CN118773195A - Nucleic acids targeting complement component 5 and uses thereof - Google Patents
Nucleic acids targeting complement component 5 and uses thereof Download PDFInfo
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- CN118773195A CN118773195A CN202410750679.XA CN202410750679A CN118773195A CN 118773195 A CN118773195 A CN 118773195A CN 202410750679 A CN202410750679 A CN 202410750679A CN 118773195 A CN118773195 A CN 118773195A
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Abstract
本发明涉及生物技术领域，公开了一种靶向补体成分5的核酸及其用途。本发明的核酸能够有效抑制C5的表达，从而能够治疗和/或预防与补体系统激活相关的疾病。
The present invention relates to the field of biotechnology, and discloses a nucleic acid targeting complement component 5 and its use. The nucleic acid of the present invention can effectively inhibit the expression of C5, thereby being able to treat and/or prevent diseases related to complement system activation.
Description
相关申请的交叉引用CROSS-REFERENCE TO RELATED APPLICATIONS
本发明要求2023年06月16日提交的申请号为202310716812.5的中国专利申请的优先权，其全部内容通过引入并入本文。The present invention claims the priority of Chinese patent application No. 202310716812.5 filed on June 16, 2023, the entire contents of which are incorporated herein by reference.
技术领域Technical Field
本发明涉及生物技术领域，具体地，涉及靶向补体成分5的核酸及其用途。The present invention relates to the field of biotechnology, in particular to a nucleic acid targeting complement component 5 and uses thereof.
背景技术Background Art
补体成分5(C5，又名C5D,C5a,C5b,CPAMD4,ECLZB)是一个在肝细胞中表达的188kDa的蛋白(Tack BF et al.)。C5是人体血补体系统的补体之一，可以被3个补体激活途径激活，经典激活途径、替代激活途径和凝集素激活途径。C5激活后被C5转化酶切成C5a和C5b，C5a是中性粒细胞的趋化因子，而C5b可以形成膜攻击复合物，导致细胞膜损伤(MerleNS et al.；DiScipio RG)。C5缺陷可能会导致反复感染。补体系统的激活也会导致多种自身免疫性疾病。目前有一款靶向C5的抗体药，Eculizumab被批准用于阵发性睡眠性血红蛋白尿(paroxysmal nocturnal hemoglobinuria，PNH)、非典型溶血性尿毒症综合征(atypical hemolytic uremic syndrome，aHUS)、全身性重症肌无力(generalizedmyasthenia gravis)和视神经脊髓炎(neuromyelitis optica)(Rother RPet al；Keating GM)，但该药价格昂贵，需要有新的药物来治疗补体相关疾病。Complement component 5 (C5, also known as C5D, C5a, C5b, CPAMD4, ECLZB) is a 188kDa protein expressed in hepatocytes (Tack BF et al.). C5 is one of the complements in the human blood complement system and can be activated by three complement activation pathways: the classical activation pathway, the alternative activation pathway, and the lectin activation pathway. After activation, C5 is cleaved into C5a and C5b by C5 convertase. C5a is a chemotactic factor for neutrophils, while C5b can form a membrane attack complex, causing cell membrane damage (MerleNS et al.; DiScipio RG). C5 deficiency may lead to recurrent infections. Activation of the complement system can also lead to a variety of autoimmune diseases. Currently, there is an antibody drug targeting C5, Eculizumab, which is approved for paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis, and neuromyelitis optica (Rother RP et al; Keating GM), but the drug is expensive and new drugs are needed to treat complement-related diseases.
RNA干扰(RNAinterference,RNAi)指在进化过程中高度保守的、由双链小干扰核糖核酸(small interference RNA，siRNA)诱发的同源mRNA高效特异性降解的现象，同时RNAi药物也存在相比抗体药效时间长的优点。因此，研究开发靶向C5的siRNA具有重要的意义。RNA interference (RNAi) refers to the highly conserved phenomenon in the evolutionary process, which is induced by double-stranded small interfering RNA (siRNA) to efficiently and specifically degrade homologous mRNA. RNAi drugs also have the advantage of having a longer duration of efficacy than antibodies. Therefore, it is of great significance to study and develop siRNA targeting C5.
发明内容Summary of the invention
本发明的目的是为了克服现有技术存在的问题，提供一种新的靶向补体成分5的核酸及其用途。The purpose of the present invention is to overcome the problems existing in the prior art and provide a novel nucleic acid targeting complement component 5 and its use.
本发明第一方面提供了一种核酸，该核酸包括正义链和反义链，其中，所述正义链含有与SEQ ID NO：1～34任意一项所示序列具有80％以上的序列同一性的序列，所述反义链含有与SEQ ID NO：35～68任意一项所示序列具有80％以上的序列同一性的序列。The first aspect of the present invention provides a nucleic acid, which includes a sense strand and an antisense strand, wherein the sense strand contains a sequence having more than 80% sequence identity with the sequence shown in any one of SEQ ID NOs: 1 to 34, and the antisense strand contains a sequence having more than 80% sequence identity with the sequence shown in any one of SEQ ID NOs: 35 to 68.
本发明第二方面提供了一种靶向药物递送系统，该靶向药物递送系统包括靶向基团、连接基团和通过连接基团与靶向基团连接的如上所述的核酸。A second aspect of the present invention provides a targeted drug delivery system, which comprises a targeting group, a linking group, and the nucleic acid as described above connected to the targeting group via the linking group.
本发明第三方面提供了一种药物组合物，该药物组合物含有如上所述的核酸或靶向药物递送系统和药学上可接受的载体。The third aspect of the present invention provides a pharmaceutical composition, which contains the nucleic acid or targeted drug delivery system as described above and a pharmaceutically acceptable carrier.
本发明第四方面提供了一种抑制细胞中补体成分5基因的表达的方法，该方法包括：使所述细胞与如上所述的核酸、靶向药物递送系统或药物组合物接触，以抑制所述细胞中的补体成分5基因的表达。The fourth aspect of the present invention provides a method for inhibiting the expression of complement component 5 gene in a cell, the method comprising: contacting the cell with the nucleic acid, targeted drug delivery system or pharmaceutical composition as described above to inhibit the expression of complement component 5 gene in the cell.
本发明第五方面提供了如上所述的核酸、靶向药物递送系统或药物组合物在以下任一方面的用途：1)治疗和/或预防与补体成分5相关的疾病；2)制备用于治疗和/或预防与补体成分5相关的疾病的药物。The fifth aspect of the present invention provides the use of the nucleic acid, targeted drug delivery system or pharmaceutical composition as described above in any of the following aspects: 1) treating and/or preventing diseases associated with complement component 5; 2) preparing drugs for treating and/or preventing diseases associated with complement component 5.
本发明的核酸能够有效降低C5水平，且能有效预防或治疗补体系统相关疾病，如阵发性睡眠性血红蛋白尿、非典型溶血性尿毒症综合征、全身性重症肌无力、视神经脊髓炎、系统性红斑狼疮等。The nucleic acid of the present invention can effectively reduce the level of C5, and can effectively prevent or treat complement system-related diseases, such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, systemic myasthenia gravis, neuromyelitis optica, systemic lupus erythematosus, etc.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
图1为本发明实施例中注射siRNA药物后第四天血清中C5蛋白水平。FIG. 1 shows the C5 protein level in serum on the fourth day after injection of siRNA drug in an embodiment of the present invention.
图2为本发明实施例中siRNA药物与现有的SN-68002的效果对比。FIG. 2 is a comparison of the effects of the siRNA drug in the embodiment of the present invention and the existing SN-68002.
图3为本发明实施例中SN-681263与SN-681274在人源C5转基因小鼠中的药效。FIG3 shows the efficacy of SN-681263 and SN-681274 in human C5 transgenic mice according to the examples of the present invention.
图4为本发明实施例中siRNA在人源C5转基因小鼠中的药效；其中，A为SN-681263的实验结果；B为SN-681274的实验结果。FIG. 4 shows the efficacy of siRNA in human C5 transgenic mice according to an embodiment of the present invention; wherein A is the experimental result of SN-681263; and B is the experimental result of SN-681274.
具体实施方式DETAILED DESCRIPTION
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明，本领域技术人员可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. Those skilled in the art may make various modifications and changes to the present invention without departing from the scope or spirit of the present invention. For example, a feature described or illustrated as part of one embodiment may be used in another embodiment to produce a further embodiment.
术语解释Explanation of terms
除非另有说明，用于披露本发明的所有术语(包括技术和科学术语)的意义与本发明所属领域普通技术人员所通常理解的相同。通过进一步的指导，随后的定义用于更好地理解本发明的教导。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.
本文所使用的术语“和/或”、“或/和”、“及/或”的选择范围包括两个或两个以上相关所列项目中任一个项目，也包括相关所列项目的任意的和所有的组合，所述任意的和所有的组合包括任意的两个相关所列项目、任意的更多个相关所列项目、或者全部相关所列项目的组合。需要说明的是，当用至少两个选自“和/或”、“或/和”、“及/或”的连词组合连接至少三个项目时，应当理解，在本申请中，该技术方案毫无疑问地包括均用“逻辑与”连接的技术方案，还毫无疑问地包括均用“逻辑或”连接的技术方案。比如，“A及/或B”包括A、B和A+B三种并列方案。又比如，“A，及/或，B，及/或，C，及/或，D”的技术方案，包括A、B、C、D中任一项(也即均用“逻辑或”连接的技术方案)，也包括A、B、C、D的任意的和所有的组合，也即包括A、B、C、D中任两项或任三项的组合，还包括A、B、C、D的四项组合(也即均用“逻辑与”连接的技术方案)。The terms "and/or", "or/and", and "and/or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and/or", "or/and", and "and/or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and/or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and/or, B, and/or, C, and/or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").
