WO2022142894A1 - 一种抑制cfd表达的干扰rna及其制备方法和应用 - Google Patents

一种抑制cfd表达的干扰rna及其制备方法和应用 Download PDF

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WO2022142894A1
WO2022142894A1 PCT/CN2021/133001 CN2021133001W WO2022142894A1 WO 2022142894 A1 WO2022142894 A1 WO 2022142894A1 CN 2021133001 W CN2021133001 W CN 2021133001W WO 2022142894 A1 WO2022142894 A1 WO 2022142894A1
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interfering rna
sirna
complement
cfd
diseases
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French (fr)
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林美娜
陈晓萌
赵宣
孙宇
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北京键凯科技股份有限公司
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Definitions

  • the present invention relates to the technical field of biomedicine, in particular, to an interfering RNA that inhibits the expression of CFD and a preparation method and application thereof.
  • Complement was first discovered by Jules Bordet as a heat-labile component in normal plasma that opsonizes and kills bacteria.
  • the complement system refers to a series of more than 20 proteins circulating in the blood and tissue fluids. Most proteins are generally inactive, but due to recognition of microbial molecular components, they are sequentially activated in an enzymatic cascade - activation of one protein enzymatically cleaves and activates the next protein in the cascade .
  • Complement can be activated through three different pathways, the classical pathway, the alternative pathway (alternative pathway) and the lectin pathway.
  • Complement factor D plays an early and central role in the activation of the alternative pathway of the complement cascade. Activation of the alternative complement pathway is initiated by spontaneous hydrolysis of the thioester bond within C3 to generate C3( H2O ), which associates with factor B to form the C3( H2O ) B complex.
  • the role of complement factor D is to cleave factor B within the C3( H2O )B complex to form Ba and Bb.
  • the Bb fragment remains associated with C3( H2O ) to form the alternative pathway C3 convertase C3( H2O )Bb.
  • C3b generated by any C3 convertase also associates with factor B to form C3bB, which is cleaved by factor D to generate the late alternative pathway
  • C3 convertase C3bBb which can be within all three defined complement pathways
  • C5b plays a role in the assembly of factors C6, C7, C8 and C9 into membrane attack complexes that destroy pathogenic cells by lysing them.
  • factor D is a rather suitable disease-suppressive target in activating the alternative complement pathway.
  • Dysfunction or overactivation of complement has been linked to certain autoimmune, inflammatory, and neurodegenerative diseases as well as ischemia-reperfusion injury and cancer.
  • activation of the alternative pathway of the complement cascade contributes to the production of C3a and C5a, both potent anaphylatoxins, which also play a role in many inflammatory diseases. Therefore, in some cases, it is desirable to reduce the response of the complement pathway, including the alternative complement pathway.
  • Down-regulation of complement activation can effectively treat several conditions, including systemic lupus erythematosus and glomerulonephritis, rheumatoid arthritis, cardiopulmonary bypass surgery and hemodialysis, ultrafiltration rejection in organ transplantation, myocardial infarction, Tissue damage from ischemia-reperfusion and adult respiratory distress syndrome.
  • inflammatory conditions and autoimmune diseases that are also closely associated with complement activation, including heat injury, severe asthma, anaphylactic shock, enteritis, urticaria, angioedema, vasculitis, multiple sclerosis, myasthenia gravis, psoriasis, Dermatomyositis, membranous proliferative glomerulonephritis, and Sjögren's syndrome.
  • age-related macular degeneration is the leading cause of vision loss in people aged fifty or older in industrialized countries. It is estimated that by 2020, the number of people living with AMD may exceed 196 million, and by 2040, the number is expected to rise to 288 million. Based on many genetic studies, there is evidence for a link between the complement cascade and macular degeneration. Individuals with mutations in the gene encoding complement factor H have a five-fold increased risk of macular degeneration, and individuals with mutations in other complement factor genes also have an increased risk of AMD. Individuals with mutant factor H also have elevated levels of C-reactive protein, a marker of inflammation. Without an appropriate functional factor H, alternative pathways of the complement cascade would be overactivated, leading to cellular damage. It is therefore desirable to inhibit the alternative pathway.
  • Patent CN108934169A discloses a composition and method for inhibiting factor D, and the disclosed aptamer can treat eye diseases by inhibiting factor D.
  • Patent CN201710229191.2 discloses a monoclonal antibody against human complement factor D and its use.
  • compositions, antibodies, etc. that can inhibit complement factor D, but does not disclose the siRNA capable of inhibiting complement factor D in the present invention.
  • a first aspect of the present invention provides an interfering RNA, the sequence of the interfering RNA comprising more than 80% homology with SEQ ID NO.1 and/or SEQ ID NO.2 or comprising SEQ ID NO. 1 and/or the nucleotide sequence shown in SEQ ID NO.2.
  • SEQ ID NO.1 The specific sequence of SEQ ID NO.1 is 5'-GCAAGAAGCCCGGGAUCUA-3'.
  • SEQ ID NO.2 The specific sequence of SEQ ID NO.2 is 5'-UAGAUCCCGGGCUUCUUGC-3'.
  • the interfering RNA inhibits the expression of CFD (complement factor D).
  • the interfering RNA comprises a sense strand and an antisense strand paired with its reverse complement.
  • the interfering RNA is selected from: siRNA, dsRNA, shRNA, aiRNA, miRNA and combinations thereof.
  • the interfering RNA is siRNA, and its sense strand comprises the nucleotide sequence shown in SEQ ID NO.1, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.2.
  • the ends of the sense strand and/or antisense strand (such as the 3' end) of the above-mentioned interfering RNA (such as siRNA) molecule may also be provided with n over-hangs to increase the activity of the interfering RNA .
  • deoxynucleosides such as deoxythymidine (dT), deoxycytidine (dC), deoxyuridine (dU), etc.
  • the above-mentioned interfering RNA molecule may further comprise at least one modified nucleotide, and the modified interfering RNA has better properties than the corresponding unmodified interfering RNA, such as higher stability, lower immunostimulatory, etc.
  • the second aspect of the present invention provides a cell comprising the above-mentioned interfering RNA.
  • the cells inhibit the expression of CFD.
  • the cells are any CFD-expressing cells, such as adipocytes, myeloid cells or hepatocytes, etc.
  • the cells are 293T cells.
  • the third aspect of the present invention provides a preparation method of the above-mentioned interfering RNA, and the preparation method includes chemical synthesis method, in vitro transcription method, enzymatic digestion method or in vivo transcription method.
