CN114686481B

CN114686481B - Interference RNA for inhibiting CFD expression and preparation method and application thereof

Info

Publication number: CN114686481B
Application number: CN202011640647.2A
Authority: CN
Inventors: 林美娜; 陈晓萌; 赵宣; 孙宇
Original assignee: Jenkem Technology Co Ltd
Current assignee: Jenkem Technology Co Ltd
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2023-08-15
Anticipated expiration: 2040-12-31
Also published as: WO2022142894A1; CN114686481A

Abstract

The application provides an interference RNA for inhibiting CFD expression and application thereof. In particular, the interfering RNA is siRNA, comprises a nucleotide sequence of GCAAGAAGCCCGGGAUCUA and/or UAGAUCCCGGGCUUCUUGC, can effectively inhibit the expression of CFD, can be used for researching the regulation of the complement activation alternative pathway, and has important value for preparing medicines for treating diseases related to complement overactivation.

Description

Interference RNA for inhibiting CFD expression and preparation method and application thereof

Technical Field

The application relates to the technical field of biological medicines, in particular to an interference RNA for inhibiting CFD expression, a preparation method and application thereof.

Background

Complement was at the earliest a thermally unstable component found by JulesBordet that acted as a opsonic and bacterial killing component in normal plasma. The complement system refers to a series of more than 20 proteins circulating in blood and interstitial fluid. Most proteins are normally not functional, but due to the recognition of the components of the microbial molecule they are activated in turn in one enzyme cascade-activation of one protein cleaves enzymatically and activates the next protein in the cascade. Complement can be activated by three different pathways, the classical pathway, the alternative pathway (alternative pathway) and the lectin pathway.

Complement Factor D (CFD) plays an early and central role in the activation of alternative pathways of the complement cascade. Activation of the alternative complement pathway results from spontaneous hydrolysis of thioester bonds within C3 to produce C3 (H ₂ O) to initiate, C3 (H) ₂ O) associates with factor B to form C3 (H) ₂ O) B complex. Complement factor D acts to cleave C3 (H ₂ O) factor B within the B complex to form Ba and Bb. Bb fragment remains identical to C3 (H ₂ O) associate to form alternative pathway C3 convertase C3 (H) ₂ O) Bb. In addition, C3B produced by any C3 convertase also associates with factor B to form C3bB, which factor D breaks down to produce the later alternative pathway C3 convertase C3bBb, which C3 convertase can provide important downstream amplification within all three defined complement pathways, ultimately leading to recruitment and assembly of other factors in the complement cascade pathway, including the cleavage of C5 into C5a and C5B. C5b plays a role in the assembly of factors C6, C7, C8 and C9 into membrane attack complexes that can destroy pathogenic cells by lysing the cells. Factor D has a very low plasma concentration in humans (1.8 μg/ml) and has been shown to be an enzyme that activates the rate limiting function of the complement alternative pathway. Factor D is therefore a fairly suitable disease-inhibiting target in activating the complement alternative pathway.

Dysfunction or excessive activation of complement has been linked to certain autoimmune, inflammatory, and neurodegenerative diseases, ischemia-reperfusion injury, and cancer. For example, activation of alternative pathways of the complement cascade contributes to the production of C3a and C5a (both potent anaphylatoxins), C3a and C5a also play a role in many inflammatory diseases. Thus, in some cases, it is desirable to reduce the response of the complement pathway, including the alternative complement pathway.

Down-regulating complement activation is effective in treating several conditions including systemic lupus erythematosus and glomerulonephritis, rheumatoid arthritis, cardiopulmonary bypass surgery and hemodialysis, ultrafiltration rejection in organ transplantation, myocardial infarction, tissue damage due to ischemia reperfusion, and adult respiratory distress syndrome. Still other inflammatory conditions and autoimmune diseases are also closely associated with complement activation, including thermal injury, severe asthma, anaphylactic shock, enteritis, urticaria, angioedema, vasculitis, multiple sclerosis, myasthenia gravis, psoriasis, dermatomyositis, membranous proliferative glomerulonephritis, and sjogren's syndrome.

