技术领域Technical Field
本发明涉及用于减少INHBE表达的siRNA、缀合物及药物组合物，属于生物医药领域。The present invention relates to siRNA, conjugate and pharmaceutical composition for reducing INHBE expression, belonging to the field of biomedicine.
背景技术Background Art
抑制素亚基βE(INHBE)是一种编码抑制素βE亚基的基因。主要在肝脏中表达，参与调节许多细胞过程，包括细胞增殖、细胞凋亡、免疫反应和激素分泌等。抑制素βE亚基同源二聚化形成分泌肽抑制素E，它是转化生长因子β(TGF-β)超家族的成员，在大量正常组织中普遍表达，在细胞增殖、凋亡、免疫反应和激素分泌过程中特别活跃，在肝脏中为最高表达。其具有旁分泌和内分泌功能，在肝细胞中局部作用以抑制细胞增殖，并作为脂肪细胞的肝因子以增加脂肪细胞标志物的表达。Inhibin subunit βE (INHBE) is a gene encoding the inhibin βE subunit. It is mainly expressed in the liver and is involved in regulating many cellular processes, including cell proliferation, apoptosis, immune response, and hormone secretion. Inhibin βE subunits homodimerize to form the secreted peptide inhibin E, which is a member of the transforming growth factor β (TGF-β) superfamily. It is ubiquitously expressed in a large number of normal tissues and is particularly active in cell proliferation, apoptosis, immune response, and hormone secretion. It is most highly expressed in the liver. It has paracrine and endocrine functions, acting locally in hepatocytes to inhibit cell proliferation, and as a hepatokine for adipocytes to increase the expression of adipocyte markers.
有研究者利用UK Biobank中超过36万个个体的全外显子测序数据，挖掘出与较低的经体重指数校正的腰臀比(WHRadj BMI)相关的十二种基因变异，发现INHBE基因中罕见的功能丧失(INHBE pLOF)是有助于更健康的脂肪分布的一个新的遗传因素。对INHBE pLOF突变体携带者的进一步分析表明，其分泌的抑制素E水平降低了约90％，显示出包括甘油三酯减少，高密度脂蛋白胆固醇增加以及空腹葡萄糖减少等有利的代谢情况。Using whole exome sequencing data from more than 360,000 individuals in UK Biobank, researchers have discovered twelve genetic variants associated with lower waist-to-hip ratios adjusted for body mass index (WHRadj BMI), and found that rare loss of function in the INHBE gene (INHBE pLOF) is a new genetic factor that contributes to healthier fat distribution. Further analysis of INHBE pLOF mutant carriers showed that their secreted inhibin E levels were reduced by about 90%, showing favorable metabolic conditions including reduced triglycerides, increased high-density lipoprotein cholesterol, and reduced fasting glucose.
流行病学和遗传学研究都表明了脂肪组织的分布在代谢健康和心血管疾病风险中发挥作用。并且以腰臀比增加为标志的腹部肥胖是心血管疾病的风险标志，同时多项全基因组关联研究也指出与腹部脂肪堆积相关的遗传变异与驱动胰岛素抵抗、血脂异常和非酒精性脂肪性肝炎等代谢性疾病的肝脂肪变性相关。Both epidemiological and genetic studies have shown that the distribution of adipose tissue plays a role in metabolic health and cardiovascular disease risk. Abdominal obesity, marked by an increased waist-to-hip ratio, is a risk marker for cardiovascular disease, while multiple genome-wide association studies have also pointed out that genetic variants associated with abdominal fat accumulation are associated with hepatic steatosis, which drives metabolic diseases such as insulin resistance, dyslipidemia, and non-alcoholic steatohepatitis.
目前对肥胖的治疗包括生活方式的改变、节食、锻炼和药物治疗，如降脂剂，如他汀类药物和其他药物。然而，这些疗法和治疗往往受到依从性的限制，并不总是有效的，往往会导致副作用并导致药物相互作用。因此，为了有效控制腹部肥胖，减少心血管疾病的发生，需要开发更多的药物来使脂肪更健康的分布。Current treatments for obesity include lifestyle changes, diet, exercise, and medications, such as lipid-lowering agents, such as statins and other drugs. However, these therapies and treatments are often limited by compliance, are not always effective, often cause side effects and lead to drug interactions. Therefore, in order to effectively control abdominal obesity and reduce the occurrence of cardiovascular disease, more drugs need to be developed to achieve a healthier distribution of fat.
发明内容Summary of the invention
为了解决上述的技术问题，本发明提供的如下siRNA及其修饰序列能够特异性地抑制抑制素亚单位βE(INHBE)基因表达，含有siRNA的药物组合物或siRNA缀合物能够特异性地靶向肝脏，从而可以抑制调节抑制素亚单位βE(INHBE)基因表达，实现与肥胖相关联疾病和/或病症的治疗。In order to solve the above-mentioned technical problems, the following siRNA and its modified sequence provided by the present invention can specifically inhibit the expression of inhibin subunit βE (INHBE) gene, and the pharmaceutical composition or siRNA conjugate containing siRNA can specifically target the liver, thereby inhibiting and regulating the expression of inhibin subunit βE (INHBE) gene, thereby achieving the treatment of diseases and/or conditions associated with obesity.
在一些实施方案中，本发明提供了第一种能够抑制抑制素亚单位βE(INHBE)基因表达的siRNA，该siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO：1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO：2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：In some embodiments, the present invention provides a first siRNA capable of inhibiting the expression of the inhibin subunit βE (INHBE) gene, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 1, and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 2, and differs by no more than 3 nucleotides:
5’-CUCUUUGCUUGAGGAUCUUUU-3’(SEQ ID NO:1)；5’-CUCUUUGCUUGAGGAUCUUUU-3’ (SEQ ID NO: 1);
5’-AAGAUCCUCAAGCAAAGAGUG-3’(SEQ ID NO:2)。5'-AAGAUCCUCAAGCAAAGAGUG-3' (SEQ ID NO: 2).
在一些实施方案中，本发明提供了第二种能够抑制抑制素亚单位βE(INHBE)基因表达的siRNA，该siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO：3所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO：4所示的核苷酸序列长度相等，且不多于3个核苷酸差异：In some embodiments, the present invention provides a second siRNA capable of inhibiting the expression of the inhibin subunit βE (INHBE) gene, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 3, and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 4, and differs by no more than 3 nucleotides:
5’-GCUUAAGAUCCGAGCCAAUUU-3’(SEQ ID NO:3)；5’-GCUUAAGAUCCGAGCCAAUUU-3’ (SEQ ID NO: 3);
5’-AUUGGCUCGGAUCUUAAGCUC-3’(SEQ ID NO:4)。5'-AUUGGCUCGGAUCUUAAGCUC-3' (SEQ ID NO: 4).
本发明提供了含有本发明的siRNA和/所述siRNA缀合物的载体或细胞。The present invention provides a vector or a cell containing the siRNA and/or the siRNA conjugate of the present invention.
本发明提供了一种药物组合物，所述药物组合物含有本发明的siRNA和/或siRNA缀合物和药学上可接受的载体。The present invention provides a pharmaceutical composition, which contains the siRNA and/or siRNA conjugate of the present invention and a pharmaceutically acceptable carrier.
在一些实施方案中，本发明提供了所述的siRNA和/或药物组合物和/或siRNA缀合物在制备用于治疗由抑制素亚单位βE(INHBE)基因表达引起的疾病和/或病症的药物中的用途。In some embodiments, the present invention provides the use of the siRNA and/or pharmaceutical composition and/or siRNA conjugate in the preparation of a medicament for treating diseases and/or disorders caused by the expression of the inhibin subunit βE (INHBE) gene.
在一些实施方案中，本发明提供了一种治疗抑制素亚单位βE(INHBE)基因表达引起的疾病和/或病症的方法，所述方法包括将有效量的本发明的siRNA和/或药物组合物和/或siRNA缀合物给予高血糖或糖尿病相关联的疾病和/或病症的受试者。In some embodiments, the present invention provides a method for treating diseases and/or conditions caused by inhibin subunit βE (INHBE) gene expression, the method comprising administering an effective amount of siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present invention to a subject suffering from a disease and/or condition associated with hyperglycemia or diabetes.
在一些实施方案中，本发明提供了一种抑制抑制素亚单位βE(INHBE)基因表达的方法，该方法包括将有效量的本发明的siRNA和/或药物组合物和/或siRNA缀合物与肝细胞接触。In some embodiments, the present invention provides a method for inhibiting the expression of inhibin subunit βE (INHBE) gene, the method comprising contacting an effective amount of siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present invention with hepatocytes.
本发明提供了一种试剂盒，所述试剂盒含有本发明的siRNA和/或药物组合物和/或siRNA缀合物。The present invention provides a kit, wherein the kit contains the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present invention.
有益效果Beneficial Effects
本发明提供的siRNA、药物组合物和siRNA缀合物具有显著增强的血浆和溶酶体稳定性，还具有较高的抑制素亚单位βE mRNA抑制活性，较低的脱靶效应，和/或能显著治疗肥胖或代谢相关联的疾病和/或病症。The siRNA, pharmaceutical composition and siRNA conjugate provided by the present invention have significantly enhanced plasma and lysosomal stability, higher inhibitory activity against inhibin subunit βE mRNA, lower off-target effects, and/or can significantly treat obesity or metabolism-related diseases and/or disorders.
在一些实施方案中，本发明提供的siRNA、药物组合物或siRNA缀合物在体外实验中显示出优异的靶基因抑制活性。在一些实施方案中，本发明提供的siRNA、药物组合物或siRNA缀合物在肝细胞中显示出至少50％、60％、70％、80％、90％或95％的靶基因表达抑制率。In some embodiments, the siRNA, pharmaceutical composition or siRNA conjugate provided by the present invention shows excellent target gene inhibition activity in in vitro experiments. In some embodiments, the siRNA, pharmaceutical composition or siRNA conjugate provided by the present invention shows at least 50%, 60%, 70%, 80%, 90% or 95% inhibition rate of target gene expression in hepatocytes.
本发明提供的siRNA、药物组合物或siRNA缀合物未显示出明显脱靶效应。脱靶效应可以是例如抑制非靶基因的基因正常表达。据认为，如果脱靶基因表达的结合/抑制与在靶基因效果相比低于50％、40％、30％、20％或10％时，该脱靶效应就是不显著的。The siRNA, pharmaceutical composition or siRNA conjugate provided by the present invention do not show obvious off-target effects. Off-target effects can be, for example, inhibition of normal gene expression of non-target genes. It is believed that if the binding/inhibition of off-target gene expression is less than 50%, 40%, 30%, 20% or 10% compared to the on-target gene effect, the off-target effect is not significant.
由此说明，本发明提供的siRNA、药物组合物以及siRNA缀合物能够抑制抑制素亚单位βE(INHBE)基因的表达，有效治疗肥胖相关联的疾病和/或病症，具有良好的应用前景。This shows that the siRNA, pharmaceutical composition and siRNA conjugate provided by the present invention can inhibit the expression of the inhibin subunit βE (INHBE) gene, effectively treat obesity-related diseases and/or symptoms, and have good application prospects.
本发明的其他特征和优点将在随后的具体实施方式部分予以详细说明。Other features and advantages of the present invention will be described in detail in the following detailed description.
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。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.
在发明中，INHBE mRNA是指具有Genbank注册号为NM_031479.5所示序列的mRNA。进一步地，若无其它说明，本发明中所使用的术语“靶基因”是指转录表达INHBE mRNA的基因，术语“靶mRNA”是指上述INHBE mRNA。In the present invention, INHBE mRNA refers to mRNA having a sequence shown in Genbank Accession No. NM_031479.5. Further, unless otherwise specified, the term "target gene" used in the present invention refers to a gene that transcribes and expresses INHBE mRNA, and the term "target mRNA" refers to the above-mentioned INHBE mRNA.
定义definition
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.
本发明所述术语“连接”，当表示两个分子之间的联系时，指两个分子通过共价键连接或者两个分子经由非共价键(例如，氢键或离子键)关联。The term "linked" as used herein, when referring to the connection between two molecules, means that the two molecules are connected by a covalent bond or the two molecules are associated via a non-covalent bond (eg, a hydrogen bond or an ionic bond).
本发明所述“寡核苷酸”是含有10-50个核苷酸或核苷酸碱基对的核苷酸序列。在本发明的一些实施方式中，寡核苷酸具有这样的核碱基序列，其与细胞内表达的靶基因中的编码序列至少部分互补。所述核苷酸可以任选被修饰。在本发明一些实施方式中，在将寡核苷酸递送至表达基因的细胞后，寡核苷酸能够在体外或体内抑制或阻断基因的表达。"Oligonucleotide" of the present invention is a nucleotide sequence containing 10-50 nucleotides or nucleotide base pairs. In some embodiments of the present invention, the oligonucleotide has such a core base sequence, which is at least partially complementary to the coding sequence in the target gene expressed in the cell. The nucleotides can be optionally modified. In some embodiments of the present invention, after the oligonucleotide is delivered to the cell expressing the gene, the oligonucleotide can inhibit or block the expression of the gene in vitro or in vivo.
本发明所述，与术语“INHBE”可互换使用的“抑制素亚单位βE”是指是一类肝细胞生长因子，参与肝细胞生长和分化的调节。INHBE mRNA序列的其他实例可使用公共数据库例如GenBank、UniProt和OMIM容易地获得。In the present invention, "inhibin subunit βE", which is used interchangeably with the term "INHBE", refers to a class of hepatocyte growth factors involved in the regulation of hepatocyte growth and differentiation. Other examples of INHBE mRNA sequences can be easily obtained using public databases such as GenBank, UniProt and OMIM.
本发明所述术语“肥胖相关疾病和/或病症”或“INHBE相关疾病”是由INHBE过度复制引起的或与之相关的疾病或障碍。术语“INHBE相关疾病”包括从INHBE基因表达或复制减少中受益的疾病、障碍或病症。INHBE相关疾病的非限制性实例包括例如肥胖、血脂异常、非酒精性脂肪肝炎。The term "obesity-related diseases and/or conditions" or "INHBE-related diseases" as used herein refers to diseases or disorders caused by or associated with excessive replication of INHBE. The term "INHBE-related disease" includes diseases, disorders or conditions that benefit from reduced expression or replication of the INHBE gene. Non-limiting examples of INHBE-related diseases include, for example, obesity, dyslipidemia, and non-alcoholic steatohepatitis.
本发明所述术语“抑制”，表示与还没有经过处理的细胞、细胞群或组织相比，用本发明所述siRNA、药物组合物以及siRNA缀合物处理该细胞、细胞群或组织时，基因表达降低。The term "inhibit" as used herein means that gene expression is reduced when the cell, cell group or tissue is treated with the siRNA, pharmaceutical composition and siRNA conjugate of the present invention, compared with cells, cell groups or tissues that have not been treated.
本发明所述术语“抑制”与“减少”、“沉默”、“下调”、“抑止”和其他类似术语可互换使用，并且包括任何水平的抑制。优选地，抑制包括统计学上显著的抑制或临床上显著的抑制。The term "inhibit" in the present invention is used interchangeably with "reduce", "silence", "downregulate", "suppress" and other similar terms, and includes any level of inhibition. Preferably, inhibition includes statistically significant inhibition or clinically significant inhibition.