  • the preparation method is a chemical synthesis method, including using 3'-cholesterol to modify CPG island as a solid support, using 2'-O-TBDMS as a protective group, using 5-ethylthio-1H-tetrazole as a protective group
  • Acetonitrile solution is used as activator
  • pyridine/water solution of iodine is used as oxidant
  • dichloromethane solution of trichloroacetic acid is used as deprotection reagent.
  • the coupling time of 6 minutes the coupling time of galactose ligand corresponds to L and S monomer is 10-20 minutes.
  • the procedure of oligonucleotide solid-phase synthesis was performed to obtain siRNA.
  • the step further comprises drying the CPG islands.
  • the step further includes extraction.
  • the fourth aspect of the present invention provides a delivery system for the above-mentioned interfering RNA, which comprises the above-mentioned interfering RNA and a carrier.
  • the above-mentioned carrier can adopt any carrier suitable for delivering the above-mentioned interfering RNA of the present invention to the target tissue or target cell, etc., such as the prior art (for example Chen Zhonghua, Zhu Desheng, Li Jun, Huang Zhanqin. "Research on non-viral siRNA carrier” "Progress”. China Pharmacology Bulletin. 2015, 31(7): 910-4; Wang Rui, Qu Bingnan, Yang Jing. "Research progress of siRNA-loaded nano-formulations". China Pharmacy. 2017, 28(31): 4452 -4455) disclosed in.
  • the prior art for example Chen Zhonghua, Zhu Desheng, Li Jun, Huang Zhanqin. "Research on non-viral siRNA carrier” "Progress”. China Pharmacology Bulletin. 2015, 31(7): 910-4; Wang Rui, Qu Bingnan, Yang Jing. "Research progress of siRNA-loaded nano-formulations". China Pharmacy. 2017, 28(31): 44
  • the above-mentioned vector is a viral vector, such as lentivirus, retrovirus, adenovirus, herpes simplex virus, and the like.
  • the above-mentioned carrier is a non-viral vector, specifically such as liposome, polymer, polypeptide, antibody, aptamer, etc. or a combination thereof; wherein, the above-mentioned interfering RNA can be chemically bonded to the non-viral vector
  • the RNA is delivered by coupling or physical mixing, and the physical mixing ratio can be 1:1-50 (such as 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1 :30, 1:35, 1:40, 1:45, 1:50).
  • the above-mentioned liposomes can be cationic lipids (such as lipofectamine series of Invitrogenn company, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)), neutral ionic liposomes (such as di-oil phosphatidylcholine (DOPC), cholesterol, etc.), anionic liposomes (such as dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE), etc.) or mixtures thereof.
  • DOTAP 1,2-dioleoyl-3-trimethylammonium propane
  • DOPC di-oil phosphatidylcholine
  • DOPG dioleoylphosphatidylglycerol
  • DOPE dioleoylphosphatidylethanolamine
  • the above-mentioned polymer may be a synthetic polymer (such as polyethyleneimine, cyclodextrin, polyethylene glycol, etc.) or a natural polymer (such as chitosan, telogen, hyaluronic acid, etc.) or its mixture.
  • a synthetic polymer such as polyethyleneimine, cyclodextrin, polyethylene glycol, etc.
  • a natural polymer such as chitosan, telogen, hyaluronic acid, etc.
  • the above-mentioned polypeptide may be a cell penetrating peptide (CPP) (eg, low molecular weight protamine, Tat peptide, transportan peptide, penetratin peptide, oligoarginine peptide, etc.).
  • CPP cell penetrating peptide
  • the above-mentioned antibody can be a single-chain antibody (eg, scFv-tp, scFv-9R, etc.).
  • a fifth aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned interfering RNA or its delivery system, and pharmaceutically acceptable excipients.
  • the sixth aspect of the present invention provides a method for inhibiting the expression of CFD, which comprises transfecting the above-mentioned interfering RNA into cells.
  • the seventh aspect of the present invention provides an application of the above-mentioned interfering RNA, the above-mentioned cell, the above-mentioned delivery system or the above-mentioned pharmaceutical composition in the preparation of a medicine for preventing and/or treating a disease related to complement overactivation.
  • said diseases related to complement hyperactivation include autoimmune diseases, inflammatory diseases, neurodegenerative diseases, ischemia-reperfusion injury, eye diseases or cancer.
  • the eighth aspect of the present invention provides the use of the above-mentioned interfering RNA, the above-mentioned cell, the above-mentioned delivery system or the above-mentioned pharmaceutical composition in a medicine related to the prevention and/or treatment of complement hyperactivation-related diseases.
  • said diseases related to complement hyperactivation include autoimmune diseases, inflammatory diseases, neurodegenerative diseases, ischemia-reperfusion injury, eye diseases or cancer.
  • the ninth aspect of the present invention provides the use of the above-mentioned interfering RNA, the above-mentioned cell, the above-mentioned delivery system or the above-mentioned pharmaceutical composition in inhibiting the expression of CFD genes.
  • a tenth aspect of the present invention provides a method for inhibiting CFD gene expression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the above-mentioned interfering RNA of the present invention, or a delivery system or pharmaceutical combination thereof the steps of the thing.
  • the eleventh aspect of the present invention provides a method for preventing and/or treating a disease related to complement hyperactivation, comprising administering to a subject a therapeutically effective amount of the above-mentioned interfering RNA or its delivery system and pharmaceutical composition of the present invention. step.
  • the present invention also provides a method for introducing the above-mentioned interfering RNA of the present invention into a cell, which comprises the step of contacting the cell with a delivery system for the interfering RNA.
  • the above-mentioned cells are in a subject.
  • the above-mentioned step of contacting the cells with the interfering RNA delivery system is a step of contacting the cells by administering the interfering RNA delivery system into a subject through a systemic route or a local route.
  • interfering RNA includes single-stranded RNA (eg, mature miRNA, ssRNAi oligonucleotides, ssDNAi oligonucleotides) or double-stranded RNA (ie, duplex RNAs such as siRNA, dsRNA, shRNA, aiRNA, or precursor miRNA), which, when the interfering RNA is in the same cell as the target gene or sequence, is capable of reducing or inhibiting the expression of the target gene or sequence (e.g., by mediating degradation and inhibiting complementarity to the interfering RNA sequence) mRNA translation).
  • single-stranded RNA eg, mature miRNA, ssRNAi oligonucleotides, ssDNAi oligonucleotides
  • double-stranded RNA ie, duplex RNAs such as siRNA, dsRNA, shRNA, aiRNA, or precursor miRNA
  • Interfering RNA therefore refers to a single-stranded RNA complementary to the target mRNA sequence or a double-stranded RNA formed from two complementary strands or from a single self-complementary strand. Specifically, interfering RNA molecules are chemically synthesized.
