CN114686481A - Interfering RNA for inhibiting CFD expression and preparation method and application thereof - Google Patents

Abstract

The invention provides an interfering RNA for inhibiting CFD expression and application thereof. Particularly, the interfering RNA is siRNA which comprises nucleotide sequences of GCAAGAAGCCCGGGAUCUA and/or UAGAUCCCGGGCUUCUUGC, can effectively inhibit the expression of CFD, can be used for researching the regulation and control of the alternative pathway of complement activation, and has important value for preparing medicines for treating diseases related to complement over-activation.

Description

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

Technical Field

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

Background

Complement was first discovered by jules bordet as a heat-labile component in normal plasma that functions to condition and kill bacteria. The complement system refers to a series of more than 20 proteins circulating in blood and interstitial fluid. Most proteins are normally inactive, but due to recognition of the molecular components of the microorganism, they are sequentially activated in an enzyme cascade-activation of one protein enzymatically cleaves and activates the next protein in the cascade. Complement can be activated by three different pathways, namely the classical pathway, the alternative pathway (the 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 linkages within C3 to yield C3 (H)₂O), C3 (H)₂O) associates with factor B to form C3 (H)₂O) B complex. The role of complement factor D is to cleave C3 (H)₂O) factor B within the B complex to form Ba and Bb. Bb fragment remains with C3 (H)₂O) to form the alternative pathway C3 convertase C3 (H)₂O) Bb. In addition, C3B, produced by any C3 convertase, also associates with factor B to form C3bB, which factor D cleaves to produce the late alternative pathway C3 convertase C3bBb, which C3 convertase can provide important events within all three defined complement pathwaysSwimming, ultimately leads to recruitment and assembly of other factors in the complement cascade, including the breakdown of C5 into C5a and C5 b. C5b plays a role in the assembly of factors C6, C7, C8 and C9 into a membrane attack complex that can destroy pathogenic cells by lysing the cells. Factor D is present in very low plasma concentrations in humans (1.8. mu.g/ml) and there is evidence that it is the enzyme that activates the rate-limiting role in the complement replacement pathway. Factor D is therefore a fairly suitable disease-inhibiting target in activating the complement alternative pathway.

Complement dysfunction or overactivation has been linked to certain autoimmune, inflammatory, and neurodegenerative diseases as well as 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), and 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.

Downregulation of complement activation can be effective in the treatment of 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. Still other inflammatory disorders and autoimmune diseases are also closely related to complement activation, including thermal injury, severe asthma, anaphylactic shock, inflammatory bowel disease, urticaria, angioedema, vasculitis, multiple sclerosis, myasthenia gravis, psoriasis, dermatomyositis, membranoproliferative glomerulonephritis, and sjogren's syndrome.

Of these, age-related macular degeneration (AMD) is the leading cause of vision loss in people of fifty years or older in industrialized countries. It is estimated that by 2020, the number of people with AMD can exceed 1.96 billion, and by 2040 this number is expected to rise to 2.88 billion. 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, as do individuals with mutations in other complement factor genes. Individuals with mutant factor H also have elevated levels of C-reactive protein, which is a marker of inflammation. Without the proper functional factor H, alternative pathways of the complement cascade would be over-activated, leading to cell damage. Inhibition of alternative pathways is therefore desirable.

Thus, by developing specific inhibitors against complement system inhibitory targets, such as factor D, e.g., sirnas that inhibit complement factor D, that down-regulate complement activation, such inhibitors would have potential utility in treating the above-mentioned diseases.

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.

Therefore, although compositions, antibodies, and the like capable of inhibiting complement factor D have been disclosed in the prior art, siRNA capable of inhibiting complement factor D in the present invention has not been disclosed.

Disclosure of Invention

In a first aspect of the invention, an interfering RNA is provided, the sequence of which comprises a nucleotide sequence shown in SEQ ID No.1 and/or SEQ ID No.2 or a nucleotide sequence having more than 80% homology with 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 that is reverse complementary paired to the sense strand.

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

In one embodiment of the invention, the interfering RNA is siRNA, wherein the 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.

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

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

In a second aspect of the invention, a cell comprising the above interfering RNA is provided.

Preferably, said cell inhibits the expression of CFD.

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

In a third aspect of the present invention, there is provided a method for preparing the above-mentioned interfering RNA, wherein the method for preparing the interfering RNA comprises a chemical synthesis method, an in vitro transcription method, an enzymatic digestion method, or an in vivo transcription method.

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

Preferably, the step further comprises drying the CPG islands.

Preferably, said step further comprises extraction.

