WO2022052738A1 - 环状RNA Cwc27下调剂在制备预防和/或治疗阿尔茨海默病的药物中的应用 - Google Patents

环状RNA Cwc27下调剂在制备预防和/或治疗阿尔茨海默病的药物中的应用 Download PDF

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WO2022052738A1
WO2022052738A1 PCT/CN2021/112434 CN2021112434W WO2022052738A1 WO 2022052738 A1 WO2022052738 A1 WO 2022052738A1 CN 2021112434 W CN2021112434 W CN 2021112434W WO 2022052738 A1 WO2022052738 A1 WO 2022052738A1
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rnacwc27
disease
circular
circcwc27
cyclic
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French (fr)
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王昊
陈红专
宋成寰
张永芳
黄婉莹
徐见容
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上海交通大学医学院
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

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  • the invention belongs to the fields of biotechnology and medicine, and specifically relates to the application of circular RNA Cwc27 (circular RNA Cwc27, circCwc27 for short) and its analogs as nucleotide drugs for Alzheimer's disease treatment and/or prevention.
  • AD Alzheimer's disease
  • SP senile plaques
  • a ⁇ ⁇ -amyloid
  • NFT neurofibrillary tangles
  • AD Alzheimer's disease
  • Circular RNAs are a class of endogenous non-coding RNA molecules that do not have a cap structure at the 5' end and a poly A tail structure at the 3' end, and form a circular structure by covalent bonds. It can be derived from exons or introns of coding genes.
  • circRNAs Circular RNAs
  • the purpose of the present invention is to provide the application of cyclic RNACwc27 targets or drugs based on cyclic RNACwc27 targets in the treatment and/or prevention of Alzheimer's disease.
  • the present invention provides the use of a cyclic RNACwc27 down-regulator in the preparation of a medicament for preventing and/or treating Alzheimer's disease, wherein the cyclic RNACwc27 down-regulator is a cyclic RNACwc27 inhibitor and/or a cyclic RNACwc27 modifiers.
  • the circular RNACwc27 down-regulating agent can reduce the transcription level of A ⁇ precursor protein APP by inhibiting the entry of Pur- ⁇ into the nucleus, reduce the expression level of APP protein, improve learning and memory ability, and has clinical application prospects for treating AD.
  • the circular RNACwc27 down-regulating agent is: an RNA fragment comprising the base sequence shown in SEQ ID NO.1; an RNA fragment comprising a base sequence complementary to the base sequence shown in SEQ ID NO.2; based on SEQ ID NO.3 is a cyclic RNACwc27 modification modified by substitution, deletion or addition of one or more nucleotides; or a polypeptide, protein or compound capable of inhibiting the expression of cyclic RNACwc27.
  • SEQ ID NO.1 is the nucleotide sequence of the down-regulating agent of circular RNACwc27
  • SEQ ID NO.2 is the nucleotide sequence at the joint of circular RNACwc27
  • SEQ ID NO.3 is the nucleoside of circular RNACwc27 itself acid sequence.
  • the sequence of circCwc27 in mouse, rat and human brain is highly conserved, basically including the base sequence shown in SEQ ID NO. It can be used as a reliable target for the treatment and/or prevention of Alzheimer's disease.
  • the cyclic RNACwc27 modifier is obtained by performing at least one of the following modifications on cyclic RNACwc27: ribose modification of any nucleotide, base modification, phosphate backbone modification, and addition of any nucleotide. Subtract, replace.
  • the medicament includes a circular RNACwc27 down-regulating agent, and a pharmaceutically acceptable carrier or adjuvant.
  • the present invention provides the use of circular RNACwc27 as a target in the development, screening or preparation of drugs for preventing and/or treating Alzheimer's disease.
  • the present invention provides the use of circular RNACwc27 as a target in preparing a drug screening model for preventing and/or treating Alzheimer's disease.
  • the present invention provides a method for screening a drug for preventing and/or treating Alzheimer's disease, the method comprising:
  • RNACwc27 is reduced, it indicates that the candidate substance is a potential substance for preventing and/or treating Alzheimer's disease.
  • Figure 1 shows the levels of circCwc27 in the cortex, hippocampus and AD patients of APP/PS1 mice, where A is the expression level of each circRNA in the cortex of APP/PS1 and control wild-type mice, and B is APP/PS1 and control wild-type mice The expression levels of circRNAs in the hippocampus of mice; C is the level of circCwc27 in the plasma of AD patients and healthy controls; it can be seen from Figure 1 that the levels of circCwc27 in the plasma of AD patients and the brain tissue of APP/PS1 mice were significantly increased.
  • Figure 2 shows the effect of circCwc27 interference virus on the expression of APP protein in the brain of APP/PS1 mice;
  • A is the electropherogram of APP protein in the hippocampus of APP/PS1 mice injected with circCwc27 interference virus or control virus, and
  • B is APP in A Statistical chart of protein expression; it can be seen from Figure 2 that the circCwc27 interference virus can significantly reduce the expression of APP protein in the brain of APP/PS1 mice.