  • RNA interfering RNA
  • a test sample eg, a biological sample from an organism of interest that expresses the target gene or a sample of cells in culture that expresses the target gene
  • an agent that silences, reduces, or inhibits the expression of the target gene.
  • the interfering RNA (eg, siRNA) is contacted, the expression of the target gene in the test sample is compared to the expression of the target gene in the sample not contacted with the interfering RNA (eg, siRNA), a control sample (eg, a sample expressing the target gene) can be compared. ) is set to a value of 100%. In specific embodiments, when the value of the test sample relative to the control sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45% , 40%, 35%, 30%, 25%, 20%, 10%, 5%, or 0%, silencing, inhibition or reduction of target gene expression is achieved.
  • siRNA interfering RNA
  • Suitable assays include, but are not limited to, testing protein or mRNA levels using techniques known to those of skill in the art, such as, for example, dot blots, Northern blots, real-time RT-PCR, in situ hybridization, ELISA, immunoprecipitation, enzyme function , and phenotypic assays known to those of skill in the art.
  • Interfering RNA includes "small interfering RNA” or “siRNA", each strand of the siRNA molecule comprising about 15 to about 60 nucleotides in length (eg, about 15-60, 15-50, 15-40, 15 in length) -30, 15-25, or 19-25 nucleotides, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length).
  • the siRNA is chemically synthesized.
  • the siRNA molecules of the present invention are capable of silencing the expression of target sequences in vitro and/or in vivo.
  • the siRNA comprises at least one modified nucleotide, eg, the siRNA comprises one, two, three, four, five, six, seven, eight, nine, Ten or more modified nucleotides.
  • dsRNA is intended to include any precursor molecule that is processed in vivo by endonucleases to produce active siRNA.
  • shRNA i.e. "small hairpin RNA” or “short hairpin RNA” includes short RNA sequences that produce tight hairpin turns that can be used by RNA interference to silence gene expression. shRNA hairpins can be cleaved into siRNA by cellular machinery.
  • microRNAs are single-stranded RNA molecules about 21-23 nucleotides in length that regulate gene expression.
  • the term "therapeutically effective amount” refers to the amount of a subject compound that will elicit the biological or medical response of the tissue, system or subject being sought by the researcher, veterinarian, medical physician or other clinician.
  • the term "therapeutically effective amount” includes an amount of active ingredient that, when administered, is sufficient to prevent the development of one or more of the signs or symptoms of the disorder or disease being treated, or to alleviate to some extent the disorder or disease being treated one or more of the signs or symptoms.
  • a therapeutically effective amount will vary depending on the active ingredient, the disease to be treated and its severity, and the age, weight, sex, etc. of the subject.
  • the subject can be a mammal, such as a human, a monkey, a dog, a rabbit, a mouse, a rat, etc.; in an embodiment of the present invention, the above-mentioned subject is a human.
  • the "autoimmune disease” in the present invention includes but is not limited to allergy, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura , Autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, myasthenia gravis, multiple sclerosis, urticaria, psoriasis, dermatomyositis, Sjögren's syndrome, pain or neurological disorders, etc.
  • the "inflammatory disease” in the present invention includes acute inflammation as well as chronic inflammation. Specifically, including but not limited to degenerative inflammation, exudative inflammation, proliferative inflammation, specific inflammation, etc., including but not limited to severe burns, endotoxemia, septic shock, adult respiratory distress syndrome, hemodialysis, Anaphylactic shock, severe asthma, angioedema, Crohn's disease, sickle cell anemia, poststreptococcal glomerulonephritis, pancreatitis, colitis, vasculitis, adverse drug reactions, drug allergies, IL- 2 induced vascular leakage syndrome or radiographic (contrast) contrast agent allergy.
  • Neurodegenerative diseases include but are not limited to Alzheimer's disease, progressive blindness or external ophthalmoplegia, multiple system atrophy, frontotemporal dementia, Huntington's chorea, corticobasal degeneration, spinocerebellar degeneration Ataxia, motor neuron disease, hereditary motor sensory neuropathy, etc.
  • ischemia-reperfusion injury in the present invention includes, but is not limited to, acute myocardial infarction, aneurysm, stroke, hemorrhagic shock, crush injury, multiple organ failure, intestinal ischemia, complement during cardiopulmonary bypass surgery Ischemia-reperfusion injury following activation or other ischemia-causing events, etc.
  • Eye diseases include, but are not limited to, macular degeneration diseases such as age-related macular degeneration (AMD) in all stages including dry and wet (non-exudative and exudative) forms, diabetic retinopathy and others Ischemia-related retinopathy, choroidal neovascularization (CNV), uveitis, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neoplasia Angiogenesis, and retinal neovascularization.
  • AMD age-related macular degeneration
  • Ischemia-related retinopathy Ischemia-related retinopathy
  • CNV choroidal neovascularization
  • uveitis diabetic macular edema
  • pathological myopia von Hippel-Lindau disease
  • CRVO central retinal vein occlusion
  • age-related macular degeneration includes non-exudative (such as intermediate dry AMD or geographic atrophy (GA)) and exudative (such as wet AMD (choroidal neovascularization (CNV)) AMD, diabetic retinopathy (DR), endophthalmitis, and uveitis, additionally non-exudative AMD may include hard drusen, soft drusen, geographic atrophy, and/or pigmented lumps, among others.
  • “Cancer” in the present invention includes but is not limited to lymphoma, B cell tumor, T cell tumor, myeloid/monocyte tumor, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrium cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, gum Plasmoblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma.
  • the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia;
  • the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma , T-cell lymphoma, and Waldenstrom macroglobulinemia;
  • the sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhab
  • Treatment means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but not necessarily Involves complete elimination of all disease-related signs, symptoms, conditions, or disorders.
  • the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides, but still have the activity described in the present invention.
  • the "homology" in the present invention means that in terms of using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs, so that the used sequences are compared with those obtained in the prior art. , with (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31% , 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48 %, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%
  • the siRNA in the present invention can effectively inhibit the expression of complement factor D, and the inhibition efficiency can reach more than 78%, which is helpful for the treatment of related diseases caused by excessive complement activation.
  • the method for suppressing CFD in the present invention is simple and quick.
  • Figure 3 The expression level of CFD mRNA in cells, where NC is the negative control group transfected with negative control siRNA (siRNA-NC), mock is the cell group added with transfection agent, blank is the addition of PBS without siRNA and transfection The blank control group of the agent, CFD is the experimental group transfected with CFDsiRNA.