Among them, age-related macular degeneration (AMD) is a major cause of vision loss in people of fifty years or older in industrialized countries. It is estimated that by 2020, the number of people suffering from AMD may exceed 1.96 billion, and by 2040, the number is expected to rise to 2.88 billion. Based on many genetic studies, evidence exists for a link between the complement cascade and macular degeneration. Individuals with mutations in genes encoding complement factor H have a five-fold increased risk of macular degeneration, as do individuals with mutations in other complement factor genes. Individuals with mutant factor H also have increased levels of C-reactive protein, a marker of inflammation. Without the appropriate functional factor H, alternative pathways of the complement cascade would be overactivated, leading to cell damage. Thus, inhibition of alternative pathways is desirable.

Thus, by developing specific inhibitors, such as siRNAs that inhibit complement factor D, against complement system inhibition targets, such as factor D, can down-regulate complement activation, such inhibitors would have potential efficacy in treating the above-mentioned diseases.

Patent CN108934169a discloses a composition and method for inhibiting factor D, the disclosed aptamer being capable of treating ocular diseases by inhibiting factor D.

Patent CN201710229191.2 discloses a monoclonal antibody against human complement factor D and uses thereof.

Thus, compositions, antibodies, etc. capable of inhibiting complement factor D are disclosed in the prior art, but siRNA capable of inhibiting complement factor D in the present application is not disclosed.

Disclosure of Invention

In a first aspect of the application, there is provided an interfering RNA, the sequence of which comprises a nucleotide sequence having more than 80% homology with SEQ ID NO.1 and/or SEQ ID NO.2 or comprising a nucleotide sequence as shown in SEQ ID NO.1 and/or SEQ ID NO. 2.

The specific sequence of SEQ ID NO.1 is 5'-GCAAGAAGCCCGGGAUCUA-3'.

The specific sequence of SEQ ID NO.2 is 5'-UAGAUCCCGGGCUUCUUGC-3'.

Preferably, the interfering RNA inhibits the expression of CFD (complement factor D).

Preferably, the interfering RNA comprises a sense strand and an antisense strand complementary to the sense strand.

Preferably, the interfering RNA is selected from the group consisting of: siRNA, dsRNA, shRNA, aiRNA, miRNA, and combinations thereof.

In one embodiment of the present application, the interfering RNA is an siRNA, the sense strand of which comprises the nucleotide sequence shown in SEQ ID NO.1, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO. 2.

Preferably, the ends (e.g., 3' ends) of the sense strand and/or antisense strand of the interfering RNA (e.g., siRNA) molecule may also be provided with n overhanging bases (Over-hang) to increase the activity of the interfering RNA. Wherein the nucleobases may be identical or different deoxynucleosides (e.g., deoxythymidine (dT), deoxycytidine (dC), deoxyuridine (dU), etc.), n is an integer from 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10), especially an integer from 2 to 4; preferably, n=2, and the pendant base may be dTdT, dTdC or du, etc.

In some embodiments, the interfering RNA molecules may further comprise at least one modified nucleotide, where the modified interfering RNA has better properties than the corresponding unmodified interfering RNA, such as higher stability, lower immunostimulatory properties, etc.

In a second aspect of the application, there is provided a cell comprising an interfering RNA as described above.

Preferably, the cells inhibit CFD expression.

Preferably, the cell is any CFD expressing cell, such as an adipocyte, a myeloid cell, a hepatocyte, or the like, and in one embodiment of the application, the cell is a 293T cell.

In a third aspect of the present application, there is provided a method for producing the above-mentioned interfering RNA, said method comprising chemical synthesis, in vitro transcription, enzymatic digestion or in vivo transcription.

Preferably, the preparation method is a chemical synthesis method, comprising the steps of performing oligonucleotide solid phase synthesis by taking a 3 '-cholesterol modified CPG island as a solid phase support, taking 2' -O-TBDMS as a protecting group, taking 5-ethylthio-1H-tetrazole acetonitrile solution as an activating agent, taking pyridine/water solution of iodine as an oxidizing agent, taking trichloroacetic acid dichloromethane solution as a deprotection reagent, and performing coupling time for 6 minutes and coupling time for 10-20 minutes of galactose ligands corresponding to L and S monomers to obtain siRNA.