  • Figure 3 shows the learning and memory ability of AD model mice after two months of APP/PS1 supplementation with circCwc27 interfering virus LV-sh-circCwc27 via brain stereotaxic injection, where A is the latency time of the mice in the treatment group and the control group to find the target quadrant, B is the area under the curve of the time for the treatment group and the control group to find the target quadrant, C is the swimming speed of the treatment group and the control group, D is the residence time of the treatment group and the control group in the target quadrant, and E is the treatment group and the control group.
  • A is the latency time of the mice in the treatment group and the control group to find the target quadrant
  • B is the area under the curve of the time for the treatment group and the control group to find the target quadrant
  • C is the swimming speed of the treatment group and the control group
  • D is the residence time of the treatment group and the control group in the target quadrant
  • E is the treatment group and the control group.
  • Figure 4 shows the amyloid pathology of AD model mice after APP/PS1 was injected with circCwc27 interfering virus LV-sh-circCwc27 for two months after stereotaxic injection in the brain, where A is the APP/PS1 mice injected with circCwc27 interfering virus or control virus Fluorescence images of amyloid plaques in the hippocampus and cortex, B is the statistical graph of amyloid plaque deposition in Figure A; as can be seen from Figure 4, compared with the control group, the AD model after localized injection supplemented with circCwc27 interfered with the virus was smaller The amyloid pathology in the mice was markedly relieved.
  • circCwc27 non-coding RNA circular RNA Cwc27
  • SEQ ID NO.3 The expression in the brain tissue of Alzheimer's disease patients and model mice is significantly increased, It is also involved in the production of amyloid pathology in the brain.
  • CircCwc27 interfering virus in the brain of AD model mice can significantly alleviate the occurrence of amyloid pathology in the brain and improve the learning and memory ability of mice, indicating that circCwc27 has clinical application in the treatment of AD prospects, thus completing the present invention.
  • preventing a disease refers to preventing the development of clinical symptoms of a disease in a mammal that may have been exposed or pretreated to the disease but has not yet experienced or displayed symptoms of the disease.
  • treating a disease can refer to inhibiting a disease, eg, preventing or reducing the progression of a disease or its clinical symptoms, or alleviating a disease, eg, regressing a disease or its clinical symptoms.
  • circCwc27 downregulators include substances that reduce the level of circCwc27.
  • the cyclic RNACwc27 downregulator is a cyclic RNACwc27 inhibitor and/or a cyclic RNACwc27 modifier.
  • the interfering RNA of circCwc27 including the RNA fragment of the base sequence shown in SEQ ID NO.1.
  • An example of a circCwc27 inhibitor includes an RNA fragment having a nucleotide sequence complementary to the circCwc27 linker on its nucleotide sequence, that is, an RNA fragment including a base sequence complementary to the base sequence shown in SEQ ID NO. 2.
  • circCwc27 modifiers refer to nucleic acid sequences designed based on the nucleotide sequence of circCwc27 and their modifications, such as circular RNACwc27 modifiers based on SEQ ID NO.3 that are substituted, deleted, or added with one or more nucleotides.
  • the modification includes one or a combination of ribose modification, base modification, and phosphate backbone modification of any nucleotide, or addition, subtraction, or substitution of any nucleotide. As long as the modified nucleotide sequence can reduce the similar functional activity of circCwc27.
  • circCwc27 downregulators also include substances that can reduce the activity and stability of circCwc27, and substances that can reduce the effective time of circCwc27.
  • circCwc27 down-regulators also include other polypeptides, proteins or compounds that can inhibit the expression of circCwc27.
  • circCwc27 down-regulator on inhibiting amyloid pathology in AD brain.
  • the application of circCwc27 down-regulator can effectively inhibit the progression of pathological phenotype in AD model mice and improve learning and memory abilities.
  • the study found that circCwc27 was significantly increased in peripheral plasma of AD patients and brain tissue of APP/PS1 mice.
  • inhibiting circCwc27 in the brain can significantly reduce the expression levels of the A ⁇ precursor protein APP and the key cleavage enzyme BACE1, thereby inhibiting the production of A ⁇ and the production of amyloid pathology.
  • the application of the present invention can be used to prepare a nucleotide analog medicine for treating Alzheimer's disease.
  • circCwc27 down-regulator can reduce the transcription levels of APP and BACE1, inhibit the amyloid precursor protein pathway, and serve as a novel nucleotide drug.
  • circCwc27 can be used as a target for the development, screening or preparation of drugs for the prevention and/or treatment of Alzheimer's disease.
  • Candidate substances can be screened by using circCwc27 as the target.
