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全球首款RNAi药物“诞生”花了20年,未来10年将“爆发” | Nature深度好文

曼话 曼话 来源:医药魔方
2019-03-15
Nature 系列
原文

1998年,美国科学家Andrew Fire和Craig Mello在Nature[1]杂志上发表了一篇开创性的论文,确定了双链RNA(dsRNA)是线虫中转录后基因沉默(PTGS)的诱因。他们将这种现象称为RNA干扰(RNAi)。

 

RNAi的发现(图1)解释了植物和真菌中令人困惑的基因沉默现象,并引发了生物学上的一场革命,最终,研究证明,非编码RNAs是多细胞生物中基因表达的主要调控因子。RNAi通路在几乎所有人类细胞中调控着mRNA的稳定性和翻译。


图1 RNAi通路发现和阐明的早期事件 (图片来源:Nature Reviews Drug Discovery)


2001年,Sayda M.Elbashir等发表的一篇Nature[2],以及Natasha J. Caplen等发表的1篇PNAS[3]证实,由21和22个核苷酸组成的dsRNAs能够在哺乳动物细胞中诱导RNAi沉默,且不会引起非特异性干扰素反应。这些小干扰RNA(siRNAs)很快就成为生物学研究中无处不在的工具,因为它们能够通过一段碱基序列轻易地抑制任何基因。

 

对药物开发人员来说,siRNAs的效力和多功能性、抑制编码蛋白质的基因的前景以及有望成为“可编程”药物的潜力都非常“诱人”。到2003年,已有很多家公司布局RNAi疗法。

 

不幸的是,第一次使用未经修饰的siRNAs进行的临床试验产生了与免疫相关的毒性,以及可疑(不确定)的RNAi效应。第二波临床试验使用了系统给药的siRNA纳米颗粒制剂,尽管取得了重要进展(如,首次证实,siRNA纳米颗粒系统给药可在人体内产生RNAi效应),但依然表现出了显著的剂量限制毒性以及疗效不足。这些研发中暴露的问题使得大部分制药公司在2010年代早期退出了RNAi领域,这给RNAi药物的研发带来了资金危机。

 

不过,尽管面临这些挑战,一些小型RNAi公司和学术研究人员并没有就此放弃。他们吸取了以往临床试验失败的惨痛教训,坚持对触发器设计、序列选择、化学修饰和递送机制进行改进。在这些方面取得的实质性进展,结合更加明智的疾病适应症选择、更好的验证干预途径、更成熟的临床开发过程以及改善的生产制造能力,创造出了一条更加安全、有效的候选RNAi药物管线。


图2 patisiran的治疗机制(图片来源:Nature Reviews Drug Discovery)

 

2018年8月10日,美国FDA批准Alnylam公司作用于肝脏的siRNA药物——ONPATTRO (patisiran),用于治疗遗传性转甲状腺素蛋白淀粉样变性(hATTR)引起的神经损伤。


hATTR是一种罕见的、遗传性、危及生命的神经退行性疾病,由周围神经系统、心脏、胃肠道和其它器官中转甲状腺素蛋白(transthyretin, TTR)淀粉样蛋白沉积引起。患者患有进行性神经病、心肌病、行走障碍和各种其它衰弱症状,诊断后中位生存期为5-15年。

 

大多数TTR在肝脏中产生。TTR中有>120个突变就能导致hATTR。Patisiran通过沉默肝细胞中野生型和突变型TTR mRNAs来降低血清中TTR蛋白的水平(图2)。Patisiran的批准给hATTR患者带来了新的希望,让RNAi药物系统性递送到肝脏组织在临床上成为现实,预示着RNAi治疗领域进入了新时代。

 

图片来源:Nature Reviews Drug Discovery

 

3月7日,来自美国Beckman研究所的3位科学家就“RNAi药物设计和研发方面的关键进展、目前临床管线的状态以及未来的发展前景”在Nature Reviews Drug Discovery[4]杂志上发表了一篇深度综述。

 

