近年来,小核酸药物研发和临床治疗取得了多项突破性成果,已成为全球投资新风口和生物制药巨头的必争之地,核酸疗法进入快车道。核酸适配体药物作为核酸疗法的重要组成部分,具有许多独特优势,在医药研发领域有着广阔的市场前景。一、核酸适配体药物介绍核酸适配体(Aptamer)是指通过指数富集的配体系统进化技术(systematic evolution of ligands by exponential enrichment,SELEX)得到的一段长度在20~100 nt左右的单链DNA或RNA序列,可以作为靶向性治疗的分子探针。SELEX技术的初始文库是单链DNA或RNA文库,由1014~1016个随机的长度为20~100个核苷酸序列的寡核苷酸组成,两端为固定引物序列,中间有一个随机区域。初始文库与靶标物质孵育后,结合的寡核苷酸从未结合序列中分离并通过PCR扩增,得到的富集池投入下一轮的筛选,不断循环最终找到高亲和力适配体。(图1)图1 SELEX筛选过程示意图(来源:核酸适体的筛选及其在生物医学领域的研究进展)核酸适配体可折叠形成发卡(hairpin)、假结(pseudoknot) 、凸环(bulge)、G-四聚体(G-quadruplex)等稳定的二级和三级空间结构。核酸适配体的这些空间构型能够与靶标物质通过静电作用、氢键作用、堆积作用、疏水作用和形状匹配等多种相互作用力形成嵌合或包被的高亲和力复合物,从而实现对靶标物质的高度特异性识别结合。(图2)图2 核酸适配体折叠识别结合靶标示意图(来源:Oligonucleotide Aptamer-Mediated Precision Therapy of Hematological Malignancies)核酸适配体能够识别、结合的靶标物质范围非常广泛,包括金属离子、小分子、氨基酸、多肽、蛋白质、生长因子、细胞粘附分子,甚至是完整的细胞、病毒等。核酸适配体具有与抗体相当的亲和力,其亲和常数可达纳摩尔或皮摩尔水平,因此被称为“化学抗体”。然而同抗体相比核酸适配体具有更大的优势,其中包括具有更好的组织穿透性、更高的热稳定性、更低的免疫原性、更易生产以及可以在不丧失活性的情况下在适配体的天然寡核苷酸骨架中引入各种化学修饰等。表1所示为核酸适配体和抗体的对比情况。表1 核酸适配体和抗体对比Criteria AptamersAntibodyBasic compositionNucleotide (four members: A, G, T/U and C)Amino acid (20 members)MaterialsNucleic acid (single-stranded DNA or RNA)• Protein (polymer peptide) •Antibodies consist of two light chains and two heavy chainsMolecular weight and/or size• 6–30kDa (20–100nt) • ~2nm• 150–180kDa • ~15nmSecondary structure•Hairpin, stem, loop, bulge, G-quadruplex or kissing complex• α-Helix and β-fold Binding pattern and/or mechanism of action • Surface recognition • Three-dimensional interactions via van der Waals forces, hydrogen bonding and electrostatic interactions similar to the way antibodies bind to antigen • Reversal of activity via complementary antidote oligonuclotides• Binding pocket (key and lock model) • Three-dimensional interaction; antibodies recognize epitopes located on the target antigen Affinity •High • Multivalent aptamers can confer increasing affinity and additional functions•High •Affinity between antibody and antigen depends on the number of identical epitopes on the target antigenSpecificity •High • The aptamer is able to identify single point mutations and conformational isomers•High •Antigens may have multiple epitopes, which allow different antibodies to bind to the same antigenPotential targets •Wide range: ions, organic and inorganic molecules, nucleic acids, peptides, proteins, toxins, viral particles, whole cells, entire organs and live animals• Limited to immunogenic molecules •No toxins or other molecules that do not cause strong immune responsesGeneration