本发明中所使用的术语“含有”、“包含”和“包括”是同义词，其是包容性或开放式的，不排除额外的、未被引述的成员、元素或方法步骤。As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.
本发明中用端点表示的数值范围包括该范围内所包含的所有数值及分数，以及所引述的端点。Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.
本发明中涉及浓度数值，其含义包括在一定范围内的波动。比如，可以在相应的精度范围内波动。比如2％，可以允许±0.1％范围内波动。对于数值较大或无需过于精细控制的数值，还允许其含义包括更大波动。比如100mM，可以允许±1％、±2％、±5％等范围内的波动。涉及分子量，允许其含义包括±10％的波动。The present invention relates to concentration values, and its meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuations within the range of ±0.1%. For values that are large or do not require too fine control, its meaning is also allowed to include greater fluctuations. For example, 100mM can allow fluctuations within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, its meaning is allowed to include fluctuations of ±10%.
本发明中，涉及“多个”、“多种”等描述，如无特别限定，指在数量上指大于等于2。In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.
本发明中，以开放式描述的技术特征中，包括所列举特征组成的封闭式技术方案，也包括包含所列举特征的开放式技术方案。In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.
本发明中，“优选”、“更好”、“更佳”、“为宜”仅为描述效果更好的实施方式或实施例，应当理解，并不构成对本发明保护范围的限制。In the present invention, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.
本发明中，“任选地”、“任选的”、“任选”、“可选地”、“可选的”、“可选”，指可有可无，也即指选自“有”或“无”两种并列方案中的任一种。如果一个技术方案中出现多处“任选”或“可选”，如无特别说明，且无矛盾之处或相互制约关系，则每项“任选”或“可选”各自独立。In the present invention, "optionally", "optional", "optionally", "optionally", "optional", "optional", "optional" means optional, that is, it means to be selected from any one of the two parallel schemes of "yes" or "no". If multiple "optional" or "optional" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" or "optional" is independent.
本发明中，术语“核酸”是指含有RNA或RNA样(例如化学修饰的RNA)寡核苷酸分子的组合物，所述寡核苷酸分子能够以序列特异性方式降解或抑制(例如，在适当的条件下降解或抑制)靶mRNA的信使RNA(mRNA)转录物的翻译。所述的核酸可以通过RNA干扰机制(即，通过与哺乳动物细胞的RNA干扰途径机制(RNA诱导沉默复合物或RISC)的相互作用而诱导RNA干扰)、或通过任何替代机制或途径来起作用。本文公开的包括正义链和反义链的核酸的下的限定范围包括但不限于：短(或小)干扰RNA(siRNA)、双链RNA(dsRNA)、微小RNA(miRNA)、短发夹RNA(shRNA)和切丁酶(dicer)底物。In the present invention, the term "nucleic acid" refers to a composition containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules, which can degrade or inhibit (e.g., degrade or inhibit under appropriate conditions) the translation of messenger RNA (mRNA) transcripts of target mRNA in a sequence-specific manner. The nucleic acid can act through an RNA interference mechanism (i.e., inducing RNA interference through interaction with the RNA interference pathway mechanism of mammalian cells (RNA-induced silencing complex or RISC)), or through any alternative mechanism or approach. The scope of the nucleic acids disclosed herein including sense and antisense strands includes, but is not limited to, short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) and dicer substrates.
本发明中，当提及给定基因的表达时，术语“沉默”、“降低”、“抑制”、“下调”或“敲减”，意指如通过基因在其中转录的细胞、细胞群、组织、器官或受试者中，由基因转录的RNA水平或者由mRNA翻译的多肽、蛋白质或蛋白质亚基的水平测量的，与未如此治疗的第二细胞、细胞群、组织、器官或受试者相比，当细胞、细胞群、组织、器官或受试者用本文所述的核酸进行治疗时，所述基因的表达是降低的。As used herein, the terms "silencing", "reducing", "inhibiting", "downregulating" or "knockdown" when referring to the expression of a given gene, mean that the expression of the gene is reduced when the cell, cell population, tissue, organ or subject is treated with a nucleic acid described herein, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein or protein subunit translated from mRNA in a cell, cell population, tissue, organ or subject in which the gene is transcribed, compared to a second cell, cell population, tissue, organ or subject not so treated.
本发明中，“完全互补的”意指在核碱基或核苷酸序列分子的杂交对中，第一寡核苷酸的邻接序列中的所有(100％)碱基，与第二寡核苷酸的邻接序列中的相同数目的碱基杂交。邻接序列可以包含第一核苷酸序列或第二核苷酸序列的全部或部分。In the present invention, "fully complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, all (100%) bases in the contiguous sequence of the first oligonucleotide hybridize with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may include all or part of the first nucleotide sequence or the second nucleotide sequence.
本发明中，“部分互补的”意指在核碱基或核苷酸序列分子的杂交对中，第一寡核苷酸的邻接序列中的至少70％但并非全部碱基，与第二寡核苷酸的邻接序列中的相同数目的碱基杂交。邻接序列可以包含第一核苷酸序列或第二核苷酸序列的全部或部分。In the present invention, "partially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 70% but not all of the bases in the contiguous sequence of the first oligonucleotide hybridize with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may include all or part of the first nucleotide sequence or the second nucleotide sequence.
本发明中，“基本上互补的”意指在核碱基或核苷酸序列分子的杂交对中，第一寡核苷酸的邻接序列中的至少85％但并非全部碱基，与第二寡核苷酸的邻接序列中的相同数目的碱基杂交。邻接序列可以包含第一核苷酸序列或第二核苷酸序列的全部或部分。In the present invention, "substantially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 85% but not all of the bases in the contiguous sequence of the first oligonucleotide hybridize with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may comprise all or part of the first nucleotide sequence or the second nucleotide sequence.
本发明中，当提及“至少部分互补的”意指在核碱基或核苷酸序列分子的杂交对中，第一寡核苷酸与第二寡核苷酸为部分互补的、基本上互补的或完全互补的。In the present invention, "at least partially complementary" means that the first oligonucleotide and the second oligonucleotide are partially complementary, substantially complementary or completely complementary in a hybridization pair of nucleobase or nucleotide sequence molecules.
本发明中，术语“治疗”意指为了提供受试者中的一种或多种疾病症状的数目、严重性和/或频率的缓解或减轻而采取的方法或步骤。所述的治疗可以包括受试者中的一种或多种疾病症状的数目、严重性和/或频率的预防、管理、预防性治疗、和/或抑制或降低。In the present invention, the term "treatment" means a method or step taken to provide relief or reduction in the number, severity and/or frequency of one or more disease symptoms in a subject. The treatment may include prevention, management, prophylactic treatment, and/or inhibition or reduction of the number, severity and/or frequency of one or more disease symptoms in a subject.
本发明中，术语“连接”意指两种化合物或分子通过共价键进行接合。除非另有说明，否则如本文使用的，术语“连接”可以指具有或不具有任何中间原子或原子群的第一化合物和第二化合物之间的连接。In the present invention, the term "connection" means that two compounds or molecules are joined by a covalent bond. Unless otherwise specified, as used herein, the term "connection" may refer to a connection between a first compound and a second compound with or without any intermediate atoms or atomic groups.
核酸Nucleic Acids
本发明提供了一种(被修饰或未被修饰的)核酸，该核酸包括正义链和反义链，所述正义链含有与SEQ ID NO：1～34任意一项所示序列具有80％(如80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)以上的序列同一性的序列，所述反义链含有与SEQ ID NO：35～68任意一项所示序列具有80％(如80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)以上的序列同一性的序列。The present invention provides a (modified or unmodified) nucleic acid, which comprises a sense strand and an antisense strand, wherein the sense strand comprises a sequence having a sequence identity of 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) or more to any one of SEQ ID NOs: 1 to 34, and the antisense strand comprises a sequence having a sequence identity of 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) or more to any one of SEQ ID NOs: 35 to 68.
在一些实施方式中，所述反义链具有15～30(如15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30)个核苷酸(碱基)。In some embodiments, the antisense strand has 15 to 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) nucleotides (bases).
在一些实施方式中，所述正义链具有15～30(如15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30)个核苷酸(碱基)。In some embodiments, the sense strand has 15 to 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) nucleotides (bases).
在具体实施时，本领域人员可以结合正义链和反义链的互补性考虑而对本发明中所提供的序列而进行组合，从而得到组合后的核酸(siRNA)。In specific implementation, those skilled in the art can combine the sequences provided in the present invention in consideration of the complementarity of the sense strand and the antisense strand, thereby obtaining a combined nucleic acid (siRNA).
在本发明的优选实施方式中，如表1所示，所述核酸选自正义链序列为SEQ ID NO：1且反义链序列为SEQ ID NO：35的siRNA-1、正义链序列为SEQ ID NO：2且反义链序列为SEQID NO：36的siRNA-2、正义链序列为SEQ ID NO：3且反义链序列为SEQ ID NO：37的siRNA-3、正义链序列为SEQ ID NO：4且反义链序列为SEQ ID NO：38的siRNA-4、正义链序列为SEQ IDNO：5且反义链序列为SEQ ID NO：39的siRNA-5……、siRNA-32、siRNA-33、siRNA-34中的至少一种。In a preferred embodiment of the present invention, as shown in Table 1, the nucleic acid is selected from at least one of siRNA-1 having a sense chain sequence of SEQ ID NO: 1 and an antisense chain sequence of SEQ ID NO: 35, siRNA-2 having a sense chain sequence of SEQ ID NO: 2 and an antisense chain sequence of SEQ ID NO: 36, siRNA-3 having a sense chain sequence of SEQ ID NO: 3 and an antisense chain sequence of SEQ ID NO: 37, siRNA-4 having a sense chain sequence of SEQ ID NO: 4 and an antisense chain sequence of SEQ ID NO: 38, siRNA-5 having a sense chain sequence of SEQ ID NO: 5 and an antisense chain sequence of SEQ ID NO: 39..., siRNA-32, siRNA-33, and siRNA-34.