  • siRNA-NC negative control siRNA
  • mock is the cell group added with transfection agent
  • blank is the addition of PBS without siRNA
  • transfection The blank control group of the agent
  • CFD is the experimental group transfected with CFDsiRNA.
  • siRNA was designed according to the characteristic of inhibiting complement factor D.
  • the sense strand of the final designed siRNA is 5'-GCAAGAAGCCCGGGAUCUA-3' (SEQ ID NO.1), and the antisense strand is 5'-UAGAUCCCGGGCUUCUUGC-3' (SEQ ID NO.2).
  • the above-mentioned siRNA was prepared by chemical synthesis method and detected and analyzed by mass spectrometry.
  • the oligonucleotides containing 2'-hydroxy ribonucleotides were synthesized according to the theoretical yield of 1 ⁇ mol.
  • a solution of 5-ethylthio-1H-tetrazole (Chemgenes product) in acetonitrile was prepared as activator (0.25M), 0.02M iodine in pyridine/water solution was prepared as oxidant, and a 3% solution of trichloroacetic acid in dichloromethane was used as deprotection reagent , placed in the designated position of the reagent corresponding to the ABI 394 DNA/RNA automatic synthesizer.
  • Set the synthesis program to input the specified oligonucleotide base sequence, and start the cycle of oligonucleotide synthesis.
  • the coupling time of each step is 6 minutes, and the coupling time of the galactose ligand corresponding to the L and S monomers is 10-20 minutes.
  • the oligonucleotide solid-phase synthesis is completed. Dry the CPG with dry nitrogen, transfer it to a 5ml EP tube, add 2ml of ammonia water/ethanol solution (3/1), and heat at 55°C for 16-18 hours. Centrifuge at 10,000 rpm for 10 min, take the supernatant, and drain concentrated ammonia/ethanol to obtain a white colloidal solid. The solid was dissolved in 200 ⁇ l of 1 M TBAF THF solution and shaken at room temperature for 20 hours. Add 0.5ml of 1M Tris-HCl buffer (pH7.4), shake at room temperature for 15 minutes, place it in a centrifugal dryer to pump to 1/2 the original volume, and remove THF.
  • the solution was extracted twice with 0.5ml of chloroform, 1ml of 0.1M TEAA loading solution was added, the mixed solution was poured into a solid phase extraction column, and the mass spectrometry detection and analysis was completed on the HTCS LC-MS system (Novatia). Nucleic acid molecular weight was calculated by normalization with Promass software after primary scan. The above method is to synthesize two single strands respectively. After the mass spectrometry identification is correct, the two single strands are mixed in an equimolar ratio and annealed to form a double strand, which is the siRNA sequence. The detection results of the sense and antisense strands of siRNA by mass spectrometry are shown in Figure 1 and Figure 2, respectively.
  • the siRNA in order to detect the inhibitory effect of the siRNA obtained in Example 1 on CFD, the siRNA was first transfected into the cultured cells, and then RNA was extracted to obtain the expression of CFD mRNA by real-time quantitative PCR.