文章介绍了RNAi的机制以及早期发现历史,总结了目前在合成RNAi触发器中使用到的基序、设计规则和化学修饰,讨论了多种药物递送途径,评估了RNAi药物管线的当前临床状态,对比了patisiran和后续候选药物,并分析了RNAi领域未来的机会和挑战。


药物设计与开发


图3 哺乳动物miRNA生物发生、合成RNAi触发过程以及RNAi沉默通路。(图片来源:Nature Reviews Drug Discovery)

 

为了利用哺乳动物的RNAi通路(图3)对假定的治疗靶点进行有效的特异性抑制,RNAi药物制剂必须克服与药效学相关的挑战(包括靶向特异性、脱靶RNAi活性、免疫传感器介导的细胞毒性)以及与药物动力学相关的、在系统循环、细胞摄取和内涵体逃逸(siRNA从内涵体逃逸到细胞质中可增强RNAi效果[5])方面的挑战。而这些挑战是通过RNAi触发器的结构基序、序列选择和化学修饰以及递送途径和赋形剂(辅料)的选择和设计来解决的。

 

结构基序

 

虽然RNAi通路酶对dsRNA分子的相容性有限制性的结构要求,但科学家们已开发出了一系列具有不同结构基序和功能特性的合成RNAi触发器(图4)。合成RNAi触发器通常是完全碱基配对的dsRNAs或短发夹RNA(shRNAs),总长度在15到30bp之间。短于15bp的dsRNAs会失去参与RNAi机制的能力,而长于30bp的dsRNAs则能够通过激活PKR通路诱导非特异性细胞毒性。


 图4  不同类型的合成RNAi触发器具有代表性的二级结构基序以及它们进入RNAi通路的主要机制。(图片来源:Nature Reviews Drug Discovery)


序列选择

 

2016年,1篇发表在Cancer Gene Therapy[6]上的综述提供了用于siRNA设计的软件包列表,并推荐了使用protocols。通过对其中涉及的一些问题的讨论,综述指出,未来,RNAi药物的开发人员可能需要在合理的靶点周围进行广泛的靶点序列筛选,以确定最佳候选药物。

 

化学修饰

 

对RNAi药物来说,化学修饰(组织靶向配体除外)有两个基本功能。首先,它们通过削弱检测dsRNA的内源性免疫传感器的激活,大大提高了安全性;其次,它们通过增强dsRNA触发器抵抗内源性核酸内切酶和外切酶降解的能力,大大提高了效力。除了这些功能外,化学修饰还可以改善序列选择性以降低脱靶RNAi活性,以及改变物理和化学性质以增强递送。


递送赋形剂

 

dsRNA触发器的化学修饰、大小、亲水性和电荷都对系统循环、外渗、组织渗透、细胞摄取以及内涵体逃逸构成了重大挑战。许多化学赋形剂(辅料)已被开发出来以克服这些障碍,包括纳米颗粒、脂质纳米颗粒(LNPs)、聚合物、树状大分子、核酸纳米结构、外泌体和GalNAc偶联蜂毒素样肽(NAGMLPs)。siRNA常见的靶向配体包括适配体、抗体、多肽和小分子(如GalNAc)(表1)。

 

表1 RNAi药物的递送方法以及赋形剂

数据来源(Nature Reviews Drug Discovery)

 

给药位置

 

除赋形剂外,给药方法和给药部位对RNAi药物的生物利用度和生物分布也有深远的影响。临床开发中的RNAi药物涉及的给药方式包括通过静脉注射和皮下注射进行系统给药、通过吸入(肺中)或特定位置注射(如眼内、脑脊液)等方式进行局部给药。

 

临床产品

目前,除已被批准上市的patisiran外,针对肝脏、肾脏和眼部适应症的多种候选药物正在进行I、II和III期临床试验(表2);此外,在接下来的两年里,一些靶向中枢神经系统(CNS)和其它非肝脏组织的IND申请“迫在眉睫”。此外,用于RNAi有效载荷(特异性增强)和赋形剂的新技术有望在未来5年内带来新的适应症突破。