  • the screened substances that can reduce the expression of circCwc27, inhibit the activity and stability of circCwc27, and/or reduce the effective action time of circCwc27 can be used as alternative drugs for the prevention and/or treatment of Alzheimer's disease.
  • a drug screening model targeting circCwc27 can be established to screen drugs targeting circCwc27.
  • a system expressing circCwc27 is treated with a candidate substance, and the expression of circCwc27 in the system is detected. If the expression of circCwc27 is decreased, it indicates that the candidate substance is a potential substance for preventing and/or treating Alzheimer's disease.
  • a pharmaceutical composition which comprises an effective amount of a circCwc27 down-regulating agent, and a pharmaceutically acceptable carrier or adjuvant.
  • Effective amount means that a circCwc27 downregulator (i) treats a particular disease, condition or disorder, (ii) attenuates, ameliorates or eliminates one or more symptoms of a particular disease, condition or disorder, or (iii) prevents or delays the present invention The amount of onset of one or more symptoms of the particular disease, condition or disorder.
  • “Pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid filler or gel substances which are suitable for human use and which must be of sufficient purity and sufficiently low toxicity. "Compatibility” as used herein means that the components of the composition can be intermixed with the active ingredient without significantly reducing the efficacy of the active ingredient.
  • Pharmaceutically acceptable carriers or excipients include but are not limited to cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as Stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc
  • the medicament or pharmaceutical composition in the present disclosure can be prepared into injection preparations, oral preparations, nasal drops, spray preparations, ointment preparations or patches, etc. according to the usual methods of pharmaceutical preparation.
  • circCwc27 down-regulators can also be combined with other drugs for the prevention and/or treatment of Alzheimer's disease.
  • Example 1 The level of circCwc27 was significantly increased in the peripheral plasma of AD patients and the brain tissue of model mice.
  • 6-month-old APP/PS1 transgenic mice and 3 wild control mice of the same age and background were selected. After anesthesia, the brains were collected by perfusion. The brain tissue was divided into two halves. One half was used for RNA extraction for circRNA sequencing analysis, and the other half was polyplexed Paraffin sections were prepared after formaldehyde fixation for in situ hybridization experiments. Relevant operations comply with ethical operating standards and procedures.
  • circRNA sequencing analysis Determine the concentration of NanoDrop ND-100, select 1-2 ⁇ g RNA for library construction, and identify the quality of the library by Agilent2100, and use qPCR for library quantification.
  • the mixed sample libraries are sequenced using Illumina 4000 sequencer , including NaOH alkali denaturation single-strand generation, Illumina flow cell in situ amplification, 150 paired-end cycle sequencing and other steps.
  • the Solexa pipeline version 1.8 software was used for image processing and base recognition, the FastQC software was used to evaluate the quality of the reads after de-linking, the reference genome was compared by the STAR software, the Backsplice junction reads were detected by CIRCexplorer2, and the expression differences were detected by the R software edgeR calculate.
  • RNAeasy TM Plus kit of Biyuntian Company was used to extract total RNA, and real-time fluorescence quantitative PCR was used.
  • the RNA was reverse transcribed into cDNA according to the instructions of the PrimeScript TM RT Master Mix kit provided by TAKARA, and the cDNA was reverse transcribed according to the TB Green TM Premix Ex.
  • the method provided in the instruction of the Taq TM kit was performed on the RT-PCR ABI7300 instrument for real-time fluorescence quantitative PCR reaction, with GAPDH as the internal reference. After the reaction is completed, the CT value in each reaction tube is calculated by the supporting computer software.
  • the relative quantification method 2 ⁇ Ct was used to calculate the relative expression levels of circRNAs.
  • Example 2 Interfering with the expression of circCwc27 reduces the level of APP protein
  • AD model mice were injected with circCwc27 interfering virus LV-sh-circCwc27 (purchased from Shanghai Jiying Biotechnology Co., Ltd.) by stereotaxic injection of APP/PS1 in the brain.
  • circCwc27 interfering RNA was coated with lentivirus as a carrier to stably reduce the expression of circCwc27 in mouse brain for a long time.
  • the sequence of circCwc27 interfering RNA is shown in SEQ ID NO. The volume is 5 ⁇ L.
  • the control group was the group of APP/PS1 mice injected with the supplemented control virus LV-sh-circCon (purchased from Shanghai Jiying Biotechnology Co., Ltd.) via stereotaxic brain.
  • the injected virus concentration was 1 ⁇ 10 9 virus/microliter, and the injection volume was 5 ⁇ L. Then, it was found by western blotting that circCwc27 interfering with the virus could significantly reduce the expression of APP protein in the brain of APP/PS1 mice, indicating that circCwc27 plays an important role in regulating A ⁇ production.