  • 293T cells were routinely cultured at 37°C under 5% CO 2 .
  • the 12-well plate was taken out of the incubator at 37°C, 5% CO2 for RNA extraction for subsequent detection.
  • Trizol lysis completely remove the cell culture medium, add 1 mL of TrizolTM Reagent, pipette 3-5 times to fully lyse the cells, and leave at room temperature for 3-5 minutes;
  • RNA quality inspection RNA content was detected by Nanodrop, and RNA integrity was detected by 1% agarose gel electrophoresis.

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Abstract

提供了一种抑制补体因子D（CFD）表达的干扰RNA及其应用。该干扰RNA可为siRNA，其包含GCAAGAAGCCCGGGAUCUA和/或UAGAUCCCGGGCUUCUUGC的核苷酸序列，其可有效抑制CFD的表达，可用于研究补体激活旁路途径的调控，可用于制备治疗与补体过度活化相关疾病的药物。

Description

一种抑制CFD表达的干扰RNA及其制备方法和应用 技术领域
本发明涉及生物医药技术领域，具体地说，涉及一种抑制CFD表达的干扰RNA及其制备方法和应用。
背景技术
补体最早是由JulesBordet发现的一种正常血浆中起到调理作用和杀死细菌的一种热不稳定的成分。补体系统是指在血液和组织液中循环的一系列20种以上的蛋白质。大多数蛋白质通常是不起作用的，但由于对微生物分子成分的识别，它们在一个酶级联中依次被激活—一种蛋白质的激活以酶的方式裂解并激活级联中的下一种蛋白质。补体可以通过三种不同的途径激活，即经典途径、旁路途径(替代途径)和凝集素途径。
补体因子D(CFD)在补体级联的替代途径的活化中起着早期和核心作用。替代补体途径的活化由C3内的硫酯键的自发水解以产生C3(H 2O)来引发，C3(H 2O)与因子B缔合形成C3(H 2O)B复合物。补体因子D的作用是断裂C3(H 2O)B复合物内的因子B以形成Ba和Bb。Bb片段保持与C3(H 2O)缔合以形成替代途径C3转化酶C3(H 2O)Bb。另外，由任何C3转化酶生成的C3b也与因子B缔合以形成C3bB，因子D将其断裂以生成后期替代途径C3转化酶C3bBb，该C3转化酶可在所有三种所定义的补体途径内提供重要的下游放大，最终导致补体级联途径中其它因子的募集和组装，包括C5断裂为C5a和C5b。C5b在因子C6、C7、C8和C9组装为膜攻击复合物中起作用，所述复合物可通过裂解细胞来破坏病原细胞。D因子在人体中的血浆浓度非常低(1.8μg/ml)，并且已有证据显示它是激活补体替代途径中具有限速作用的酶。因此，D因子在激活补体替代途径中是相当合适的疾病抑制靶标。
补体的功能障碍或过度活化已经与某些自身免疫、炎性、和神经变性疾病以及缺血-再灌注损伤和癌症联系起来。例如，补体级联的替代途径的活化有助于C3a和C5a(二者 均为强效过敏毒素)的产生，C3a和C5a也在许多炎性疾病中起作用。因此，在一些情况下，期望减少补体途径的响应，包括替代补体途径。
若将补体的活化下调可以有效治疗下列几种病症，包括系统性红斑狼疮和肾小球肾炎、类风湿性关节炎、心肺血管绕道手术和血液透析、器官移植中的超滤排斥、心肌梗死、缺血再灌注造成的组织损伤和成人呼吸窘迫综合征。还有其他炎性病症和自身免疫疾病也与补体激活密切相关，包括热损伤、严重哮喘、过敏性休克、肠炎、荨麻疹、血管性水肿、血管炎、多发性硬化、重症肌无力、牛皮癣、皮肌炎、膜性增生性肾小球肾炎和干燥综合征。
其中，老年性黄斑变性(AMD)是工业化国家中五十岁或以上人群视力丧失的主要原因。据估计，到2020年，患有AMD的人数可能会超过1.96亿，并且到2040年，该数字预计会上升至2.88亿。基于许多遗传研究，存在补体级联与黄斑变性之间的联系的证据。在编码补体因子H的基因中有突变的个体具有五倍的增加的黄斑变性风险，在其它补体因子基因中有突变的个体也具有增加的AMD风险。具有突变因子H的个体也有着增高的C-反应蛋白水平，C-反应蛋白是炎症的标志物。没有适当的功能因子H，补体级联的替代途径将过度活化，导致细胞损伤。因此期望抑制替代途径。
因此，藉由针对补体系统抑制靶标，如D因子，发展出特异性的抑制剂，例如抑制补体因子D的siRNA，能对补体的活化功能下调，则此类抑制剂将具有治疗上述疾病的潜在功效。
专利CN108934169A公开了一种用于抑制因子D的组合物和方法，其所公开的适体能通过抑制因子D治疗眼部疾病。
专利CN201710229191.2公开了一种抗人补体D因子的单克隆抗体及其用途。
因此，现有技术中公开了能抑制补体因子D的组合物、抗体等，但是并没有公开本发明中能抑制补体因子D的siRNA。
发明内容
本发明的第一方面，提供了一种干扰RNA，所述的干扰RNA的序列包含与SEQ ID NO.1和/或SEQ ID NO.2具有80％以上的同源性或者包含SEQ ID NO.1和/或SEQ ID NO.2所示的核苷酸序列。
SEQ ID NO.1的具体序列为5′-GCAAGAAGCCCGGGAUCUA-3′。
SEQ ID NO.2的具体序列为5′-UAGAUCCCGGGCUUCUUGC-3′。
优选的，所述的干扰RNA抑制CFD(补体因子D)的表达。
优选的，所述的干扰RNA包含正义链和与其反向互补配对的反义链。
优选的，所述的干扰RNA选自：siRNA、dsRNA、shRNA、aiRNA、miRNA及其组合。
在本发明的一个实施方式中，所述的干扰RNA为siRNA，其正义链包含SEQ ID NO.1所示核苷酸序列，反义链包含SEQ ID NO.2所示核苷酸序列。
优选的，上述干扰RNA(如siRNA)分子的正义链和/或反义链的末端(如3′末端)还可设有n个悬挂碱基(Over-hang)，以增加该干扰RNA的活性。其中，悬挂碱基可以为相同或不同的脱氧核苷(如脱氧胸苷(dT)、脱氧胞苷(dC)、脱氧尿苷(dU)等)，n为1-10的整数(如1、2、3、4、5、6、7、8、9、10)，特别是2-4的整数；优选的，n＝2，悬挂碱基可以为dTdT、dTdC或dUdU等等。
在一些实施方案中，上述干扰RNA分子中还可包含至少一个修饰的核苷酸，修饰的干扰RNA具有比相应未修饰的干扰RNA具有更佳的性质，如更高的稳定性，更低的免疫刺激性等。