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

Specifically, any vector suitable for delivering the above-described interfering RNA of the present invention to a target tissue or a target cell or the like can be used as the vector, such as those disclosed in the prior art (e.g., Chenzhonghua, Zhude Sheng, Li Jun, Huang Zhang Du. "research progress on non-viral siRNA vector". China pharmacological report 2015, 31 (7): 910-4; WangRui, Polygala japonica, Yang Jing. "research progress on siRNA-carrying Nanoprotein. pharmacy 2017, 28 (31): 4452 4455).

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

In another embodiment of the present invention, the above-mentioned vector is a non-viral vector, such as specifically a liposome, a polymer, a polypeptide, an antibody, an aptamer, etc. or a combination thereof; wherein the interfering RNA can be delivered by chemical bond coupling or physical mixing with the non-viral vector, and the ratio of physical mixing 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).

Specifically, the liposome may be a cationic lipid (e.g., lipofectamine series, 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP) available from invitrogen), a neutral ion liposome (e.g., Dioleoylphosphatidylcholine (DOPC), cholesterol, etc.), an anionic liposome (e.g., Dioleoylphosphatidylglycerol (DOPG), Dioleoylphosphatidylethanolamine (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, transportan peptide, pentatin peptide, oligo-arginine peptide, etc.).

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

In a fifth aspect of the present invention, a pharmaceutical composition is provided, which comprises the above-mentioned interfering RNA or its delivery system, and a pharmaceutically acceptable excipient.

In a sixth aspect of the present invention, there is provided a method for inhibiting CFD expression, the method comprising transfecting the above-described interfering RNA into a cell.

In a seventh aspect of the present invention, there is provided a use 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 medicament for preventing and/or treating a disease associated with excessive complement activation.

Preferably, the complement overactivation-associated disease includes an autoimmune disease, an inflammatory disease, a neurodegenerative disease, ischemia-reperfusion injury, an ocular disease or cancer.

In an eighth aspect of the present invention, there is provided 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 medicament for preventing and/or treating a disease associated with excessive complement activation.

Preferably, the complement overactivation-associated disease includes an autoimmune disease, an inflammatory disease, a neurodegenerative disease, ischemia-reperfusion injury, an ocular disease or cancer.

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

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

In the eleventh aspect of the present invention, a method for preventing and/or treating a disease associated with complement overactivation is provided, which comprises the step of administering a therapeutically effective amount of the above-mentioned interfering RNA or its delivery system, pharmaceutical composition of the present invention to a subject.

In a twelfth aspect of the present invention, the present invention also provides a method for introducing the above-described 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.

Specifically, the above cell is in a subject.

Specifically, the above step of contacting the cell with the delivery system of interfering RNA is a step of administering the delivery system of interfering RNA into the body of the subject via a systemic route or a local route to contact the cell.

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 invention pertains.

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 inhibition of translation of mRNA complementary to the interfering RNA sequence) when the interfering RNA is in the same cell as the target gene or sequence. Interfering RNA thus refers to single-stranded RNA complementary to a target mRNA sequence or 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 invention to silence, reduce, or inhibit expression of a target gene (e.g., a CFD gene). To examine 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 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 samples 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 the target gene is achieved when the test sample has a value of about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 10%, 5%, or 0% relative to the control sample. 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 blot, Northern blot, real-time RT-PCR, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art.

Interfering RNAs include "small interfering RNAs" or "sirnas," where each strand of the siRNA molecule comprises nucleotides of about 15 to about 60 in length (e.g., nucleotides of about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 in length, or nucleotides of 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 invention 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. The shRNA hairpin structure can be cleaved by the cellular machinery to siRNA.

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

In the present invention, 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 following active ingredients: when administered, it is sufficient to prevent the development of, or 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, as well as the age, weight, sex, etc. of the subject.

In the present invention, the subject may be a mammal, e.g., a human, monkey, dog, rabbit, mouse, rat, etc.; in one embodiment of the present invention, the subject is a human.

The "autoimmune disease" according to 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, sjogren's syndrome, pain, or neurological disorder, etc.

The "inflammatory disease" as defined in the present invention includes acute inflammation and also includes chronic inflammation. Specifically, the inflammation includes but is not limited to degenerative inflammation, exudative inflammation, proliferative inflammation, specific inflammation, etc., including but not limited to severe burn, endotoxemia, septic shock, adult respiratory distress syndrome, hemodialysis, anaphylactic shock, severe asthma, angioedema, Crohn's disease, sickle cell anemia, streptococcal post-infection glomerulonephritis, pancreatitis, enteritis, vasculitis, adverse drug reactions, drug allergies, IL-2 induced vascular leak syndrome, or radiographic (contrast) contrast agent allergies, etc.