在一些优选的实施方式中，所述反义链含有与SEQ ID NO：35～68任意一项所示序列相差不超过0个、1个、2个或3个核苷酸的至少15、16、17、18、19、20、21、22或23个连续核苷酸，所述正义链含有与所述反义链至少部分互补的(如部分互补的、基本上互补的或完全互补的)核苷酸序列。In some preferred embodiments, the antisense strand contains at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides that differ from the sequence shown in any one of SEQ ID NOs: 35 to 68 by no more than 0, 1, 2 or 3 nucleotides, and the positive strand contains a nucleotide sequence that is at least partially complementary (such as partially complementary, substantially complementary or completely complementary) to the antisense strand.
在一些优选的实施方式中，所述正义链含有与SEQ ID NO：1～34任意一项所示序列相差不超过0个、1个、2个或3个核苷酸的至少15、16、17、18、19、20或21个连续核苷酸。In some preferred embodiments, the sense strand contains at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ from the sequence shown in any one of SEQ ID NOs: 1 to 34 by no more than 0, 1, 2 or 3 nucleotides.
本发明中，所述正义链和反义链可以具有相同的长度，也可以具有不同的长度。In the present invention, the sense strand and the antisense strand may have the same length or different lengths.
上述核酸中所有核苷酸基团可以是未经化学修饰的，也可以含有至少一个修饰的核苷酸基团，且修饰可以在任意位置的核苷酸上。All nucleotide groups in the above nucleic acid may be chemically unmodified, or may contain at least one modified nucleotide group, and the modification may be on the nucleotide at any position.
在一些实施方式中，所述正义链和反义链可以是彼此部分互补、基本上互补或完全互补的。In some embodiments, the sense and antisense strands may be partially complementary, substantially complementary, or fully complementary to each other.
在一些优选的实施方式中，所述反义链含有与SEQ ID NO：35、36、37、38、39、41、43中任意一项所示序列相差0个、1个或2个核苷酸的核苷酸序列，所述正义链含有与所述反义链至少部分互补的(如部分互补的、基本上互补的或完全互补的)核苷酸序列。当反义链具有上述序列时，双链RNA对C5具有显著更优的抑制效果。In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in any one of SEQ ID NOs: 35, 36, 37, 38, 39, 41, and 43 by 0, 1, or 2 nucleotides, and the sense strand contains a nucleotide sequence that is at least partially complementary (such as partially complementary, substantially complementary, or completely complementary) to the antisense strand. When the antisense strand has the above sequence, the double-stranded RNA has a significantly better inhibitory effect on C5.
在一些优选的实施方式中，所述正义链含有与SEQ ID NO：1、2、3、4、5、7、9中任意一项所示序列相差0个、1个或2个核苷酸的核苷酸序列。In some preferred embodiments, the sense strand contains a nucleotide sequence that differs from the sequence shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 7, and 9 by 0, 1, or 2 nucleotides.
在一些进一步优选的实施方式中，所述反义链含有与SEQ ID NO：35、36中任意一项所示序列相差0个、1个或2个核苷酸的核苷酸序列，所述正义链含有与所述反义链至少部分互补的(如部分互补的、基本上互补的或完全互补的)核苷酸序列。当反义链具有该序列时，经过不同修饰的双链RNA对C5的抑制效果均进一步更优。In some further preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in any one of SEQ ID NOs: 35 and 36 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that is at least partially complementary (such as partially complementary, substantially complementary or completely complementary) to the antisense strand. When the antisense strand has this sequence, the inhibitory effect of double-stranded RNAs with different modifications on C5 is further improved.
在一些优选的实施方式中，所述正义链含有与SEQ ID NO：1、2中任意一项所示序列相差0个、1个或2个核苷酸的核苷酸序列。In some preferred embodiments, the sense strand contains a nucleotide sequence that differs from the sequence shown in any one of SEQ ID NOs: 1 and 2 by 0, 1 or 2 nucleotides.
在一些最优选的实施方式中，所述反义链含有与SEQ ID NO：36中所示序列相差0个、1个或2个核苷酸的核苷酸序列，所述正义链含有与所述反义链至少部分互补的(如部分互补的、基本上互补的或完全互补的)核苷酸序列。当反义链具有该序列时，经过不同修饰的双链RNA对C5的抑制效果均最优。In some most preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 36 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that is at least partially complementary (such as partially complementary, substantially complementary or completely complementary) to the antisense strand. When the antisense strand has this sequence, the inhibitory effects of double-stranded RNAs with different modifications on C5 are optimal.
在一些优选的实施方式中，所述正义链含有与SEQ ID NO：2中任意一项所示序列相差0个、1个或2个核苷酸的核苷酸序列。In some preferred embodiments, the sense strand contains a nucleotide sequence that differs from the sequence shown in any one of SEQ ID NO:2 by 0, 1 or 2 nucleotides.
在一些实施方式中，所述核酸中的正义链或反义链与本发明所提及的对应序列的序列同一性低于100％或相差1个以上核苷酸时，仍然具有与所述对应序列相近或相当的对C5的抑制效果。例如，在所述核酸的反义链的3’端所连接的UU替换为AA，CC，GG或UG等，或任何两个核酸的组合。这样的核酸序列也属于本发明的保护范围。In some embodiments, when the sequence identity of the sense strand or antisense strand in the nucleic acid with the corresponding sequence mentioned in the present invention is less than 100% or differs by more than 1 nucleotide, it still has a similar or equivalent inhibitory effect on C5 as the corresponding sequence. For example, the UU connected to the 3' end of the antisense strand of the nucleic acid is replaced by AA, CC, GG or UG, etc., or a combination of any two nucleic acids. Such nucleic acid sequences also fall within the scope of protection of the present invention.
本发明中所提到的上述关于裸序列(即未经修饰的序列)的技术方案，其效果优势并不依赖于修饰方法或靶向载体的选择。以下对其可以适用的修饰方案及进一步优选的修饰方案进行展开介绍：The above-mentioned technical solution for naked sequence (i.e., unmodified sequence) mentioned in the present invention has an effect advantage that does not depend on the selection of modification method or targeting vector. The following is an introduction to the applicable modification scheme and further preferred modification scheme:
根据本发明所述的核酸，其中，所述核酸含有核苷酸基团作为基本结构单元，所述核苷酸基团含有磷酸基团、核糖基团和碱基，优选情况下，所述核酸含有至少一个修饰的核苷酸基团。修饰后的核酸对C5的抑制效率不低于50％(如50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)。According to the nucleic acid of the present invention, the nucleic acid contains a nucleotide group as a basic structural unit, and the nucleotide group contains a phosphate group, a ribose group and a base. Preferably, the nucleic acid contains at least one modified nucleotide group. The inhibition efficiency of the modified nucleic acid on C5 is not less than 50% (such as 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).
根据本发明所述的核酸，其中，所述修饰的核苷酸基团为磷酸基团和/或核糖基团被修饰的核苷酸基团。带修饰的位点可以在正义链和/或反义链的第1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30位核苷酸中的至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30位上。According to the nucleic acid of the present invention, wherein the modified nucleotide group is a nucleotide group in which a phosphate group and/or a ribose group is modified. The modified site can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 of the 1st, 2nd, 3rd, 4th, 5th, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30th nucleotide of the sense strand and/or the antisense strand.
例如，磷酸基团的修饰是指对磷酸基团中的氧进行修饰，包括硫代磷酸修饰(Phosphorthioate)和硼烷化磷酸盐修饰(Boranophosphate)等。如下式所示分别用硫、硼烷、胺基、烷基或烷氧基置换磷酸基团中的氧。这几种修饰都能稳定核酸的结构，保持碱基配对的高特异性和高亲和力。For example, modification of the phosphate group refers to modification of the oxygen in the phosphate group, including phosphorothioate modification and boranophosphate modification. As shown in the following formula, the oxygen in the phosphate group is replaced by sulfur, borane, amine, alkyl or alkoxy. These modifications can stabilize the structure of nucleic acids and maintain high specificity and high affinity of base pairing.
以上结构式中，BASE表示碱基A、U、C、G或T。X可以是氧(O)或硫(S)。R在以上结构中可以是相同的，也可以是不同的，比如：氢(H)、氟(F)、甲氧基(OME)或甲氧乙基(MOE)、羟基、烯丙基、乙基胺基、炔丙基、氨基、氰基乙基、乙酰基等，R’和R”可以各自独立地是氢(H)、甲基(CH3)、乙基(CH2CH3)、丙基(CH2CH2CH3)、异丙基(CH(CH3)2)、烯丙基、炔丙基、酰氧基苄基、酰氧乙基。In the above structural formula, BASE represents the base A, U, C, G or T. X can be oxygen (O) or sulfur (S). R in the above structure can be the same or different, such as: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, amino, cyanoethyl, acetyl, etc., and R' and R" can each independently be hydrogen (H), methyl (CH3), ethyl (CH2CH3), propyl (CH2CH2CH3), isopropyl (CH(CH3)2), allyl, propargyl, acyloxybenzyl, acyloxyethyl.