  • Example 3 circCwc27 inhibitor alleviates amyloid pathology and behavioral characterization in AD model mice
  • mice 6-month-old APP/PS1 male mice were purchased from the Model Animal Center of Nanjing University. Mice were randomly divided into WT-LV-sh-circCon group (wild-type mice injected with control virus group), WT-LV-sh-circCwc27 group (wild-type mice injected with circCwc27 interference virus group), APP/PS1-LV -sh-circCon (APP/PS1 mice injected with control virus group) group and APP/PS1-LV-sh-circCwc27 group (APP/PS1 mice injected with circCwc27 interference virus group).
  • WT-LV-sh-circCon group wild-type mice injected with control virus group
  • WT-LV-sh-circCwc27 group wild-type mice injected with circCwc27 interference virus group
  • APP/PS1-LV -sh-circCon APP/PS1 mice injected with control virus group
  • mice were anesthetized by intraperitoneal injection of 3% sodium pentobarbital, their heads were fixed on the stereotaxic injector in the prone position, and 5 ⁇ l of LV-sh-circCwc27 (purchased from Shanghai Jiying Biotechnology Co., Ltd.) solution was drawn with a micro syringe.
  • the concentration of the solution was 1 x 109 virus/microliter, and the brain tissue was inserted vertically at the skull hole.
  • the microinjection pump was used to enter the third ventricle of the mouse at a speed of 0.5 ⁇ l/min (front and rear: -0.3 mm, sagittal suture left/right: 1.0 mm, depth: 2.2 mm), and the syringe was slowly drawn out after the injection was completed.
  • the area around the skull injection site was sterilized, and the skin was sutured and then returned to the mouse cage to continue feeding. Two months later, the learning and memory abilities of the mice were tested by the Morris water maze.
  • the brains were sacrificed and sliced, and the amyloid pathology in the brain was detected (amyloid A ⁇ antibody was purchased from Cell Signaling Technology, product number 2450T).

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Abstract

本发明公开环状RNA Cwc27下调剂在制备预防和/或治疗阿尔茨海默病的药物中的应用。所述环状RNACwc27下调剂为环状RNACwc27抑制剂和/或环状RNACwc27修饰物。所述环状RNACwc27下调剂能够通过抑制Pur-α入核，从而降低Aβ前体蛋白APP的转录水平，减少APP蛋白的表达水平，改善学习和记忆能力，具有治疗AD的临床应用前景。

Description

环状RNA Cwc27下调剂在制备预防和/或治疗阿尔茨海默病的药物中的应用 技术领域
