the luminance signal with Multi-Detection Microplate Reader.

   24 小时后，收获细胞并制备细胞裂解液。然后，将 10μL 细胞裂解液转移到读板中，依次向样品中添加萤火虫和海肾荧光素酶的底物。使用多检测微孔板读板仪检测发光信号

5. For RT-qPCR assay, transfect the siRNAs into the cells that endogenously express the target gene at desired concentrations (typically no less than three concentrations, e.g., 100, 20, and 5 nM).

   对于 RT-qPCR 检测，将 siRNA 以期望的浓度（通常不少于三个浓度，例如 100、20 和 5 nM）转染到内源性表达目标基因的细胞中。

6. 24 or 48 h later, harvest the cells and extract the total RNA according to the standard procedures. Verify the quality of total RNA by separating in agarose gel and analyzing with Microvolume Spectrophotometers.

   24 或 48 小时后，收获细胞并按照标准程序提取总 RNA。通过琼脂糖凝胶电泳分离和微量体积分光光度计分析验证总 RNA 的质量。

7. Prepare the cDNA with 1μg total RNA in a 20μL standard reaction solution, then use 80μL ddH₂O to dilute the cDNA solution.

   使用 1μg 总 RNA 在 20μL 标准反应液中制备 cDNA，然后使用 80μL ddH₂O 稀释 cDNA 溶液。

8. Perform the quantitative PCR with a 20μL reaction solution containing 5μL of cDNA template by using a PCR machine .

   使用含有 5μL cDNA 模板的 20μL 反应液进行定量 PCR 反应，使用 PCR 仪进行检测。

9. Finally, calculate the inhibition efficiency with the Ct values by normalizing to the control group (e.g., mock without transfection siRNA, or scrambled siRNA-treated sample, or untreated sample) (see Notes 10-12).

   最后，通过标准化对照组（例如未转染 siRNA 的空白对照组、处理过无序列 siRNA 的样品或未处理样品）的 Ct 值计算抑制效率（参见注释 10-12）

3.2.4 Off-Target Effect Evaluation 脱靶效应评估

1. Assess the off-target effect of siRNAs with either psiCheck reporter system or RNA-seq technology.

   siRNA 的脱靶效应可以使用 psiCheck 报告系统或 RNA-seq 技术进行评估

2. For psiCheck-based off-target effect evaluation, transfect siRNAs into Hek293A at concentration gradients ranged from 50 nM, 0.5 nM, 0.25 nM, 0.125 nM, 0.0625 nM, 0.0313 nM, 0.0156 nM, 0.0078 nM, 0.0039 nM, 0.002 nM, 0.001 nM to 0.0005 nM.

   对于基于 psiCheck 的脱靶效应评估，将 siRNA 转染到 Hek293A 细胞中，浓度梯度范围为 50 nM、0.5 nM、0.25 nM、0.125 nM、0.0625 nM、0.0313 nM、0.0156 nM、0.0078 nM、0.0039 nM、0.002 nM、0.001 nM 和 0.0005 nM

3. Record the enzymes’ activities of Renilla and Firefly luciferases with Multi-Detection Microplate Reader. Then generate the concentration-activity curves and calculate the IC₅₀s with GraphPad Prism software.

   使用多功能检测微孔板读板仪记录海肾和萤火虫萤光素酶的酶活性。然后利用 GraphPad Prism 软件生成浓度-活性曲线并计算 IC₅₀ 值

4. With this reporter-based assay, the off-target effects resulted from complete-match of the passenger strand with the target or from seed-match of either the guide or the passenger strand with the target can be quickly determined.

   这种基于报告系统的检测可以快速确定由乘客链完全匹配靶序列或引导链/乘客链种子序列匹配靶序列引起的脱靶效应。

5. Alternatively, apply high throughput RNA-seq, a newly developed technology to evaluate the expression inhibition of numerous endogenously expressing genes both in vitro and in vivo (Fig. 7b).

   RNA-seq 是一种新开发的技术，可以用于评估体外和体内大量内源表达基因的表达抑制 (图 7b)

6. In this circumstance, prepare total RNA with high quality from either cultured cells (cell lines or primary cells) or tissue samples. Perform the RNA-seq usually by Contract Research Organization (CRO). Analyze the data by ourselves (if feasible) or the researchers of CRO. Finally, figure out the expression levels of all on-target genes and those off-target transcripts.