核糖基团的修饰是指对核糖基团中2′-羟基(2′-OH)的修饰。在核糖基团的2′-羟基位置引入某些取代基如甲氧基或氟后，使核酸不易被核糖核酸酶切割，由此增加了核酸的稳定性，使核酸具有更强的抵抗核酸酶水解的性能。对核苷酸戊糖中2′-羟基的修饰包括2′-氟修饰(2′-fluoromodification,如2’-arabino-fluoro modification)、2′-甲氧基修饰(2′-OME)、2′-甲氧乙基修饰(2′-MOE)、2′-2,4-二硝基苯酚修饰(2′-DNPmodification)、环锁乙基修饰(2′,4′-constrained ethylmodification)、2′-氨基修饰(2′-Amino modification)、2′-脱氧修饰(2′-Deoxy modification)、BNA、无环核酸修饰、错位核酸修饰、L型核酸修饰等。BNA(内环桥接核苷酸)是指受约束的或不能接近的核苷酸。BNA可以含有五元环、六元环、或七元环的具有“固定的”C 3'-内切糖缩拢的桥联结构。通常将该桥并入该核糖环的2'-、4'-位处以提供2',4'-BNA核苷酸，如锁乙基修饰(LNA)、环锁乙基修饰(ENA)和乙基锁核酸修饰(cET BNA)。无环核酸是核苷酸的糖环被打开形成的核苷酸，如解锁核酸(UNA)核苷酸和甘油核酸(GNA)核苷酸。错位核酸修饰是指3’,5’-磷酸键链接被2’,5’-磷酸键链所代替。L型核酸修饰是指天然存在的D型核酸被替换为其镜像立体对应体L型核酸。The modification of the ribose group refers to the modification of the 2′-hydroxyl (2′-OH) in the ribose group. After introducing certain substituents such as methoxy or fluorine at the 2′-hydroxyl position of the ribose group, the nucleic acid is not easily cut by ribonucleases, thereby increasing the stability of the nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis. The modification of the 2′-hydroxyl in the pentose of the nucleotide includes 2′-fluoro modification (2′-fluoromodification, such as 2′-arabino-fluoro modification), 2′-methoxy modification (2′-OME), 2′-methoxyethyl modification (2′-MOE), 2′-2,4-dinitrophenol modification (2′-DNP modification), 2′,4′-constrained ethyl modification, 2′-amino modification (2′-Amino modification), 2′-deoxy modification (2′-Deoxy modification), BNA, acyclic nucleic acid modification, misaligned nucleic acid modification, L-type nucleic acid modification, etc. BNA (endocyclic bridged nucleotide) refers to a constrained or inaccessible nucleotide. BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C 3'-endo sugar condensed bridge structure. The bridge is usually incorporated into the 2'-, 4'-position of the ribose ring to provide a 2', 4'-BNA nucleotide, such as a locked ethyl modification (LNA), a ring locked ethyl modification (ENA) and an ethyl locked nucleic acid modification (cET BNA). Acyclic nucleic acids are nucleotides formed by opening the sugar ring of the nucleotide, such as unlocked nucleic acid (UNA) nucleotides and glycerol nucleic acid (GNA) nucleotides. Misplaced nucleic acid modification refers to the replacement of a 3', 5'-phosphate bond link by a 2', 5'-phosphate bond chain. L-type nucleic acid modification refers to the replacement of a naturally occurring D-type nucleic acid with its mirror stereo equivalent, an L-type nucleic acid.
其中，BASE表示碱基A、U、C、G或T。R在以上结构中可以是相同的，也可以是不同的，比如：氢(H)、氟(F)、甲氧基(OME)或甲氧乙基(MOE)、羟基、烯丙基、乙基胺基、炔丙基、氰基乙基、乙酰基等。Wherein, BASE represents the base A, U, C, G or T. R in the above structure can be the same or different, such as: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl, acetyl, etc.
根据本发明所述的核酸，其中，优选情况下，核糖基团被修饰的核苷酸基团为核糖基团的2’-OH被甲氧基或氟取代的核苷酸基团。According to the nucleic acid of the present invention, preferably, the nucleotide group in which the ribose group is modified is a nucleotide group in which the 2'-OH of the ribose group is substituted by a methoxy group or a fluorine group.
根据本发明的一种特别优选的实施方式，其中，所述核酸的正义链中含有尿嘧啶碱基或胞嘧啶碱基的核苷酸基团为所述核糖基团被修饰的核苷酸基团，即，所述核酸的正义链中含有尿嘧啶碱基或胞嘧啶碱基的核苷酸基团中的核糖基团的2’-OH被甲氧基或氟取代。更优选地，所述核酸的正义链和反义链的3’端均可以连接有dTdT；或者，所述核酸的反义链的3’端可以连接有AA或UU或任何两个核酸的组合(可以是但不局限于CC，GG或UG)，使序列具有特异性对mRNA降解的诱因。具有以上修饰的核酸表现出更为优异的体内抑制效果，且上述修饰能够进一步降低本发明的核酸在体内的免疫原性。According to a particularly preferred embodiment of the present invention, the nucleotide group containing uracil base or cytosine base in the sense strand of the nucleic acid is a nucleotide group in which the ribose group is modified, that is, the 2'-OH of the ribose group in the nucleotide group containing uracil base or cytosine base in the sense strand of the nucleic acid is replaced by methoxy or fluorine. More preferably, the 3' ends of the sense strand and the antisense strand of the nucleic acid can be connected with dTdT; or, the 3' end of the antisense strand of the nucleic acid can be connected with AA or UU or a combination of any two nucleic acids (which can be but is not limited to CC, GG or UG), so that the sequence has a specific inducement to mRNA degradation. The nucleic acid with the above modification shows a more excellent inhibitory effect in vivo, and the above modification can further reduce the immunogenicity of the nucleic acid of the present invention in vivo.
本发明的核酸还可以包括在反义链5’端连接一单磷酸核苷的修饰。由于siRNA导链终端的5’-单磷酸对于RISC识别是重要的。其中5’-羟基的磷酸化对siRNA能否有效地装载到细胞内部的Ago2上起了一定程度的作用。在siRNA中的导链5’端单磷酸盐与Argonaute-2(Ago2)有H键的相互作用，从而确保对mRNA的靶标有准确定位和精确切割。常用的5’-单磷酸核苷的衍生物有如下几种，此类磷酸核苷的衍生物已被证实在生物代谢介质中具有一定稳定性，对促进siRNA导链装载到细胞内部的Ago2有一定作用(NucleicAcids Research,2015,43,2993–3011)。根据本发明所述的核酸，其中，优选情况下，反式-乙烯基磷酸酯(VP)作为首选，也可以包括上面所述以外的单磷酸核苷的衍生物。The nucleic acid of the present invention may also include a modification of a monophosphate nucleoside connected to the 5' end of the antisense strand. Since the 5'-monophosphate at the terminal of the siRNA guide strand is important for RISC recognition. Among them, the phosphorylation of the 5'-hydroxyl group plays a certain role in whether the siRNA can be effectively loaded onto the Ago2 inside the cell. The monophosphate at the 5' end of the guide strand in the siRNA has an H bond interaction with Argonaute-2 (Ago2), thereby ensuring accurate positioning and precise cutting of the mRNA target. Commonly used 5'-monophosphate nucleoside derivatives are as follows. Such phosphate nucleoside derivatives have been proven to have certain stability in biological metabolic media and have a certain effect on promoting the loading of the siRNA guide strand into the Ago2 inside the cell (Nucleic Acids Research, 2015, 43, 2993-3011). According to the nucleic acid of the present invention, wherein, preferably, trans-vinyl phosphate (VP) is the first choice, and monophosphate nucleoside derivatives other than those described above may also be included.
以上结构中，BASE表示碱基A、U、C、G或T。R在以上结构中可以是相同的，也可以是不同的，比如：氢(H)、氟(F)、甲氧基(OME)或甲氧乙基(MOE)、羟基、烯丙基、乙基胺基、炔丙基、氰基乙基、氨基、乙酰基等。In the above structures, BASE represents the base A, U, C, G or T. R in the above structures can be the same or different, such as hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl, amino, acetyl, etc.
在一些实施方式中，所述核酸中的至少一个核苷酸是修饰的核苷酸或包括修饰的间键。In some embodiments, at least one nucleotide in the nucleic acid is a modified nucleotide or includes a modified linkage.
在一些优选的实施方式中，所述修饰的核苷酸选自2'-O-甲基核苷酸、2'-氟核苷酸、2'-脱氧核苷酸、2',3'-开环核苷酸模拟物、锁定的核苷酸、2'-F-阿拉伯糖核苷酸、2'-甲氧基乙基核苷酸、脱碱基核苷酸、核糖醇、反向核苷酸、反向2'-O-甲基核苷酸、反向2'-脱氧核苷酸、2'-氨基修饰的核苷酸、2'-烷基修饰的核苷酸、吗啉代核苷酸、含有乙烯基膦酸酯的核苷酸、含有环丙基膦酸酯的核苷酸和3'-O-甲基核苷酸中的一种或多种；所述修饰的核苷酸进一步优选自2'-O-甲基核苷酸和2'-氟核苷酸中的一种或多种。In some preferred embodiments, the modified nucleotide is selected from one or more of 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-open ring nucleotide mimics, locked nucleotides, 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitols, reverse nucleotides, reverse 2'-O-methyl nucleotides, reverse 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, nucleotides containing vinyl phosphonate, nucleotides containing cyclopropyl phosphonate and 3'-O-methyl nucleotides; the modified nucleotide is further preferably selected from one or more of 2'-O-methyl nucleotides and 2'-fluoro nucleotides.