本发明属于生物技术和医学领域，具体涉及环状RNACwc27(circular RNA Cwc27，简称circCwc27)及其类似物作为阿尔茨海默病治疗和/或预防的核苷酸药物的应用。
背景技术
阿尔茨海默病(Alzheimer’s disease，AD)是一种最常见的神经变性疾病，因其高患病率、高致残率及高死亡率而成为当今社会中一种严重影响人类健康的重大疾病。现有的研究发现AD的主要病理学改变表现为β-淀粉样蛋白(Aβ)聚集形成的老年斑(SP)、Tau蛋白异常聚集形成的神经原纤维缠结(NFT)及脑内神经元的大量丢失。针对AD，临床上至今还缺乏有效的早期诊断手段和治疗措施。在发达国家和我国，AD已成为继心脏病、癌症和卒中之后人类的第4位死因。目前，AD的发病机制依然不清楚，但是普遍认为淀粉样前体蛋白降解通路的激活，导致Aβ产生异常增加和聚集，是导致AD发生的关键因素。因此阐明AD发病机制和寻找新型的治疗策略及靶点成为目前急需解决的关键所在。
环状RNA(circRNA)是一类不具有5’端帽子结构和3’端poly A尾结构，以共价键形成环状结构的内源性非编码RNA分子。其可以来源于编码基因的外显子或内显子。20世纪70年代，科学家们在针对RNA病毒的研究中首次发现了circRNA的存在，之后通过RNA测序技术和生物信息学的帮助，大量circRNA分子陆续被发现。最开始的研究认为circRNA是RNA剪切形成的副产物，但近年来科学家们逐渐发现由于circRNA存在保守性、稳定性、以及表达特异性等特征，使得其具有重要的研究价值。最近的研究表明，分布于核内的circRNA能调节其亲本基因的转录，胞质内的circRNA能够充当miRNA的海绵，RNA结合蛋白的隔绝子等，甚至发现小部分circRNA也能翻译蛋白质。近年来，国际上对于circRNA在AD发病作用的研究日渐增多，目前国内外研究主要集中在circRNA表达谱的建立以寻找在AD中异常表达的circRNA，而对于这些异常表达的circRNA在AD中的功能角色方面的研究还少见报道。在中枢神经系统中，circCwc27的作用依然并不清楚。同时经对现有技术的国内外文献检索，至今未见有关circRNA(包括circCwc27)与AD治疗的研究报道。
发明内容
针对上述问题，本发明的目的在于提供环状RNACwc27靶点或基于环状RNACwc27靶点的药物在治疗和/或预防阿尔茨海默病中的应用。
第一方面，本发明提供环状RNACwc27下调剂在制备预防和/或治疗阿尔茨海默病的药物中的应用，其中所述环状RNACwc27下调剂为环状RNACwc27抑制剂和/或环状RNACwc27修饰物。
所述环状RNACwc27下调剂能够通过抑制Pur-α入核，从而降低Aβ前体蛋白APP的转录水平，减少APP蛋白的表达水平，改善学习和记忆能力，具有治疗AD的临床应用前景。
所述环状RNACwc27下调剂为：包括如SEQ ID NO.1所示的碱基序列的RNA片段；包括与如SEQ ID NO.2所示的碱基序列互补的碱基序列的RNA片段；基于SEQ ID NO.3经过取代、缺失或者添加一个或多个核苷酸的环状RNACwc27修饰物；或者能够抑制环状RNACwc27表达的多肽、蛋白质或者化合物。其中，SEQ ID NO.1是环状RNACwc27的下调剂的核苷酸序列，SEQ ID NO.2是环状RNACwc27接头处的核苷酸序列，SEQ ID NO.3是环状RNACwc27自身的核苷酸序列。
circCwc27在小鼠、大鼠和人脑内的序列高度保守，基本均包含如SEQ ID NO.3所示的碱基序列，针对circCwc27在小鼠上取得的研究成果更具有临床转化意义，因此circCwc27可作为阿尔茨海默病的治疗和/或预防的可靠靶点。
较佳地，所述环状RNACwc27修饰物是通过对环状RNACwc27进行如下修饰中的至少一种而得：任意核苷酸的核糖修饰、碱基修饰、磷酸骨架修饰、任意核苷酸的增减、替换。
较佳地，所述药物包括环状RNACwc27下调剂、以及药学上可接受的载体或辅料。
第二方面，本发明提供环状RNACwc27作为靶点在开发、筛选或制备用于预防和/或治疗阿尔茨海默病的药物中的应用。
第三方面，本发明提供环状RNACwc27作为靶点在制备用于预防和/或治疗阿尔茨海默病的筛药模型中的应用。
第四方面，本发明提供一种筛选预防和/或治疗阿尔茨海默病的药物的方法，所述方法包括：
用候选物质处理表达环状RNACwc27表达的体系；和
检测所述体系中环状RNACwc27的表达；
若环状RNACwc27的表达降低，则表明该候选物质是预防和/或治疗阿尔茨海默病的潜在物 质。
附图说明
图1示出在APP/PS1小鼠皮层、海马及AD患者血浆内circCwc27水平，其中A为APP/PS1及对照野生型小鼠皮层内各circRNA的表达水平，B为APP/PS1及对照野生型小鼠海马内各circRNA的表达水平；C为AD患者及健康对照者血浆内circCwc27水平；从图1可以看出在AD患者血浆和APP/PS1小鼠脑组织内的circCwc27水平显著升高。
图2示出circCwc27干扰病毒对APP/PS1小鼠脑内APP蛋白表达的影响；其中A为APP/PS1小鼠注射circCwc27干扰病毒或对照病毒后海马内APP蛋白的电泳图，B为A中APP蛋白表达量的统计图；从图2可以看出circCwc27干扰病毒能够显著降低APP/PS1小鼠脑内APP蛋白的表达。