   首先，需从培养的细胞 (细胞系或原代细胞) 或组织样本中提取高质量的总 RNA。通常由专业合同研究组织 (CRO) 进行 RNA-seq 检测。数据分析可以由研究者自己完成 (如果可行) 或由 CRO 的研究人员完成。最后，分析所有靶基因和脱靶转录物的表达水平

7. Assess the off-target effect resulted from the immune response, such as activation of the TLRs pathway, by measuring the cytokine levels and the expression of relevant genes (e.g., TNF-α, IL-6, IL-12, IFN-γ, IFIT genes).

   通过测量细胞因子水平和相关基因 (例如 TNF-α、IL-6、IL-12、IFN-γ、IFIT 基因) 的表达来评估由免疫反应引起的脱靶效应，例如 TLRs 通路激活

3.3 siRNA Chemical Modification siRNA 化学修饰

1. It is well known that chemical modification of siRNA is extremely essential for siRNA therapeutic development because it can significantly enhance siRNA’s capability of resistance to enzymes’ attacking, improve siRNA’s specificity, and reduce potential off-target effects. Sophisticated modification strategies have been proposed and investigated for both siRNA with canonical structure or Dicer-substrate siRNA (DsiRNA) (Fig. 4). Chemical structures of the representative modification building blocks are shown in Fig. 5.

   siRNA 的化学修饰对于其治疗药物的开发非常重要，因为这种修饰可以显著提高 siRNA 抵抗酶攻击的能力，提高其特异性，并减少潜在的离靶效应。对于具有常规结构的 siRNA 或 Dicer 底物 siRNA（DsiRNA），已经提出并研究了多种复杂的修饰策略（见图 4）。常用修饰结构式如图 5 所示。

2. Select siRNA modification design from Fig. 4. Alternatively, test additional designs based on these patterns or other novel designs.

   从图 4 中选择 siRNA 的修饰设计。此外，还可以基于这些模式或其他新设计进行额外的测试。

3. Among these designs, 2′-fluoro (2′-F), 2′-O-methoxy (2′-OMe), DNA, phosphorothioate, and inverted base (idT/A) are primarily employed to enhance siRNA’s stability , while unlocked nucleic acid (UNA) and glycol nucleic acid (GNA) positioned at 7 of the antisense strand are used to reduce the off-target effect mediated by seed region (Fig. 7a).

   在这些设计中，2′-氟（2′-F）、2′-O-甲氧基（2′-OMe）、DNA、硫代磷酸酯和倒置碱基（idT/A）主要用于增强 siRNA 的稳定性，而解锁核苷酸（UNA）和乙二醇核苷酸（GNA）定位于反义链的第 7 位，用于减少由种子区介导的离靶效应（见图 7a）

Fig. 4 Examples of siRNA chemical modification design. GalNAc, N-acetylgalactosamine, can be placed at 3′-end or 5′-end of the sense strand. For DsiRNA, they can be positioned at the unpaired G-A-A-A nucleotides. Abbreviations: 2′-F 2′-fluoro, 2′-OMe 2′-methoxy, UNA unlocked nucleic acid, GNA glycol nucleic acid, SS sense strand, AS antisense strand

Fig. 5 Chemical structures of siRNA modification building blocks. R=H or OH, for RNA or DNA, respectively

3.4 In Vivo Delivery of GalNAc-siRNA Conjugate GalNAc-siRNA 偶联物的体内递送

1. As aforementioned, GalNAc-siRNA conjugate constitutes one of the most powerful and successful delivery platforms for liver-targeted transportation of siRNA. Therefore, dozens of siRNA therapeutic pipelines have been established, aiming to treat diverse diseases including hyperlipidemia, hepatitis B, amyloidosis, etc.