本发明的第二方面，提供了一种包含上述干扰RNA的细胞。
优选的，所述的细胞抑制CFD的表达。
优选的，所述的细胞为任何表达CFD的细胞，例如脂肪细胞、髓系细胞或肝细胞等等，在本发明的一个具体实施方式中，所述的细胞为293T细胞。
本发明的第三方面，提供了一种上述干扰RNA的制备方法，所述的制备方法包 括化学合成法、体外转录法、酶消化法或体内转录法。
优选的，所述的制备方法为化学合成法，包括以3’-胆固醇修饰CPG岛作为固相支持物，以2’-O-TBDMS作为保护基，以5-乙硫基-1H-四唑乙腈溶液作为活化剂，以碘的吡啶/水溶液作为氧化剂，以三氯乙酸二氯甲烷溶液作为脱保护试剂，按照偶合时间6分钟，半乳糖配体对应L和S单体偶合时间10-20分钟的程序进行寡核苷酸固相合成，获得siRNA。
优选的，所述的步骤还包括干燥CPG岛。
优选的，所述的步骤还包括萃取。
本发明的第四方面，提供了一种上述干扰RNA的递送系统，其包含上述干扰RNA和载体。
具体地，上述载体可以采用任何适于将本发明上述干扰RNA递送于靶组织或靶细胞等的载体，如现有技术(例如陈中华，朱德生，李军，黄展勤.“非病毒siRNA载体研究进展”.中国药理学通报.2015，31(7)：910-4；王锐，曲炳楠，杨婧.“载siRNA的纳米制剂研究进展”.中国药房.2017，28(31)：4452-4455)中公开的那些。
在本发明的一个实施方式中，上述载体为病毒载体，具体如慢病毒、逆转录病毒、腺病毒、单纯疱疹病毒等。
在本发明的另一个实施方式中，上述载体为非病毒载体，具体如脂质体、聚合物、多肽、抗体、适配体等或其组合；其中，上述干扰RNA可以与非病毒载体通过化学键偶联或物理混合的方式进行RNA的递送，物理混合的比例可以为1:1-50(如1:1、1:5、1:10、1:15、1:20、1:25、1:30、1:35、1:40、1:45、1:50)。
具体地，上述脂质体可以为阳离子脂质(如Invitrogenn公司的lipofectamine系列、1,2-二油酰基-3-三甲基铵丙烷(DOTAP))、中性离子脂质体(如二油酰磷 脂酰胆碱(DOPC)、胆固醇等)、阴离子脂质体(如二油酰磷脂酰甘油(DOPG)、二油酰磷脂酰乙醇胺(DOPE)等)或其混合物。
具体地，上述聚合物可以为合成型聚合物(如聚乙烯亚胺、环糊精、聚乙二醇等)或天然型聚合物(如壳聚糖、端胶原、透明质酸等)或其混合物。
具体地，上述多肽可以为细胞穿透肽(CPP)(如低分子量鱼精蛋白、Tat肽、transportan肽、penetratin肽、寡聚精氨酸肽等)。
具体地，上述抗体可以为单链抗体(如scFv-tp、scFv-9R等)。
本发明的第五方面，提供了一种药物组合物，其包括上述干扰RNA或其递送系统，以及药学上可接受的辅料。
本发明的第六方面，提供了一种CFD表达的抑制方法，所述的抑制方法包括将上述的干扰RNA转染到细胞中。
本发明的第七方面，提供了一种上述的干扰RNA、上述的细胞、上述的递送系统或上述的药物组合物在制备预防和/或治疗补体过度活化相关疾病的药物中的应用。
优选的，所述的补体过度活化相关疾病包括自身免疫疾病、炎性疾病、神经变性疾病、缺血-再灌注损伤、眼疾病或癌症。
本发明的第八方面，提供了上述的干扰RNA、上述的细胞、上述的递送系统或上述的药物组合物在涉及用于预防和/或治疗补体过度活化相关疾病的药物中的应用。
优选的，所述的补体过度活化相关疾病包括自身免疫疾病、炎性疾病、神经变性疾病、缺血-再灌注损伤、眼疾病或癌症。
本发明的第九方面，提供了一种上述的干扰RNA、上述的细胞、上述的递送系统或上述的药物组合物在抑制CFD基因表达中的应用。
本发明的第十方面，提供了一种在需要其的受试者中抑制CFD基因表达的方法， 其包括向该受试者施用治疗有效量的本发明上述干扰RNA或其递送系统、药物组合物的步骤。
本发明的第十一方面，提供了一种预防和/或治疗补体过度活化相关疾病的方法，其包括向受试者施用治疗有效量的本发明上述干扰RNA或其递送系统、药物组合物的步骤。
本发明的第十二方面，本发明还提供一种将本发明上述干扰RNA引入细胞的方法，其包括使该细胞与该干扰RNA的递送系统接触的步骤。
具体地，上述细胞在受试者体内。
具体地，上述使细胞与干扰RNA的递送系统接触的步骤为将干扰RNA的递送系统通过全身途径或局部途径施用至受试者体内来接触所述细胞的步骤。
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
如在本文中所使用的术语“干扰RNA”包括单链RNA(例如，成熟miRNA、ssRNAi寡核苷酸、ssDNAi寡核苷酸)或双链RNA(即，双链体RNA如siRNA、dsRNA、shRNA、aiRNA、或前体miRNA)，其在当干扰RNA与靶基因或序列处于相同细胞中时，能够降低或抑制靶基因或序列的表达(例如，通过介导降解和抑制与干扰RNA序列互补的mRNA的翻译)。干扰RNA因此是指与靶mRNA序列互补的单链RNA或者由两条互补链或由单条自互补链形成的双链RNA。具体地，干扰RNA分子是化学合成的。
短语“抑制靶基因的表达”是指本发明的干扰RNA(例如，siRNA)沉默、降低、或抑制靶基因(例如，CFD基因)的表达的能力。为了检验基因沉默的程度，使测试样品(例如，来自表达靶基因的目标生物体的生物样品或在培养物中的表达靶基因的细胞的样品)与沉默、降低、或抑制靶基因的表达的干扰RNA(例如，siRNA)接触，将测试样品中靶基因的表达与未与干扰RNA(例如，siRNA)接触的样品中靶基因的表达进行对比，可以将对照样品(例如，表达靶基因的样品)设定为100％ 的值。在具体的实施方式中，当测试样品相对于对照样品的值为约95％、90％、85％、80％、75％、70％、65％、60％、55％、50％、45％、40％、35％、30％、25％、20％、10％、5％、或0％时，实现靶基因的表达的沉默、抑制或降低。适合的测定包括但不限于，利用本领域技术人员已知的技术检验蛋白质或mRNA水平，诸如，例如点印迹、Northern印迹、real-time RT-PCR、原位杂交、ELISA、免疫沉淀、酶功能、以及本领域技术人员已知的表型测定。
干扰RNA包括“小干扰RNA”或“siRNA”，siRNA分子的每条链包含长度为约15至约60的核苷酸(例如，长度为约15-60、15-50、15-40、15-30、15-25、或19-25的核苷酸，或者长度为15、16、17、18、19、20、21、22、23、24或25的核苷酸)。在一个具体实施方式中，siRNA是化学合成的。本发明的siRNA分子能够体外和/或体内沉默靶序列的表达。在其他实施方式中，siRNA包含至少一个修饰的核苷酸，例如siRNA在双链区中包含一个、两个、三个、四个、五个、六个、七个、八个、九个、十个或更多个修饰的核苷酸。
如在本文中所用，术语“dsRNA”意在包括在体内由核酸内切酶加工以产生活性siRNA的任何前体分子。