如本发明所用，短语“抑制INHBE的表达”或“抑制INHBE基因的表达”包括抑制任何INHBE基因(例如，在INHBE中由INHBE表达的INHBE基因、细胞中由表达构建体表达的INHBE基因)以及编码INHBE蛋白的INHBE基因的变体或突变体的表达。该术语包括编码一种或多种INHBE蛋白的任何INHBE转录以及INHBE基因的变体和突变体的敲减。As used herein, the phrase "inhibiting the expression of INHBE" or "inhibiting the expression of an INHBE gene" includes inhibiting the expression of any INHBE gene (e.g., an INHBE gene expressed by an INHBE in an INHBE, an INHBE gene expressed by an expression construct in a cell) and a variant or mutant of an INHBE gene encoding an INHBE protein. The term includes knockdown of any INHBE transcript encoding one or more INHBE proteins and variants and mutants of an INHBE gene.
所述正义链和反义链中的每个核苷酸各自独立地为修饰或未修饰的核苷酸。在本发明的上下文中，除非另有说明，“缀合”是指两个或多个各自具有特定功能的化学部分之间以共价连接的方式彼此连接；相应地，“缀合物”是指该各个化学部分之间通过共价连接而形成的化合物。进一步地，“siRNA缀合物”表示一个或多个具有特定功能的化学部分共价连接至siRNA上而形成的化合物。在下文中，有时也将本发明的siRNA缀合物简称为“缀合物”。siRNA缀合物应根据上下文，理解为siRNA缀合物的总称，第一种siRNA缀合物或第二种siRNA缀合物，或siRNA正义链缀合物或siRNA反义链缀合物。Each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide. In the context of the present invention, unless otherwise specified, "conjugation" refers to the connection between two or more chemical moieties, each having a specific function, in a covalently linked manner; accordingly, "conjugate" refers to a compound formed by covalently linking the chemical moieties. Further, "siRNA conjugate" means a compound formed by covalently linking one or more chemical moieties having a specific function to siRNA. In the following, the siRNA conjugate of the present invention is sometimes referred to as a "conjugate". The siRNA conjugate should be understood as a general term for siRNA conjugates, the first siRNA conjugate or the second siRNA conjugate, or the siRNA sense chain conjugate or the siRNA antisense chain conjugate, depending on the context.
在一些实施例中，所述缀合基团可以连接在核苷酸的磷酸基团、2’-位羟基、5’-位羟基或者碱基上。在一些实施例中，所述缀合基团还可以连接在3’-位羟基上，此时核苷酸之间采用2’-5’磷酸二酯键连接。当缀合基团连接在siRNA链的末端时，所述缀合基团通常连接在核苷酸的磷酸基团上；当缀合基团连接在siRNA的内部序列时，所述缀合基团通常连接在核糖糖环或者碱基上。各种连接方式可以参考文献：Muthiah Manoharan et.al.siRNAconjugates carrying sequentially assembled trivalent N-acetylgalactosaminelinked through nucleosides elicit robust gene silencing in vivo inhepatocytes.ACS Chemical biology,2015,10(5):1181-7.In some embodiments, the conjugate group can be connected to the phosphate group, 2'-hydroxyl group, 5'-hydroxyl group or base of the nucleotide. In some embodiments, the conjugate group can also be connected to the 3'-hydroxyl group, in which case the nucleotides are connected by a 2'-5' phosphodiester bond. When the conjugate group is connected to the end of the siRNA chain, the conjugate group is usually connected to the phosphate group of the nucleotide; when the conjugate group is connected to the internal sequence of the siRNA, the conjugate group is usually connected to the ribose sugar ring or the base. Various connection methods can be referred to in the literature: Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo inhepatocytes.ACS Chemical biology, 2015, 10(5): 1181-7.
在一些实施方案中，所述siRNA与缀合基团间可以通过酸不稳定的、或可还原的化学键相连，在细胞内涵体的酸性环境下，这些化学键可降解，从而使siRNA成为自由状态。对于不可降解的缀合方式，缀合基团可连接在siRNA的正义链，从而尽量降低缀合对siRNA活性的影响。In some embodiments, the siRNA and the conjugated group can be connected by acid-labile or reducible chemical bonds, which can be degraded in the acidic environment of the cell endosome, thereby making the siRNA free. For non-degradable conjugation methods, the conjugated group can be connected to the sense strand of the siRNA, thereby minimizing the effect of conjugation on the activity of the siRNA.
在上文及下文中，如无特别说明，通常，“G”、“C”、“A”、“T”和“U”各自代表含有鸟嘌呤、胞嘧啶、腺嘌呤、胸腺嘧啶和尿嘧啶作为碱基的核苷酸。然而，应理解，术语“核糖核苷酸”或“核苷酸”也可指如下文进一步详述的修饰的核苷酸，核苷酸类似物(surrogatereplacement moiety)。In the above and below, unless otherwise specified, generally, "G", "C", "A", "T" and "U" represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as bases, respectively. However, it should be understood that the term "ribonucleotide" or "nucleotide" may also refer to modified nucleotides, nucleotide analogs (surrogate replacement moieties) as described in further detail below.
其中a、c、g和u分别为2’O甲基腺苷3’磷酸、2’O甲基胞苷3’磷酸、2’O甲基鸟苷3’磷酸和2’O甲基尿苷3’磷酸；Where a, c, g, and u are 2’O-methyladenosine 3’ phosphate, 2’O-methylcytidine 3’ phosphate, 2’O-methylguanosine 3’ phosphate, and 2’O-methyluridine 3’ phosphate, respectively;
Af、Cf、Gf和Uf分别为2’氟代腺苷3’磷酸、2’氟代胞苷3’磷酸、2’氟代鸟苷3’磷酸和2’氟代尿苷3’磷酸；Af, Cf, Gf, and Uf are 2’-fluoroadenosine 3’ phosphate, 2’-fluorocytidine 3’ phosphate, 2’-fluoroguanosine 3’ phosphate, and 2’-fluorouridine 3’ phosphate, respectively;
dA、dC、dG和dT分别为2’脱氧腺苷3’磷酸、2’脱氧胞苷3’磷酸、2’脱氧鸟苷3’磷酸和2’脱氧胸苷3’磷酸；dA, dC, dG, and dT are 2’deoxyadenosine 3’ phosphate, 2’deoxycytidine 3’ phosphate, 2’deoxyguanosine 3’ phosphate, and 2’deoxythymidine 3’ phosphate, respectively;
(Agn)是腺苷二醇核酸(GNA)；并且s是硫代磷酸酯键。(Agn) is adenosine diol nucleic acid (GNA); and s is a phosphorothioate bond.
在本发明的上下文中，表述“互补”或“反向互补”可互相替代使用，并具有本领域技术人员周知的含义，即，在双链核酸分子中，一条链的碱基各自与另一条链上的碱基以互补的方式相配对。在DNA中，嘌呤碱基腺嘌呤(A)始终与嘧啶碱基胸腺嘧啶(T)(或者在RNA中为尿嘧啶(U))相配对；嘌呤碱基鸟嘌呤(C)始终与嘧啶碱基胞嘧啶(G)相配对。每个碱基对都包括一个嘌呤和一个嘧啶。当一条链上的腺嘌呤始终与另一条链上的胸腺嘧啶(或尿嘧啶)配对，以及鸟嘌呤始终与胞嘧啶配对时，两条链被认为是彼此相互补的，以及从其互补链的序列中可以推断出该链的序列。与此相应地，“错配”在本领域中意指在双链核酸中，对应位置上的碱基并未以互补的形式配对存在。In the context of the present invention, the expressions "complementary" or "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one chain are each paired with the bases on the other chain in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one chain is always paired with thymine (or uracil) on the other chain, and guanine is always paired with cytosine, the two chains are considered to be complementary to each other, and the sequence of the chain can be inferred from the sequence of its complementary chain. Correspondingly, "mismatch" means in the art that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary form.
在上文及下文中，如无特别说明，“基本上反向互补”是指所涉及的两段核苷酸序列之间存在不多于3个的碱基错配；“实质上反向互补”是指两段核苷酸序列之间存在不多于1个的碱基错配；“完全反向互补”是指两段核苷酸序列之间不存在碱基错配。在上文及下文中，一个核苷酸序列与另外一个核苷酸序列存在“核苷酸差异”，是指前者与后者相比，相同位置的核苷酸的碱基种类发生了改变，例如，在后者中一个核苷酸碱基为A时，在前者的相同位置处的对应核苷酸碱基为U、C、G或者T的情况下，认定为两个核苷酸序列之间在该位置处存在核苷酸差异。在一些实施方案中，以无碱基核苷酸或其等同物代替原位置的核苷酸时，也可认为在该位置处产生了核苷酸差异。In the above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there are no more than 1 base mismatch between the two nucleotide sequences; "completely reverse complementary" means that there is no base mismatch between the two nucleotide sequences. In the above and below, the existence of "nucleotide difference" between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position of the former is changed compared with the latter. For example, when a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position of the former is U, C, G or T, it is determined that there is a nucleotide difference at that position between the two nucleotide sequences. In some embodiments, when the nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, it can also be considered that a nucleotide difference has occurred at that position.
在上文及下文中，特别是在描述本发明的siRNA、含siRNA的药物组合物或siRNA缀合物的制备方法时，除非特别说明，所述核苷单体(nucleoside monomer)指，根据欲制备的siRNA或siRNA缀合物中核苷酸的种类和顺序，亚磷酰胺固相合成中使用的修饰或未修饰的核苷亚磷酰胺单体(unmodified or modified RNA phosphoramidites,有时RNAphosphoramidites也称为Nucleoside phosphoramidites)。亚磷酰胺固相合成为本领域技术人员所公知的RNA合成中所用的方法。本发明所用的核苷单体均可通过商业化购买得到。In the above and below, particularly when describing the preparation method of siRNA of the present invention, pharmaceutical composition containing siRNA or siRNA conjugate, unless otherwise specified, the nucleoside monomer (nucleoside monomer) refers to the modified or unmodified nucleoside phosphoramidite monomer (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites are also referred to as Nucleoside phosphoramidites) used in phosphoramidite solid phase synthesis according to the type and order of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphoramidite solid phase synthesis is a method used in RNA synthesis known to those skilled in the art. The nucleoside monomer used in the present invention can be obtained by commercial purchase.
如本文所使用的，“任选的”或“任选地”是指其后描述的事件或状况可以发生或不发生，并且该描述包括事件或状况发生的情况和不发生的情况。例如，“任选地取代”的“烷基”包括下文定义的“烷基”和“取代烷基”本领域技术人员将理解的是，对于包含一个或多个取代基的任何基团，这些基团不打算引入空间上不切实际、合成上不可行和/或本身不稳定的任何取代或取代模式。As used herein, "optional" or "optionally" means that the event or situation described thereafter may or may not occur, and the description includes instances where the event or situation occurs and instances where it does not occur. For example, "optionally substituted" "alkyl" includes "alkyl" and "substituted alkyl" as defined below. It will be understood by those skilled in the art that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically unfeasible, and/or inherently unstable.
“受试者”一词，如本文所使用的，指任何动物，例如哺乳动物或有袋动物。本发明的受试者包括但不限于人类、非人灵长类(例如，恒河猴或其他类型的猕猴)、小鼠、猪、马、牛、大鼠或任何种类的家禽。The term "subject", as used herein, refers to any animal, such as a mammal or marsupial. Subjects of the present invention include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, cattle, rats, or poultry of any kind.
如本文所使用的，“治疗”是指获得有益的或期望的结果的方法，包括但不限于治疗益处。“治疗益处”意味着根除或改善被治疗的潜在障碍。此外，治疗益处通过根除或改善与潜在障碍相关的一个或多个生理症状，从而在受试者中观察到改善而获得，尽管受试者可能仍然受到潜在障碍的折磨。As used herein, "treatment" refers to an approach to obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. In addition, a therapeutic benefit is obtained by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.
在一方面，本发明提供了第一种至第三种能够抑制INHBE基因表达的siRNA。以下依次对其进行详细描述。In one aspect, the present invention provides first to third siRNAs capable of inhibiting the expression of INHBE gene, which are described in detail below.
本发明的siRNA含有核苷酸基团作为基本结构单元，本领域技术人员公知，所述核苷酸基团含有磷酸基团、核糖基团和碱基，在此不再赘述。The siRNA of the present invention contains a nucleotide group as a basic structural unit. It is well known to those skilled in the art that the nucleotide group contains a phosphate group, a ribose group and a base, which will not be described in detail here.
第一种siRNAThe first siRNA
按照本发明，所述siRNA可以是第一种siRNA。According to the present invention, the siRNA may be the first siRNA.
所述第一种siRNA含有正义链和反义链，所述第一种siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：The first siRNA comprises a sense strand and an antisense strand, each nucleotide in the first siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 1, and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 2, and differs by no more than 3 nucleotides:
5’-CUCUUUGCUUGAGGAUCUUUU-3’(SEQ ID NO:1)；5’-CUCUUUGCUUGAGGAUCUUUU-3’ (SEQ ID NO: 1);
5’-AAGAUCCUCAAGCAAAGAGUG-3’(SEQ ID NO:2)。5'-AAGAUCCUCAAGCAAAGAGUG-3' (SEQ ID NO: 2).
在一些实施例中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。在一些实施方案中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列之间不多于1个核苷酸差异。In some embodiments, the sense strand comprises only nucleotide sequence I, and the antisense strand comprises only nucleotide sequence II. In some embodiments, there is no more than one nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1, and/or there is no more than one nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2.
在一些实施例中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个的碱基错配；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有碱基错配。In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary, or completely reverse complementary; the substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences; the substantially reverse complementary means that there are no more than 1 base mismatch between the two nucleotide sequences; and the completely reverse complementary means that there are no base mismatches between the two nucleotide sequences.
第二种siRNAThe second siRNA
按照本发明，所述siRNA可以是第二种siRNA。According to the present invention, the siRNA may be a second siRNA.
所述第二种siRNA含有正义链和反义链，所述第二种siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO:3所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:4所示的核苷酸序列长度相等，且不多于3个核苷酸差异：The second siRNA comprises a sense strand and an antisense strand, and each nucleotide in the second siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:3 and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:4 and differs by no more than 3 nucleotides:
5’-GCUUAAGAUCCGAGCCAAUUU-3’(SEQ ID NO:3)；5’-GCUUAAGAUCCGAGCCAAUUU-3’ (SEQ ID NO: 3);
5’-AUUGGCUCGGAUCUUAAGCUC-3’(SEQ ID NO:4)。5'-AUUGGCUCGGAUCUUAAGCUC-3' (SEQ ID NO: 4).
在一些实施例中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。在一些实施方案中，所述核苷酸序列I与SEQ ID NO:3所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:4所示的核苷酸序列之间不多于1个核苷酸差异。In some embodiments, the sense strand comprises only nucleotide sequence I, and the antisense strand comprises only nucleotide sequence II. In some embodiments, there is no more than one nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:3, and/or there is no more than one nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:4.
在一些实施例中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补。In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary, or completely reverse complementary.