   如前所述，GalNAc-siRNA 偶联物是目前最强大且最成功的 siRNA 肝脏靶向运输平台之一。因此，已经建立了几十条 siRNA 治疗管线，旨在治疗包括高脂血症、乙型肝炎、淀粉样变性等多种疾病。

2. Display GalNAc moieties at the 3′-end or 5′-end of the sense strand of siRNA. Alnylam Pharmaceuticals, Silence Therapeutics, Arbutus Biopharma, etc., conjugate trivalent GalNAc at the 3′-end of siRNA sense strand, while Arrowhead Pharmaceuticals demonstrate there is no significant difference between 3′-conjugation and 5′-conjugation. Dicerna Pharmaceuticals position tetravalent GalNAc at the unpaired nucleotides of the DsiRNA structure. A challenging task for this field is to develop proprietary linker structures. A series of different linkers have been designed, and some of them can deliver oligonucleotides to hepatocytes efficiently. The linker chemistry developed by Alnylam is shown in Fig. 2g.

   GalNAc 配体可以修饰在 siRNA 双链乘客链的 3' 端或 5' 端。Alnylam Pharmaceuticals、Silence Therapeutics、Arbutus Biopharma 等公司在 siRNA 正义链的 3′末端偶联三价 GalNAc，而 Arrowhead Pharmaceuticals 则表明 3′-端偶联和 5′-端偶联之间没有显著差异。Dicerna Pharmaceuticals 则将四价 GalNAc 定位于 DsiRNA 结构中未配对的核苷酸。该领域的一个挑战性任务是开发专有的连接子结构。已经设计了一系列不同的连接子，其中一些可以有效地将寡核苷酸递送到肝细胞。Alnylam 开发的连接子化学结构如图 2g 所示。

3.5 In Vivo Activity of GalNAc-siRNA Conjugate GalNAc-siRNA 偶联物的体内活性

1. Subcutaneously (SC) administer GalNAc-siRNA conjugate into the mice (male C57BL/6, 6–8 weeks, weighing 18–22 g) at 0.5–10 mg/kg (see Note 13).

   将 GalNAc-siRNA 偶联物以皮下注射（SC）的方式注射到小鼠体内（雄性 C57BL/6，6–8 周龄，体重 18–22 克），剂量为 0.5–10 mg/kg（参见注释 13）。

2. Sacrifice animals by cervical dislocation, harvest the liver tissues 3 days or 1 week after administration and keep them in RNAlater (see Note 14).

   注射后 3 天或 1 周，通过颈椎脱位法处死小鼠，收集肝组织并将其保存在 RNAlater中（参见注释 14）。

3. Homogenize the tissues and extract the total RNAs according to the same protocol with that of the cultured cells.

   将组织均质化，并按照与培养细胞相同的协议提取总 RNA。

4. Prepare cDNAs and quantify the expression levels of the target gene by real-time qPCR (see Note 12).

   制备 cDNA，并通过实时 qPCR 定量目标基因的表达水平（参见注释 12）。

5. Administer multiple doses of GalNAc-siRNA to achieve potent and durable gene silence and treatment effect. Examples of gene silence performances with single or multiple doses of GalNAc-siRNA are shown in Fig. 6.

   多次给药 GalNAc-siRNA，以实现强效和持久的基因沉默和治疗效果。单次或多次给药 GalNAc-siRNA 的基因沉默性能示例如图 6 所示。

6. As an optional choice, quantify siRNAs exposed in liver tissue and load into the RISC by stem-loop reverse transcription followed by Taqman PCR (Fig. 7c, d).

   作为一个可选方案，通过茎环逆转录后接 Taqman PCR 定量肝组织中暴露的 siRNA 并加载到 RISC 中（图 7c、d）。

Fig. 6 Gene silencing by GalNAc-siRNA conjugate targeting mouse transthyretin (mTTR) in C57BL/6 mice. (a) mRNA expression of mTTR in liver tissues after treated with single dose of GalNAc-siRNA conjugate (dark blue; 0.2, 1, and 5 mg/kg, n = 5). (b) Long-term reduction of TTR protein in mice during weekly subcutaneous dosing at 1 mg/kg. The TTR protein was analyzed with serum samples by ELISA (enzyme-linked immunosorbent assay). Data were shown as mean ± standard error.通过 GalNAc-siRNA 结合物靶向 C57BL/6 小鼠中的小鼠转甲状腺素蛋白 (mTTR) 进行基因沉默。(a) 用单剂量 GalNAc-siRNA 结合物 (深蓝色；0.2、1 和 5 mg/kg，n = 5) 治疗后肝组织中 mTTR 的 mRNA 表达。(b) 每周皮下给药 1 mg/kg 期间小鼠 TTR 蛋白的长期减少。通过 ELISA (酶联免疫吸附测定) 分析血清样本中的 TTR 蛋白。数据以平均值 ± 标准误差显示。 (The figure is adapted and modified from the literature. Copyright © 2014 American Chemical Society)