当然,需要指出的是,RNAi药物的药物动力学、药效学和毒性限制策略方面仍有很大的改进空间。一些新技术的出现有望实现这一目标,如改善内涵体逃逸的技术、增加RNAi疗法有效性和安全性的抗体偶联技术、降低LNPs毒性的技术、改善递送方式的技术、快速逆转RNAi活性的技术(许多RNAi药物一次给药后疗效持续数周,可能会引发毒副作用)、限制RNAi药物在特定细胞中发挥作用的技术、丰富临床前动物模型的技术等。


但综合来说,目前的进展表明,未来10年,RNAi疗法有非常多值得期待的地方。


表2 部分目前处于临床试验阶段的RNAi疗法

数据来源(Nature Reviews Drug Discovery)

 

总结

 

从1998年发现RNAi现象,到2006年两位发现者共同获得诺贝尔生理学或医学奖,再到2018年全球首款RNAi药物获批,这20年的开发历史证明了坚持的力量。毫无疑问的是,随着相关技术的不断进步,未来RNAi疗法会取得更多的突破。


相关论文:

[1] Andrew Fire et al. Potent and specificgenetic interference by double-stranded RNA in Caenorhabditis elegans. Nature(1998).

[2] Sayda M. Elbashir et al. Duplexes of21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature(2001).

[3] Natasha J. Caplen et al. Specificinhibition of gene expression by small double-stranded RNAs in invertebrate andvertebrate systems. PNAS (2001).

[4] Ryan L. Setten et al. Thecurrent state and future directions of RNAi-based therapeutics. Nature ReviewsDrug Discovery (2019).

[5] 杨江勇等. 细胞内siRNA传递:应用修饰的细胞穿透肽从内涵体逃逸. 《国际药学研究杂志》(2009).

[6]E Fakhr et al. Precise and efficient siRNAdesign: a key point in competent gene silencing. Cancer Gene Therapy(2016).

 

参考资料:

1# RNAi疗法重大里程碑,FDA批准首款hATTR药物Onpattro


机器翻译

In 1998, American scientists Andrew Fire and Craig Mello published a groundbreaking paper in Nature [1] that determined that double-stranded RNA (dsRNA) is the cause of post-transcriptional gene silencing (PTGS) in nematodes. They call this phenomenon RNA interference (RNAi).

The discovery of RNAi (Figure 1) explains the confusing gene silencing in plants and fungi and triggers a biological Revolution, and finally, studies have shown that non-coding RNAs are the major regulators of gene expression in multicellular organisms. The RNAi pathway regulates mRNA stability and translation in almost all human cells.

Figure 1 RNAi pathway Early events discovered and clarified (Source: Nature Reviews Drug Discovery)

In 2001, a Nature[2] published by Sayda M. Elbashir et al., and a PNAS published by Natasha J. Caplen et al. [3] confirmed that dsRNAs consisting of 21 and 22 nucleotides are capable of inducing RNAi silencing in mammalian cells without causing non-specific interferon responses. These small interfering RNAs (siRNAs) quickly became biological studies. Tools that are ubiquitous because they can pass A base sequence easily inhibits any gene.

The efficacy and versatility of siRNAs for drug developers. The prospect of inhibiting genes encoding proteins and the potential to become "programmable" drugs Very "tempting." By 2003, many companies had deployed RNAi therapy.

Unfortunately, the first clinical trials using unmodified siRNAs produced immune-related toxicity. And suspicious (uncertained) RNAi effects. The second wave of clinical trials used systemically administered siRNA nanoparticle formulations, although significant advances have been made (eg, for the first time, siRNA nanoparticle system administration produces RNAi in humans). Effects), but still exhibit significant dose-limiting toxicity and insufficient efficacy. These developmental exposures have caused most pharmaceutical companies to withdraw from the RNAi field in the early 2010s, which has brought a financial crisis to the development of RNAi drugs. /p>

However, despite these challenges, some small RNAi companies and academic researchers have not given up on this. They have learned the painful teachings of previous clinical trial failures. Adhere to trigger design. Sequence selection. Improvements in chemical modification and delivery mechanisms. Substantial advances in these areas, combined with more sensible disease indication options. Better validation interventions. More mature clinical development processes and Improved manufacturing capabilities have created a safer and more effective candidate RNAi drug pipeline.