在一些优选的实施方式中，所述修饰的间键优选自硫代磷酸酯核苷酸间键和甲基膦酸酯核苷酸间键中的一种或多种；所述修饰的间键进一步优选自硫代磷酸酯单酯核苷酸间键、硫代磷酸二酯核苷酸间键中的一种或多种。In some preferred embodiments, the modified inter-linkage is preferably selected from one or more of phosphorothioate inter-nucleotide linkages and methylphosphonate inter-nucleotide linkages; the modified inter-linkage is further preferably selected from one or more of phosphorothioate monoester inter-nucleotide linkages and phosphorothioate diester inter-nucleotide linkages.
在一些实施方式中，所述反义链5'末端和3'末端处分别含有2个硫代磷酸酯核苷酸间键，且含有4～6个2'-氟核苷酸，其余核苷酸均为2'-O-甲基核苷酸。In some embodiments, the antisense strand contains two phosphorothioate internucleotide bonds at the 5' end and the 3' end, respectively, and contains 4 to 6 2'-fluoro nucleotides, and the remaining nucleotides are 2'-O-methyl nucleotides.
在一些实施方式中，所述正义链的5'末端含有2个硫代磷酸酯核苷酸间键，且含有2～4个2'-氟核苷酸，其余核苷酸均为2'-O-甲基核苷酸。In some embodiments, the 5' end of the sense strand contains two phosphorothioate internucleotide bonds and 2 to 4 2'-fluoro nucleotides, and the remaining nucleotides are all 2'-O-methyl nucleotides.
在一些优选的实施方式中，所述反义链在从5'端计数的第2、4、6、14和16处核苷酸为2'-氟核苷酸。In some preferred embodiments, the antisense strand has 2'-fluoro nucleotides at the 2nd, 4th, 6th, 14th and 16th nucleotides counted from the 5' end.
在一些优选的实施方式中，所述正义链在从5'端计数的第7、9和11处核苷酸为2'-氟核苷酸。In some preferred embodiments, the sense strand has 2'-fluoro nucleotides at the 7th, 9th and 11th nucleotides counted from the 5' end.
在一些优选的实施方式中，所述反义链具有如表2中任意一种所示的反义链序列相差不超过0个、1个、2个或3个核苷酸的至少15、16、17、18、19、20、21、22或23个连续核苷酸，所述正义链具有如表2中任意一种所示的正义链序列相差不超过0个、1个、2个或3个核苷酸的至少15、16、17、18、19、20或21个连续核苷酸。In some preferred embodiments, the antisense strand has at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides that differ from the antisense strand sequence of any one of Table 2 by no more than 0, 1, 2 or 3 nucleotides, and the sense strand has at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ from the sense strand sequence of any one of Table 2 by no more than 0, 1, 2 or 3 nucleotides.
在一些优选的实施方式中，所述正义链和反义链形成具有表3中任意一种所示的siRNA。In some preferred embodiments, the sense strand and the antisense strand form a siRNA having any one of the sequences shown in Table 3.
在一些实施方式中，所述反义链从5'端到3'端含有与以下核苷酸序列相差0个、1个或2个核苷酸的核苷酸序列：In some embodiments, the antisense strand contains a nucleotide sequence from the 5' end to the 3' end that differs from the following nucleotide sequence by 0, 1 or 2 nucleotides:
SN-51263：asUfsuAfgAfcggaaucUfuGfaagucsusu；SN-51263: asUfsuAfgAfcggaaucUfuGfaagucsusu;
SN-51274：gsUfsgUfuUfugcauugCfuGfuuugcsusu；SN-51274:gsUfsgUfuUfugcauugCfuGfuuugcsusu;
SN-51276：usAfsgUfaUfgacaguuCfuUfugacususu；SN-51276:usAfsgUfaUfgacaguuCfuUfugacususu;
SN-51277：usUfsuGfaUfgccagguAfuUfucugcsusu；SN-51277:usUfsuGfaUfgccagguAfuUfucugcsusu;
SN-51279：usUfsuGfaUfgccagguAfuUfucugcsusu；SN-51279:usUfsuGfaUfgccagguAfuUfucugcsusu;
SN-51283：asGfsaGfaGfgacauauUfuGfaugccsusu；SN-51283: asGfsaGfaGfgacauauUfuGfaugccsusu;
SN-51293：usUfsuGfaCfauuaaacUfcCfagcacsusu；SN-51293:usUfsuGfaCfauuaaacUfcCfagcacsusu;
所述正义链含有与所述反义链至少部分互补的核苷酸序列。The sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand.
在一些实施方式中，所述正义链从5'端到3'端含有如以下任意一项核苷酸序列相差0个、1个或2个核苷酸的核苷酸序列：In some embodiments, the sense strand contains a nucleotide sequence from the 5' end to the 3' end that differs by 0, 1 or 2 nucleotides from any of the following nucleotide sequences:
SN-21263：gsascuucAfaGfaUfuccgucuaau；SN-21263:gsascuucAfaGfaUfuccgucuaau;
SN-21274：gscsaaacAfgCfaAfugcaaaacac；SN-21274: gscsaaacAfgCfaAfugcaaaacac;
SN-21276：asgsucaaAfgAfaCfugucauacua；SN-21276: asgsucaaAfgAfaCfugucauacua;
SN-21277：gsuscaaaGfaAfcUfgucauacuac；SN-21277:gsuscaaaGfaAfcUfgucauacuac;
SN-21279：gscsagaaAfuAfcCfuggcaucaaa；SN-21279:gscsagaaAfuAfcCfuggcaucaaa;
SN-21283：gsgscaucAfaAfuAfuguccucucu；SN-21283:gsgscaucAfaAfuAfuguccucucu;
SN-21293：gsusgcugGfaGfuUfuaaugucaaa。SN-21293:gsusgcugGfaGfuUfuaaugucaaa.
在本发明的各序列中，以小写字母表示的核苷酸代表该核苷酸为2'-O-甲基核苷酸；f表示其左侧相邻的一个核苷酸为2’-氟核苷酸；s代表左右相邻的两个核苷酸之间通过硫代磷酸二酯键连接。In each sequence of the present invention, a nucleotide represented by a lowercase letter represents that the nucleotide is a 2'-O-methyl nucleotide; f represents that the adjacent nucleotide on its left is a 2'-fluoro nucleotide; s represents that the two adjacent nucleotides on the left and right are connected by a phosphorothioate diester bond.
在一些实施方式中，所述反义链从5'端到3'端含有与以下核苷酸序列相差0个、1个或2个核苷酸的核苷酸序列：In some embodiments, the antisense strand contains a nucleotide sequence from the 5' end to the 3' end that differs from the following nucleotide sequence by 0, 1 or 2 nucleotides:
SN-51263：usCfsuUfcUfaccauguCfaAfcuucususu；SN-51263:usCfsuUfcUfaccauguCfaAfcuucususu;
SN-51274：gsUfsgUfuUfugcauugCfuGfuuugcsusu；SN-51274:gsUfsgUfuUfugcauugCfuGfuuugcsusu;
所述正义链含有与所述反义链至少部分互补的核苷酸序列。The sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand.
在一些优选的实施方式中，所述正义链从5'端到3'端含有如以下任意一项核苷酸序列相差0个、1个或2个核苷酸的核苷酸序列：In some preferred embodiments, the sense strand contains a nucleotide sequence from the 5' end to the 3' end that differs by 0, 1 or 2 nucleotides from any of the following nucleotide sequences:
SN-21263：gsascuucAfaGfaUfuccgucuaau；SN-21263:gsascuucAfaGfaUfuccgucuaau;
SN-21274：gscsaaacAfgCfaAfugcaaaacac。SN-21274: gscsaaacAfgCfaAfugcaaaacac.
在具体实施时，本领域人员可以结合对正义链和反义链的互补性的考虑，而对上述所提到的正义链和反义链的序列进行组合。在一种优选的实施方式中，将编号倒数三位数一致的正义链和反义链序列进行组合，以得到相应的核酸(siRNA)。比如，将SN-21263和SN-51263组合，将SN-21274和SN-51274组合，以得到相应的核酸(siRNA)。In specific implementation, those skilled in the art can combine the sense strand and antisense strand sequences mentioned above in consideration of the complementarity of the sense strand and the antisense strand. In a preferred embodiment, the sense strand and antisense strand sequences with the same last three digits of the number are combined to obtain the corresponding nucleic acid (siRNA). For example, SN-21263 and SN-51263 are combined, and SN-21274 and SN-51274 are combined to obtain the corresponding nucleic acid (siRNA).
根据本发明所述的核酸，可以通过本领域常规的方法获得，例如通过固相合成和液相合成得到，所述固相合成已经有商业的订制服务，因此可以通过商购获得。所述修饰的核苷酸基团可以通过具有相应修饰的核苷酸单体引入。The nucleic acid according to the present invention can be obtained by conventional methods in the art, such as solid phase synthesis and liquid phase synthesis, and the solid phase synthesis has commercial custom services and can therefore be obtained commercially. The modified nucleotide group can be introduced by a nucleotide monomer having a corresponding modification.
基于如上合成的核酸(siRNA)，本发明可以进一步构建与上述siRNA具有相同或相似功能的shRNA表达质粒，构建该表达质粒的方法为本领域技术人员所公知，在此不再赘述。Based on the nucleic acid (siRNA) synthesized as above, the present invention can further construct an shRNA expression plasmid having the same or similar function as the above siRNA. The method for constructing the expression plasmid is well known to those skilled in the art and will not be described in detail here.
本发明还提供了如上所述的核酸的靶向基因位点。在一些实施方式中，所述靶向基因位点如表1第1列中的任意一项所标注。The present invention also provides a targeted gene site of the nucleic acid as described above. In some embodiments, the targeted gene site is marked as any one of the items in column 1 of Table 1.