如前所述，本发明公开的siRNA中的核苷酸各自独立地为修饰或未修饰的核苷酸。在一些实施例中，本发明的siRNA中的每个核苷酸均为未经修饰的核苷酸。在一些实施方案中，本发明的siRNA中的部分或全部核苷酸为修饰的核苷酸，核苷酸基团上的这些修饰不会导致本发明的siRNA缀合物抑制INHBE基因表达的功能明显削弱或丧失。As described above, the nucleotides in the siRNA disclosed in the present invention are each independently a modified or unmodified nucleotide. In some embodiments, each nucleotide in the siRNA of the present invention is an unmodified nucleotide. In some embodiments, some or all of the nucleotides in the siRNA of the present invention are modified nucleotides, and these modifications on the nucleotide groups do not result in a significant weakening or loss of the function of the siRNA conjugate of the present invention to inhibit the expression of the INHBE gene.
在一些实施方案中，本发明的siRNA可以为表1列出的未修饰siRNA中的任意一种。In some embodiments, the siRNA of the present invention can be any one of the unmodified siRNAs listed in Table 1.
在一些实施方案中，本发明公开的siRNA至少含有1个修饰的核苷酸。在本发明公开的上下文中，所使用的术语“修饰的核苷酸”是指核苷酸的核糖基2’位羟基被其他基团取代形成的核苷酸或核苷酸类似物，或者具有经修饰的碱基的核苷酸。所述修饰的核苷酸不会导致siRNA抑制基因表达的功能明显削弱或丧失。例如，可以选择J.K.Watts,G.F.Deleavey,and M.J.Damha,Chemically modified siRNA:tools andapplications.Drug Discov Today,2008,13(19-20):842-55中公开的修饰的核苷酸。In some embodiments, the siRNA disclosed in the present invention contains at least one modified nucleotide. In the context disclosed in the present invention, the term "modified nucleotide" used refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribose group of the nucleotide with other groups, or a nucleotide with a modified base. The modified nucleotide will not cause the function of siRNA to inhibit gene expression to be significantly weakened or lost. For example, the modified nucleotide disclosed in J.K.Watts, G.F.Deleavey, and M.J.Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13 (19-20): 842-55 can be selected.
在一些实施方案中，本发明提供的siRNA的正义链或所述反义链中的至少一个核苷酸为修饰的核苷酸，和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基。换而言之，所述正义链和所述反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基和/或核糖基的至少一部分为具有修饰基团的磷酸酯基和/或具有修饰基团的核糖基。In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided by the present invention is a modified nucleotide, and/or at least one phosphate group is a phosphate group with a modified group. In other words, at least a portion of the phosphate group and/or ribose group in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand is a phosphate group with a modified group and/or a ribose group with a modified group.
在一些实施方案中，所述正义链和/或所述反义链中的全部核苷酸均为修饰的核苷酸。在一些实施方案中，本发明公开提供的siRNA的正义链和所述反义链中的每一个核苷酸独立地为氟代修饰的核苷酸或非氟代修饰的核苷酸。In some embodiments, all nucleotides in the sense strand and/or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand of the siRNA disclosed in the present invention is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.
在本发明公开的上下文中，“氟代修饰的核苷酸”指核苷酸的核糖基2’位的羟基被氟取代形成的核苷酸，其具有以下式(1)所示的结构。“非氟代修饰的核苷酸”指核苷酸的核糖基2’位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物。在一些实施方案中，每一个非氟代修饰的核苷酸独立地选自核苷酸的核糖基2’位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物中的一种。In the context of the present disclosure, "fluorinated modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and has a structure shown in the following formula (1). "Non-fluorinated modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group. In some embodiments, each non-fluorinated modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group.
这些核糖基2’位的羟基被非氟基团取代形成的核苷酸是本领域技术人员所公知的，这些核苷酸可以选自2’-烷氧基修饰的核苷酸、2’-经取代的烷氧基修饰的核苷酸、2’-烷基修饰的核苷酸、2’-经取代的烷基修饰的核苷酸、2’-氨基修饰的核苷酸、2’-经取代的氨基修饰的核苷酸、2’-脱氧核苷酸中的一种。The nucleotides formed by replacing the hydroxyl group at the 2' position of these ribose groups with non-fluorine groups are well known to those skilled in the art, and these nucleotides can be selected from 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, and 2'-deoxynucleotides.
在一些实施方案中，2’-烷氧基修饰的核苷酸为2’-甲氧基(2’-OMe)修饰的核苷酸，如式(2)所示。在一些实施方案中，2’-经取代的烷氧基修饰的核苷酸，可以是2’-甲氧基乙基(2’-ΜΟΕ)修饰的核苷酸，如式(3)所示。在一些实施方案中，2’-氨基(2’-ΝΗ2)修饰的核苷酸如式(4)所示。在一些实施方案中，2’-脱氧核苷酸(DNA)如式(5)所示：In some embodiments, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe) modified nucleotide, as shown in formula (2). In some embodiments, the 2'-substituted alkoxy modified nucleotide can be a 2'-methoxyethyl (2'-MOE) modified nucleotide, as shown in formula (3). In some embodiments, the 2'-amino (2'-NH2 ) modified nucleotide is as shown in formula (4). In some embodiments, the 2'-deoxynucleotide (DNA) is as shown in formula (5):
核苷酸类似物指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的基团。在一些实施方案中，核苷酸类似物可以是异核苷酸、桥联的核苷酸或无环核苷酸。Nucleotide analogs refer to groups that can replace nucleotides in nucleic acids, but have structures different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxyribonucleotides. In some embodiments, nucleotide analogs can be isonucleotides, bridged nucleotides or acyclic nucleotides.
桥联的核苷酸(bridged nucleic acid，简称BNA)可以含有五无环、六元环或七元环的具有“固定的”C3’-内切糖缩拢的桥联结构。通常将该桥掺入到该核糖的2’-、4’-位处以提供一个2’，4’-BNA核苷酸。在一些实施例中，BNA可以是LNA、ENA、cETBNA等，其中，LNA如式(6)所示，ENA如式(7)所示，cETBNA如式(8)所示。The bridged nucleic acid (BNA) may contain a five-membered ring, a six-membered ring or a seven-membered ring with a "fixed" C3'-endosugar condensed bridge structure. The bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2', 4'-BNA nucleotide. In some embodiments, BNA may be LNA, ENA, cETBNA, etc., wherein LNA is shown in formula (6), ENA is shown in formula (7), and cETBNA is shown in formula (8).
无环核苷酸是核苷酸的糖环被打开形成的一类核苷酸。在一些实施方案中，无环核苷酸可以是解锁核酸(UNA)或甘油核酸(GNA)，其中，UNA如式(9)所示，GNA如式(10)所示：Acyclic nucleotides are a type of nucleotides formed by opening the sugar ring of a nucleotide. In some embodiments, the acyclic nucleotide may be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA), wherein UNA is as shown in formula (9) and GNA is as shown in formula (10):
上述式(9)和式(10)中，Ra选自H、OH或烷氧基(O-烷基)。In the above formula (9) and formula (10),Ra is selected from H, OH or alkoxy (O-alkyl).
异核苷酸是指核苷酸中碱基在核糖环上的位置发生改变而形成的化合物。在一些实施方案中，异核苷酸可以是碱基从核糖环的1’-位移动至2’-位或3’-位而形成的化合物，如式(11)或(12)所示：Isonucleotides refer to compounds formed by a change in the position of a base on the ribose ring in a nucleotide. In some embodiments, an isonucleotide may be a compound formed by a base moving from the 1'-position to the 2'-position or the 3'-position of the ribose ring, as shown in formula (11) or (12):
上述式(11)、式(12)所示化合物中，Base表示核酸碱基，例如A、U、G、C或T；Rb选自H、OH、F或者如上所述的非氟基团。In the compounds represented by the above formula (11) and formula (12), Base represents a nucleic acid base, such as A, U, G, C or T; and Rb is selected from H, OH, F or the non-fluorine group as described above.
在一些实施方案中，核苷酸类似物选自异核苷酸、LNA、ENA、cET、UNA和GNA中的一种。在一些实施方案中，每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸，在上文和下文中，所述甲氧基修饰的核苷酸指核糖基的2’-羟基被甲氧基取代而形成的核苷酸。In some embodiments, the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA. In some embodiments, each non-fluorinated modified nucleotide is a methoxy-modified nucleotide, and in the above and below, the methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.
在上文及下文中，“氟代修饰的核苷酸”、“2’-氟修饰的核苷酸”、“核糖基团的2’-羟基被氟取代的核苷酸”和“具有2’-氟代核糖基的核苷酸”意义相同，均指核苷酸的2’-羟基被氟取代，而形成的具有如式(1)所示结构的化合物；“甲氧基修饰的核苷酸”、“2’-甲氧基修饰的核苷酸”、“核糖基团的2’-羟基被甲氧基取代的核苷酸”和“具有2’-甲氧基核糖基的核苷酸”意义相同，均指核苷酸核糖基团的2’-羟基被甲氧基取代而形成的具有如式(2)所示结构的化合物。In the above and below, “fluorinated modified nucleotides”, “2’-fluorinated modified nucleotides”, “nucleotides in which the 2’-hydroxyl group of the ribose group is substituted by fluorine” and “nucleotides having a 2’-fluorinated ribose group” have the same meaning, and all refer to compounds having a structure as shown in formula (1) formed by replacing the 2’-hydroxyl group of the nucleotide with fluorine; “methoxy-modified nucleotides”, “2’-methoxy-modified nucleotides”, “nucleotides in which the 2’-hydroxyl group of the ribose group is substituted by methoxy” and “nucleotides having a 2’-methoxyribose group” have the same meaning, and all refer to compounds having a structure as shown in formula (2) formed by replacing the 2’-hydroxyl group of the ribose group of the nucleotide with a methoxy group.
在一些实施方案中，本发明提供的siRNA的正义链或所述反义链还可以包括碱基修饰或替换。In some embodiments, the sense strand or the antisense strand of the siRNA provided by the present invention may further include base modification or substitution.
具有上述修饰的siRNA可使血液中的核糖核酸酶不易切割核酸，从而增加核酸的稳定性，使核酸具有更强的抵抗核酸酶水解的性能。同时，上述修饰的siRNA具有较高的抑制靶mRNA的活性。The modified siRNA can make it difficult for ribonucleases in the blood to cut nucleic acids, thereby increasing the stability of nucleic acids and making them more resistant to nuclease hydrolysis. At the same time, the modified siRNA has a higher activity in inhibiting target mRNA.
在一些实施方案中，本发明提供的siRNA的正义链和反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基中的至少一部分为具有修饰基团的磷酸酯基。在一些实施方案中，具有修饰基团的磷酸酯基为磷酸酯基中的磷酸二酯键中的至少一个氧原子被硫原子取代而形成的硫代磷酸酯基；在一些实施方案中，所述具有修饰基团的磷酸酯基为具有如式(13)所示结构的硫代磷酸酯基：In some embodiments, at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand of the siRNA provided by the present invention is a phosphate group having a modified group. In some embodiments, the phosphate group having a modified group is a thiophosphate group formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom; in some embodiments, the phosphate group having a modified group is a thiophosphate group having a structure as shown in formula (13):
这种修饰能稳定siRNA的双链结构，保持碱基配对的高特异性和高亲和力。This modification can stabilize the double-stranded structure of siRNA and maintain high specificity and high affinity of base pairing.
在一些实施方案中，本发明提供的siRNA中，硫代磷酸酯基连接存在于由以下位置组成的组中的至少一处：正义链或反义链任意一端的第一个和第二个核苷酸之间；正义链或反义链任意一端的第二个和第三个核苷酸之间；或上述的任意组合。在一些实施方案中，硫代磷酸酯基连接存在于除正义链5’末端以外的全部上述位置处。在一些实施方案中，硫代磷酸酯基连接存在于除正义链3’末端以外的全部上述位置处。In some embodiments, in the siRNA provided by the present invention, the phosphorothioate linkage is present in at least one of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination thereof. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 5' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 3' end of the sense strand.
在一些实施方案中，所述siRNA反义链的5’末端核苷酸为5’-磷酸核苷酸或5’-磷酸类似物修饰的核苷酸。In some embodiments, the 5' terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
常用的所述5’-磷酸核苷酸或5’-磷酸类似物修饰的核苷酸是本领域技术人员所公知的，如5’-磷酸核苷酸可具有如式(14)结构：The commonly used 5'-phosphate nucleotides or 5'-phosphate analogue modified nucleotides are well known to those skilled in the art. For example, the 5'-phosphate nucleotides may have a structure as shown in formula (14):
再如，Anastasia Khvorova and Jonathan K.Watts,The chemical evolutionof oligonucleotide therapies of clinical utility.Nature Biotechnology,2017,35(3):238-48中公开了如下4种5’-磷酸类似物修饰的核苷酸：For example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48 disclose the following four 5'-phosphate analogue-modified nucleotides:
其中，R选自Η、OH、甲氧基、氟；Base表示核酸碱基，选自A、U、C、G或T。Wherein, R is selected from Ｈ, OH, methoxy, fluorine; Base represents a nucleic acid base, selected from A, U, C, G or T.
在一些实施方案中，5’-磷酸核苷酸为式(14)所示的含有5’-磷酸修饰的核苷酸，5’-磷酸类似物修饰的核苷酸为含有乙烯基磷酸酯修饰的核苷酸，如式(15)所示，或者为硫代磷酸酯修饰的核苷酸，如式(17)所示。In some embodiments, the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification as shown in formula (14), and the nucleotide modified with a 5'-phosphate analog is a nucleotide containing a vinyl phosphate modification as shown in formula (15), or a nucleotide modified with a thiophosphate as shown in formula (17).
表2中显示了本文公开的修饰的核苷酸单体的缩写。Abbreviations for the modified nucleomonomers disclosed herein are shown in Table 2.
表2.本发明公开的核苷酸单体的缩写Table 2. Abbreviations of nucleotide monomers disclosed in the present invention
在一些实施方案中，本发明提供的siRNA为表3列出的siRNA中的任意一种。In some embodiments, the siRNA provided by the present invention is any one of the siRNAs listed in Table 3.
本发明提供的上述siRNA不仅具有显著增强的血浆和溶酶体稳定性，还具有很高的靶mRNA抑制活性。The siRNA provided by the present invention not only has significantly enhanced plasma and lysosomal stability, but also has high target mRNA inhibitory activity.
本发明提供的siRNA可以通过本领域常规的siRNA制备方法(例如固相合成)得到。其中，固相合成已经有商业化定制服务。可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本发明所述的siRNA中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。The siRNA provided by the present invention can be obtained by conventional siRNA preparation methods in the art (e.g., solid phase synthesis). Among them, solid phase synthesis has commercial customization services. The modified nucleotide groups can be introduced into the siRNA of the present invention by using nucleoside monomers with corresponding modifications. The method for preparing nucleoside monomers with corresponding modifications and the method for introducing the modified nucleotide groups into siRNA are also well known to those skilled in the art.
siRNA缀合物siRNA conjugates
本发明提供了一种siRNA缀合物，所述siRNA缀合物含有上述siRNA以及缀合连接至该siRNA的缀合基团。The present invention provides a siRNA conjugate, which contains the above siRNA and a conjugation group conjugated to the siRNA.