and discovery• In vitro SELEX (2–15 selection rounds) • ~2–8 weeks •Aptamer can be selected in hours or days via high-throughput automated SELEX• In vivo biological system • ~6 months or longerManufacturing and costs •Chemical solid-phase synthesis •Controllable and completely in vitro procedure • 2 days for milligrams; 2 weeks for grams •No or low risk of contamination • Facile regulatory affairs and cGMP • Low cost for DNA; high cost for long RNA (>60nt) with special modifications •Costs lowered with the development of new technology• In vivo (animal-based production) • Potential contamination due to cells or animal-based production • 3 months for 5–20 grams • From mammalian cells: high cost • From transgenic plants or animals: low cost Batch-to-batch variation•None or low• Significant Physical and thermal stability •Very stable and long shelf-life • Resistant to high temperature (even up to 95°C) and cycles of denaturation and renaturation •Aptamers can be lyophilized for long-term storage and transport at room temperature •Unstable and limited shelf-life • Susceptible to temperature (even at room temperature or 37°C) and irreversible denaturation • Requires refrigeration for storage and transport Chemical modification and conjugation •Convenient and controllable •Various types available, including sugar, backbone, base and other modifications •Aptamers can be rationally modified without loss of binding affinity • Restricted and uncontrollable • Limited types and chemical reactions • Stochastic modifications very likely to cause negative consequences, such as loss of activity Tissue uptake and penetration• Faster • Slower Immunogenicity•None or low immunogenicity•High immunogenicity • Increased immune reaction with repeated dosingNuclease degradation•Vulnerable • Limited half-life in vivo (~10min for unmodified version)• Resistant and not affected by nucleases in vivoKidney filtration • Faster • Short circulation time in vivo (~30min for unconjugated version)• Slower • Long circulation time (up to 1 month) Patents and distribution• Exclusive patents in SELEX technology • Limited initial distribution• Expired protection or no early patents • More widespread distribution Development and market• The development pathway is less explored • Insufficient education and investment (R&D support) •Commercialization has focused on diagnostic-based aptamer products•Well-developed infrastructure •Abundant support from finance and education • Rapid and sustained increase in drug market sharecGMP, current good manufacturing practice; nt, nucleotide; SELEX, systematic evolution of ligands by exponential enrichment.