Preferably, the step further comprises drying the CPG island.

Preferably, the step further comprises extraction.

In a fourth aspect of the present application, there is provided a delivery system for the above-described interfering RNA comprising the above-described interfering RNA and a vector.

Specifically, the above-mentioned vector may employ any vector suitable for delivering the above-mentioned interfering RNA of the present application to a target tissue or a target cell or the like, such as those disclosed in the prior art (e.g., chen Zhonghua, zhu Desheng, li Jun, huang Zhanqin. "progress of non-viral siRNA vector research". Chinese pharmacological bulletin.2015, 31 (7): 910-4; wang Rui, qu Bingnan, yang. "progress of siRNA-carrying nanopreparation research". Chinese pharmacy. 2017, 28 (31): 4452-4455).

In one embodiment of the present application, the vector is a viral vector, specifically, lentivirus, retrovirus, adenovirus, herpes simplex virus, and the like.

In another embodiment of the present application, the above-mentioned vector is a non-viral vector, such as a liposome, a polymer, a polypeptide, an antibody, an aptamer, etc., or a combination thereof; wherein, the interfering RNA can be delivered by coupling with a non-viral vector through chemical bond or by physical mixing, and the ratio of physical mixing can be 1:1-50 (e.g. 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50).

Specifically, the liposome may be a cationic liposome (e.g., lipofectamine series of Invitrogen corporation, 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP)), a neutral ionic liposome (e.g., dioleoyl phosphatidylcholine (DOPC), cholesterol, etc.), an anionic liposome (e.g., dioleoyl phosphatidylglycerol (DOPG), dioleoyl phosphatidylethanolamine (DOPE), etc.), or a mixture thereof.

Specifically, the polymer may be a synthetic polymer (e.g., polyethyleneimine, cyclodextrin, polyethylene glycol, etc.) or a natural polymer (e.g., chitosan, telogen, hyaluronic acid, etc.), or a mixture thereof.

Specifically, the polypeptide may be a Cell Penetrating Peptide (CPP) (e.g., low molecular weight protamine, tat peptide, transport peptide, pentratin peptide, oligoarginine peptide, etc.).

Specifically, the antibody may be a single chain antibody (e.g., scFv-tp, scFv-9R, etc.).

In a fifth aspect of the application, a pharmaceutical composition is provided, comprising the interfering RNA or delivery system thereof as described above, and a pharmaceutically acceptable adjuvant.

In a sixth aspect of the application, there is provided a method of inhibiting CFD expression, said method comprising transfecting the interfering RNA described above into a cell.

In a seventh aspect, the present application provides a use of the interfering RNA, the cell, the delivery system or the pharmaceutical composition described above for the preparation of a medicament for preventing and/or treating a disease associated with excessive activation of complement.

Preferably, the complement activation-related disease includes autoimmune disease, inflammatory disease, neurodegenerative disease, ischemia-reperfusion injury, eye disease or cancer.

In an eighth aspect of the application, there is provided the use of an interfering RNA as described above, a cell as described above, a delivery system as described above or a pharmaceutical composition as described above in a medicament for the prevention and/or treatment of a disease associated with excessive activation of complement.

In a ninth aspect of the application, there is provided the use of an interfering RNA as described above, a cell as described above, a delivery system as described above or a pharmaceutical composition as described above for inhibiting CFD gene expression.

In a tenth aspect of the application, there is provided a method of inhibiting CFD gene expression in a subject in need thereof comprising the step of administering to the subject a therapeutically effective amount of an interfering RNA of the application described above or a delivery system, pharmaceutical composition thereof.

In an eleventh aspect of the present application, there is provided a method for preventing and/or treating a disease associated with excessive activation of complement, comprising the step of administering to a subject a therapeutically effective amount of the above-described interfering RNA of the present application or a delivery system, pharmaceutical composition thereof.

In a twelfth aspect of the application, the application also provides a method of introducing the interfering RNA of the application described above into a cell, comprising the step of contacting the cell with a delivery system for the interfering RNA.