"neurodegenerative diseases" as used herein include, but are not limited to, Alzheimer's disease, progressive blindness or ophthalmoplegia, multiple system atrophy, frontotemporal dementia, Huntington's chorea, corticobasal degeneration, spinocerebellar ataxia, motor neuron disease, hereditary motor sensory neuropathy, and the like.

The term "ischemia-reperfusion injury" as used herein includes, but is not limited to, acute myocardial infarction, aneurysm, stroke, hemorrhagic shock, crush injury, multiple organ failure, intestinal ischemia, ischemia-reperfusion injury following complement activation during cardiopulmonary bypass surgery or other ischemia-causing event, and the like.

"ocular diseases" as described herein include, but are not limited to, macular degenerative 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. Wherein the senile macular degeneration (AMD) includes non-exudative (e.g. intermediate (intermediate) dry AMD or Geographic Atrophy (GA)) and exudative (e.g. wet AMD (choroidal neovascularization (CNV)) AMD, Diabetic Retinopathy (DR), endophthalmitis and uveitis, and further the non-exudative AMD may include hard drusen, soft drusen, geographic atrophy and/or pigment agglomeration, etc.

"cancer" as referred to herein includes, but is not limited to, lymphoma, B cell tumor, T cell tumor, myeloid/monocytic tumor, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial 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, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; said 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's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

"treating" 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 complete elimination of all disease-related signs, symptoms, conditions, or disorders.

As used herein to describe a sequence of a protein or nucleic acid, the "comprising" of the invention 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 possess the activity described herein.

"homology" as used herein means that, in the context of using a protein sequence or a nucleotide sequence, one skilled in the art can adjust the sequence as needed to obtain a sequence having (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%, 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% homology.

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

Drawings

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

FIG. 1: a CFD siRNA sense strand mass spectrum map;

FIG. 2: mass spectrum profile of CFD siRNA antisense strand;

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

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Example 1: synthesis of siRNA

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

Next, the above siRNA was prepared by chemical synthesis and analyzed by mass spectrometry, and the oligonucleotides containing 2' -hydroxy ribonucleotides in this example were synthesized in a theoretical yield of 1. mu. mol.

Firstly, 1 mu mol of universal solid phase support 3 '-cholesterol modified CPG island (product of Chemomenes) is weighed, and the monomer of 2' -O-TBDMS protective group protected RNA phosphoramidite is dissolved in anhydrous acetonitrile solution to ensure that the concentration of the monomer reaches 0.2M. 5-ethylthio-1H-tetrazole (a product of Chemcene) acetonitrile solution is prepared to be used as an activating agent (0.25M), 0.02M iodine pyridine/water solution is prepared to be used as an oxidizing agent, 3 percent trichloroacetic acid dichloromethane solution is prepared to be used as a deprotection reagent, and the deprotection reagent is placed at a reagent designated position corresponding to an ABI 394 model DNA/RNA automatic synthesizer. Setting a synthesis program, inputting a specified oligonucleotide base sequence, starting cyclic oligonucleotide synthesis, wherein 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. After automatic circulation, the oligonucleotide solid phase synthesis is completed. The 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 draining off concentrated ammonia water/ethanol to obtain white colloidal solid. The solid was dissolved in 200. mu.l of 1M TBAF THF and shaken at room temperature for 20 hours. 0.5ml of 1M Tris-HCl buffer (pH7.4) was added thereto, shaken at room temperature for 15 minutes, and then placed in a centrifugal pump to pump the mixture to 1/2 in volume, and THF was removed.

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

Example 2 inhibitory Effect of CFD siRNA

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

1 cell culture

Cell name: 293T

a)293T cells were routinely cultured at 37 ℃ in 5% CO₂Under the conditions of (a).

b) mu.L of OPTI-MEM medium was diluted to 5. mu.L of CFD siRNA (or siRNA NC at 20. mu.M concentration), and 50. mu.L of OPTI-MEM medium was diluted to 3. mu.L of Lipofectamine 3000_TMThe transfection reagent and the two are mixed, shaken gently and kept stand for 15 min. Also, Mock and Blank controls were set.

c) Add 108. mu.L of the above mixture to each well.

d) 293T cells in logarithmic growth phase were taken at 1.2X 10 per well⁵Cells were seeded in 12-well plates in 892. mu.L volumes per well, resulting in a total volume of 1000. mu.L per well. The siRNA (or siRNA NC) transfection concentrations were all 100 nM.

e) 48h after transfection, 12-well plates were incubated at 37 ℃ in 5% CO₂Taken out of the incubator and used for extracting RNA for subsequent detection.