来源:Aptamers as targeted therapeutics: current potential and challenges凭借其诸多独特的优势,核酸适配体在药物研发领域发展前景广阔。美国 FDA 在 2004年通过了第一个用于治疗年龄相关的黄斑变性的核酸适配体药物哌加他尼钠 ( pegaptanib sodium,商品名为Macugen) ,将核酸适配体药物的研发推向一个新的高度。现已有11种核酸适配体药物进入临床试验阶段(表2),未来必定会有更多的核酸适配体药物被研发出来,并最终用于治疗疾病。表2 进入临床阶段的核酸适配体药物药物名称性质靶标治疗疾病临床阶段时间研究单位PegaptanibRNAVEGFAMD上市(现已退市)2004Eyetech/Pfizer公司ZimuraRNAC5AMDⅠ期2016Ophthotech公司FovistaDNAPDGFAMDⅢ期2016Ophthotech公司AS1411DNA核仁蛋白肾癌Ⅱ期2008Antisoma研究中心NOX-A12RNACXCL12CLLⅠ期2012NOXXON制药公司NOX-E36RNACCL2DNⅠ期2015NOXXON制药公司ARC1779DNAVWFTTPⅡ期2008Archemix公司ARC19499RNATFPI血友病Ⅰ期2010维也纳医科大学NU172DNA凝血酶心脏病Ⅱ期2013ARCA生物制药公司REG1RNAFIXa冠状动脉疾病Ⅱ期2007Regado生物科学公司NOX-H94RNA铁调素ACIⅡ期2013NOXXON制药公司数据来源:核酸适配体药物的研究进展二、核酸适配体药物体外代谢研究策略和传统小分子药物一样,在药物临床前研究阶段,通常需要对Aptamer药物进行体外代谢研究,并提供代谢途径及其产物相关信息。Aptamer药物的代谢酶主要为核酸外切酶和核酸内切酶,广泛分布于血液和各个组织器官,一个合适的体外代谢研究体系,能够最大程度地预测体内的情况,对于Aptamer药物的早期筛选非常重要。表3所示为常用的Aptamer药物体外代谢研究体系。表3 Aptamer药物的体外代谢研究体系生物基质试验类型备注血清/血浆代谢稳定性,血浆蛋白结合试验金属螯合剂抗凝的血浆不适合代谢稳定性研究。肝S9代谢稳定性,代谢产物鉴定肝S9一定程度上可以代替肝组织匀浆使用。肝微粒体代谢稳定性肝微粒体的核酸酶活性较低,可以根据实际情况进行选择。肝组织匀浆代谢稳定性,代谢产物鉴定酶体系比较全面,推荐用于Aptamer药物的体外筛选评价。肝细胞代谢稳定性,代谢产物鉴定肝细胞酶体系最为完善,适用于肝靶向的Aptamer药物研究。 溶酶体 代谢稳定性溶酶体具有丰富的酶体系,包括核酸酶和各种水解酶等,是研究Aptamer药物代谢稳定性的高效实验体系。三、IPHASE相关产品为满足客户对于Aptamer药物体外代谢研究的需求,IPHASE作为体外研究生物试剂引领者,凭借先进的设备,专业的技术人员和多年研发的经验,开发出来多种用于Aptamer药物体外代谢研究的产品,助力广大客户进行Aptamer药物早期筛选研究。类别分类 亚细胞组分肝溶酶体酸化肝匀浆液肝/肠/肾/肺S9肝/肠/肾/肺微粒体肝/肠/肾/肺胞质液原代肝细胞悬浮肝细胞贴壁肝细胞专属血浆血浆稳定性血浆蛋白结合IPHASE/汇智和源凭借多年的研发经验,推出了多领域、多种类的高端科研试剂,为药物早期研发提供筛选工具,为生命科学领域的探索提供新材料、新方法和新手段,为食品、药品、化学品等的遗传毒性研究提供便捷产品,望广大科研工作者来电咨询,咨询热线400-127-6686。参考文献:[1] Shuanghui Yang, Huan Li, Ling Xu, et al. Oligonucleotide Aptamer-Mediated Precision Therapy of Hematological Malignancies. Molecular Therapy: Nucleic Acids. 2018, 13, 164-165.[2] Jiehua Zhou, John Rossi. Aptamers as targeted therapeutics: Current potential and challenges. Nat. Rev. Drug Discovery. 2017, 16(3): 181-202.[3] 靳贵晓,李娟,杨黄浩. 核酸适体的筛选及其在生物医学领域的研究进展[J].福州大学学报,2016,44(6):920-934.[4] 刘珊,肖楠,王睿,等. 核酸适配体药物的研究进展[J].生命科学仪器,2020,18:16-24.发 文 章 得 奖 励凡使用本公司产品,在国内及国际刊物上发表论文(论文发表日起一年内),并注明产品属于IPHASE BIOSCIENCES Co.,Ltd. / 汇智和源生物技术(苏州)有限公司所有,即可申请奖励。根据发表刊物影响因子不同,给予不同金额奖品:非SCI论文及IF≤5分,500元礼品;5分<IF≤8分 800元礼品;8分<IF≤10分 1000元礼品;IF≥10分 2000元礼品;注:①礼品卡也可兑换同等金额产品购买抵用券; ②如遇我司公司名称书写不规范或不是第一作者 等情况,对应给予奖品金额将发放50%;活动多多,礼品丰厚,快来参与吧!关 于 我 们汇智和源,致力于为创新药研发企业及生命科学研究机构提供高品质的生物试剂,IPHASE为公司核心品牌,品牌宗旨“Innovative Reagents For Innovative Research”。