Specifically, the above-described cells are in a subject.

Specifically, the step of contacting the cells with the delivery system of the interfering RNA is a step of administering the delivery system of the interfering RNA into the subject by a systemic route or a local route to contact the cells.

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application relates.

The term "interfering RNA" as used herein includes single-stranded RNA (e.g., mature miRNA, ssRNAi oligonucleotide, ssDNAi oligonucleotide) or double-stranded RNA (i.e., duplex RNA such as siRNA, dsRNA, shRNA, aiRNA, or precursor miRNA) that is capable of reducing or inhibiting expression of a target gene or sequence (e.g., by mediating degradation and inhibiting translation of mRNA complementary to the interfering RNA sequence) when the interfering RNA is in the same cell as the target gene or sequence. An interfering RNA is thus 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.

The phrase "inhibiting expression of a target gene" refers to the ability of an interfering RNA (e.g., siRNA) of the application to silence, reduce, or inhibit expression of a target gene (e.g., CFD gene). To test the extent of gene silencing, a test sample (e.g., a biological sample from a target organism expressing a target gene or a sample of cells expressing a target gene in culture) is contacted with an interfering RNA (e.g., siRNA) that silences, reduces, or inhibits the expression of the target gene, the expression of the target gene in the test sample is compared to the expression of the target gene in a sample that is not contacted with the interfering RNA (e.g., siRNA), and a control sample (e.g., a sample expressing the target gene) can be set to a value of 100%. In specific embodiments, silencing, inhibition, or reduction of expression of a target gene is achieved 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%. Suitable assays include, but are not limited to, assaying protein or mRNA levels using techniques known to those skilled in the art, such as, for example, dot blotting, northern blotting, real-time RT-PCR, in situ hybridization, ELISA, immunoprecipitation, enzymatic function, and phenotypic assays known to those skilled in the art.

The interfering RNA includes a "small interfering RNA" or "siRNA", where each strand of the siRNA molecule comprises nucleotides from about 15 to about 60 in length (e.g., nucleotides from about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 in length, or nucleotides 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 in length). In a specific embodiment, the siRNA is chemically synthesized. The siRNA molecules of the application are capable of silencing expression of a target sequence in vitro and/or in vivo. In other embodiments, the siRNA comprises at least one modified nucleotide, e.g., the siRNA comprises one, two, three, four, five, six, seven, eight, nine, ten or more modified nucleotides in the double-stranded region.

As used herein, the term "dsRNA" is intended to include any precursor molecule that is processed in vivo by an endonuclease to produce an active siRNA.

As used herein, the term "shRNA," i.e., "small hairpin RNA" or "short hairpin RNA," includes short RNA sequences that produce tight hairpin turns (hairpin turns) that can be used to silence gene expression by RNA interference. shRNA hairpin structures can be cleaved by cellular machinery into siRNA.

Typically, micrornas (mirnas)) are single stranded RNA molecules of about 21-23 nucleotides in length that regulate gene expression.

In the present application, the term "therapeutically effective amount" refers to the amount of a subject compound that will elicit the biological or medical response of a tissue, system or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes the amount of the active ingredient: when administered, it is sufficient to prevent the development of, or to alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the active ingredient, the disease to be treated and its severity, the age, weight, sex, etc. of the subject.

In the present application, the subject may be a mammal, such as a human, monkey, dog, rabbit, mouse, rat, etc.; in one embodiment of the application, the subject is a human.

The "autoimmune diseases" described herein include, but are not limited to, allergies, 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, sjogren's syndrome, pain or neurological disorders, and the like.

The "inflammatory disease" as used herein includes acute inflammation as well as chronic inflammation. In particular, including but not limited to degenerative, exudative, proliferative, specific, 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, post-streptococcal glomerulonephritis, pancreatitis, enteritis, vasculitis, adverse drug reactions, drug allergies, IL-2 induced vascular leak syndrome, or radiographic (contrast) contrast agent allergies, etc.