Figure 2 patisiran treatment mechanism (Source: Nature Reviews Drug Discovery)

2018 On August 10, the US FDA approved Alnylam's siRNA drug, ONPATTRO (patisiran), for the treatment of hereditary transthyretin amyloidosis (hATTR)-induced neurological damage.

hATTR is a rare. Hereditary. Life-threatening neurodegenerative disease caused by deposition of transthyretin (TTR) amyloid in the peripheral gastrointestinal tract and other organs. Progressive neuropathy. Cardiomyopathy. Walking disorders and various other debilitating symptoms, the median survival after diagnosis is 5-15 years.

Most TTR is produced in the liver. >120 mutations can cause hATTR.Patisiran to reduce serum TTR protein levels by silencing wild-type and mutant TTR mRNAs in hepatocytes (Fig. 2). Patisiran's approval brings new hope to hATTR patients, allowing The systematic delivery of RNAi drugs to liver tissue has become a reality in the clinic, indicating that the field of RNAi therapy has entered a new era.

Source: Nature Reviews Drug Discovery

March 7, Three scientists from the Beckman Institute in the United States published an in-depth review in the journal Nature Reviews Drug Discovery [4] on "Key advances in RNAi drug design and development. Current state of clinical pipelines and future development prospects." /p>

The article introduces the mechanism of RNAi and the history of early discovery, summarizes the motifs currently used in the synthesis of RNAi triggers. Design rules and chemical modifications, discusses various drug delivery routes, and evaluates RNAi drugs. The current clinical status of the pipeline, comparing patisiran and subsequent drug candidates, and analyzing future opportunities and challenges in the RNAi field.

Drug Design and Development

Figure 3 Mammalian miRNA Creatures Occurs. Synthetic RNAi triggering process and RNAi silencing pathway. (Source: Nature Reviews Drug Discovery)

Efficient specific inhibition of putative therapeutic targets in order to utilize the mammalian RNAi pathway (Figure 3) , RNAi pharmaceutical preparations must overcome the challenges associated with pharmacodynamics (including targeting specificity. Off-target RNAi activity. Immunosensor-mediated cytotoxicity) and pharmacokinetics. In systemic circulation. Cell uptake and endosomal escape (The escape of siRNA from endosomes to the cytoplasm enhances the RNAi effect [5]). These challenges are through the structural motif of RNAi triggers. Sequence selection and chemical modification, as well as delivery routes and excipients (excipients) Selection and design to solve.

Structure motifs

Although RNAi pathway enzymes have restrictive structural requirements for the compatibility of dsRNA molecules, scientists have developed a A series of synthetic RNAi flip-flops with different structural motifs and functional properties (Figure 4). Synthetic RNAi flip-flops are typically fully base paired dsRNAs or short hairpin RNAs (shRNAs) with a total length between 15 and 30 bp. 15 bp dsRNAs lose their ability to participate in the RNAi machinery, while dsRNAs longer than 30 bp can induce non-specific cytotoxicity by activating the PKR pathway.

Figure 4 Different types of synthetic RNAi triggers are representative of secondary Structural motifs and their main mechanisms of entry into the RNAi pathway. (Source: Nature Reviews Drug Discovery)

Sequence Selection

2016, 1 published in Cancer Gene Therapy [6] The review provides a list of software packages for siRNA design and recommends the use of protocols. By discussing some of the issues involved, the review points out that in the future, developers of RNAi drugs may need to be widely around reasonable targets. Screening of target sequences to determine the best drug candidate.