表1Table 1
注：第1列指靶向基因的首个碱基在C5基因序列中的位置，以此类推；第3、5列中数字表示序列编号，例如，“1”表示SEQ ID NO：1。Note: The first column refers to the position of the first base of the targeted gene in the C5 gene sequence, and so on; the numbers in the third and fifth columns represent the sequence numbers, for example, "1" represents SEQ ID NO: 1.
其中，靶向基因的参考序列为人补体成分5的编码序列NM_001317163.2。Among them, the reference sequence of the targeted gene is the coding sequence of human complement component 5 NM_001317163.2.
靶向药物递送系统Targeted drug delivery system
本发明还提供了一种靶向药物递送系统，其特征在于，该靶向药物递送系统包括靶向基团、连接基团和通过连接基团与靶向基团连接的如上所述的核酸。The present invention also provides a targeted drug delivery system, characterized in that the targeted drug delivery system comprises a targeting group, a linking group and the nucleic acid as described above connected to the targeting group via the linking group.
结合本领域常识而将本发明的核酸(siRNA)在应用于不同的靶向药物递送系统时，均具有较优的抑制效果。换言之，本发明中想要保护的裸序列以及修饰后的序列，其效果并不依赖于靶向载体的选择。为了进一步提高siRNA的生物利用度和治疗效果，本发明还对靶向药物递送系统进行了优化，并得到了如下的技术方案。In combination with common sense in the art, nucleic acid (siRNA) of the present invention has a better inhibitory effect when applied to different targeted drug delivery systems. In other words, the naked sequence and modified sequence that are intended to be protected in the present invention do not rely on the selection of the targeting vector for their effect. In order to further improve the bioavailability and therapeutic effect of siRNA, the present invention also optimizes the targeted drug delivery system and obtains the following technical scheme.
在一些实施方式中，所述连接基团与所述核酸的正义链或反义链的3'端或5'端连接。In some embodiments, the linker is linked to the 3' end or the 5' end of the sense strand or the antisense strand of the nucleic acid.
在一些具体实施方式中，所述连接基团与所述核酸的正义链的3'端连接。In some embodiments, the linker is attached to the 3' end of the sense strand of the nucleic acid.
在一些实施方式中，该靶向药物递送系统包括配体和与所述配体连接的所述的核酸。In some embodiments, the targeted drug delivery system comprises a ligand and the nucleic acid linked to the ligand.
在一些实施方式中，所述配体为是GalNAc衍生物。In some embodiments, the ligand is a GalNAc derivative.
在一些实施方式中，所述配体是通过单链、双链或三链支链接头连接的一种或多种GalNAc衍生物。In some embodiments, the ligand is one or more GalNAc derivatives connected by single-stranded, double-stranded, or triple-stranded branched linkers.
在一些优选的实施方式中，该靶向药物递送系统的结构如下式I所示：In some preferred embodiments, the structure of the targeted drug delivery system is shown in Formula I below:
式I中，Nu代表所述核酸(siRNA)。在一些实施方式中，该系统中的化合物部分可以通过磷酸二酯键与siRNA的正义链的5’端或3’端相连，也可以通过磷酸二酯键与siRNA的反义链的5’端或3’端相连。该靶向药物递送系统利用其左侧的结构特性能提高核酸药物(Nu)的细胞穿透能力，增强其在细胞内的稳定性，且制备工艺简单，实用性强。In formula I, Nu represents the nucleic acid (siRNA). In some embodiments, the compound part in the system can be connected to the 5' end or 3' end of the sense strand of the siRNA through a phosphodiester bond, and can also be connected to the 5' end or 3' end of the antisense strand of the siRNA through a phosphodiester bond. The targeted drug delivery system utilizes the structural characteristics on its left side to improve the cell penetration ability of the nucleic acid drug (Nu), enhance its stability in the cell, and has a simple preparation process and strong practicality.
药物组合物Pharmaceutical composition
本发明还提供了一种药物组合物，该药物组合物含有如上所述的核酸或靶向药物递送系统以及药学上可接受的载体。The present invention also provides a pharmaceutical composition, which contains the nucleic acid or targeted drug delivery system as described above and a pharmaceutically acceptable carrier.
可以通过常规的方法由所述核酸和所述药学上可接受的载体制备得到所述药物组合物。例如，所述药物组合物可以为注射液。所述注射液可以用于皮下、肌肉或静脉的注射。The pharmaceutical composition can be prepared from the nucleic acid and the pharmaceutically acceptable carrier by conventional methods. For example, the pharmaceutical composition can be an injection. The injection can be used for subcutaneous, intramuscular or intravenous injection.
根据本发明所述的药物组合物，其中，对核酸或靶向药物递送系统以及药学上可接受的载体的量没有特别的要求，一般地，相对于1重量份的所述核酸(或1重量份的以核酸计的靶向药物递送系统)，所述药学上可接受的载体的含量可以为1-100000重量份(如1重量份、5重量份、10重量份、50重量份、100重量份、500重量份、1000重量份、5000重量份、10000重量份、50000重量份、100000重量份或以上任意两个数值之间的任意值)。According to the pharmaceutical composition of the present invention, there is no special requirement for the amount of the nucleic acid or the targeted drug delivery system and the pharmaceutically acceptable carrier. Generally, relative to 1 part by weight of the nucleic acid (or 1 part by weight of the targeted drug delivery system calculated as nucleic acid), the content of the pharmaceutically acceptable carrier can be 1-100000 parts by weight (such as 1 part by weight, 5 parts by weight, 10 parts by weight, 50 parts by weight, 100 parts by weight, 500 parts by weight, 1000 parts by weight, 5000 parts by weight, 10000 parts by weight, 50000 parts by weight, 100000 parts by weight or any value between any two of the above values).
根据本发明所述的药物组合物，其中，所述药学上可接受的载体可以为本领域常规采用的各种载体，例如，可以包括pH值缓冲液、保护剂和渗透压调节剂中的至少一种。所述pH值缓冲液可以为pH值为7.5-8.5的三羟甲基胺基甲烷盐酸盐缓冲液和/或pH为5.5-8.5的磷酸盐缓冲液，优选为pH为5.5-8.5的磷酸盐缓冲液。所述保护剂可以为肌醇、山梨醇和蔗糖中的至少一种。以所述药物组合物的总重量为基准，所述保护剂的含量可以为0.01-30重量％(如0.01重量％、0.05重量％、0.1重量％、0.5重量％、1重量％、5重量％、10重量％、15重量％、20重量％、25重量％、30重量％或以上任意两个数值之间的任意值)。所述渗透压调节剂可以为氯化钠和/或氯化钾。所述渗透压调节剂的含量使所述药物组合物的渗透压为200-700毫渗摩尔/千克。根据所需渗透压，本领域技术人员可以确定所述渗透压调节剂的含量。According to the pharmaceutical composition of the present invention, wherein the pharmaceutically acceptable carrier can be various carriers conventionally used in the art, for example, it can include at least one of a pH buffer, a protective agent and an osmotic pressure regulator. The pH buffer can be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH value of 7.5-8.5 and/or a phosphate buffer with a pH value of 5.5-8.5, preferably a phosphate buffer with a pH value of 5.5-8.5. The protective agent can be at least one of inositol, sorbitol and sucrose. Based on the gross weight of the pharmaceutical composition, the content of the protective agent can be 0.01-30% by weight (such as 0.01% by weight, 0.05% by weight, 0.1% by weight, 0.5% by weight, 1% by weight, 5% by weight, 10% by weight, 15% by weight, 20% by weight, 25% by weight, 30% by weight or any value between any two of the above values). The osmotic pressure regulator can be sodium chloride and/or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 mOsmole/kg. According to the required osmotic pressure, those skilled in the art can determine the content of the osmotic pressure regulator.
根据本发明一种优选的实施方式，所述药学上可接受的载体为脂质体。脂质体可以为任何一种能包裹核酸的脂质体，其直径可以是25-1000nm，可以包括但不限于胆固醇及其类似物或衍生物。According to a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is a liposome. The liposome can be any liposome capable of encapsulating nucleic acid, and its diameter can be 25-1000 nm, and can include but not limited to cholesterol and its analogs or derivatives.
本发明所述药物组合物的使用剂量可以为本领域常规的剂量，所述剂量可以根据各种参数、尤其根据受试者的年龄、体重和性别来确定。例如，对于雌性，3-4月龄，体重25-30g的小鼠，以所述药物组合物中的所述核酸的量计，所述药物组合物的用量可以为0.01-100mg/kg体重，优选为1-10mg/kg体重。The dosage of the pharmaceutical composition of the present invention can be a conventional dosage in the art, and the dosage can be determined according to various parameters, especially according to the age, weight and gender of the subject. For example, for female, 3-4 month old mice weighing 25-30 g, based on the amount of the nucleic acid in the pharmaceutical composition, the dosage of the pharmaceutical composition can be 0.01-100 mg/kg body weight, preferably 1-10 mg/kg body weight.
方法及用途Methods and uses
本发明还提供一种抑制细胞中补体成分5基因的表达的方法，该方法包括：使所述细胞与如上所述的核酸、靶向药物递送系统或药物组合物接触，以抑制所述细胞中的补体成分5基因的表达。The present invention also provides a method for inhibiting the expression of complement component 5 gene in a cell, the method comprising: contacting the cell with the nucleic acid, targeted drug delivery system or pharmaceutical composition as described above to inhibit the expression of complement component 5 gene in the cell.
在一些实施方式中，所述细胞在受试者体内，例如，人类受试者，例如，患有补体成分5相关疾病的受试者。In some embodiments, the cell is in a subject, e.g., a human subject, e.g., a subject suffering from a complement component 5-associated disease.
在一些实施方式中，所述细胞位于体外。所述方法基于研究目的、或用于构建动物模型。In some embodiments, the cell is in vitro. The method is for research purposes or for constructing an animal model.