图3示出AD模型小鼠APP/PS1经脑立体定位注射补充circCwc27干扰病毒LV-sh-circCwc27两月后的学习和记忆能力，其中A是治疗组与对照组小鼠寻找目标象限潜伏时间，B是治疗组与对照组小鼠寻找目标象限时间的曲线下面积，C是治疗组与对照组小鼠的游泳速度，D是治疗组与对照组小鼠在目标象限的停留时间，E是治疗组与对照组小鼠的运动轨迹图；从图3可以看出，与对照组相比，处理组学习和记忆能力显著提高。
图4示出AD模型小鼠APP/PS1经脑立体定位注射补充circCwc27干扰病毒LV-sh-circCwc27两月后淀粉样蛋白病理情况，其中A为APP/PS1小鼠注射circCwc27干扰病毒或对照病毒后海马及皮层内淀粉样斑块的萤光图，B为A图中淀粉样斑块沉积的统计图；从图4可以看出，与对照组相比，定位注射补充circCwc27干扰病毒后AD模型小鼠的淀粉样蛋白病理出现显著缓解。
具体实施方式
以下结合附图和下述实施方式进一步说明本发明，应理解，附图和下述实施方式仅用于说明本发明，而非限制本发明。
本发明人经研究发现，非编码RNA环状RNA Cwc27(简称circCwc27，其碱基序列如SEQ ID NO.3所示。在阿尔茨海默病患者和模型小鼠脑组织中表达显著升高，并参与脑内淀粉样蛋白病理的产生。针对AD模型小鼠脑内给予circCwc27干扰病毒可以显著缓解脑内淀粉样蛋白病理的发生，改善小鼠学习和记忆能力，表明circCwc27具有治疗AD的临床应用前景，由此完成本发明。
在此公开了circCwc27下调剂在制备预防和/或治疗阿尔茨海默病的药物中的应用。
术语“预防疾病”例如是指在可能暴露或预先处置于疾病但尚未经历或显示疾病症状 的哺乳动物中使疾病临床症状不发展。
术语“治疗疾病”可指抑制疾病，例如阻止或降低疾病或其临床症状的发展，或者缓解疾病，例如使疾病或其临床症状退化。
circCwc27下调剂包括能够降低circCwc27水平的物质。所述环状RNACwc27下调剂为环状RNACwc27抑制剂和/或环状RNACwc27修饰物。例如circCwc27的干扰RNA(包括如SEQ ID NO.1所示的碱基序列的RNA片段。
circCwc27抑制剂的一个示例包括其核苷酸序列上具有与circCwc27接头互补的核苷酸序列，即、包括与如SEQ ID NO.2所示的碱基序列互补的碱基序列的RNA片段。
circCwc27修饰物是指基于circCwc27的核苷酸序列设计核酸序列及其修饰物，例如基于SEQ ID NO.3经过取代、缺失或者添加一个或多个核苷酸的环状RNACwc27修饰物。所述修饰包括任意核苷酸的核糖修饰、碱基修饰和磷酸骨架修饰中的一种或几种的组合或任意核苷酸的增减、替换。只要修饰后的核苷酸序列能满足降低circCwc27类似的功能活性。
另外，应理解，circCwc27下调剂也包括能够降低circCwc27活性和稳定性的物质、能够降低circCwc27有效作用时间的物质。例如，circCwc27下调剂还包括其他能够抑制环状RNACwc27表达的多肽、蛋白质或者化合物。
在此公开了circCwc27下调剂在抑制AD脑内淀粉样蛋白病理的效应，应用circCwc27下调剂能够有效抑制AD模型小鼠病理表型的进展，提高学习和记忆能力。研究发现，circCwc27在AD患者外周血浆及APP/PS1小鼠脑组织显著升高。针对AD模型小鼠，通过脑内抑制circCwc27，能够显著减少Aβ前体蛋白APP和关键裂解酶BACE1的表达水平，从而抑制Aβ的产生和淀粉样蛋白病理的产生。本发明所述的应用中可为制备治疗阿尔茨海默病的一种核苷酸类似物的药物。
circCwc27下调剂能够降低APP和BACE1转录水平，抑制淀粉样前体蛋白途径，作为新型的核苷酸药物。
针对AD动物模型APP/PS1小鼠，通过脑内注射circCwc27干扰慢病毒作为治疗药物，结果证明，抑制circCwc27能够显著减少Aβ的产生和淀粉样蛋白病理的形成，促进学习和记忆能力的缓解。
circCwc27可以作为靶点，用于开发、筛选或制备用于预防和/或治疗阿尔茨海默病的药物。可以将circCwc27作为作用对象，对候选物质进行筛选。筛选出的能够降低circCwc27表达、抑制circCwc27活性和稳定性、和/或减少circCwc27有效作用时间的物质可以作为备选的预防和/或治疗阿尔茨海默病的药物。
可以建立一种以circCwc27为靶点的筛药模型来筛选以circCwc27为作用对象的药物。
一实施方式中，用候选物质处理表达circCwc27的体系，检测所述体系中circCwc27的表达，若circCwc27的表达降低，则表明该候选物质是预防和/或治疗阿尔茨海默病的潜在物质。
在此公开一种药物组合物，其包括有效量circCwc27下调剂、以及药学上可接受的载体或辅料。
“有效量”意指circCwc27下调剂(i)治疗特定疾病、病症或障碍，(ii)减弱、改善或消除特定疾病、病症或障碍的一种或多种症状，或(iii)预防或延迟本文所述特定疾病、病症或障碍的一种或多种症状发作的量。