"neurodegenerative disorders" as described herein include, but are not limited to, alzheimer's disease, progressive blindness or extraocular muscle paralysis, multisystem atrophy, frontotemporal dementia, huntington's chorea, corticobasal degeneration, spinocerebellar ataxia, motor neuron disease, hereditary motor sensory neuropathy, and the like.

"ischemia-reperfusion injury" as used herein includes, but is not limited to, acute myocardial infarction, aneurysms, stroke, hemorrhagic shock, crush injury, multiple organ failure, bowel ischemia, complement activation during cardiopulmonary bypass surgery, or other post-ischemic event ischemia-reperfusion injury, and the like.

The "eye diseases" described herein include, but are not limited to, macular degeneration diseases such as all stages of age-related macular degeneration (AMD) including dry and wet (non-exudative and exudative) forms, diabetic retinopathy and other ischemia-related retinopathies, choroidal Neovascularization (CNV), uveitis, diabetic macular edema, pathologic myopia, von Hippel-Lindau disease, histoplasmosis of the eye, central Retinal Vein Occlusion (CRVO), corneal neovascularization, and retinal neovascularization. Among them, age-related macular degeneration (AMD) includes non-exudative (e.g., intermediate dry AMD or geographic atrophy (geographic atrophy, GA)) and exudative (e.g., wet AMD (choroidal neovascularization (CNV)) AMD, diabetic Retinopathy (DR), endophthalmitis, and uveitis, and further non-exudative AMD may include hard drusen, soft drusen, geographic atrophy, and/or pigment agglomeration, etc.

The "cancer" as described herein includes, but is not limited to, lymphomas, B-cell tumors, T-cell tumors, bone marrow/monocyte tumors, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, renal cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcomas. Wherein the leukemia is selected from acute lymphoblastic (lymphoblastic) leukemia, acute myelogenous 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's macroglobulinemia; the sarcoma is selected from osteosarcoma, ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

The term "treatment" as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

The term "comprising" as used herein to describe the sequence of a protein or nucleic acid may consist of the sequence or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described herein.

"homology" as used herein means that a person skilled in the art can adjust the sequence according to actual work requirements, using sequences having (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%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%, 99.1%,99.2%,99.3%,99.4%, 99.6%,99.7%, 99.9% as compared with sequences obtained in the prior art.

The siRNA of the application can effectively inhibit the expression of complement factor D, and the inhibition efficiency can reach more than 78%, thereby being beneficial to treating related diseases caused by excessive activation of complement. The CFD inhibition method is simple and quick.

Drawings

Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein:

fig. 1: CFD siRNA sense strand mass spectrum;

fig. 2: mass spectrum of CFD siRNA antisense strand;

fig. 3: expression level of CFD mRNA in cells, where NC is a negative control group transfected with negative control siRNA (siRNA-NC), mock is a group of cells added with transfection agent, blank is a blank group added with PBS without siRNA and transfection agent, and CFD is an experimental group transfected with CFD siRNA.

Detailed Description

The technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

Example 1: synthesis of siRNA

In this example, the sequence of siRNA was designed based on the property of inhibiting complement factor D. The final design of siRNA sense strand 5'-GCAAGAAGCCCGGGAUCUA-3' (SEQ ID NO. 1) and antisense strand 5'-UAGAUCCCGGGCUUCUUGC-3' (SEQ ID NO. 2).

Next, the above siRNA was prepared by chemical synthesis and detected and analyzed by mass spectrometry, and the oligonucleotides of the ribonucleotide containing 2' -hydroxy group in this example were all completed according to the theoretical yield of 1. Mu. Mol synthesis specification.