在一些实施方式中，使所述细胞与所述核酸接触抑制C5的表达的至少50％、60％、70％、80％、90％、95％(例如，与所述细胞首次与所述核酸接触之前的C5的表达水平相比；例如，在向所述受试者施用第一剂量的所述核酸之前)。在某些实施例中，抑制C5的表达使受试者血清样品中的C5蛋白水平降低至少50％、60％、70％、80％、90％或95％，例如，与所述细胞首次与所述核酸接触之前的C5的表达水平相比。In some embodiments, contacting the cell with the nucleic acid inhibits expression of C5 by at least 50%, 60%, 70%, 80%, 90%, 95% (e.g., compared to the expression level of C5 before the cell was first contacted with the nucleic acid; e.g., before the first dose of the nucleic acid was administered to the subject). In certain embodiments, inhibiting expression of C5 reduces the level of C5 protein in a serum sample of the subject by at least 50%, 60%, 70%, 80%, 90%, or 95%, e.g., compared to the expression level of C5 before the cell was first contacted with the nucleic acid.
本发明还提供如上所述的核酸、如上所述的靶向药物递送系统或如上所述的药物组合物在以下任一方面的用途：1)治疗和/或预防与补体成分5相关的疾病；2)制备用于治疗和/或预防与补体成分5相关的疾病的药物。The present invention also provides the use of the nucleic acid as described above, the targeted drug delivery system as described above, or the pharmaceutical composition as described above in any of the following aspects: 1) treating and/or preventing diseases associated with complement component 5; 2) preparing drugs for treating and/or preventing diseases associated with complement component 5.
在一些实施方式中，所述疾病为：(i)与补体成分5增强或升高相关的疾病；或(ii)将受益于补体成分5表达减少的疾病。In some embodiments, the disease is: (i) a disease associated with enhanced or elevated complement component 5; or (ii) a disease that would benefit from decreased complement component 5 expression.
在一些实施方式中，所述疾病为补体系统激活引起的相关疾病。In some embodiments, the disease is a disease associated with complement system activation.
在一些实施方式中，所述疾病选自以下疾病中的一种或几种：阵发性睡眠性血红蛋白尿症(PNH)、非典型溶血性尿毒症综合征(aHUS)、哮喘、类风湿性关节炎(RA)；抗磷脂抗体综合征；狼疮肾炎；缺血-再灌注损伤；典型的或感染性溶血性尿毒症综合征(tHUS)；致密沉积物病(DDD)；视神经脊髓炎(NMO)；多灶性运动神经病(MMN)；多发性硬化(MS)；黄斑变性(例如，年龄相关的黄斑变性(AMD))；溶血、肝脏酶升高以及血小板低(HELLP)综合征；血栓性血小板减少性紫癜(TTP)；自发性流产；寡免疫血管炎；大疱性表皮松解症；习惯性流产；先兆子痫、创伤性脑损伤、重症肌无力、冷凝集素病、皮肌炎大疱性类天疱疮、志贺毒素大肠杆菌相关的溶血性尿毒症综合征、C3肾病、抗嗜中性粒细胞细胞质抗体相关联的血管炎、体液和血管移植排斥、移植物功能异常、心肌梗塞、异源移植、脓毒症、冠状动脉病、皮肌炎、格雷夫斯病、动脉粥样硬化、阿耳茨海默氏病、全身性炎症反应脓毒症、脓毒性休克、脊髓损伤、肾小球性肾炎、桥本氏甲状腺炎、I型糖尿病、牛皮癣、天疱疮、自身免疫性溶血性贫血(AIHA)、ITP、肺出血肾炎综合征、德戈斯病、抗磷脂综合征(APS)、灾难性APS(CAPS)、心血管病症、心肌炎、脑血管疾病、外周血管疾病、肾血管疾病、肠系膜/肠血管疾病、血管炎、亨诺赫-舍恩莱因紫癜肾炎、系统性红斑狼疮(SLE)、全身性红斑狼疮相关联的血管炎、与类风湿性关节炎相关联的血管炎、免疫复合物血管炎、高安氏病、扩张性心肌病、糖尿病性血管病、川畸氏病(动脉炎)、静脉气体栓子(VGE)以及支架放置、旋转式动脉粥样斑块去除术、膜性肾病、格-巴二氏综合征和经皮穿刺冠状动脉成形术(PTCA)后的再狭窄。In some embodiments, the disease is selected from one or more of the following diseases: paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma, rheumatoid arthritis (RA); antiphospholipid antibody syndrome; lupus nephritis; ischemia-reperfusion injury; typical or infectious hemolytic uremic syndrome (tHUS); dense deposit disease (DDD); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); macular degeneration (e.g., senile age-related macular degeneration (AMD); hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous abortion; pauci-immune vasculitis; epidermolysis bullosa; recurrent abortions; preeclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis, bullous pemphigoid, Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome, C3 nephropathy, anti-neutrophil cytoplasmic antibody-associated vasculitis, humoral and vascular transplant rejection, allograft dysfunction Common, myocardial infarction, allogeneic transplantation, sepsis, coronary artery disease, dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory response sepsis, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), ITP, Goodpasture's syndrome, Degos' disease, antiphospholipid syndrome (APS), catastrophic APS (CAPS), cardiovascular disorders, myocarditis, cerebrovascular disease, peripheral vascular disease, kidney Vascular disease, mesenteric/intestinal vascular disease, vasculitis, Henoch-Schönlein purpura nephritis, systemic lupus erythematosus (SLE), vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic vasculopathy, Kawasaki's disease (arteritis), venous gas embolism (VGE) and restenosis after stent placement, rotational atherectomy, membranous nephropathy, Guillain-Barre syndrome, and percutaneous transluminal coronary angioplasty (PTCA).
本发明中，所述受试者可以为哺乳动物，包含灵长类动物(如人、非人灵长类动物，例如猴子和黑猩猩)、非灵长类动物(如牛、猪、马、山羊、兔子、绵羊、仓鼠、豚鼠、猫、狗、大鼠或小鼠)或鸟类。在一些实施方式中，所述受试者优选为灵长类动物，更优选为人。可以通过多种途径给药，取决于是需要局部治疗还是全身治疗。给药的方式可以为但不限于静脉内给药、动脉内给药、皮下给药、腹膜内给药、经皮给药(如通过植入器械)和软组织内给药。给药量可以参照前述，在此不再赘述。In the present invention, the subject may be a mammal, including primates (such as humans, non-human primates, such as monkeys and chimpanzees), non-primates (such as cattle, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats or mice) or birds. In some embodiments, the subject is preferably a primate, more preferably a human. Administration can be by a variety of routes, depending on whether local or systemic treatment is required. The mode of administration can be, but is not limited to, intravenous administration, intra-arterial administration, subcutaneous administration, intraperitoneal administration, transdermal administration (such as by implantation of a device) and administration into soft tissue. The dosage can refer to the above, and will not be repeated here.
在一些实施方式中，所述核酸、所述的靶向药物递送系统或所述的药物组合物通过皮下给药、静脉内给药和/或肌肉内给药而施用于受试者。In some embodiments, the nucleic acid, the targeted drug delivery system or the pharmaceutical composition is administered to the subject by subcutaneous administration, intravenous administration and/or intramuscular administration.
实施例Example
下面将结合实施例对本发明的实施方案进行详细描述。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，优先参考本发明中给出的指引，还可以按照本领域的实验手册或常规条件，还可以参考本领域已知的其它实验方法，或者按照制造厂商所建议的条件。Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.
下述的具体实施例中，涉及原料组分的量度参数，如无特别说明，可能存在称量精度范围内的细微偏差。涉及温度和时间参数，允许仪器测试精度或操作精度导致的可接受的偏差。In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.
实施例1siRNA的体外筛选Example 1 In vitro screening of siRNA
在筛选得到表1所示的正义链和反义链序列后，对各正义链和反义链进行修饰，修饰后的序列如表2所示。After screening to obtain the sense strand and antisense strand sequences shown in Table 1, each sense strand and antisense strand was modified, and the modified sequences are shown in Table 2.
表2Table 2
表中，a/c/g/u＝2’-OMe核苷酸；Af/Cf/Gf/Uf＝2’-F核苷酸；s＝硫代磷酸二酯键。In the table, a/c/g/u = 2'-OMe nucleotides; Af/Cf/Gf/Uf = 2'-F nucleotides; s = phosphorothioate diester bond.
通过固相合成法，将表2中的正义链和反义链形成表3中修饰后的双链siRNA。The sense strand and antisense strand in Table 2 were synthesized into the modified double-stranded siRNA in Table 3 by solid phase synthesis.
表3Table 3
表中，a/c/g/u＝2’-OMe核苷酸；Af/Cf/Gf/Uf＝2’-F核苷酸；s＝硫代磷酸二酯键。In the table, a/c/g/u = 2'-OMe nucleotides; Af/Cf/Gf/Uf = 2'-F nucleotides; s = phosphorothioate diester bond.