一般来说，所述缀合基团包含药学上可接受的至少一个靶向基团和任选的接头(linker)，并且，所述siRNA、所述接头和所述靶向基团依次连接。在一些实施方案中，所述靶向基团为2-4个。所述siRNA分子可以非共价或共价缀合至所述缀合基团，例如可以共价缀合至所述缀合基团。siRNA与缀合基团的缀合位点可以在siRNA正义链的3’端或5’端，还可以在siRNA的内部序列中。在一些实施方案中，所述siRNA与缀合基团的缀合位点在siRNA正义链的3’末端或5’末端。In general, the conjugated group comprises at least one pharmaceutically acceptable targeting group and an optional linker, and the siRNA, the linker and the targeting group are connected in sequence. In some embodiments, the targeting group is 2-4. The siRNA molecule can be non-covalently or covalently conjugated to the conjugated group, for example, it can be covalently conjugated to the conjugated group. The conjugation site of siRNA and conjugated group can be at the 3' end or 5' end of the siRNA sense strand, or in the internal sequence of siRNA. In some embodiments, the conjugation site of siRNA and conjugated group is at the 3' end or 5' end of the siRNA sense strand.
在一些实施方案中，所述缀合基团可以连接在核苷酸的磷酸基团、2’-位羟基或者碱基上。在一些实施方案中，所述缀合基团还可以连接在3’-位羟基上，此时核苷酸之间采用2’-5’磷酸二酯键连接。当缀合基团连接在siRNA链的末端时，所述缀合基团通常连接在核苷酸的磷酸基团上；当缀合基团连接在siRNA的内部序列时，所述缀合基团通常连接在核糖糖环或者碱基上。各种连接方式可以参考文献：Muthiah Manoharan et.al.siRNAconjugates carrying sequentially assembled trivalent N-acetylgalactosaminelinked through nucleosides elicit robust gene silencing in vivo inhepatocytes.ACS Chemical biology,2015,10(5):1181-7.In some embodiments, the conjugate group can be connected to the phosphate group, 2'-hydroxyl group or base of the nucleotide. In some embodiments, the conjugate group can also be connected to the 3'-hydroxyl group, in which case the nucleotides are connected by a 2'-5' phosphodiester bond. When the conjugate group is connected to the end of the siRNA chain, the conjugate group is usually connected to the phosphate group of the nucleotide; when the conjugate group is connected to the internal sequence of the siRNA, the conjugate group is usually connected to the ribose sugar ring or the base. Various connection methods can be referred to in the literature: Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo inhepatocytes.ACS Chemical biology, 2015, 10(5): 1181-7.
在一些实施方案中，所述siRNA与缀合基团间可以通过酸不稳定的或可还原的化学键相连，在细胞内涵体的酸性环境下，这些化学键可降解，从而使siRNA成为自由状态。对于不可降解的缀合方式，缀合基团可连接在siRNA的正义链，从而尽量降低缀合对siRNA活性的影响。In some embodiments, the siRNA and the conjugated group can be connected by acid-labile or reducible chemical bonds, which can be degraded in the acidic environment of the cell endosome, thereby making the siRNA free. For non-degradable conjugation methods, the conjugated group can be connected to the sense strand of the siRNA, thereby minimizing the effect of conjugation on the activity of the siRNA.
在一些实施方案中，所述药学上可接受的靶向基团可以是siRNA给药领域常规使用的配体，例如WO2009082607A2中描述的各种配体，以引用的方式将其全部公开内容并入本文。In some embodiments, the pharmaceutically acceptable targeting group may be a ligand conventionally used in the field of siRNA administration, such as various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.
在一些实施方案中，靶向基团包含脱唾液酸糖蛋白受体配体。在一些实施方案中，脱唾液酸糖蛋白受体配体包括一种或多种半乳糖衍生物或由其组成。如本发明所用，术语“半乳糖衍生物”包括半乳糖和对脱唾液酸糖蛋白受体的亲和力等于或大于半乳糖的亲和力的乳糖衍生物。半乳糖衍生物包括但不限于：半乳糖、半乳糖胺、N甲酰半乳糖胺、N乙酰半乳糖胺、N丙酰基半乳糖胺、N正丁酰基半乳糖胺和N异丁酰基半乳糖胺(参见例如：Iobst,S.T.和Drickamer,K.J.B.C.1996年，第271卷，第6686页)。可用于寡核苷酸和其他分子在体内靶向肝脏的半乳糖衍生物和半乳糖衍生物簇是本领域已知的(参见，例如，Baenziger和Fiete，1980,Cell,22,611620；Connolly et al.,1982,J.Biol.Chem.,257,939945)。半乳糖衍生物已被用于通过其与肝细胞表面上表达的脱唾液酸糖蛋白受体(ASGPr)的结合而在体内将分子靶向肝细胞。ASGPr配体与ASGPr(s)的结合有利于细胞特异性靶向肝细胞以及内吞分子进入肝细胞。ASGPr配体可以是单体(例如，具有单个半乳糖衍生物)或多聚体(例如，具有多个半乳糖衍生物)。可使用本领域已知的方法将半乳糖衍生物或半乳糖衍生物簇连接至siRNA的3’端或5’端。In some embodiments, the targeting group comprises an asialoglycoprotein receptor ligand. In some embodiments, the asialoglycoprotein receptor ligand comprises one or more galactose derivatives or is composed of it. As used in the present invention, the term "galactose derivative" includes galactose and a lactose derivative whose affinity for the asialoglycoprotein receptor is equal to or greater than the affinity of galactose. Galactose derivatives include but are not limited to: galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine and N-isobutyrylgalactosamine (see, for example: Iobst, S.T. and Drickamer, K.J.B.C. 1996, Vol. 271, p. 6686). Galactose derivatives and galactose derivative clusters that can be used for oligonucleotides and other molecules to target the liver in vivo are known in the art (see, e.g., Baenziger and Fiete, 1980, Cell, 22, 611620; Connolly et al., 1982, J. Biol. Chem., 257, 939945). Galactose derivatives have been used to target molecules to hepatocytes in vivo through their binding to the asialoglycoprotein receptor (ASGPr) expressed on the surface of hepatocytes. The binding of ASGPr ligands to ASGPr (s) facilitates cell-specific targeting to hepatocytes and endocytosis of molecules into hepatocytes. ASGPr ligands can be monomers (e.g., having a single galactose derivative) or polymers (e.g., having multiple galactose derivatives). Galactose derivatives or galactose derivative clusters can be attached to the 3' end or 5' end of siRNA using methods known in the art.
在一些实施方案中，所述siRNA缀合物中药学上可接受的靶向基团可以是半乳糖或N-乙酰半乳糖胺，其中，半乳糖或N-乙酰半乳糖胺分子可以是一价、二价、三价、四价。应当理解的是，所述的一价、二价、三价、四价分别指siRNA分子与含有作为靶向基团的半乳糖或N-乙酰半乳糖胺分子的缀合基团形成siRNA缀合物后，该siRNA缀合物中siRNA分子与半乳糖或N-乙酰半乳糖胺分子的摩尔比为1:1、1:2、1:3或1:4。在一些实施方案中，所述药学上可接受的靶向基团是N-乙酰半乳糖胺。在一些实施方案中，当本发明所述的siRNA与含有N-乙酰半乳糖胺的缀合基团缀合时，N-乙酰半乳糖胺分子是三价或四价。在一些实施方案中，当本发明所述的siRNA与含有N-乙酰半乳糖胺的缀合基团缀合时，N-乙酰半乳糖胺分子是三价。In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the monovalent, divalent, trivalent, and tetravalent refer to the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate after the siRNA molecule and the conjugated group containing the galactose or N-acetylgalactosamine molecule as the targeting group form the siRNA conjugate respectively: 1, 1: 2, 1: 3, or 1: 4. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA of the present invention is conjugated to a conjugated group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described herein is conjugated to a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.
靶向基团可经由合适的接头与siRNA分子相连，本领域技术人员可以根据靶向基团的具体类型选择合适的接头。这些接头、靶向基团的种类以及与siRNA的连接方式，可参见WO2015006740A2的公开内容，通过引用的方式将其整体内容并入本发明。The targeting group can be connected to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group. These linkers, the types of targeting groups and the connection mode with siRNA can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated into the present invention by reference.
在一些实施方案中，当所述靶向基团为N-乙酰半乳糖胺时，合适的接头可以为如式(19)所示的结构：In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be a structure as shown in formula (19):
其中，m为1-3的整数；Wherein, m is an integer from 1 to 3;
LA为具有如式(20)所示结构的包含酰胺键的链状部分，每个所述LA在其两端分别与一个所述靶向基团和所述LC部分通过醚键相连接：LA is a chain portion containing an amide bond having a structure as shown in formula (20), and each of theLA is connected to one of the targeting groups and theLC portion via an ether bond at both ends thereof:
LB为具有如式(21)所示结构的包含N-酰基吡咯烷的链状部分，所述链状部分在其一端具有羰基并与所述LC部分通过酰胺键相连接，在另一端具有氧原子并与所述siRNA通过磷酸酯键相连接：LB is a chain portion containing N-acylpyrrolidine having a structure as shown in formula (21), wherein the chain portion has a carbonyl group at one end and is connected to theLC portion via an amide bond, and has an oxygen atom at the other end and is connected to the siRNA via a phosphate bond:
LC为基于羟甲基氨基甲烷、二羟甲基氨基甲烷或三羟甲基氨基甲烷的2-4价连接基团，所述LC经由氧原子与各个所述LA部分通过醚键相连接，并且经由氮原子与所述LB部分通过酰胺键相连接。LC is a 2-4 valent linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trihydroxymethylaminomethane, and is connected to eachLA part through an ether bond via an oxygen atom, and is connected to theLB part through anamide bond via a nitrogen atom.
在一些实施方案中，作为接头的-(LA)3三羟甲基氨基甲烷-LB-连接N-乙酰半乳糖胺分子和siRNA分子所形成的siRNA缀合物，其结构如下式(22)所示：In some embodiments, -(LA )3 tris(hydroxymethylaminomethane)-LB - as a linker connects the N-acetylgalactosamine molecule and the siRNA molecule to form a siRNA conjugate, the structure of which is shown in the following formula (22):
式中，双螺旋结构表示siRNA。In the formula, the double helix structure represents siRNA.
同样，siRNA与缀合基团的缀合位点可以在siRNA正义链的3’端或5’端，还可以在siRNA的内部序列中。Likewise, the conjugation site between siRNA and the conjugation group can be at the 3' end or 5' end of the siRNA sense strand, or in the internal sequence of the siRNA.
在一些实施方案中，本发明所述siRNA的正义链3’末端通过接头-(LA)3三羟甲基氨基甲烷-LB-与三个N-乙酰半乳糖胺(GalNAc)分子共价缀合，得到siRNA分子与GalNAc分子的摩尔比为1:3的siRNA缀合物，下文也可将其称为(GalNAc)3-siRNA，其结构如下式(23)所示：In some embodiments, the 3' end of the sense strand of the siRNA of the present invention is covalently conjugated to three N-acetylgalactosamine (GalNAc) molecules via a linker -(LA )3 tris(hydroxymethylaminomethane)-LB- to obtain a siRNA conjugate having a molar ratio of 1:3 between siRNA molecules and GalNAc molecules, which may also be referred to as (GalNAc)3 -siRNAhereinafter , and has a structure as shown in the following formula (23):
其中，双螺旋结构表示所述siRNA，并且所述接头连接至所述siRNA的正义链3’末端。Wherein, the double helix structure represents the siRNA, and the linker is connected to the 3' end of the positive strand of the siRNA.
在一些实施方案中，所述接头连接至所述siRNA的正义链5’末端。In some embodiments, the linker is attached to the 5' end of the sense strand of the siRNA.
在一些实施方案中，所述siRNA缀合物具有如式(24)、(25)或(26)所示的结构In some embodiments, the siRNA conjugate has a structure as shown in formula (24), (25) or (26):
其中，R2为式(S1)所示结构的基团：Wherein, R2 is a group having a structure represented by formula (S1):
其中，E1为OH或SH，Nu为本发明的siRNA；Wherein, E1 is OH or SH, and Nu is the siRNA of the present invention;
R1是长度为1-20个碳原子的直链亚烷基或环状亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的任何一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中R1可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10)烷基(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10)烷基苯基)、氰基、-CO2H、C(O)O(C1-C10)烷基)、-CON(C1-C10)烷基(C1-C10烷基)、-CONH(C1-C10)烷基、-CONH2、-NH C(O)(C1-C10)烷基)、-NHC(O)(苯基)、-N(C1-C10)烷基、-N(C1-C10烷基)C(O)(C1-C10)烷基)、-N(C1-C10)烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤烷基、-OC(O)C1-C10烷基、-SO2(C1-C10)烷基、-SO2(苯基)、-SO2(C1-C10)卤代烷基)、-SO2NH2,-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10)烷基)、-NHSO2(苯基)和-NHSO2(C1-C)卤代烷基)；R1 is a straight chain alkylene or cyclic alkylene group having a length of 1 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2 , C2 -C10 alkenylene, C2 -C10 alkynylene, C6 -C10 arylene, C3 -C18 heterocyclylene and C5 -C10 heteroarylene; and wherein R1 may optionally have any one or more substituents selected from the group consisting of: C1 -C10 alkyl, C6 -C10 aryl, C5 -C10 heteroaryl, C1 -C10 haloalkyl, -OC1 -C10 alkyl, -OC1 -C10 alkylphenyl, -C1 -C10 alkyl-OH, -OC1 -C10 haloalkyl, -SC1 -C10 alkyl, -SC1 -C10 alkylphenyl, -C1 -C10 alkyl-SH, -SC1 -C10 haloalkyl, halogen substituent, -OH, -SH, -NH2 , -C1 -C10 alkyl-NH2 , -N(C1 -C10 ) alkyl(C1 -C10 alkyl), -NH(C1 -C10 alkyl), -N(C1 -C10 alkyl)(C1 -C10 alkylphenyl), -NH(C1 -C10 alkylphenyl), cyano, -CO2 H, C(O)O(C1 -C10 ) alkyl), -CON(C1 -C10 ) alkyl(C1 -C10 alkyl), -CONH(C1 -C10 ) alkyl, -CONH2 , -NH C(O)(C1 -C10 -NHC(O)(phenyl), -N(C1 -C10 )alkyl, -N(C1 -C10 )alkyl)C(O)(C1 -C10 )alkyl), -N(C1 -C10 )alkyl)C(O)(phenyl), -C(O)C1 -C10alkyl , -C(O)C1-C 10alkylphenyl, -C(O)C1-C 10haloalkyl, -OC(O)C1-C 10alkyl, -SO2 (C1 -C10 )alkyl, -SO2 (phenyl), -SO2 (C1 -C10 )haloalkyl), -SO2 NH2 , -SO2 NH(C1-C 10alkyl), -SO2 NH(phenyl), -NHSO2 (C1 -C10 )alkyl), -NHSO2 (phenyl) and -NHSO2 (C1 -C)haloalkyl);
每个L1独立的是长度为1-40个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的任何一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中R1可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10)烷基(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10)烷基苯基)、氰基、-CO2H、C(O)O(C1-C10)烷基)、-CON(C1-C10)烷基(C1-C10烷基)、-CONH(C1-C10)烷基、-CONH2、-NH C(O)(C1-C10)烷基)、-NHC(O)(苯基)、-N(C1-C10)烷基、-N(C1-C10烷基)C(O)(C1-C10)烷基)、-N(C1-C10)烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤烷基、-OC(O)C1-C10烷基、-SO2(C1-C10)烷基、-SO2(苯基)、-SO2(C1-C10)卤代烷基)、-SO2NH2,-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10)烷基)、-NHSO2(苯基)和-NHSO2(C1-C10)卤代烷基)；Each L1 is independently a straight chain alkylene group having a length of 1 to 40 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2 , C2 -C10 alkenylene, C2 -C10 alkynylene, C6 -C10 arylene, C3 -C18 heterocyclylene and C5 -C10 heteroarylene; and wherein R1 may optionally have any one or more substituents selected from the group consisting of: C1 -C10 alkyl, C6 -C10 aryl, C5 -C10 heteroaryl, C1 -C10 haloalkyl, -OC1 -C10 alkyl, -OC1 -C10 alkylphenyl, -C 1 -C10 alkyl-OH , -OC1 -C10 haloalkyl, -SC1 -C 10 -C10 alkyl), -C1 -C10 alkylphenyl, -C1 -C10 alkyl-SH, -C1 -C10 haloalkyl, halogen substituent, -OH, -SH, -NH2 , -C1 -C10 alkyl-NH2 , -N(C1 -C10 ) alkyl(C1 -C10 alkyl), -NH(C1 -C10 alkyl), -N(C1 -C10 alkyl)(C1 -C10 alkylphenyl), -NH(C1 -C10 alkylphenyl), cyano, -CO2 H, C(O)O(C1 -C10 ) alkyl), -CON(C1 -C10 ) alkyl(C1 -C10 alkyl), -CONH(C1 -C10 ) alkyl, -CONH2 , -NH C(O)(C1 -C10 -NHC(O)(phenyl), -N(C1 -C10 )alkyl, -N(C1 -C10 )alkyl)C(O)(C1 -C10 )alkyl), -N(C1 -C10 )alkyl)C(O)(phenyl), -C(O)C1 -C10alkyl , -C(O)C1-C 10alkylphenyl, -C(O)C1-C 10haloalkyl, -OC(O)C1-C 10alkyl, -SO2 (C1 -C10 )alkyl, -SO2 (phenyl), -SO2 (C1 -C10 )haloalkyl), -SO2 NH2 , -SO2 NH(C1-C 10alkyl), -SO2 NH(phenyl), -NHSO2 (C1 -C10 )alkyl), -NHSO2 (phenyl) and -NHSO2 (C1 -C10 )haloalkyl);
在一些实施方案中，L1可选自于由A1-A14基团或其任意连接组合所组成的组，其中A1-A14的结构和定义如下所示：In some embodiments,L1 can be selected from the group consisting of A1-A14 groups or any combination thereof, wherein the structures and definitions of A1-A14 are as follows:
其中，每个k1独立地为1-20的整数；Wherein, each k1 is independently an integer from 1 to 20;
每个k2独立地为1-20的整数；Each k2 is independently an integer from 1 to 20;
每个Rc独立地为C1-C10烷基；Each Rc is independently C1 -C10 alkyl;
每个Rd选自于由A15-A19及其任意组合所组成的组：EachRd is selected from the group consisting of A15-A19 and any combination thereof:
每个Re独立地为C1-C10烷基；表示基团共价连接的位点。EachRe is independently a C1 -C10 alkyl group; Indicates the site to which a group is covalently attached.