Fig. 7 The in vivo performances of GNA-modified GalNAc-sbiRNA conjugate targeting hydroxyacid oxidase 1 (HAO1). (a) The modification design of siRNA. (b) Off-target effect evaluated by RNA-seq in rat hepatocytes. (c, d) siRNA amount in liver tissue (c) and RISC (d) that quantified by stem-loop RT-qPCR. (e) Glutamate dehydrogenase (GLDH) levels measured with rat serum specimen. (f) H&E staining of the liver tissues collected at necropsy. Error bars represent standard deviation of the mean. *p < 0.05.(The figure is adopted from the literature, Copyright 2018 Nature Publishing Group) GNA 修饰的 GalNAc-siRNA 结合物靶向羟基酸氧化酶 1 (HAO1) 的体内性能。(a) siRNA 的修饰设计。(b) 通过 RNA-seq 在大鼠肝细胞中评估的脱靶效应。(c, d) 通过茎环 RT-qPCR 量化的肝组织 (c) 和 RISC (d) 中的 siRNA 量。(e) 用大鼠血清样本测量谷氨酸脱氢酶 (GLDH) 水平。(f) 尸检时收集的肝组织的 H&E 染色。误差线表示平均值的标准差。p* < 0.05。

3.6 In Vivo Toxicity of GalNAc-siRNA Conjugate GalNAc-siRNA 偶联物的体内毒性

1. According to the guidelines of toxicity assessment , a series of studies need to be performed for Investigational New Drug (IND) application. These assessments include but not limited to acute toxicity test, long-term toxicity test, genetic toxicity test, reproductive toxicity test, and carcinogenic toxicity test. Herein, we mainly discuss some preliminary toxicity assessments.

   根据毒性评估指南，为了新药临床试验申请（IND），需要进行一系列研究。这些评估包括但不限于急性毒性试验、长期毒性试验、遗传毒性试验、生殖毒性试验和致癌毒性试验。在此，我们主要讨论一些初步的毒性评估。

2. Subcutaneously administer GalNAc-siRNA conjugate to toxicology-relevant animal species, such as rat and nonhuman primate (NHP), at toxicological doses (see Notes 15 and 16).

   皮下注射 GalNAc-siRNA 缀合物到用于毒性研究的动物物种，例如大鼠和非人类灵长类 (NHP)，剂量为毒理学剂量 (参见注释 15 和 16)。

3. Collect plasma or serum samples at 3–6 h or 24 h after administration.

   给药后 3-6 小时或 24 小时收集血浆或血清样本

4. Examine the cytokines, such as TNF-α, IFN-γ, IL-1β, IL-2, IL-5, IL-6, IL-12(p70), MCP-1, KC, GM-CSF using Luminex® assays (high-throughput multiplex bead-based assays) with Luminex 100 system or ELISA.

   使用 Luminex 100 系统或 ELISA，通过 Luminex®测定法（高通量多重微球测定法）检查细胞因子，例如 TNF-α, IFN-γ, IL-1β, IL-2, IL-5, IL-6, IL-12(p70), MCP-1, KC, GM-CSF

5. Count the blood cell with whole blood samples to analyze the profiles of the key components in the blood, such as red blood cells, white blood cells, hemoglobin, hematocrit, and platelets.