2RNA extraction

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

b) adding 0.2 volume (0.2mL/1mL Trizol) of chloroform, shaking by votex for 15s, and standing at room temperature for 5 min;

c) centrifuging at 12000rpm for 15min at 4 deg.C, allowing stratification to occur, and carefully pipetting the upper aqueous phase (the volume of the aqueous phase is about 60% of the volume of Magzol) into a new 1.5mL centrifuge tube;

d) adding isopropanol (about 0.6mL) with the same volume as the supernatant, and uniformly mixing by reversing the upper part and the lower part, and precipitating at-20 ℃ for more than 1 h;

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

f) adding 1mL of 75% ethanol, gently blowing and sucking to float the precipitate, and centrifuging at 12000rpm for 5min at 4 ℃;

g) repeating the step f;

h) removing supernatant, centrifuging for a short time, drying with 10 μ L gun, opening the cover of the centrifuge tube, drying the precipitate to translucence, and adding appropriate amount of RNase-free H₂And dissolving the O.

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

3Q-PCR detection process

(1) Reverse transcription of RNA

a) Taking total RNA extracted from a sample as a template, establishing the following reaction system:

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

(2) Quantification of

a) The reaction system was set up as follows:

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

TABLE 1 primer sequences

b) PCR amplification was performed according to the following procedure

Pre-denaturation at 95 ℃ for 10min, and then entering the following cycle

*95℃ 10s

60℃ 20s

70℃ 10s

Reading board

Return to run 40 cycles

Preparing a melting curve: read plate between 70 ℃ and 95 ℃ every 0.5 ℃ and stop for 5 s.

4 inhibiting effect

The relative expression amount of CFD mRNA was calculated by the Δ Δ Ct method using GAPDH as an internal standard gene. Compared with the transfection NC siRNA, CFD siRNA has 78% inhibition rate on CFD mRNA. The expression levels of mRNA in each group of cells are shown in Table 2 or FIG. 3.

TABLE 2 mRNA expression levels

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Sequence listing

<110> Beijing Kekai science and technology GmbH

<120> interfering RNA for inhibiting CFD expression and preparation method and application thereof

<130> 1

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gcaagaagcc cgggaucua 19

<210> 2

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

uagaucccgg gcuucuugc 19

<210> 3

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tctgacttca acagcgacac 20

<210> 4

<211> 20

<212> DNA/RNA

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<400> 4

gccaaattcg ttgtcatacc 20

<210> 5

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tcacccaagc aacaaagtcc 20

<210> 6

<211> 25

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtaggtgctc aataaagacc aacca 25

Claims (10)

1. An interfering RNA, characterized in that the sequence of the interfering RNA comprises the nucleotide sequence shown in SEQ ID NO.1 and/or SEQ ID NO. 2.

2. The interfering RNA of claim 1, wherein the interfering RNA is selected from the group consisting of: s iRNA, dsRNA, shRNA, airRNA, miRNA and combinations thereof.

3. The interfering RNA of claim 1 or 2, wherein the interfering RNA is siRNA, and the 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.

4. The interfering RNA of any one of claims 1-3, further comprising a dangling base that is dTdT, dTdC, or dUdU.

5. A cell comprising the interfering RNA of any one of claims 1-4.

6. A method of producing the interfering RNA of any one of claims 1 to 4, wherein the method comprises a chemical synthesis method, an in vitro transcription method, an enzymatic digestion method or an in vivo transcription method, preferably the method is a chemical synthesis method, further preferably the method comprises a step of performing oligonucleotide solid phase synthesis by using 3 '-cholesterol-modified CPG islands as a solid support, 2' -O-TBDMS as a protecting group, 5-ethylthio-1H-tetrazole acetonitrile solution as an activating agent, iodine in pyridine/water solution as an oxidizing agent, trichloroacetic acid in dichloromethane solution as a deprotection reagent according to a sequence of coupling time 6 minutes, galactose ligand corresponding to L and S monomers coupling time 10-20 minutes, to obtain S iRNA.

7. A delivery system for interfering RNA comprising the interfering RNA of any one of claims 1-4 and a vector; preferably, the vector is a viral vector or a non-viral vector.

8. A pharmaceutical composition comprising the interfering RNA of any one of claims 1-4 or the delivery system of claim 7, and a pharmaceutically acceptable excipient.

9. A method for inhibiting the expression of CFD, comprising transfecting the interfering RNA of any one of claims 1-4 into a cell.

10. Use of the interfering RNA of any one of claims 1-4, the cell of claim 5, the delivery system of claim 7, the pharmaceutical composition of claim 8 for modulating the alternative complement activation pathway or for the manufacture of a medicament for the treatment of a disease associated with complement overactivation; preferably, the complement hyperactivation-related disease is selected from the group consisting of an autoimmune disease, an inflammatory disease, a neurodegenerative disease, ischemia-reperfusion injury, an ocular disease, or cancer.

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