技术人员应当理解的是，尽管为了方便起见，L1被定义为线性亚烷基，但是它可能不是线性基团或者名称不同，例如由于上述替换和/或取代而产生的胺或烯基。为了本发明内容的目的，L1的长度是连接两个连接点的链中的原子数。为此目的，将替换所述直链亚烷基的碳原子而得到的环(如亚杂环基或亚杂芳基)计为一个原子。It will be appreciated by the skilled person that althoughL is defined as a linear alkylene for convenience, it may not be a linear group or may be named differently, such as an amine or alkenyl resulting from the above replacement and/or substitution. For the purposes of the present disclosure, the length ofL is the number of atoms in the chain connecting the two points of attachment. For this purpose, a ring (such as a heterocyclylene or heteroarylene) resulting from the replacement of a carbon atom of the linear alkylene is counted as one atom.
M1表示靶向基团，其定义和可选择的范围与上述靶向基团相同。在一些实施方案中，每个M1独立地选自对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体中的一种。M1 represents a targeting group, and its definition and selectable range are the same as those of the above-mentioned targeting group. In some embodiments, eachM1 is independently selected from one of the ligands having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.
R2为式(S1)所示结构的基团，其中，E1为OH或SH。R2 is a group having a structure represented by formula (S1), whereinE1 is OH or SH.
R1的选择是为了实现与含氮骨架上的N原子与S1的连接。R1可以是任何能够以适当方式将S1基团连接至含氮骨架上的N原子的连接基团。在一些实施方案中，在通过固相合成的工艺制备式(24)、(25)或(26)所示的siRNA缀合物的情况下，R1基团中需要同时含有与含氮骨架上的N原子连接的连接位点和与R2中的P原子相连接的连接位点。在一些实施方案中，R1中所述与含氮骨架上的N原子连接的位点与N原子形成酰胺键，所述与R2上的P原子连接的位点与P原子形成磷酸酯键。The selection ofR1 is to achieve the connection with the N atom on the nitrogen-containing skeleton and S1.R1 can be any connecting group that can connect the S1 group to the N atom on the nitrogen-containing skeleton in an appropriate manner. In some embodiments, when the siRNA conjugate shown in formula (24), (25) or (26) is prepared by a solid phase synthesis process, theR1 group needs to contain both a connecting site for connecting to the N atom on the nitrogen-containing skeleton and a connecting site for connecting to the P atom inR2 . In some embodiments, the site inR1 that connects to the N atom on the nitrogen-containing skeleton forms an amide bond with the N atom, and the site that connects to the P atom onR2 forms a phosphate bond with the P atom.
在一些实施方案中，siRNA缀合物具有式(Z1-Nu)、(Z2-Nu)、(Z3-Nu)、(Z4-Nu)、(Z5-Nu)、(Z6-Nu)、(Z7-Nu)、(Z8-Nu)、(Z9-Nu)、(Z10-Nu)、(Z11-Nu)、(Z12-Nu)、(Z13-Nu)、(Z14-Nu)、(Z15-Nu)、(Z16-Nu)、(Z17-Nu)、(Z18-Nu)、(Z19-Nu)、(Z20-Nu)、(Z21-Nu)、(Z22-Nu)、(Z23-Nu)、(Z24-Nu)、(Z25-Nu)、(Z26-Nu)、(Z27-Nu)、(Z28-Nu)、(Z29-Nu)、(Z30-Nu)、(Z31-Nu)、(Z32-Nu)所示的结构，其中Z1-Z32为缀合基团，Nu为本发明的siRNA。In some embodiments, the siRNA conjugate has the formula (Z1-Nu), (Z2-Nu), (Z3-Nu), (Z4-Nu), (Z5-Nu), (Z6-Nu), (Z7-Nu), (Z8-Nu), (Z9-Nu), (Z10-Nu), (Z11-Nu), (Z12-Nu), (Z13-Nu), (Z14-Nu), (Z15-Nu), (Z16-Nu), (Z17-Nu), (Z18-Nu), (Z19-Nu), (Z20-Nu), (Z21-Nu), (Z22-Nu), (Z23-Nu), (Z24-Nu), (Z25-Nu), (Z26-Nu), (Z27-Nu), (Z28-Nu), (Z29-Nu), (Z30-Nu), (Z31-Nu), (Z32-Nu), (Z33-Nu), (Z34-Nu), (Z35-Nu), (Z36-Nu), (Z37-Nu), (Z38-Nu), (Z39-Nu), (Z40-Nu), (Z41-Nu), (Z42-Nu), (Z43-Nu), (Z44-Nu), (Z45-Nu), (Z46-Nu), (Z47-Nu), (Z48-Nu), (Z49-Nu), (Z50-Nu), (Z51-Nu), (Z52-Nu), (Z53-Nu), (Z54-Nu), (Z55-Nu), (Z56-Nu), (Z57-Nu), (Z58-Nu), (Z59-Nu), (Z60-Nu), (Z61-Nu), (Z62-Nu), (Z63-Nu), (Z The structures shown in (Z18-Nu), (Z19-Nu), (Z20-Nu), (Z21-Nu), (Z22-Nu), (Z23-Nu), (Z24-Nu), (Z25-Nu), (Z26-Nu), (Z27-Nu), (Z28-Nu), (Z29-Nu), (Z30-Nu), (Z31-Nu), and (Z32-Nu), wherein Z1-Z32 are conjugated groups and Nu is the siRNA of the present invention.
在一些实施方案中，式S1中的P原子可以连接到siRNA序列中任何可能的位置，例如，式S1中的P原子可以连接到siRNA正义链或反义链的任何一个核苷酸上；在一些实施方案中，式S1中的P原子连接到siRNA正义链的任何一个核苷酸上。在一些实施方案中，式S1中的P原子连接到siRNA正义链或反义链的末端；在一些实施方案中，式S1中的P原子连接到siRNA正义链的3’末端。在连接至siRNA的正义链的上述位置的情况下，(24)、(25)或(26)所示的siRNA缀合物进入细胞后，在解旋时，可以释放出单独的siRNA反义链，以阻断INHBEmRNA翻译蛋白质的过程，抑制INHBE基因表达。In some embodiments, the P atom in formula S1 can be linked to any possible position in the siRNA sequence, for example, the P atom in formula S1 can be linked to any nucleotide of the sense strand or antisense strand of the siRNA; in some embodiments, the P atom in formula S1 is linked to any nucleotide of the sense strand of the siRNA. In some embodiments, the P atom in formula S1 is linked to the end of the sense strand or antisense strand of the siRNA; in some embodiments, the P atom in formula S1 is linked to the 3' end of the sense strand of the siRNA. In the case of being linked to the above-mentioned position of the sense strand of the siRNA, after the siRNA conjugates shown in (24), (25) or (26) enter the cell, when unwinding, the separate siRNA antisense strand can be released to block the process of INHBE mRNA translation into protein and inhibit the expression of the INHBE gene.
在一些实施方案中，式S1中的P原子可以连接到siRNA中的核苷酸上任何可能的位置，例如，核苷酸的5’位、核苷酸的2’位、核苷酸的3’位或核苷酸的碱基上。在一些实施方案中，式S1中的P原子可通过形成磷酸二酯键连接至所述siRNA中的核苷酸的2’位、3’位或5’位。在一些实施方案中，式S1中的P原子连接在siRNA正义链3’末端核苷酸的3’羟基脱氢后形成的氧原子上(此时，S1中的P原子也可以看作是siRNA中含有的磷酸基团中的P原子)，或者式S1中的P原子通过取代siRNA正义链中的一个核苷酸的2’-羟基中的氢与核苷酸连接，或者式S1中的P原子通过取代siRNA正义链5’末端核苷酸的5’羟基中的氢与核苷酸连接。In some embodiments, the P atom in formula S1 can be connected to any possible position on the nucleotide in the siRNA, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide, or the base of the nucleotide. In some embodiments, the P atom in formula S1 can be connected to the 2' position, 3' position, or 5' position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments, the P atom in formula S1 is connected to the oxygen atom formed after dehydrogenation of the 3' hydroxyl group of the nucleotide at the 3' end of the siRNA sense chain (at this time, the P atom in S1 can also be regarded as the P atom in the phosphate group contained in the siRNA), or the P atom in formula S1 is connected to the nucleotide by replacing the hydrogen in the 2'-hydroxyl group of a nucleotide in the siRNA sense chain, or the P atom in formula S1 is connected to the nucleotide by replacing the hydrogen in the 5' hydroxyl group of the nucleotide at the 5' end of the siRNA sense chain.
本发明的siRNA缀合物在具有显著提高的血浆中稳定性、低脱靶效应的同时，还表现出较高的INHBE mRNA沉默活性。在一些实施方案中，本发明的siRNA可以为表1或表3示出的siRNA中的任意一种。含有这些siRNA的siRNA缀合物表现出更高的INHBE mRNA沉默活性。The siRNA conjugates of the present invention have significantly improved stability in plasma and low off-target effects, while also showing higher INHBE mRNA silencing activity. In some embodiments, the siRNA of the present invention can be any one of the siRNAs shown in Table 1 or Table 3. The siRNA conjugates containing these siRNAs show higher INHBE mRNA silencing activity.
本发明所述siRNA或siRNA缀合物中，每个相邻核苷酸之间由磷酸二酯键或硫代磷酸二酯键连接，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子带有负电荷，它可以以羟基或巯基的形式存在，羟基或巯基中的氢离子也可以部分或全部被阳离子取代。所述阳离子可以是任意的阳离子，如金属阳离子、铵离子NH4+、有机铵阳离子中的一种。出于提高溶解性考虑，在一种实施方案中，所述阳离子选自碱金属离子、三级胺形成的铵阳离子和季铵阳离子中的一种或多种。碱金属离子可以是和/或Na+，三级胺形成的阳离子可以是三乙胺形成的铵离子和/或N，N-二异丙基乙胺形成的铵离子。因此，本发明所述siRNA或siRNA缀合物可以至少部分以盐的形式存在。在一种方式中，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子至少部分与钠离子结合，本发明所述siRNA或siRNA缀合物以钠盐或部分钠盐的形式存在。In the siRNA or siRNA conjugate of the present invention, each adjacent nucleotide is connected by a phosphodiester bond or a thiophosphodiester bond, and the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the thiophosphodiester bond carries a negative charge, which can exist in the form of a hydroxyl or a sulfhydryl group, and the hydrogen ions in the hydroxyl or sulfhydryl group can also be partially or completely replaced by cations. The cation can be any cation, such as a metal cation, an ammonium ion NH4+ , or an organic ammonium cation. In order to improve solubility, in one embodiment, the cation is selected from one or more of an alkali metal ion, an ammonium cation formed by a tertiary amine, and a quaternary ammonium cation. The alkali metal ion can be and/or Na+, and the cation formed by the tertiary amine can be an ammonium ion formed by triethylamine and/or an ammonium ion formed by N, N-diisopropylethylamine. Therefore, the siRNA or siRNA conjugate of the present invention can exist at least partially in the form of a salt. In one embodiment, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the thiophosphodiester bond is at least partially bound to a sodium ion, and the siRNA or siRNA conjugate of the present invention exists in the form of a sodium salt or a partial sodium salt.
本领域技术人员知晓的是，可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本发明所述的siRNA中。制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。所有修饰的核苷单体均可以商购得到或者采用已知方法制备得到。It is known to those skilled in the art that the modified nucleotide groups can be introduced into the siRNA of the present invention by using nucleoside monomers with corresponding modifications. The method of preparing nucleoside monomers with corresponding modifications and the method of introducing the modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or prepared by known methods.
式(24)、(25)或(26)所示的siRNA缀合物的制备Preparation of siRNA conjugates represented by formula (24), (25) or (26)
可以采用任意合理的合成路线制备式(24)、(25)或(26)所示的siRNA缀合物。Any reasonable synthetic route can be used to prepare the siRNA conjugates represented by formula (24), (25) or (26).