   用全血样品进行血细胞计数，分析血液中关键成分的含量，例如红细胞、白细胞、血红蛋白、血细胞比容和血小板

6. In another study, do not collect any sample within 2 weeks after siRNA injection. Instead, perform clinical observations for 2 weeks. In this case, record clinical symptoms, including animal appearance, eating, drinking, excretion, behavior, and response to stimulation during this process. Finally, as an optional choice, collect the blood samples, measure parameters revealing the liver and kidney functions (e.g., aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total bilirubin (TBIL), glutamate dehydrogenase (GLDH), serum creatinine (CREA), blood urea nitrogen (BUN)), and analyze cytokines and the blood cells. Evaluate the pathological changes of the tissue samples.

   在另一项研究中，在 siRNA 注射后两周内不收集任何样本。相反，进行为期两周的临床观察。在此过程中记录临床症状，包括动物的外观、进食、饮水、排泄、行为和对刺激的反应。最后，作为一个可选方案，收集血样，测量揭示肝肾功能的参数（例如，天冬氨酸转氨酶（AST）、丙氨酸转氨酶（ALT）、碱性磷酸酶（ALP）、总胆红素（TBIL）、谷氨酸脱氢酶（GLDH）、血清肌酐（CREA）、血尿氮（BUN）），分析细胞因子和血细胞。评估组织样本的病理变化。

7. An example of toxicity evaluation of GalNAc-siRNA conjugate is shown in Fig. 7e, f . It has been demonstrated that GNA positioned at 7′ of siRNA guide strand can significantly erase the seed region-mediated off-target effect and therefore dramatically alleviate the hepatotoxicity.

   图 7e, f 展示了 GalNAc-siRNA 缀合物毒性评价的一个例子。该研究表明，定位于 siRNA 引导链 7' 端的 GNA 可以显著降低种子区介导的脱靶效应，从而大大减轻肝毒性。

👀

4 Notes 注释

1. Use other alignment tools.

   使用其他比对工具。

2. Some commonly used online siRNA design tools are shown as follows:

   一些常用的在线 siRNA 设计工具如下

   (a) siDESIGN Center https://horizondiscovery.com/products/tools/siDESIGN-Center.

   (b) siDirect 2.0 (http://sidirect2.rnai.jp/).

   (c) Block-iT RNAi Designer (https://rnaidesigner.invitrogen.com/rnaiexpress/setOption.do?designOption=sirna ).

   (d) DSIR (http://www.bioinfo.ensmp.fr/dsir/).

   (e) GenScript siRNA Target Finder (https://www.genscript.com/tools/sirna-target-finder).

   (f) RNAi Design Tool (https://sg.idtdna.com/site/order/designtool/index/DSIRNA_CUSTOM).

   (g) siDRM (http://c1.accurascience.com/siDRM/index.php).

   (h) siRNA Selection Server (http://jura.wi.mit.edu/bioc/siRNAext/ ).

   (i) OptiRNA (http://optirna.unl.edu/).

3. Alternatively, design siRNAs separately with the transcripts for targeting conserved regions across species. Combine siRNA lists reported by different tools, and analyze their sequence features to determine if there is any siRNA targeting the conserved regions.

   或者，使用转录本分别设计 siRNA，以靶向跨物种的保守区域。结合不同工具报告的 siRNA 列表，并分析其序列特征以确定是否有任何 siRNA 靶向保守区域。

4. Combine the siRNA sequences and verify if there is any siRNA that is reported by more than one tool when using multiple tools to design siRNA. Prefer siRNA(s) reported by more than one tool, especially if the algorithms are different from each other.

   在使用多种工具设计 siRNA 时，结合 siRNA 序列并验证是否有任何 siRNA 被多个工具报告。优先选择由多个工具报告的 siRNA，尤其是当算法彼此不同时。

5. Typically, select less than ten siRNAs for further activity evaluation for basic research because higher than 80% silencing efficiency usually can be achieved within these siRNAs. There is no necessity to consider the intellectual property issues if we do not intend to submit a patent application. Otherwise, select dozens or even thousands of siRNAs for screening and evaluation.

   通常，选择少于十个 siRNA 进行进一步的活性评估，用于基础研究，因为这些 siRNA 通常可以实现高于 80% 的沉默效率。如果我们不打算申请专利，就没有必要考虑知识产权问题。否则，选择几十甚至几千个 siRNA 进行筛选和评估。

6. Alternatively, chemically modified siRNA. Perform the following tests with the modified siRNAs. The advantage of using modified siRNA is saving time and cost. However, a potential risk is that we may miss some siRNAs with high activity when they are not modified. Because chemical modification may influence the activity although the modification chemistry is rationally designed and optimized.