如在本文中所用，术语“shRNA”即“小发夹RNA”或“短发夹RNA”包括产生紧密发夹转角(hairpin turn)的短RNA序列，所述发夹转角可以用于通过RNA干扰来沉默基因表达。shRNA发夹结构可由细胞机器裂解为siRNA。
通常，微RNA(miRNA))是长度为约21-23个核苷酸的调节基因表达的单链RNA分子。
本发明中，术语“治疗有效量”指的是将引起由研究者、兽医、医学医生或其他临床医生正在寻找的组织、系统或对象的生物学或医学应答的对象化合物的量。术语“治疗有效量”包括以下的活性成分的量：当被施用时，其足以预防治疗的紊乱或疾病的迹象或症状中的一个或多个的发展，或以一定程度减轻治疗的紊乱或疾 病的迹象或症状中的一个或多个。治疗有效量将根据活性成分、待治疗的疾病及其严重程度、以及受试者的年龄、体重、性别等而变化。
本发明中，受试者可以为哺乳动物，如，人类、猴、狗、兔、小鼠、大鼠等；在本发明的一个实施例中，上述受试者为人类。
本发明所述的“自身免疫疾病”包括但不限于过敏、哮喘、心肌炎、肾炎、肝炎、系统性红斑狼疮、类风湿性关节炎、硬皮病、甲状腺功能亢进、原发性血小板减少性紫癜、自身免疫性溶血性贫血、溃疡性结肠炎、自身免疫性肝病、糖尿病、重症肌无力、多发性硬化、荨麻疹、牛皮癣、皮肌炎、干燥综合症、疼痛或神经障碍等。
本发明所述的“炎性疾病”包括急性炎症，也包括慢性炎症。具体的，包括但不限于变质性炎症、渗出性炎症、增生性炎症、特异性炎症等，包括但不限于重度烧伤、内毒素血症、感染性休克、成人呼吸窘迫综合征、血液透析、过敏性休克、严重的哮喘、血管性水肿、克罗恩氏病、镰状细胞贫血、链球菌感染后肾小球肾炎、胰腺炎、肠炎、血管炎、不良药物反应、药物变态反应、IL-2诱导的血管渗漏综合征或放射摄影(对比)造影剂变态反应等。
本发明所述的“神经变性疾病”包括但不限于阿尔茨海默氏病、进行性失明或眼外肌麻痹、多系统萎缩、额颞叶痴呆、Huntington舞蹈病，皮质基底节变性、脊髓小脑共济失调、运动神经元病、遗传性运动感觉神经病等。
本发明所述的“缺血-再灌注损伤”包括但不限于急性心肌梗死、动脉瘤、中风、出血性休克、挤压伤、多器官功能衰竭、肠缺血、心肺旁路手术期间的补体活化或其它引起缺血的事件后缺血-再灌注损伤等。
本发明所述的“眼疾病”包括但不限于黄斑变性性疾病如所有阶段的老年性黄斑变性(AMD)包括干性和湿性(非渗出性和渗出性)形式、糖尿病视网膜病和其它缺血相关视网膜病、脉络膜新血管形成(CNV)、葡萄膜炎、糖尿病黄斑水肿、病理性近视、von Hippel-Lindau病、眼的组织胞浆菌病、视网膜中央静脉阻塞(CRVO)、角膜新血管形成、和视网膜新血管形成。其中老年性黄斑变性(AMD)包括非渗出性(例如中期(intermediate)干性AMD或地图样萎缩(geographic atrophy，GA))和渗出性(例如湿性AMD(脉络膜新血管形成(CNV))AMD、糖尿病视网膜病(DR)、眼内炎和葡萄膜炎，另外非渗出性AMD可包括硬玻璃疣、软玻璃疣、地图样萎缩和/或色素结块等。
本发明所述的“癌症”包括但不限于淋巴瘤、B细胞肿瘤、T细胞肿瘤、骨髓/单核细 胞肿瘤、非小细胞肺癌、白血病、卵巢癌、鼻咽癌、乳腺癌、子宫内膜癌、结肠癌、直肠癌、胃癌、膀胱癌、肺癌、支气管癌、骨癌、前列腺癌、胰腺癌、肝和胆管癌、食管癌、肾癌、甲状腺癌、头颈部癌、睾丸癌、胶质母细胞瘤、星形细胞瘤、黑色素瘤、骨髓增生异常综合征、以及肉瘤。其中，所述的白血病选自急性淋巴细胞性(成淋巴细胞性)白血病、急性骨髓性白血病、髓性白血病、慢性淋巴细胞性白血病、多发性骨髓瘤、浆细胞白血病、以及慢性骨髓性白血病；所述淋巴瘤选自霍奇金淋巴瘤和非霍奇金淋巴瘤，包括B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、边缘区B细胞淋巴瘤、T细胞淋巴瘤、和瓦尔登斯特伦巨球蛋白血症；所述肉瘤选自骨肉瘤、尤文肉瘤、平滑肌肉瘤、滑膜肉瘤、软组织肉瘤、血管肉瘤、脂肪肉瘤、纤维肉瘤、横纹肌肉瘤、及软骨肉瘤。
本发明所述的“治疗”表示在疾病已开始发展后减缓、中断、阻止、控制、停止、减轻、或逆转一种体征、症状、失调、病症、或疾病的进展或严重性，但不一定涉及所有疾病相关体征、症状、病症、或失调的完全消除。
本发明所述的“包含”在本申请中用于描述蛋白质或核酸的序列时，所述蛋白质或核酸可以是由所述序列组成，或者在所述蛋白质或核酸的一端或两端可以具有额外的氨基酸或核苷酸，但仍然具有本发明所述的活性。
本发明所述“同源性”，是指在使用蛋白序列或核苷酸序列的方面，本领域技术人员可以根据实际工作需要对序列进行调整，使使用序列与现有技术获得的序列相比，具有(包括但不限于)1％，2％，3％，4％，5％，6％，7％，8％，9％，10％，11％，12％，13％，14％，15％，16％，17％，18％，19％，20％，21％，22％，23％，24％，25％，26％，27％，28％，29％，30％，31％，32％，33％，34％，35％，36％，37％，38％，39％，40％，41％，42％，43％，44％，45％，46％，47％，48％，49％，50％，51％，52％，53％，54％，55％，56％，57％，58％，59％，60％，70％，80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，99.1％，99.2％，99.3％，99.4％，99.5％，99.6％，99.7％，99.8％，99.9％的同源性。
本发明中的siRNA能有效的抑制补体因子D的表达，抑制效率可达78％以上，有助于治疗补体过度活化引起的相关疾病。本发明中的CFD的抑制方法简单快捷。
附图说明
以下，结合附图来详细说明本发明的实施例，其中：
图1：CFD siRNA正义链质谱图谱；
图2：CFD siRNA反义链的质谱图谱；
图3：CFD mRNA在细胞中的表达水平，其中，NC为转染阴性对照siRNA(siRNA-NC)的阴性对照组，mock为加入转染剂细胞组，blank为加入PBS而无siRNA和转染剂的空白对照组，CFD为转染CFDsiRNA的实验组。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：siRNA的合成
本实施例中首先根据能抑制补体因子D的特性，设计siRNA的序列。最终设计出的siRNA的正义链为5′-GCAAGAAGCCCGGGAUCUA-3′(SEQ ID NO.1)，反义链为5′-UAGAUCCCGGGCUUCUUGC-3′(SEQ ID NO.2)。
接下来利用化学合成法制备上述的siRNA并利用质谱进行检测分析，本实施例中含有2’-羟基的核糖核苷酸的寡聚核苷酸均按照理论产量1μmol合成规格完成。