Firstly, 1 mu mol of a general solid support 3 '-cholesterol modified CPG island (Chemgenes product) and a monomer of a 2' -O-TBDMS protecting group protected RNA phosphoramidite are weighed and dissolved in anhydrous acetonitrile solution to enable the concentration to reach 0.2M. Preparing 5-ethylthio-1H-tetrazole (Chemgenes product) acetonitrile solution as an activator (0.25M), preparing pyridine/water solution of 0.02M iodine as an oxidant, and 3% trichloroacetic acid dichloromethane solution as a deprotection reagent, and placing the solution at a reagent designated position corresponding to an ABI 394 type DNA/RNA automatic synthesizer. Setting a synthesis program, inputting a designated oligonucleotide base sequence, starting to synthesize the cyclic oligonucleotide, wherein the coupling time of each step is 6 minutes, and the coupling time of galactose ligands corresponding to L and S monomers is 10-20 minutes. After automatic circulation, the oligonucleotide solid phase synthesis is completed. CPG was dried with dry nitrogen, transferred to a 5ml EP tube, 2ml of ammonia/ethanol solution (3/1) was added and heated at 55℃for 16-18 hours. Centrifuging at 10000rpm for 10min, collecting supernatant, and pumping out concentrated ammonia water/ethanol to obtain white colloidal solid. The solid was dissolved in 200. Mu.l of 1M TBAF in THF and shaken at room temperature for 20 hours. 0.5ml of 1M Tris-HCl buffer (pH 7.4) was added, and the mixture was shaken at room temperature for 15 minutes, and the mixture was pumped to a volume of 1/2 of the original volume by a centrifugal pump, and THF was removed.

The solution was extracted 2 times with 0.5ml chloroform, 1ml of 0.1M TEAA loading solution was added, and the mixed solution was poured into a solid phase extraction column, and mass spectrometry analysis was completed on a HTCS LC-MS system (Novat). The nucleic acid molecular weight was normalized by Promass software after the primary scan. The method synthesizes two single chains respectively, and after the mass spectrum identification is correct, the two single chains are mixed in an equimolar ratio and annealed into double chains, namely the siRNA sequence. The results of mass spectrometry detection of the sense strand and the antisense strand of the siRNA are shown in FIG. 1 and FIG. 2, respectively.

EXAMPLE 2 inhibition of CFD siRNA

In this example, in order to examine the CFD inhibition effect of the siRNA obtained in example 1, the siRNA was first transfected into cultured cells, and then RNA was extracted and expression of CFD mRNA was obtained by real-time quantitative PCR.

1 cell culture

Cell name: 293T (293T)

a) 293T cells were routinely cultured at 37℃with 5% CO ₂ Is a member of the group (a) and (b).

b) Diluting 5. Mu.L CFD siRNA (or siRNA NC, concentration 20. Mu.M) with 50. Mu.L OPTI-MEM culture medium, diluting 3. Mu.L Lipofectamine 3000 with 50. Mu.L OPTI-MEM culture medium _TM Transfection reagent, the two are mixed, gently shaken and stood for 15min. Furthermore, mock and Blank control groups were set.

c) The above-mentioned 108. Mu.L of the mixture was added to each well.

d) 293T cells in logarithmic growth phase were taken at 1.2X10 per well ⁵ Cells were seeded in 12-well plates at 892 μl per well volume, resulting in a total volume per well of 1000 μl. The siRNA (or siRNA NC) transfection concentrations were 100nM.

e) After 48h of transfection, 12-well plates were incubated from 37℃with 5% CO ₂ Is taken out of the incubator of (2) and used for extracting RNA for subsequent detection.

2RNA extraction

a) Trizol cleavage: thoroughly removing the cell culture solution, adding 1mL TrizolTM Reagent, sucking and beating for 3-5 times by a liquid-transfering gun, fully cracking the cells, and standing for 3-5 minutes at room temperature;

b) Adding 0.2 volume (0.2 mL/1mL Trizol) chloroform, voltex shaking for 15s, and standing at room temperature for 5min;

c) Centrifugation at 12000rpm for 15min at 4℃and delamination occurred, carefully pipetting the upper aqueous phase (aqueous phase volume about 60% of the Magzol volume) into a new 1.5mL centrifuge tube;

d) Adding isopropanol (about 0.6 mL) with the same volume as the supernatant, mixing the mixture upside down, and precipitating at-20 ℃ for more than 1 h;

e) Centrifuging at 12000rpm at 4deg.C for 30min to obtain white precipitate at the bottom of the tube, and removing supernatant;

f) Adding 1mL of 75% ethanol, slightly blowing and sucking to float the precipitate, and centrifuging at 12000rpm at 4deg.C for 5min;

g) Repeating the step f;

h) Removing supernatant, centrifuging briefly, sucking with 10 μl gun, opening the cover of the centrifuge tube, drying, and adding appropriate amount of RNase-free H when the precipitate is semitransparent ₂ O is dissolved.