   或者，化学修饰的 siRNA。用修饰的 siRNA 进行以下测试。使用修饰的 siRNA 的优点是节省时间和成本。然而，一个潜在的风险是，我们可能会错过一些未经修饰的高活性 siRNA。因为尽管修饰化学是合理设计和优化的，化学修饰也可能影响活性。

7. If the library of siRNAs is not large, for instance, less than 20 siRNAs, directly use RT-qPCR for activity evaluation instead of using luciferase reporter system.

   如果 siRNA 库不大，例如少于 20 个 siRNA，则直接使用 RT-qPCR 进行活性评估，而不是使用荧光素酶报告系统。

8. Evaluate the activity of siRNA with psiCheck reporter vector, because it is demonstrated that there is no significant difference between the siRNAs targeting the CDS region and those targeting the 3′-UTR, regarding to their silencing activities, as evaluated by reporter system.

   使用 psiCheck 报告载体评估 siRNA 的活性，因为已证明，靶向 CDS 区域的 siRNA 和靶向 3'-UTR 区域的 siRNA 在沉默活性方面没有显著差异，如报告系统所评估的

9. Use other nucleic acid transfection reagents, e.g., Lipofectamine 3000 and Lipofectamine RNAiMAX.

   使用其他核酸转染试剂，例如 Lipofectamine 3000 和 Lipofectamine RNAiMAX。

10. Typically, use a scrambled siRNA in both luciferase assay and PCR assays, and normalize the activity of tested siRNA to that of the scrambled siRNA. However, if the Ct values of untreated sample, mock sample, and scrambled siRNA-treated sample are consistent, use all the above negative controls for normalization.

    通常，在荧光素酶测定和 PCR 测定中使用乱序 siRNA，并将测试的 siRNA 的活性标准化为乱序 siRNA 的活性。但是，如果未处理样品、模拟样品和乱序 siRNA 处理样品的 Ct 值一致，则使用上述所有阴性对照进行标准化。

11. The sequence of a scrambled siRNA that is widely used: sense strand: UUCUCCGAACGUGUCACGUdTdT, antisense strand: ACGUGACACGUUCGGAGAAdTdT. Bioinformatics analysis shows that this siRNA target no transcript for human, mouse, rat, and monkey.

    一个广泛使用的 siRNA 序列：正链：UUCUCCGAACGUGUCACGUdTdT，反链：ACGUGACACGUUCGGAGAAdTdT。生物信息学分析表明该 siRNA 针对人、小鼠、大鼠和猴没有转录本。

12. Determine the protein expression level with Western Blotting. In this case, extract total protein and perform the assay according to the well-established standard protocols.

    用 Western Blotting 检测蛋白表达水平。此时，提取总蛋白并按照成熟的标准方案进行检测。

13. Change the dosage according to requirements of the assay. If the activity of the tested siRNA is extremely high, use low dose. If the duration of single dose of siRNA is intended to be observed, use relatively higher dose.

    据检测要求改变剂量。如果测试的 siRNA 活性极高，则使用低剂量。如果要观察单剂量 siRNA 的持续时间，则使用相对较高的剂量。

14. If the duration of siRNA potency is intended to be evaluated, choose longer period, e.g., 1 month, even half year.

    如果要评估 siRNA 效力的持续时间，则选择较长的时间，例如 1 个月，甚至半年。

15. Determine the toxicological doses that are normally higher than the treatment doses by using dose range finding (DRF) study.

    利用剂量范围探索（DRF）研究确定通常高于治疗剂量的毒理学剂量。

16. Currently, siRNA therapeutics are managed as chemical drugs around the world and prefer toxicology study in NHP. Meanwhile, recommend safety evaluations in mice or rats.

    目前，siRNA 疗法在世界范围内作为化学药物进行管理，优先在 NHP 中进行毒理学研究。同时，建议在小鼠或大鼠中进行安全性评估。