“药学上可以接受的载体”指的是一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组分能和有效成分相互掺杂，而不明显降低有效成分的药效。药学上可接受的载体或辅料包括但不限于纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如
Figure PCTCN2021112434-appb-000001
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本公开中的药物或药物组合物可按照药物制备的常用方法制成注射制剂、口服制剂、滴鼻剂、喷雾制剂、软膏制剂或贴剂等。
circCwc27下调剂也可以与其它预防和/或治疗阿尔茨海默病的药物进行联合用药。
下面进一步例举实施例以详细说明本发明。同样应理解，以下实施例只用于对本发明进行进一步说明，不能理解为对本发明保护范围的限制，本领域的技术人员根据本发明的上述内容作出的一些非本质的改进和调整均属于本发明的保护范围。下述示例具体的工艺参数等也仅是合适范围中的一个示例，即本领域技术人员可以通过本文的说明做合适的范围内选择，而并非要限定于下文示例的具体数值。
实施例1：在AD患者外周血浆和模型小鼠脑组织中，circCwc27水平显著升高。
选择6月龄APP/PS1转基因小鼠及相同年龄和背景的野生对照鼠各3只，麻醉后灌注取脑，脑组织分成两半，一半用于RNA抽提进行circRNA测序分析，另一半多聚甲醛固定后制作石蜡切片进行原位杂交实验。相关操作符合伦理学操作标准和流程。
circRNA测序分析：通过NanoDrop ND-100浓度测定，选取1-2μg RNA进行文库构建，构建好的文库通过Agilent2100鉴定文库质量，并用qPCR进行文库定量，混合好的不同样品文库使用Illumina 4000测序仪进行测序，包括NaOH碱变性单链生成，Illumina flow cell原位扩增，150双端循环测序等步骤。利用Solexa pipeline version 1.8软件进行图像处理和碱基识别，FastQC软件对去接头后的reads质量进行评估，通过STAR软件比对到参考基因组，使用CIRCexplorer2进行Backsplice junction reads检测，利用R软件edgeR进行表达差异计算。
RT-PCR：实验使用碧云天公司RNAeasy TM Plus试剂盒说明书提取总RNA，实时荧光定量PCR，根据TAKARA公司提供PrimeScript TM RT Master Mix试剂盒说明书将RNA反转录成cDNA，按照TB Green TM Premix Ex Taq TM试剂盒说明书提供的方法在RT-PCR ABI7300仪上进行实时荧光定量PCR反应，以GAPDH为内参照。反应结束后由配套的计算机软件计算各反应管内的CT值。采用相对定量方法2 ΔΔCt来计算circRNA的相对表达水平。
蛋白免疫印迹实验：PC12细胞(购自江苏凯基生物技术股份有限公司)转染si-circCwc27(购自吉满生物科技(上海)有限公司)及对照后72小时，用RIPA裂解液裂解细胞，20min后将裂解液转移至1.5mL离心管中并标记，用BCA试剂盒蛋白定量。蛋白变性后，经SDS-PAGE系统电泳分离后转PVDF膜，5％脱脂奶粉(0.1％TBST稀释)室温封闭1h，TBST洗3次，每次5min，4℃分别孵育APP及内参蛋白GAPDH抗体(购自Abcam，APP抗体货号ab32136，GAPDH抗体货号ab8245)(1∶1000)过夜，TBST洗涤3次，每次5min，孵育二抗HRP标记抗体(购自碧云天)(1∶5 000)2h，TBST洗涤3次，最后加ECL发光液、曝光、显影，用Image J软件进行灰度分析。
实施例1中通过circRNA测序分析及RT-PCR实验表明，如图1所示，在AD患者外周血浆和APP/PS1模型小鼠脑组织(海马和皮层)中，circCwc27水平显著升高( *p<0.05, **p<0.01与WT组；n＝6, ***p<0.001与对照组；n(对照组)＝18，n(AD)＝20，数据以平均值±SEM表示)。
实施例2：干扰circCwc27的表达降低APP蛋白水平
AD模型小鼠APP/PS1经脑立体定位注射补充circCwc27干扰病毒LV-sh-circCwc27(购自上海吉荧生物技术有限公司)。以慢病毒为载体包被circCwc27干扰RNA，用于长期稳定降低小鼠脑内的circCwc27表达，circCwc27干扰RNA序列如SEQ ID NO.1所示，病毒浓度为1×10 9病毒/微升，注射量为5μL。对照组为APP/PS1小鼠经脑立体定位注射补充 对照病毒LV-sh-circCon(购自上海吉荧生物技术有限公司)组，注射病毒浓度为1×10 9病毒/微升，注射量为5μL。而后经蛋白质免疫印迹实验发现，circCwc27干扰病毒能够显著降低APP/PS1小鼠脑内APP蛋白的表达，这表明circCwc27在调节Aβ生成中具有重要作用。
图2显示出circCwc27干扰病毒能够显著降低APP/PS1小鼠脑内APP蛋白的表达(***p<0.001APP/PS1-LV-sh-circ与对照组相比；n＝4,数据以平均值±SEM表示)。
实施例3：circCwc27抑制剂缓解AD模型小鼠脑内淀粉样蛋白病理和行为表征