首先称取1μmol通用固相支持物3’-胆固醇修饰CPG岛(Chemgenes产品)，2’-O-TBDMS保护基保护RNA亚磷酰胺的单体溶解于无水乙腈溶液中，使其浓度达到0.2M。配制5-乙硫基-1H-四唑(Chemgenes产品)乙腈溶液作为活化剂(0.25M)，配制0.02M碘的吡啶/水溶液作为氧化剂，以及3％三氯乙酸二氯甲烷溶液作为脱保护试剂，放置于ABI 394型号DNA/RNA自动合成仪对应的试剂指定位置。设置合成程序输入指定的寡聚核苷酸碱基序列，开始循环寡聚核苷酸合成，每步偶合时间6分钟，半乳糖配体对应L和S单体偶合时间10-20分钟。经自动循环后，完成寡核苷酸固相合成。以干燥氮气吹干CPG，转移到 5ml EP管中，加入氨水/乙醇溶液(3/1)2ml，55℃加热16～18小时。在10000rpm的转速下离心10min，取上清液，抽干浓氨水/乙醇后得到白色胶状固体。将固体溶于200μl 1M TBAF THF溶液，室温震荡20小时。加入0.5ml 1M Tris-HCl缓冲液(pH7.4)，室温震荡15分钟，置于离心抽干机抽至体积为原体积1/2，除去THF。
溶液用0.5ml氯仿萃取2次，加入1ml 0.1M TEAA上样液，将混合溶液倒入固相萃取柱，在HTCS LC-MS system(Novatia)系统上，完成质谱检测分析。一级扫描后以Promass软件归一化计算核酸分子量。上述方法分别合成两条单链，质谱鉴定正确后，两条单链以等摩尔比例混合，退火成双链，即为siRNA序列。siRNA的正义链和反义链质谱检测结果分别如图1和图2所示。
实施例2 CFD siRNA的抑制效果
本实施例中，为了检测实施例1中得到的siRNA对CFD的抑制效果，首先将siRNA转染至培养好的细胞中，然后提取RNA通过实时定量PCR获得CFDmRNA的表达情况。
1细胞培养
细胞名称：293T
a)293T细胞常规培养于37℃，5％的CO 2的条件下。
b)取50μL OPTI-MEM培养基稀释5μL CFD siRNA(或siRNA NC，浓度均为20μM)，50μL OPTI-MEM培养基稀释3μL Lipofectamine 3000 TM转染试剂，二者混合，轻轻摇匀，静置15min。此外，设置Mock及Blank对照组。
c)各孔加入上述108μL混合液。
d)取对数生长期的293T细胞，以每孔1.2×10 5细胞接种于12孔板中，每孔体积892μL，最终每孔总体积1000μL。siRNA(或siRNA NC)转染浓度均为100nM。
e)转染48h后将12孔板从37℃，5％CO 2的培养箱中取出，用于提取RNA进行后续检测。
2 RNA提取
a)Trizol裂解：彻底去除细胞培养液，加入1mLTrizolTM Reagent，移液枪吸打3-5 次,让细胞充分裂解,室温放置3～5分钟；
b)加入0.2体积(0.2mL/1mL Trizol)的氯仿，votex震荡15s，室温静置5min；
c)4℃，12000rpm离心15min，出现分层，小心吸取上层水相(水相体积约占Magzol体积的60％)到新的1.5mL离心管中；
d)加入与上清等体积的异丙醇(约0.6mL)，上下颠倒混匀，-20℃沉淀1h以上；
e)4℃，12000rpm离心30min，管底可见白色沉淀，去掉上清；
f)加75％的乙醇1mL，轻轻吹吸使沉淀飘浮，4℃12000rpm离心5min；
g)重复步骤f；
h)去掉上清后短暂离心，使用10μL枪吸干，将离心管盖打开干燥，待沉淀干燥至半透明状即加入适量RNase-free H 2O溶解。
I)RNA质检，Nanodrop检测RNA含量，1％琼脂糖凝胶电泳检测RNA的完整性。
3Q-PCR检测流程
(1)RNA反转录
a)以样品提取的总RNA作为模板，建立如下反应体系：
Figure PCTCN2021133001-appb-000001
b)以上体系混匀，离心收集液体至管底，42℃60min，72℃10min；产物即为cDNA模板
(2)定量
a)建立反应体系如下：
Figure PCTCN2021133001-appb-000002
其中CFD引物和内标基因GAPDH引物序列见表1
表1引物序列
Figure PCTCN2021133001-appb-000003
b)按如下程序进行PCR扩增
95℃预变性10min，然后进入如下循环
*95℃10s
60℃20s
70℃10s
读板
返回*共进行40个循环
制作熔解曲线：70℃至95℃之间，每0.5℃读板一次并停5s。
4抑制效果
以GAPDH为内标基因，通过ΔΔCt法计算CFDmRNA的相对表达量。与转染NC siRNA相比，CFDsiRNA对CFDmRNA的抑制率达78％。各组细胞mRNA的表达水平见表2或图3。
表2 mRNA表达水平
Figure PCTCN2021133001-appb-000004
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。

Claims (10)

  1. 一种干扰RNA，其特征在于，所述的干扰RNA的序列包含SEQ ID NO.1和/或SEQ ID NO.2所示的核苷酸序列。
  2. 根据权利要求1所述的干扰RNA，其特征在于，所述干扰RNA选自：siRNA、dsRNA、shRNA、aiRNA、miRNA及其组合。
  3. 根据权利要求1或2所述的干扰RNA，其特征在于，所述干扰RNA为siRNA，其正义链包含SEQ ID NO.1所示核苷酸序列，反义链包含SEQ ID NO.2所示核苷酸序列。
  4. 根据权利要求1-3任一所述的干扰RNA，其特征在于,所述干扰RNA还包含悬挂碱基,所述悬挂碱基为dTdT、dTdC或dUdU。
  5. 一种包含权利要求1-4任一所述的干扰RNA的细胞。
  6. 一种权利要求1-4任一所述的干扰RNA的制备方法，其特征在于，所述的制备方法包括化学合成法、体外转录法、酶消化法或体内转录法，优选的，所述的制备方法为化学合成法，进一步优选的，所述的制备方法包括以3’-胆固醇修饰CPG岛作为固相支持物，以2’-O-TBDMS作为保护基，以5-乙硫基-1H-四唑乙腈溶液作为活化剂，以碘的吡啶/水溶液作为氧化剂，以三氯乙酸二氯甲烷溶液作为脱保护试剂，按照偶合时间6分钟，半乳糖配体对应L和S单体偶合时间10-20分钟的程序进行寡核苷酸固相合成，获得siRNA。
  7. 一种干扰RNA的递送系统，其包含权利要求1-4任一项所述的干扰RNA和载体；优选的，所述载体为病毒载体或非病毒载体。
  8. 一种药物组合物，其包含权利要求1-4任一项所述的干扰RNA或权利要求7所述的递送系统，以及药学上可接受的辅料。
  9. 一种CFD表达的抑制方法，其特征在于，所述的抑制方法包括将权利要求1-4任一所述的干扰RNA转染到细胞中。
  10. 一种权利要求1-4任一项所述的干扰RNA、权利要求5所述的细胞、权利要求7所述的递送系统、权利要求8所述的药物组合物在调控补体激活旁路途径或 制备治疗补体过度活化相关疾病的药物中的应用；优选的，所述的补体过度活化相关疾病选自自身免疫疾病、炎性疾病、神经变性疾病、缺血-再灌注损伤、眼疾病或癌症。
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