I) RNA quality detection, RNA content detection by Nanodrop and RNA integrity detection by 1% agarose gel electrophoresis.

3Q-PCR detection flow

(1) RNA reverse transcription

a) Taking total RNA extracted from a sample as a template, and establishing a reaction system as follows:

b) Mixing the above systems, centrifuging to collect liquid to the bottom of the tube, and standing at 42deg.C for 60min and at 72deg.C for 10min; the product is cDNA template

(2) Quantification of

a) The reaction system is established as follows:

wherein the CFD primer and the internal standard gene GAPDH primer sequences are shown in Table 1

TABLE 1 primer sequences

b) PCR amplification was performed as follows

Pre-denatured at 95 ℃ for 10min, and then enters the following circulation

*95℃ 10s

60℃ 20s

70℃ 10s

Reading board

Return for a total of 40 cycles

Preparing a melting curve: plates were read between 70℃and 95℃and stopped for 5s every 0.5 ℃.

4 inhibitory Effect

The relative expression level of CFD mRNA was calculated by the ΔΔCt method using GAPDH as an internal reference gene. Compared with transfected NC siRNA, the inhibition rate of CFD siRNA to CFD mRNA reaches 78 percent. The expression levels of mRNA from each cell group are shown in Table 2 or FIG. 3.

TABLE 2 mRNA expression levels

The preferred embodiments of the present application have been described in detail above, but the present application is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the scope of the technical concept of the present application, and all the simple modifications belong to the protection scope of the present application.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Sequence listing

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Claims

1. An interfering RNA, wherein the interfering RNA is an siRNA, and the sense strand comprises a nucleotide sequence shown in SEQ ID No.1, and the antisense strand comprises a nucleotide sequence shown in SEQ ID No. 2.

2. The interfering RNA of claim 1 further comprising a dangling base, the dangling base being dTdT, dTdC or du.

3. A cell comprising the interfering RNA of any one of claims 1-2.

4. A method for producing an interfering RNA according to any one of claims 1 to 2, wherein the method comprises chemical synthesis, in vitro transcription, enzymatic digestion or in vivo transcription.

5. The method of claim 4, wherein the method is a chemical synthesis method.

6. The preparation method according to claim 5, wherein the preparation method comprises performing oligonucleotide solid phase synthesis according to a procedure of coupling time of 6 minutes, galactose ligand corresponding to L and S monomer coupling time of 10-20 minutes using 3 '-cholesterol modified CPG island as solid phase support, 2' -O-TBDMS as protecting group, 5-ethylthio-1H-tetrazole acetonitrile solution as activator, pyridine/water solution of iodine as oxidant, trichloroacetic acid dichloromethane solution as deprotection reagent, and obtaining siRNA.

7. A delivery system for an interfering RNA comprising the interfering RNA of any one of claims 1-2 and a vector.

8. The delivery system of claim 7, wherein the vector is a viral vector or a non-viral vector.

9. A pharmaceutical composition comprising the interfering RNA of any one of claims 1-2 or the delivery system of any one of claims 7-8, and a pharmaceutically acceptable adjuvant.

10. A method of inhibiting CFD expression, said method comprising transfecting the interfering RNA of any one of claims 1-2 into a cell, said method of inhibiting not comprising a diagnostic or therapeutic method of disease.

11. Use of an interfering RNA according to any one of claims 1-2, a cell according to claim 3, a delivery system according to any one of claims 7-8, a pharmaceutical composition according to claim 9 for modulating the alternative complement activation pathway or for the manufacture of a medicament for the treatment of a disease associated with excessive complement activation, said use excluding a diagnostic or therapeutic method of the disease.

12. The use according to claim 11, wherein the complement hyperactivation-associated disease is selected from autoimmune disease, inflammatory disease, neurodegenerative disease, ischemia-reperfusion injury, eye disease or cancer.