该实验经过上海交通大学医学院动物实验伦理委员会批准，按照动物实验操作指南实施。6月龄APP/PS1雄性小鼠购于南京大学模式动物中心。将小鼠随机分为WT-LV-sh-circCon组(野生型小鼠注射对照病毒组)，WT-LV-sh-circCwc27组(野生型小鼠注射circCwc27干扰病毒组)，APP/PS1-LV-sh-circCon(APP/PS1小鼠小鼠注射对照病毒组)组和APP/PS1-LV-sh-circCwc27组(APP/PS1小鼠注射circCwc27干扰病毒组)。3％戊巴比妥钠腹腔注射麻醉小鼠，俯卧位将其头部固定于立体定位注射仪上，用微量注射器吸取5μl LV-sh-circCwc27(购自上海吉荧生物技术有限公司)溶液，溶液的浓度是1×10 9病毒/微升，在颅骨孔处竖直插入脑组织。用微量注射泵以0.5μl/min速度入小鼠第三脑室(前卤后：-0.3mm，矢状缝左/右：1.0mm，深度：2.2mm)，待注射完毕后将注射器缓慢抽出。小鼠立体定位注射术后将颅骨注射点周围消毒，缝合皮肤后放回鼠笼中继续饲养。两个月后通过Morris水迷宫检测小鼠学习和记忆能力。行为学后处死取脑，固定切片后检测脑内淀粉样蛋白病理(淀粉样蛋白Aβ抗体购自Cell Signaling Technology，货号2450T)。
如图3所示，行为学实验结果表明，circCwc27抑制剂可以显著缩短APP/PS1小鼠的到达平台的潜伏期，并提高目标象限的停留时间。从对照组小鼠的运动轨迹图可以看出，与对照组相比，处理组学习和记忆能力显著提高( *p<0.05, **p<0.01, ***p<0.001WT-LV-sh-circ与对照组相比； #p<0.05APP/PS1-LV-sh-cir与对照组相比，n＝7-10,数据以平均值±SEM表示)。
如图4所示，脑组织病理检测结果表明，circCwc27抑制剂治疗可以降低脑内淀粉样斑块的沉积，缓解脑内淀粉样蛋白病理的严重程度( ***p<0.001APP/PS1-LV-sh-circ与对照组相比，n＝4,数据以平均值±SEM表示)。

Claims (7)

  1. 环状RNACwc27下调剂在制备预防和/或治疗阿尔茨海默病的药物中的应用，其特征在于，所述环状RNACwc27下调剂为环状RNACwc27抑制剂和/或环状RNACwc27修饰物。
  2. 根据权利要求1所述的应用，其特征在于，所述环状RNACwc27下调剂为：
    包括如SEQ ID NO.1所示的碱基序列的RNA片段；
    包括与如SEQ ID NO.2所示的碱基序列互补的碱基序列的RNA片段；基于SEQ ID NO.3经过取代、缺失或者添加一个或多个核苷酸的环状RNACwc27修饰物；或者
    能够抑制环状RNACwc27表达的多肽、蛋白质或者化合物。
  3. 根据权利要求1或2所述的应用，其特征在于，所述环状RNACwc27修饰物是通过对环状RNACwc27进行如下修饰中的至少一种而得：任意核苷酸的核糖修饰、碱基修饰、磷酸骨架修饰、任意核苷酸的增减、替换。
  4. 根据权利要求1至3中任一项所述的应用，其特征在于，所述药物包括环状RNACwc27下调剂、以及药学上可接受的载体或辅料。
  5. 环状RNACwc27作为靶点在开发、筛选或制备用于预防和/或治疗阿尔茨海默病的药物中的应用。
  6. 环状RNACwc27作为靶点在制备用于预防和/或治疗阿尔茨海默病的筛药模型中的应用。
  7. 一种筛选预防和/或治疗阿尔茨海默病的药物的方法，所述方法包括：
    用候选物质处理表达环状RNACwc27的体系；和
    检测所述体系中环状RNACwc27的表达；
    若环状RNACwc27的表达降低，则表明该候选物质是预防和/或治疗阿尔茨海默病的潜在物质。
PCT/CN2021/112434 2020-09-09 2021-08-13 环状RNA Cwc27下调剂在制备预防和/或治疗阿尔茨海默病的药物中的应用 WO2022052738A1 (zh)

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XU MINGCHU; XIE YAJING (ANGELA); ABOUZEID HANA; GORDON CHRISTOPHER T.; FIORENTINO ALESSIA; SUN ZIXI; LEHMAN ANNA; OSMAN IHAB S.; D: "Mutations in the Spliceosome ComponentCWC27Cause Retinal Degeneration with or without Additional Developmental Anomalies", THE AMERICAN JOURNAL OF HUMAN GENETICS, AMERICAN SOCIETY OF HUMAN GENETICS , CHICAGO , IL, US, vol. 100, no. 4, 9 March 2017 (2017-03-09), US , pages 592 - 604, XP029970711, ISSN: 0002-9297, DOI: 10.1016/j.ajhg.2017.02.008 *

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