在96孔细胞培养皿内加入0.5毫升细胞培养液(DMEM,10％小牛血清,1％青霉素+链霉素溶液)含104Hep3B(Procell,Cat#CL-0102)细胞，在37℃,5％CO2的细胞培养器内过夜培养。在Opti-MEM培养液内加入RNAiMAX(1.5微升/孔)和表3中的小干扰核酸(siRNA)，加入细胞培养孔使最后每孔浓度为1nM或10nM，继续在37℃,5％ CO2的细胞培养期内培养48小时。为提取RNA，将细胞培养上清液吸净，用PBS冲洗，吸净后，加入50μL配制好的裂解液(按Cells-to-CT试剂盒(ThermoFisher Scientific,Cat#4391851c)建议)混匀，静置10min后，加入2.5μL Stop solution终止2min。根据High Capacity cDNA ReverseTranscription Kits(Thermo Fisher,货号：4368814)的建议做RT-PCR,每个反应内含10ml裂解后液体。用实时荧光PCR法测基因表达定量，人C5的TaqMan探针为Hs01004342_m1，内参基因(人HPRT1)的探针为Hs02800695_m1(Thermo Fisher Scientific,Waltham,MA,USA)。PCR条件为95℃ 20秒1个循环，95℃ 1秒和60℃20秒40个循环，实时荧光PCR仪为QuantStudioTM6Pro实时荧光定量PCR系统(Thermo Fisher)。C5基因表达是以2^-ΔΔCt计算，人HPRT1基因表达作为内参。C5基因表达量以和仅有RNAiMAX的细胞组为对照的相对值表达。结果见表4。0.5 ml of cell culture medium (DMEM, 10% calf serum, 1% penicillin + streptomycin solution) containing 10 4 Hep3B (Procell, Cat#CL-0102) cells were added to a 96-well cell culture dish and cultured overnight in a cell culture incubator at 37°C, 5% CO2. RNAiMAX (1.5 μL/well) and small interfering nucleic acids (siRNA) listed in Table 3 were added to the Opti-MEM culture medium and added to the cell culture wells to make the final concentration of each well 1 nM or 10 nM. The cells were cultured for 48 hours at 37°C, 5% CO2. To extract RNA, the cell culture supernatant was aspirated and washed with PBS. After aspiration, 50 μL of the prepared lysis solution (as recommended by the Cells-to-CT kit (ThermoFisher Scientific, Cat#4391851c)) was added and mixed. After standing for 10 minutes, 2.5 μL of Stop solution was added to terminate the process for 2 minutes. RT-PCR was performed according to the recommendations of High Capacity cDNA Reverse Transcription Kits (Thermo Fisher, Cat. No. 4368814), and each reaction contained 10 ml of lysed liquid. Gene expression was measured quantitatively by real-time fluorescence PCR, and the TaqMan probe for human C5 was Hs01004342_m1, and the probe for the internal reference gene (human HPRT1) was Hs02800695_m1 (Thermo Fisher Scientific, Waltham, MA, USA). The PCR conditions were 1 cycle of 95°C for 20 seconds, 40 cycles of 95°C for 1 second and 60°C for 20 seconds, and the real-time fluorescence PCR instrument was QuantStudioTM6Pro Real-time Fluorescence Quantitative PCR System (Thermo Fisher). C5 gene expression was calculated by 2^-ΔΔCt, and human HPRT1 gene expression was used as an internal reference. C5 gene expression was expressed as a relative value to the control group with only RNAiMAX. The results are shown in Table 4.
表4.siRNA在Hep3B细胞中对C5表达的敲低作用Table 4. Knockdown effect of siRNA on C5 expression in Hep3B cells
实施例2 siRNA的体内效果验证Example 2 Verification of the in vivo effect of siRNA
为了更进一步验证siRNA在体内的活性，按表5对siRNA加了肝细胞靶向的偶合物Tri-GalNAc(其具体结构见前述式I)。其中，作为对比的SN-68002根据US9850488B2的公开内容制备得到。In order to further verify the activity of siRNA in vivo, the hepatocyte-targeting conjugate Tri-GalNAc (its specific structure is shown in the above formula I) was added to the siRNA according to Table 5. Among them, SN-68002 used as a comparison was prepared according to the disclosure of US9850488B2.
表5Table 5
a/c/g/u＝2’-OMe核苷酸；Af/Cf/Gf/Uf＝2’-F核苷酸；s＝硫代磷酸二酯键。a/c/g/u = 2'-OMe nucleotides; Af/Cf/Gf/Uf = 2'-F nucleotides; s = phosphorothioate diester bond.
将人源C5转基因小鼠在第0天分别皮下注射了3mg/kg表5中的siRNA药物，且以PBS作为对照组，并在注射后第4天对血液中的人C5蛋白进行了观察，结果如图1。Human C5 transgenic mice were subcutaneously injected with 3 mg/kg of siRNA drugs listed in Table 5 on day 0, and PBS was used as the control group. The human C5 protein in the blood was observed on day 4 after injection. The results are shown in Figure 1.
由图1可以看出，表5中的siRNA药物均能够明显降低C5蛋白的表达水平。其中，SN-681263和SN-681274药物的在药效上显著优于其它药物。As can be seen from Figure 1, the siRNA drugs in Table 5 can significantly reduce the expression level of C5 protein. Among them, SN-681263 and SN-681274 drugs are significantly better than other drugs in terms of efficacy.
另外，按表6中所列siRNA药物，SN-681263和SN-681274又与SN-68002做了比较，在人源C5转基因小鼠中皮下注射3mg/kg后第4天检测血液中人C5蛋白，可以从图2中看出，SN-681263和SN-681274药物的药效也优于SN-68002药物。In addition, according to the siRNA drugs listed in Table 6, SN-681263 and SN-681274 were compared with SN-68002, and human C5 protein in the blood was detected on the 4th day after subcutaneous injection of 3 mg/kg in human C5 transgenic mice. As can be seen from Figure 2, the efficacy of SN-681263 and SN-681274 drugs is also better than that of SN-68002 drug.
表6Table 6
其中，表6中SN-681263和SN-681274所使用的Tri-galNac为式I所示的结构，SN-68002所使用的Tri-galNac为Alnylam Tri-GalNAc(L96)。式I所示的结构和L96在分类上均属于三触角N-乙酰半乳糖胺(TriantennaryN-Acetylgalactosamine)，因此，基于上述效果数据也能够确认本发明修饰后的序列所具有的优势。而本发明中的裸序列以及修饰后的序列的效果优势并不依赖于靶向载体的选择，在上述实施例中所使用的Tri-GalNAc更换为其他可用载体(如前述的L96)后，将所得到的siRNA药物与SN-68002相比，在体外细胞试验及体内实验中仍然具有显著效果优势。Among them, the Tri-galNac used in SN-681263 and SN-681274 in Table 6 is the structure shown in Formula I, and the Tri-galNac used in SN-68002 is Alnylam Tri-GalNAc (L96). The structure shown in Formula I and L96 are both classified as triantennary N-acetylgalactosamine. Therefore, based on the above effect data, the advantages of the modified sequence of the present invention can also be confirmed. The effect advantages of the naked sequence and the modified sequence in the present invention do not depend on the selection of the targeting vector. After the Tri-GalNAc used in the above examples is replaced with other available vectors (such as the aforementioned L96), the obtained siRNA drug still has a significant effect advantage compared with SN-68002 in in vitro cell tests and in vivo experiments.
为了进一步验证药效，将人源C5转基因小鼠在第0天分别皮下注射了3mg/kg的SN-681263或SN-681274药物，且以PBS作为对照组，并对血液中的人C5蛋白进行了追踪观察，结果如图3。To further verify the efficacy of the drug, human C5 transgenic mice were subcutaneously injected with 3 mg/kg of SN-681263 or SN-681274 on day 0, and PBS was used as the control group. The human C5 protein in the blood was tracked and observed. The results are shown in Figure 3.
由图3可知，SN-681263和SN-681274药物均能够明显降低C5蛋白的表达水平，且具有较为长久的药效，其中，SN-681274的药效更优。As shown in Figure 3, both SN-681263 and SN-681274 can significantly reduce the expression level of C5 protein and have a relatively long-lasting efficacy, among which SN-681274 has a better efficacy.
为了进一步验证SN-681263和SN-681274的药效，将人源C5转基因小鼠在第0天皮下注射了0.3，1，3mg/kg的SN-681263或SN-681274药物，且以PBS作为对照组，并对血液中的人C5蛋白进行了追踪观察，结果如图4中的A(SN-681263)和B(SN-681274)所示。To further verify the efficacy of SN-681263 and SN-681274, human C5 transgenic mice were subcutaneously injected with 0.3, 1, and 3 mg/kg of SN-681263 or SN-681274 on day 0, and PBS was used as the control group. The human C5 protein in the blood was tracked and observed. The results are shown in A (SN-681263) and B (SN-681274) in Figure 4.
从图4中可以看到SN-681263和SN-681274都能剂量依赖性地降低血清中C5蛋白水平，SN-681274能维持至少28天长久的药效。As can be seen from FIG4 , both SN-681263 and SN-681274 can reduce the level of C5 protein in serum in a dose-dependent manner, and SN-681274 can maintain a long-term efficacy for at least 28 days.
实施例3siRNA的序列优势验证Example 3 siRNA sequence advantage verification
为了比较未经修饰的siRNA在细胞中对C5基因表达的沉默效应，将表7中的siRNA以1nM和10nM浓度转染Hep3B细胞后孵育48小时，利用RT-qPCR方法检测C5基因沉默，可以从表8结果中看出，SN-121274在两个剂量上相较SN-12002均有更好的C5基因表达沉默药效。In order to compare the silencing effect of unmodified siRNA on C5 gene expression in cells, Hep3B cells were transfected with siRNA in Table 7 at concentrations of 1 nM and 10 nM and incubated for 48 hours. C5 gene silencing was detected by RT-qPCR. It can be seen from the results in Table 8 that SN-121274 has better C5 gene expression silencing efficacy than SN-12002 at both doses.
表7Table 7
表8Table 8
可见，本发明的siRNA在序列层面即具有明显效果优势，在沉默C5基因方面的效果显著优于已有的优势序列。It can be seen that the siRNA of the present invention has obvious effect advantages at the sequence level, and its effect in silencing the C5 gene is significantly better than that of the existing advantageous sequences.
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.
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