在一些实施方案中，式(24)、(25)或(26)所示的siRNA缀合物可以采用如下方法制备，该方法包括在亚磷酰胺固相合成的条件下，分别按照siRNA正义链和反义链的核苷酸种类和顺序，按照3’到5’的方向将核苷单体依次连接，每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；分离出siRNA的正义链和反义链，退火，其中，所述siRNA为上述本发明的siRNA；并且，该方法还包括在偶联反应条件和偶联试剂存在下，将式(27)、(28)或(29)所示的化合物与核苷单体或连接在固相载体上的核苷酸序列接触，使式(27)、(28)或(29)所示的化合物经偶联反应连接至核苷酸序列。下文中，式(27)、(28)或(29)所示的化合物也称作缀合分子。In some embodiments, the siRNA conjugate shown in formula (24), (25) or (26) can be prepared by the following method, which includes connecting nucleoside monomers in sequence from 3' to 5' according to the nucleotide types and sequences of the sense and antisense strands of the siRNA under the conditions of phosphoramidite solid phase synthesis, wherein the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; separating the sense and antisense strands of the siRNA and annealing, wherein the siRNA is the siRNA of the present invention described above; and the method also includes contacting the compound shown in formula (27), (28) or (29) with the nucleoside monomer or the nucleotide sequence connected to the solid phase carrier under coupling reaction conditions and in the presence of a coupling agent, so that the compound shown in formula (27), (28) or (29) is connected to the nucleotide sequence through a coupling reaction. Hereinafter, the compound shown in formula (27), (28) or (29) is also referred to as a conjugated molecule.
其中：in:
R3为能够结合至式(24)、(25)或(26)所示的化合物中Nu代表的siRNA的基团。在一些实施方案中，R3为能够通过共价键结合至Nu代表的siRNA的基团。在一些实施方案中，R3为能够经反应而通过磷酸二酯键缀合至Nu代表的siRNA的任意官能团的基团；R3 is a group capable of binding to the siRNA represented by Nu in the compound represented by formula (24), (25) or (26). In some embodiments, R3 is a group capable of binding to the siRNA represented by Nu through a covalent bond. In some embodiments,R3 is a group capable of reacting and conjugating to any functional group of the siRNA represented by Nu through a phosphodiester bond;
每个T1独立地是M1中全部活性羟基被YCOO-基团取代而形成的基团，其中，每个Y独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种；在一些实施方案中，Y为甲基。L1的定义和可选择的范围如前所述。Each T1 is independently a group formed by replacing all active hydroxyl groups in M1 with YCOO- groups, wherein each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl and alkylphenyl; in some embodiments, Y is methyl. The definition and optional range ofL 1 are as described above.
R3的选择是为了实现与含氮骨架上的N原子的连接，并且为合成式(24)、(25)或(26)所示的siRNA缀合物提供合适的反应位点。在一些实施方案中，R3中包括R1连接基团或经保护的R1连接基团，以及可通过反应与siRNA形成S1所示结构的官能团。R3 is selected to achieve connection with the N atom on the nitrogen-containing skeleton and to provide a suitable reaction site for synthesizing the siRNA conjugates shown in formula (24), (25) or (26). In some embodiments,R3 includes anR1 linking group or a protectedR1 linking group, and a functional group that can react with siRNA to form the structure shown in S1.
在一些实施方案中，R3包含可与Nu代表的siRNA或核苷单体上的基团形成亚磷酸酯的第1官能团以及可与羟基或氨基反应形成共价键的第2官能团或者含有由所述共价键连接的固相载体。在一些实施方案中，所述第1官能团为亚磷酰胺、羟基或被保护的羟基。在一些实施方案中，所述第2官能团为亚磷酰胺、羧基或羧酸盐。在一些实施方案中，所述第2官能团为经由共价键连接至分子其他部分的固相载体，所述共价键由羟基或氨基形成。在一些实施方案中，所述固相载体经由磷酸酯键、羧酸酯键或酰胺键连接。在一些实施方案中，所述固相载体为树脂。In some embodiments, R3 comprises a first functional group that can form a phosphite with a group on the siRNA or nucleoside monomer represented by Nu and a second functional group that can react with a hydroxyl or amino group to form a covalent bond or contains a solid support connected by the covalent bond. In some embodiments, the first functional group is a phosphoramidite, a hydroxyl or a protected hydroxyl. In some embodiments, the second functional group is a phosphoramidite, a carboxyl or a carboxylate. In some embodiments, the second functional group is a solid support connected to the rest of the molecule via a covalent bond, and the covalent bond is formed by a hydroxyl or amino group. In some embodiments, the solid support is connected via a phosphate bond, a carboxylate bond or an amide bond. In some embodiments, the solid support is a resin.
在一些实施方案中，所述第1官能团含有羟基、-ORm或式(C3)所示的基团；所述第2官能团含有式(C1)、(C2)、(C3)、(C1’)或(C3’)所示的结构：In some embodiments, the first functional group contains a hydroxyl group, -ORm, or a group represented by formula (C3); the second functional group contains a structure represented by formula (C1), (C2), (C3), (C1') or (C3'):
式中，q1为1-4的整数，X为O或NH，M+为阳离子，Rm为羟基保护基团，SPS表示固相载体，表示基团连接共价部分的位点。In the formula, q1 is an integer of 1-4, X is O or NH, M+ is a cation,Rm is a hydroxyl protecting group, SPS represents a solid phase support, Denotes the site at which a group is attached to a covalent moiety.
在一些实施方案中，所述第1官能团含有亚磷酰胺基团，如式(C3)所示，该亚磷酰胺基团可以与核苷酸上的任意位置的羟基，如2’位羟基、3’位羟基或5’位羟基发生偶联反应形成亚磷酸酯，并经氧化或硫化形成式S1所示的磷酸二醋键或硫代磷酸酯键，将缀合分子缀合至siRNA。此时，即使所述第2官能团并不存在，式(27)、(28)或(29)所示化合物也能够缀合至核苷酸，不影响式(24)、(25)、(26)所示的siRNA缀合物的获得。在此情况下，在经由亚磷酰胺固相合成等方法获得siRNA的正义链或反义链后，使式(27)、(28)或(29)所示化合物与核苷酸序列中末端核苷酸上的羟基反应，并在后续的氧化或硫化过程中形成磷酸二酯键连接或硫代磷酸酯连接，将式(27)、(28)或(29)所示化合物缀合至siRNA。In some embodiments, the first functional group contains a phosphoramidite group, as shown in formula (C3), which can react with a hydroxyl group at any position on the nucleotide, such as the 2' hydroxyl group, the 3' hydroxyl group or the 5' hydroxyl group to form a phosphite, and then oxidize or sulfurize to form a phosphodiester bond or a phosphorothioate bond as shown in formula S1, thereby conjugating the conjugate molecule to the siRNA. In this case, even if the second functional group does not exist, the compound shown in formula (27), (28) or (29) can be conjugated to the nucleotide, without affecting the acquisition of the siRNA conjugate shown in formula (24), (25) or (26). In this case, after obtaining the sense strand or antisense strand of siRNA by phosphoramidite solid phase synthesis or the like, the compound represented by formula (27), (28) or (29) is reacted with the hydroxyl group on the terminal nucleotide in the nucleotide sequence, and a phosphodiester bond or a phosphorothioate bond is formed in a subsequent oxidation or sulfurization process to conjugate the compound represented by formula (27), (28) or (29) to the siRNA.
在一些实施方案中，所述第1官能团含有被保护的羟基。在一些实施方案中，所述第2官能团包含可与固相载体反应的基团，所述反应提供包含固相载体的缀合分子。在一些实施方案中，所述第2官能团含有羧基、羧酸盐或亚磷酰胺，如式(C1)、(C2)或(C3)所示，当所述第2官能团包含羧基或羧酸盐时，式(27)、(28)或(29)所示化合物与固相载体，例如树脂上的羟基或氨基进行酯化反应或酰胺化反应，形成经羧酸酯键连接的包含固相载体的缀合分子。当所述第2官能团包含亚磷酰胺官能团时，式(27)、(28)或(29)所示化合物与通用固相载体，例如树脂上的羟基发生偶联反应，并经氧化形成经磷酸二酯键连接的包含固相载体的缀合分子。随后，以上述连接固相载体后的产物作为起始，按照亚磷酰胺固相合成方法依次连接核苷单体，获得连接有缀合基团的siRNA的正义链或反义链。在亚磷酰胺固相合成过程中，所述第1官能团发生脱保护，随后在偶联反应条件下与核苷单体上的亚磷酰胺基团发生偶联。In some embodiments, the first functional group contains a protected hydroxyl group. In some embodiments, the second functional group contains a group that can react with a solid phase carrier, and the reaction provides a conjugated molecule containing a solid phase carrier. In some embodiments, the second functional group contains a carboxyl group, a carboxylate or a phosphoramidite, as shown in formula (C1), (C2) or (C3). When the second functional group contains a carboxyl group or a carboxylate, the compound shown in formula (27), (28) or (29) undergoes an esterification reaction or an amidation reaction with a solid phase carrier, such as a hydroxyl group or an amino group on a resin, to form a conjugated molecule containing a solid phase carrier connected via a carboxylate ester bond. When the second functional group contains a phosphoramidite functional group, the compound shown in formula (27), (28) or (29) undergoes a coupling reaction with a universal solid phase carrier, such as a hydroxyl group on a resin, and is oxidized to form a conjugated molecule containing a solid phase carrier connected via a phosphodiester bond. Subsequently, starting from the product after the solid phase support is connected, the nucleoside monomers are sequentially connected according to the phosphoramidite solid phase synthesis method to obtain the sense strand or antisense strand of the siRNA connected with the conjugated group. During the phosphoramidite solid phase synthesis process, the first functional group is deprotected and then coupled with the phosphoramidite group on the nucleoside monomer under the coupling reaction conditions.
在一些实施方案中，所述第1官能团含有羟基或被保护的羟基；所述第2官能团含有经羧酸酯键连接的固相载体或经酰胺键连接的固相载体或者经磷酸酯键连接的固相载体，如式(C1’)或(C3’)所示。此时，由式(27)、(28)、(29)所示化合物代替固相载体作为起始，按照亚磷酰胺固相合成方法依次连接核苷单体，获得连接有缀合基团的siRNA的正义链或反义链。In some embodiments, the first functional group contains a hydroxyl group or a protected hydroxyl group; the second functional group contains a solid phase carrier connected via a carboxylate bond, a solid phase carrier connected via an amide bond, or a solid phase carrier connected via a phosphate bond, as shown in formula (C1') or (C3'). In this case, the solid phase carrier is replaced by the compound shown in formula (27), (28), or (29) as a starting point, and the nucleoside monomers are sequentially connected according to the phosphoramidite solid phase synthesis method to obtain the sense strand or antisense strand of the siRNA connected with the conjugated group.
在一些实施方案中，每个T1独立地是M1。在一些实施方案中，每个S1独立地M1中至少一个活性羟基被羟基保护基团保护而形成的基团。在一些实施方案中，被保护的羟基可以式YCOO-表示，其中，每个Y独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种；在一些实施方案中，Y为甲基。In some embodiments, each T1 is independently M1. In some embodiments, each S1 is independently a group formed by protecting at least one active hydroxyl group in M1 by a hydroxyl protecting group. In some embodiments, the protected hydroxyl group can be represented by the formula YCOO-, wherein each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl and alkylphenyl; in some embodiments, Y is methyl.
在一些实施方案中，Rm是MMTr(4-甲氧基三苯甲基)、DMTr(4,4’-双甲氧基三苯甲基)、TMTr(4,4’,4”-三甲氧基三苯甲基)中的一种或多种。在一些实施方案中，Rm可以是DMTr，即4,4'-双甲氧基三苯甲基(4,4'-dimethoxytrityl)。In some embodiments, Rm is one or more of MMTr (4-methoxytrityl), DMTr (4,4'-bismethoxytrityl), TMTr (4,4',4"-trimethoxytrityl). In some embodiments, Rm may be DMTr, i.e., 4,4'-bismethoxytrityl.
相应地，除非另有说明，以下涉及缀合物和/或缀合分子的制备的描述中，当提及“脱保护”、“偶联”、“盖帽”、“氧化”、“硫化”等反应时，应当理解为本领域公知的亚磷酰胺核酸固相合成方法中所涉及的反应条件和试剂也同样适用于这些反应。示例性的反应条件和试剂将在后文详细描述。Accordingly, unless otherwise indicated, in the following descriptions of the preparation of conjugates and/or conjugated molecules, when referring to reactions such as "deprotection", "coupling", "capping", "oxidation", and "sulfurization", it should be understood that the reaction conditions and reagents involved in the phosphoramidite nucleic acid solid phase synthesis method known in the art are also applicable to these reactions. Exemplary reaction conditions and reagents will be described in detail later.
如前所述，式(24)、(25)或(26)所示的siRNA缀合物的制备方法还包括如下步骤：合成siRNA的另一链(例如，当上述步骤合成了连接有缀合分子的siRNA正义链时，还包括按照固相合成方法合成siRNA的反义链，反之亦然)，分离正义链和反义链，以及退火。具体地，在分离步骤中，连接至核苷酸序列和/或缀合分子的固相载体被切割下来，同时必要的保护基团被脱除(此时，式(27)、(28)或(29)所示化合物中的各S1基团转化为对应的M1靶向基团)，获得连接有缀合分子的siRNA正义链(或反义链)以及对应的反义链(或正义链)，正义链与反义链退火形成双链RNA结构，获得式(24)、(25)或(26)所示的siRNA缀合物。As mentioned above, the method for preparing the siRNA conjugate shown in formula (24), (25) or (26) further includes the following steps: synthesizing the other strand of siRNA (for example, when the above step synthesizes the siRNA sense strand connected to the conjugated molecule, it also includes synthesizing the antisense strand of siRNA according to the solid phase synthesis method, and vice versa), separating the sense strand and the antisense strand, and annealing. Specifically, in the separation step, the solid phase carrier connected to the nucleotide sequence and/or the conjugated molecule is cut off, and the necessary protective groups are removed (at this time, each S1 group in the compound shown in formula (27), (28) or (29) is converted into the corresponding M1 targeting group), and the siRNA sense strand (or antisense strand) connected to the conjugated molecule and the corresponding antisense strand (or sense strand) are obtained, and the sense strand and the antisense strand are annealed to form a double-stranded RNA structure to obtain the siRNA conjugate shown in formula (24), (25) or (26).