Chemical modification

For RNAi drugs, chemical modification (except for tissue targeting ligands) has two basic functions. First, they greatly improve safety by attenuating the activation of endogenous immunosensors that detect dsRNA. Second, they greatly enhance the ability of dsRNA triggers to resist endogenous endonuclease and exonuclease degradation. Effective In addition to these functions, chemical modifications can improve sequence selectivity to reduce off-target RNAi activity, as well as alter physical and chemical properties to enhance delivery.

Distributive Excipients

dsRNA Triggering Chemical modification of the device. Size. Hydrophilicity and charge are cyclic to the system. Extravasation. Tissue penetration. Cell uptake and endosomal escape constitute a major challenge. Many chemical excipients (auxiliaries) have been developed to overcome these obstacles. , including nanoparticles. Lipid nanoparticles (LNPs). Polymers. Dendrimers. Nucleic acid nanostructures. Exosomes and GalNAc-coupled melittin-like peptides (NAGMLPs). Common targeting ligands for siRNA include adaptation Antibody. Polypeptides and small molecules (such as GalNAc) (Table 1).

Table 1 RNAi drug delivery methods and excipients

Data Sources (Nature Reviews Drug Discovery)

Dosing position

In addition to excipients, the method of administration and site of administration also have profound effects on the bioavailability and biodistribution of RNAi drugs. RNAi in clinical development 1. The drug involved in the administration includes systemic administration by intravenous injection and subcutaneous injection. Local administration by inhalation (in the lungs) or injection at a specific location (eg intraocular cerebrospinal fluid).

Clinical products

At present, in addition to the patisiran that has been approved for marketing Multiple drug candidates for liver and kidney indications are undergoing Phase I.II and III clinical trials (Table 2). In addition, in the next two years, some targeted central nervous system (CNS) and Other non-liver tissue IND applications are “imminent.” In addition, new technologies for RNAi payload (specificity enhancement) and excipients are expected to lead to new indication breakthroughs in the next five years.

Of course, it should be pointed out that there is still much room for improvement in pharmacokinetics and pharmacodynamics and toxicity limitation strategies of RNAi drugs. The emergence of some new technologies is expected to achieve this goal, such as techniques to improve the escape of endosomes. Antibody Coupling Techniques for the Effectiveness and Safety of RNAi Therapy. Techniques for reducing the toxicity of LNPs. Techniques for improving delivery methods. Techniques for rapidly reversing RNAi activity (many RNAi drugs last for several weeks after one administration may cause toxic side effects) . Limit RNAi Techniques for the function of specific cells in specific cells. Enrich the technology of preclinical animal models.

In summary, however, current progress indicates that RNAi therapy has a lot to look forward to in the next 10 years.

Table 2 Partial RNAi Therapy Currently in Clinical Trial

Nature Reviews Drug Discovery

Summary

From 1998 The RNAi phenomenon was discovered in the year. By 2006, the two discoverers jointly won the Nobel Prize in Physiology or Medicine, and by 2018 the world's first RNAi drug was approved. This 20-year development history proves the strength of persistence. There is no doubt that With the continuous advancement of related technologies, future RNAi therapy will achieve more breakthroughs.

Related papers:

1]Andrew Fire et al. Potent and specificgenetic interference by double- Stranded RNA in Caenorhabditis elegans. Nature (1998).

2] Sayda M. Elbashir et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature (2001).

3]Natasha J. Caplen et al. Specificinhibition of gene expression by small double-stranded RNAs in invertebrate andvertebrate systems. PNAS (2001).

4]Ryan L. Setten et al. Thecurrent state and future directions of RNAi-based therapeutics. Nature ReviewsDrug Discovery(2019).

< p>5] Yang Jiangyong et al. Intracellular siRNA delivery: application of modified cell-penetrating peptides to escape from endosomes. International Journal of Pharmaceutical Research (2009).

6]E Fakhr et al. Precise and Effective siRNAdesign: a key point in competent gene silencing. Cancer Gene Therapy (2016).

Reference:

1# A major milestone in RNAi therapy, the FDA approved the first hATTR drug Onpattro

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