在一些实施方案中，式(24)、(25)或(26)所示的siRNA缀合物的制备方法包含以下步骤：在偶联反应条件和偶联试剂存在下，将式(27)、(28)或(29)所示的化合物与正义链或反义链的3’端的第一个核苷单体接触，使式(27)、(28)或(29)所示的化合物连接上序列中第一个核苷酸，在亚磷酰胺固相合成的条件下，按照预期的正义链或反义链核苷酸种类和顺序，以3’到5’的方向将核苷单体依次连接，合成siRNA的正义链或反义链；其中，式(27)、(28)或(29)所示化合物为R2中含有第1官能团和第2官能团，第1官能团含有被保护的羟基，第2官能团具有如式(C1’)或(C3’)所示结构的化合物，与第一个核苷单体连接前，式(27)、(28)或(29)所示化合物经过脱保护；每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；得到连接有缀合基团的核酸的正义链或反义链；在亚嶙酰胺固相合成的条件下，按照反义链或正义链核苷酸种类和顺序，按照3’到5’的方向将核苷单体依次连接，合成核酸的反义链或正义链；每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；脱除保护基并与固相载体切割，分离纯化获得正义链和反义链，退火。In some embodiments, the method for preparing the siRNA conjugate represented by formula (24), (25) or (26) comprises the following steps: contacting the compound represented by formula (27), (28) or (29) with the first nucleoside monomer at the 3' end of the sense chain or antisense chain under coupling reaction conditions and in the presence of a coupling reagent, so that the compound represented by formula (27), (28) or (29) is connected to the first nucleotide in the sequence, and under phosphoramidite solid phase synthesis conditions, the nucleoside monomers are sequentially connected in the 3' to 5' direction according to the expected sense chain or antisense chain nucleotide type and sequence to synthesize the sense chain or antisense chain of siRNA; wherein the compound represented by formula (27), (28) or (29) is a compound wherein R2 contains a first functional group and a second functional group, and the first functional group The compound of formula (27), (28) or (29) is deprotected before being connected to the first nucleoside monomer; the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; the sense strand or antisense strand of the nucleic acid connected with the conjugated group is obtained; under the conditions of amide solid phase synthesis, the nucleoside monomers are sequentially connected in the direction of 3' to 5' according to the type and sequence of nucleotides in the antisense strand or sense strand to synthesize the antisense strand or sense strand of the nucleic acid; the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; the protecting group is removed and cut with the solid phase carrier, the sense strand and the antisense strand are separated and purified, and annealed.
在一些实施方案中，式(24)、(25)或(26)所示的siRNA缀合物的制备方法包含以下步骤：按照该双链siRNA中正义链或反义链的核苷酸种类和顺序，按照3’到5’的方向将核苷单体依次连接，合成正义链和反义链，每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应，得到连接在固相载体上的正义链和连接在固相载体上的反义链；在偶联反应条件和偶联试剂存在下，将式(27)、(28)或(29)所示的化合物与连接在固相载体上的正义链或连接在固相载体上的反义链接触，将式(27)、(28)或(29)所示化合物连接至正义链或反义链，其中，式(27)、(28)或(29)所示化合物是R3中含有第1官能团，第1官能团为亚磷酰胺基团的式(27)、(28)或(29)所示化合物；脱除保护基并与固相载体切割，分别分离纯化，获得siRNA的正义链或反义链，退火，其中，所述siRNA的正义链或反义链上连接有缀合基团。In some embodiments, the method for preparing the siRNA conjugate shown in formula (24), (25) or (26) comprises the following steps: according to the nucleotide type and order of the sense strand or antisense strand in the double-stranded siRNA, the nucleoside monomers are sequentially connected in the 3' to 5' direction to synthesize the sense strand and the antisense strand, and the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization to obtain the sense strand connected to the solid phase support and the antisense strand connected to the solid phase support; under coupling reaction conditions and in the presence of a coupling reagent, the compound shown in formula (27), (28) or (29) is contacted with the sense strand connected to the solid phase support or the antisense strand connected to the solid phase support, and the compound shown in formula (27), (28) or (29) is connected to the sense strand or the antisense strand, wherein the compound shown in formula (27), (28) or (29) is R3 contains a first functional group, wherein the first functional group is a compound represented by formula (27), (28) or (29) which is a phosphoramidite group; removing the protecting group and cutting with the solid phase carrier, separating and purifying respectively to obtain the sense chain or antisense chain of siRNA, and annealing, wherein the sense chain or antisense chain of the siRNA is connected with a conjugated group.
在一些实施方案中，式S1中的P原子连接至siRNA中的正义链的3’末端，式(24)、(25)或(26)所示的siRNA缀合物的制备方法包括：In some embodiments, the P atom in formula S1 is connected to the 3' end of the sense strand in the siRNA, and the method for preparing the siRNA conjugate shown in formula (24), (25) or (26) comprises:
(1)脱除式(27)、(28)或(29)所示化合物(其中，式(27)、(28)或(29)所示化合物为R3中含有第1官能团和第2官能团，第1官能团含有被保护的羟基ORm，第2官能团具有如式(C1’)或(C3’)所示结构的化合物)中的羟基保护基团Rm；在偶联反应条件和偶联试剂存在下，将脱保护得到的产物与核苷单体接触，得到通过缀合分子连接至固相载体的核苷单体；(1) removing the hydroxyl protecting group R m in the compound represented by formula (27), (28) or (29) (wherein the compound represented by formula (27), (28) or (29) is a compound in whichR3 contains a first functional group and a second functional group, the first functional group contains a protected hydroxyl group ORm , and the second functional group has a structure represented by formula (C1′) or (C3′)); contacting the deprotected product with a nucleoside monomer under coupling reaction conditions and in the presence of a coupling reagent to obtain a nucleoside monomer connected to a solid phase carrier via a conjugated molecule;
(2)以该通过缀合分子连接至固相载体的核苷单体起始，按照3’-5’的方向通过亚磷酰胺固相合成方法合成siRNA的正义链；(2) starting with the nucleoside monomer connected to the solid phase carrier via the conjugated molecule, synthesizing the positive strand of the siRNA via a phosphoramidite solid phase synthesis method in the 3'-5' direction;
(3)通过亚磷酰胺固相合成方法，合成siRNA的反义链；(3) synthesizing the antisense strand of siRNA by phosphoramidite solid phase synthesis method;
(4)分离出siRNA的正义链和反义链并退火，获得式(24)、(25)或(26)所示的siRNA缀合物。(4) Separating the sense strand and antisense strand of siRNA and annealing them to obtain the siRNA conjugate represented by formula (24), (25) or (26).
在获得所述缀合物后，在一些实施方案中，还可利用例如液质联用色谱等方法，通过分子量检测等方式对所合成的式(24)、(25)或(26)所示的siRNA缀合物进行表征，确定所合成的siRNA缀合物为目标设计的式(24)、(25)或(26)所示的siRNA缀合物，且所合成的siRNA的序列为期望的siRNA的序列。After obtaining the conjugate, in some embodiments, the synthesized siRNA conjugate shown in formula (24), (25) or (26) can be characterized by molecular weight detection and the like using methods such as liquid chromatography-mass spectrometry to determine whether the synthesized siRNA conjugate is the target designed siRNA conjugate shown in formula (24), (25) or (26), and the sequence of the synthesized siRNA is the desired siRNA sequence.
在一些实施方案中，所述固相载体为本领域中公知的可用于核酸固相合成的固相载体。In some embodiments, the solid phase support is a solid phase support known in the art and can be used for solid phase synthesis of nucleic acids.
药物组合物Pharmaceutical composition
本发明还包括包含本发明的siRNA缀合物的药物组合物和制剂。在一些实施方案中，本文提供了包含如本文所述的siRNA缀合物和药学上可接受的载体的药物组合物。在一些实施方案中，本文中所述药物组合物还包括盐酸小檗碱或二甲双胍。The present invention also includes pharmaceutical compositions and formulations comprising the siRNA conjugates of the present invention. In some embodiments, provided herein are pharmaceutical compositions comprising the siRNA conjugates as described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions described herein further include berberine hydrochloride or metformin.
包含siRNA缀合物的药物组合物可用于治疗与INHBE基因的表达或活性相关的疾病或病症。此类药物组合物是基于递送方式配制的。一个实例是配制用于通过肠胃外递送(例如通过皮下(SC)、肌内(IM)或静脉内(IV)递送)进行系统性施用的组合物。在某些实施方案中，本发明提供了配制用于器官特异性(例如，肝)动脉内、肿瘤内、真皮内、玻璃体内注射、眼外用、眼用(滴眼液)、雾化、眼局部或其他局部途径、栓剂或口服施用的组合物。在优选的实施方案中，组合物皮下施用。Pharmaceutical compositions comprising siRNA conjugates can be used to treat diseases or conditions associated with the expression or activity of INHBE genes. Such pharmaceutical compositions are formulated based on the mode of delivery. An example is a composition formulated for systemic administration by parenteral delivery (e.g., by subcutaneous (SC), intramuscular (IM) or intravenous (IV) delivery). In certain embodiments, the invention provides compositions formulated for organ-specific (e.g., liver) intraarterial, intratumoral, intradermal, intravitreal injection, external eye, ophthalmic (eye drops), atomization, ocular topical or other local routes, suppositories or oral administration. In a preferred embodiment, the composition is administered subcutaneously.
本发明的药物组合物可以以足以抑制INHBE基因表达的剂量施用。在一些实施例中，以如下剂量施用siRNA缀合物：每剂约0.5mg/kg至50mg/kg，或0.3mg/kg至20mg/kg，或3mg/kg至10mg/kg，或优选每剂3mg/kg至10mg/kg。例如，siRNA缀合物可以以每单一剂量约0.5mg/kg、1mg/kg、1.5mg/kg、2mg/kg、3mg/kg、10mg/kg、20mg/kg、30mg/kg、40mg/kg或50mg/kg的剂量施用。The pharmaceutical composition of the present invention can be administered at a dose sufficient to inhibit INHBE gene expression. In some embodiments, the siRNA conjugate is administered at a dose of about 0.5 mg/kg to 50 mg/kg per dose, or 0.3 mg/kg to 20 mg/kg, or 3 mg/kg to 10 mg/kg, or preferably 3 mg/kg to 10 mg/kg per dose. For example, the siRNA conjugate can be administered at a dose of about 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg or 50 mg/kg per single dose.
组合物也可以制备并以不依赖于体重的对于受试者的固定剂量包装。可以通过将每公斤体重乘以平均受试者的体重来计算示例性剂量水平。例如，一般成年人的平均体重认为是约70公斤。Compositions can also be prepared and packaged in fixed doses for subjects that are independent of body weight. Exemplary dosage levels can be calculated by multiplying per kilogram of body weight by the average subject's body weight. For example, the average body weight of a typical adult is considered to be about 70 kilograms.
重复剂量方案可以包括定期施用治疗量的siRNA缀合物，例如每月一次、每隔一月一次或每三个月一次。在优选的实施方案中，以不超过每月一次的频率施用siRNA缀合物。在初始治疗方案后，可以较低频率施用治疗。Repeated dosage regimens can include regular administration of therapeutic amounts of siRNA conjugates, such as once a month, once every other month, or once every three months. In a preferred embodiment, the siRNA conjugate is administered at a frequency no greater than once a month. After the initial treatment regimen, treatment can be administered at a lower frequency.
技术人员将理解，某些因素可影响有效治疗受试者所需的剂量和时机，包括但不限于疾病或病症的严重程度、先前的治疗、受试者的总体健康状况或年龄以及存在的其他疾病。此外，用治疗有效量的组合物治疗受试者可以包括单一治疗或一系列治疗。如本文其他地方所描述的，可以使用常规方法或基于使用适当动物模型的体内测试，来估计本发明涵盖的单个siRNA缀合物的有效剂量和体内半衰期。The skilled artisan will appreciate that certain factors may affect the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or condition, previous treatment, the subject's general health or age, and the presence of other diseases. In addition, treating a subject with a therapeutically effective amount of a composition may include a single treatment or a series of treatments. As described elsewhere herein, the effective dosage and in vivo half-life of a single siRNA conjugate encompassed by the present invention may be estimated using conventional methods or based on in vivo testing using appropriate animal models.
A.赋形剂A. Excipients
“药物载体”或“药物赋形剂”是用于将一种或多种核酸递送给动物的药学上可接受的溶剂、助悬剂或任何其他药学上惰性的媒介。这样的试剂是本领域众所周知的。A "pharmaceutical carrier" or "pharmaceutical excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmaceutically inert vehicle for delivering one or more nucleic acids to an animal. Such agents are well known in the art.
B.其他组分B. Other components
本发明的组合物可以以本领域已确立的使用水平另外包含药物组合物中常规存在的其他辅助组分。因此，例如，该组合物可以包含另外的、相容的药物活性物质，例如止痒剂、收敛剂、局部麻醉剂或消炎剂，或者可以包含可用于物理配制本发明的组合物的各种剂型的另外的物质，例如防腐剂、抗氧化剂和稳定剂。然而，当添加时，此类物质不应不适当地干扰本发明组合物的组分的生物活性。可以对制剂进行灭菌，且如果需要，可以与不会有害地与制剂的核酸相互作用的助剂混合，例如是防腐剂、稳定剂、湿润剂、乳化剂、影响渗透压的盐或缓冲剂等。Composition of the present invention can comprise other auxiliary components conventionally present in pharmaceutical composition in addition with the use level that this area has established.Therefore, for example, said composition can comprise other, compatible pharmaceutical active substances, for example antipruritic, astringent, local anesthetic or anti-inflammatory, or can comprise other material of various dosage forms that can be used for physical preparation composition of the present invention, for example preservative, antioxidant and stabilizing agent.However, when adding, such material should not disturb the biological activity of the component of the present composition inappropriately.Preparation can be sterilized, and if necessary, can be mixed with the auxiliary agent that can not be harmfully interacted with the nucleic acid of preparation, for example, preservative, stabilizing agent, wetting agent, emulsifying agent, salt or buffer agent that influences osmotic pressure etc.
在一些实施方案中，本发明中表征的药物组合物包括(a)一种或多种siRNA缀合物化合物和(b)一种或多种通过非RNAi机制发挥功能并且可用于治疗肥胖或心血管相关疾病的药剂。In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more siRNA conjugate compounds and (b) one or more agents that function via non-RNAi mechanisms and are useful for treating obesity or cardiovascular-related diseases.
如上所述，除了它们的施用之外，本文所表征的siRNA缀合物可以与有效治疗肥胖或心血管相关疾病的其他已知药剂组合施用。无论如何，给药医师可以基于使用本领域已知或本文所述的标准功效测量法所观察到的结果来调整siRNA缀合物施用的量和时机。As described above, in addition to their administration, the siRNA conjugates characterized herein can be administered in combination with other known agents effective in treating obesity or cardiovascular-related diseases. In any case, the administering physician can adjust the amount and timing of siRNA conjugate administration based on the results observed using standard efficacy measurements known in the art or described herein.
实施例Example
除非特别说明，以下实施例中所用到的试剂、培养基均为市售商品，所用到的核酸电泳、real-time PCR等操作均参照Molecular Cloning(Cold Spring Harbor LaboratoryPress(1989))所记载的方法进行。所用如式(27)、(28)或(29)的缀合分子购自南京雷正医药科技有限公司。Unless otherwise specified, the reagents and culture media used in the following examples are all commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are all performed according to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). The conjugated molecules of formula (27), (28) or (29) used were purchased from Nanjing Leizheng Pharmaceutical Technology Co., Ltd.
实施例1siRNA的设计Example 1 Design of siRNA
采用oligowalk在线设计一组靶向人类INHBE基因的siRNA(人类：NCBIrefseqIDNM_031479.5；NCBI GeneID:83729)。人类NM_031479.5REFSEQ mRNA，具有2460个碱基长度。同时，为了避免任何序列产生的毒性，还需排除与人源基因相似的序列。A set of siRNA targeting human INHBE gene (human: NCBIrefseqIDNM_031479.5; NCBI GeneID: 83729) was designed online using oligowalk. Human NM_031479.5REFSEQ mRNA has a length of 2460 bases. At the same time, in order to avoid any sequence toxicity, sequences similar to human genes should also be excluded.
靶向INHBE未修饰的siRNA的正义及反义链核苷酸序列的详细列表示于表1。靶向INHBE修饰的siRNA的正义及反义链核苷酸序列的详细列表示于表3。The details of the sense and antisense strand nucleotide sequences of the siRNA targeting INHBE unmodified are shown in Table 1. The details of the sense and antisense strand nucleotide sequences of the siRNA targeting INHBE modified are shown in Table 3.
实施例2siRNA或siRNA缀合物的制备Example 2 Preparation of siRNA or siRNA conjugates
合成：按照亚磷酰胺固相合成技术合成正义链和反义链序列，在Mermade 192合成仪(BioAutomation)上使用固相载体介导的亚磷酰胺化学方法以1μmol规模合成。固相载体是加载有定制GalNAc配体分子的可控多孔玻璃(CPG，)或通用固相载体。辅助合成试剂，2’-F和2’-O-甲基RNA亚磷酰胺等均为商品化可得试剂。使用相应的亚磷酰胺引入2’-F、2’-O-甲基、GNA(二醇核酸)、5’-磷酸和无碱基修饰。在GalNAc修饰的CPG支持物上进行3’GalNAc缀合单链的合成。CPG通用固相载体用于合成反义单链，或5’GalNAc缀合单链的合成。使用5-乙硫基-1H-四唑(ETT)作为活化剂(在乙腈中，0.6M)，所有亚磷酰胺(溶于无水乙腈中，100mM)的偶联时间为5分钟。使用50mM 3-((二甲基氨基-亚甲基)氨基)-3H-1,2,4-二噻唑-3-硫酮(DDTT)于无水乙腈/吡啶(vv＝1/1)中的溶液产生硫代磷酸酯键，反应时间3分钟。所有序列在最后脱除DMT基团后即合成。Synthesis: The sense and antisense sequences were synthesized using phosphoramidite solid phase synthesis technology on a Mermade 192 synthesizer (BioAutomation) using solid phase support-mediated phosphoramidite chemistry at a 1 μmol scale. The solid phase support is a controlled pore glass (CPG, ) or universal solid phase carrier. Auxiliary synthesis reagents, 2'-F and 2'-O-methyl RNA phosphoramidites, etc. are all commercially available reagents. The corresponding phosphoramidites are used to introduce 2'-F, 2'-O-methyl, GNA (diol nucleic acid), 5'-phosphate and base-free modifications. The synthesis of 3'GalNAc-conjugated single strands is carried out on a GalNAc-modified CPG support. The CPG universal solid phase carrier is used to synthesize antisense single strands, or the synthesis of 5'GalNAc-conjugated single strands. 5-Ethylthio-1H-tetrazole (ETT) is used as an activator (in acetonitrile, 0.6M), and the coupling time of all phosphoramidites (dissolved in anhydrous acetonitrile, 100mM) is 5 minutes. Phosphorothioate bonds were generated using 50 mM 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile/pyridine (vv=1/1) for 3 minutes. All sequences were synthesized after the final removal of the DMT group.
CPG上结合的低聚体的切割和去保护：在固相合成终止后，通过用含20％二乙胺的乙腈溶液处理30分钟去除保护基，而没有从CPG上切下寡核苷酸。随后，干燥的CPG在40℃下用浓氨水处理18小时。在离心之后，上清液被转移至新的管中并且用氨水洗涤CPG。浓缩合并的溶液得到固体混合物。Cleavage and deprotection of oligomers bound to CPG: After termination of solid phase synthesis, the protecting groups were removed by treatment with acetonitrile solution containing 20% diethylamine for 30 minutes without cutting the oligonucleotide from CPG. Subsequently, the dried CPG was treated with concentrated aqueous ammonia for 18 hours at 40°C. After centrifugation, the supernatant was transferred to a new tube and the CPG was washed with aqueous ammonia. The combined solution was concentrated to obtain a solid mixture.
纯化：通过使用NanoQ阴离子交换HPLC纯化。缓冲液A是10mM高氯酸钠溶液，20mMTris，1mM EDTA，pH 7.4和含有乙腈20％，以及缓冲液B，500mM高氯酸钠，20mM Tris，1mMEDTA，pH7.4和含有乙腈20％。分离得到目标产物，并用反相C18柱脱盐。Purification: Purification was performed by using NanoQ anion exchange HPLC. Buffer A was 10 mM sodium perchlorate solution, 20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile, and buffer B was 500 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile. The desired product was isolated and desalted using a reverse phase C18 column.
寡核糖核苷酸的退火产生siRNA缀合物：把待退火的RNA寡聚体用无菌RNaseFreeH2O(无RNA水解酶)配制成200μΜ的溶液。如下设置退火反应体系，将总体积为100μL的上述溶液(双链体浓度为10nmol)放置95℃水浴锅10分钟(≥100nmol需求量需要高温20分钟)→迅速放入60℃水浴自然降温→退火完成后的溶液在4℃保存。通过合并等摩尔的RNA溶液混合互补链。Annealing of oligoribonucleotides to produce siRNA conjugates: The RNA oligomers to be annealed are prepared into a 200 μM solution using sterile RNaseFreeH2 O (without RNA hydrolase). The annealing reaction system is set up as follows: a total volume of 100 μL of the above solution (duplex concentration is 10 nmol) is placed in a 95°C water bath for 10 minutes (≥100 nmol requires high temperature for 20 minutes) → quickly placed in a 60°C water bath to cool naturally → the annealed solution is stored at 4°C. Complementary chains are mixed by combining equimolar RNA solutions.
表4显示了使用上述方法合成的INHBE siRNA缀合物。Table 4 shows the INHBE siRNA conjugates synthesized using the above method.
表4.修饰的INHBE siRNA缀合物核苷酸序列Table 4. Modified INHBE siRNA conjugate nucleotide sequences
实施例3siRNA体外活性测试Example 3 siRNA in vitro activity test
通过qPCR定量检测HepG2细胞中INHBE mRNA含量，以化合物的IC50值为指标，来评价siRNA缀合物对INHBE的抑制活性。The INHBE mRNA content in HepG2 cells was quantitatively detected by qPCR, and theIC50 value of the compound was used as an indicator to evaluate the inhibitory activity of the siRNA conjugate on INHBE.
实验材料及试剂：Experimental materials and reagents:
细胞系：HepG2细胞(中国科学院干细胞库提供)。Cell line: HepG2 cells (provided by Stem Cell Bank, Chinese Academy of Sciences).
HepG2细胞培养基(DMEM，Invitrogen-11330032；10％血清，Invitrogen-10099141；100units/mL青霉素和100μg/mL链霉素，Hyclone-SV30010；1％非必需氨基酸，Invitrogen-11140050；2mM L-谷氨酷胺，Invitrogen-25030081；1mM丙酮酸钠，Gibco-11360-070；500μg/mL Geneticin，Invitrogen-10131027)。HepG2 cell culture medium (DMEM, Invitrogen-11330032; 10% serum, Invitrogen-10099141; 100 units/mL penicillin and 100 μg/mL streptomycin, Hyclone-SV30010; 1% non-essential amino acids, Invitrogen-11140050; 2 mM L-glutamine, Invitrogen-25030081; 1 mM sodium pyruvate, Gibco-11360-070; 500 μg/mL Geneticin, Invitrogen-10131027).
试剂：胰酶(Invitrogen-25300062)；DMSO(Sigma-D2650-100ML)；转染试剂Lipofectamine RNAiMAX(Invitrogen-13778-150)；MEM Medium(HyClone-SH30024.01)；ULtraPure Distilled Water(DNAse,RNAse,Free)(Invitrogen-10977-015)；Opti-MEM I(1X)(Gibco-31985-070)；Phosphate Buffered Saline(PBS)(Gibco)；PrimeScriptTMRTreagent Kit with gDNA Eraser(takara-RR047A)；ChamQ Universal SYBR qPCR MasterMix(vyzme-Q711-02)。Reagents: trypsin (Invitrogen-25300062); DMSO (Sigma-D2650-100ML); transfection reagent Lipofectamine RNAiMAX (Invitrogen-13778-150); MEM Medium (HyClone-SH30024.01); ULtraPure Distilled Water (DNAse, RNAse, Free) (Invitrogen-10977-015); Opti-MEM I (1X) (Gibco-31985-070); Phosphate Buffered Saline (PBS) (Gibco); PrimeScriptTM RTreagent Kit with gDNA Eraser (takara-RR047A); ChamQ Universal SYBR qPCR MasterMix (vyzme-Q711-02).
耗材与仪器：48孔细胞培养板(Coming-3599)；CO2培养箱(HERA-CELL-240)；Microplate(Axygen-PCR-96-FLT-C)；qPCR equipment(QIANGE)。Consumables and instruments: 48-well cell culture plate (Corning-3599); CO2 incubator (HERA-CELL-240); Microplate (Axygen-PCR-96-FLT-C); qPCR equipment (QIANGE).
实验步骤：Experimental steps:
siRNA或siRNA缀合物通过转染进入HepG2细胞，过程如下所述：取HepG2细胞，先用PBS洗涤后，加入胰蛋白酶进行消化，调整细胞到合适的密度，24h后应用转染试剂Lipofectamine RNAiMax将siRNA转入细胞，以每孔10,000个细胞的密度接种到48孔板中，每孔HepG2细胞培养基为500μL。细胞置于5％CO2、37℃孵箱中培养48h。转染后48小时，收集细胞，提取RNA，RT-PCR检测细胞内总INHBE-RNA。siRNA or siRNA conjugates were transfected into HepG2 cells as follows: HepG2 cells were washed with PBS, then digested with trypsin, and the cells were adjusted to an appropriate density. After 24 hours, siRNA was transferred into the cells using the transfection reagent Lipofectamine RNAiMax, and the cells were seeded into 48-well plates at a density of 10,000 cells per well, with 500 μL of HepG2 cell culture medium per well. The cells were cultured in a 5% CO2 , 37°C incubator for 48 hours. 48 hours after transfection, the cells were collected, RNA was extracted, and total intracellular INHBE-RNA was detected by RT-PCR.
受试siRNA测试2个浓度点，3复孔。对照设置为NM_001330751.2，测试4个浓度点，2复孔。The test siRNA was tested at 2 concentrations and 3 replicates. The control was set to NM_001330751.2 and tested at 4 concentrations and 2 replicates.
检测INHBE RNA步骤简述如下：应用trizol法提取细胞中的总RNA，参照反转录试剂盒(takara)说明书，加入随机引物反转录成cDNA，然后qPCR检测样品中的INHBE cDNA。同时，GAPDH引物和探针特异性检测GAPDH cDNA。The steps for detecting INHBE RNA are briefly described as follows: the total RNA from the cells was extracted using the trizol method, and random primers were added for reverse transcription into cDNA according to the instructions of the reverse transcription kit (takara), and then qPCR was used to detect INHBE cDNA in the sample. At the same time, GAPDH primers and probes were used to specifically detect GAPDH cDNA.
PCR反应程序为：95℃ 2分钟，然后进入循环模式，95℃ 10秒，随后60℃，30秒，共40个循环。依据各样品的Ct值计算样品中的INHBE RNA含量。The PCR reaction program was: 95°C for 2 minutes, then enter the cycle mode, 95°C for 10 seconds, then 60°C for 30 seconds, for a total of 40 cycles. The INHBE RNA content in the sample was calculated based on the Ct value of each sample.
PCR引物如下：PCR primers are as follows:
Human INHBE-Forward 5-ACTACAGCCAGGGAGTGTGG-3；Human INHBE-Forward 5-ACTACAGCCAGGGAGTGTGG-3;
Human INHBE-Reverse 5-AGTGAGCAGGGAGCTGTAGG-3。Human INHBE-Reverse 5-AGTGAGCAGGGAGCTGTAGG-3.
Human GAPDH-Forward 5-GGAGCGAGATCCCTCCAAAAT-3；Human GAPDH-Forward 5-GGAGCGAGATCCCTCCAAAAT-3;
Human GAPDH-Reverse 5-GGCTGTTGTCATACTTCTCATGG-3。Human GAPDH-Reverse 5-GGCTGTTGTCATACTTCTCATGG-3.
每个样品目的基因INHBE mRNA的表达水平通过ΔΔCt相对定量法进行计算。目的基因相对表达量使用2-ΔΔCT表示，计算公式如下：The expression level of the target gene INHBE mRNA in each sample was calculated by the ΔΔCt relative quantitative method. The relative expression of the target gene was expressed as 2-ΔΔCT , and the calculation formula was as follows:
a)Ct值根据Quant Studio 7软件的默认设置自动计算。将Ct值导出为Excel文件。a) Ct values were automatically calculated according to the default settings of Quant Studio 7 software. Ct values were exported as Excel files.
b)使用以下公式计算基因的相对表达量：b) Calculate the relative expression of genes using the following formula:
ΔCt＝Ct(目的基因)-Ct(gapdh)，ΔCt＝Ct(target gene)-Ct(gapdh),
ΔΔCt＝ΔCt(检测样品)-ΔCt(Mock)，ΔΔCt＝ΔCt(test sample)-ΔCt(Mock),
相对于Mock的mRNA表达＝2-ΔΔCt，mRNA expression relative to Mock = 2- ΔΔCt ,
其中，Mock代表添加等浓度的Lipofectamine RNAiMax，并设置siRNA的阴性对照(NC)，阴性对照序列为：正义链为ACUACUGAGUGACAGUAGAUU，反义链为UCUACUGUCACUCAGUAGUUU。Among them, Mock represents the addition of equal concentrations of Lipofectamine RNAiMax and the setting of the negative control (NC) of siRNA. The negative control sequence is: the positive chain is ACUACUGAGUGACAGUAGAUU, and the antisense chain is UCUACUGUCACUCAGUAGUUU.
抑制率如下计算：(1-(2-ΔΔCt))*100％。表5显示了本发明的siRNA对INHBE的抑制活性。The inhibition rate was calculated as follows: (1-(2-ΔΔCt ))*100%. Table 5 shows the inhibitory activity of the siRNA of the present invention on INHBE.
表5.本发明的siRNA对INHBE RNA的抑制率Table 5. Inhibition rate of siRNA of the present invention on INHBE RNA
利用上述相同的方法转染至HepG2细胞中，测定本发明的siRNA缀合物对INHBE的抑制活性(IC50)使用GraphPad进行数据拟合得出IC50数值。表6显示了本发明的siRNA缀合物对INHBE的抑制活性(IC50)。The cells were transfected into HepG2 cells using the same method as above, and the inhibitory activity (IC50 ) of the siRNA conjugates of the present invention on INHBE was determined. GraphPad was used for data fitting to obtain the IC50 value. Table 6 shows the inhibitory activity (IC50 ) of the siRNA conjugates of the present invention on INHBE.
表6.本发明的siRNA缀合物对INHBE的抑制活性(IC50)Table 6. Inhibitory activity (IC50 ) of the siRNA conjugates of the present invention on INHBE
从表6可以看出，本发明提供的siRNA缀合物在HepG2细胞中具有较高的INHBE抑制活性。As can be seen from Table 6, the siRNA conjugate provided by the present invention has a higher INHBE inhibitory activity in HepG2 cells.
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。Although the present invention has been disclosed as above in the preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
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