WO2024061356A1 - Interfering rna for inhibiting tubb2 gene expression and use thereof - Google Patents

Interfering rna for inhibiting tubb2 gene expression and use thereof Download PDF

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WO2024061356A1
WO2024061356A1 PCT/CN2023/120769 CN2023120769W WO2024061356A1 WO 2024061356 A1 WO2024061356 A1 WO 2024061356A1 CN 2023120769 W CN2023120769 W CN 2023120769W WO 2024061356 A1 WO2024061356 A1 WO 2024061356A1
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cancer
antibody
tumors
interfering rna
delivery system
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French (fr)
Chinese (zh)
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贾宏丽
林美娜
杨脉
陈晓萌
赵宣
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北京键凯科技股份有限公司
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12N2310/141—MicroRNAs, miRNAs
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention relates to the technical fields of molecular biology and biomedicine, and specifically relates to an interfering RNA that effectively inhibits TUBB2 expression, especially siRNA, and its application.
  • Tubulin is an important part of the eukaryotic cytoskeleton and is widely present in microtubules of living cells.
  • Microtubules are the basic elements of the eukaryotic cytoskeleton and are mainly composed of tubulin ⁇ and ⁇ polymers. Under normal circumstances, there is a dynamic balance between microtubules and tubulin dimers. Microtubules play a vital role in cell support and cell movement, helping cells resist compression forces so that they can maintain their intended shape and structure; in addition, microtubules are also essential for processes such as cell movement, chromosome segregation during mitosis, and intracellular transport of organelles.
  • tubulin In humans, there are five subgroups of tubulin: ⁇ -tubulin, ⁇ -tubulin, ⁇ -tubulin, ⁇ and ⁇ -tubulin, and ⁇ -tubulin.
  • ⁇ -Tubulin is of particular interest because it forms a type of microtubule that is expressed only in neurons. Additionally, all drugs that bind to human tubulin also bind to beta-tubulin, making it a therapeutic target.
  • Antimitotic chemotherapy drugs target tubulin, the major protein in the mitotic spindle, and tubulin isoform composition is considered a determinant of tumor progression diagnosis and cellular response to chemotherapy. This means that the subtype composition of normal tissue and tumor tissue is different, which provides a theoretical basis for the development of chemotherapy drugs for tumors.
  • Common anti-tubulin drugs currently include vinblastine, paclitaxel, etc. Among them, paclitaxel can cause the dynamic balance between microtubules and tubulin dimers to lose, induce and promote tubulin polymerization, prevent depolymerization, and stabilize microtubules.
  • Vinblastine mainly inhibits the polymerization of tubulin, hinders the formation of spindle microtubules, and stops nuclear division in metaphase; it can also cause nuclear collapse and vacuolar pyknosis; it acts on the cell membrane, interfering with the movement of amino acids in the cell membrane, causing Protein synthesis is inhibited; RNA synthesis can be inhibited by inhibiting the activity of RNA polymerase, killing cells in the G1 phase. These effects prevent cells from forming spindles and spindle fibers during mitosis, inhibiting cell division and proliferation, thus exerting anti-tumor effects. Therefore, by inhibiting or promoting tubulin polymerization, it interferes with the balance of microtubules, thereby affecting cell mitosis and inhibiting cell mitosis. cell proliferation.
  • ⁇ 2 tubulin exists in the nucleus and can also exist in non-microtubule forms and can significantly affect cell behavior.
  • ⁇ 2-tubulin is involved in the development of several types of tumors, such as neuroepithelial tumors and brain tumors, colon cancer, and prostate cancer.
  • ⁇ 2-tubulin was found localized in the nucleus, suggesting that it could interact with the antimicrotubule drugs paclitaxel and vinblastine.
  • ⁇ 2-tubulin in the cytoplasm that is not assembled into microtubules can interact with voltage-dependent anion channels (VDAC) to provide energy to muscle cells.
  • VDAC voltage-dependent anion channels
  • this application screens out interfering RNA sequences with high inhibitory activity, which are expected to be prepared into RNA drugs for the treatment of tumors.
  • siRNA molecules are larger and carry a large number of negative charges, making it difficult for them to pass through the similarly negatively charged cell membrane. Therefore, finding a suitable delivery system is an important part of the process of developing siRNA drugs.
  • This application provides two feasible delivery methods: lipid nanoparticles (LNP) and antibody-conjugated siRNA (Antibody–siRNA conjugates, ARC).
  • a first aspect of the invention provides an interfering RNA targeting the tubulin ⁇ 2 (TUBB2) gene.
  • the interfering RNA inhibits the expression of TUBB2.
  • the target site sequence of the interfering RNA includes any one or more than two nucleotide sequences shown in SEQ ID NO: 1-13.
  • the target site sequence of the interfering RNA is as shown in any one of the nucleotide sequences in SEQ ID NO: 1-13.
  • the target site sequence of the interfering RNA includes any one or more of the nucleotide sequences shown in SEQ ID NO: 1-10 or 12.
  • the target site sequence of the interfering RNA is as shown in any one of the nucleotide sequences in SEQ ID NO: 1-10 or 12.
  • the target site sequence of the interference RNA includes any one or two or more nucleotide sequences shown in SEQ ID NO: 1-3 or 5-6 or 8-10.
  • the target site sequence of the interfering RNA is as shown in any one of the nucleotide sequences in SEQ ID NO: 1-3 or 5-6 or 8-10.
  • the target site sequence of the interfering RNA includes SEQ ID NO: 2, 8 and/or 10.
  • the interfering RNA includes siRNA, dsRNA, shRNA, aiRNA or miRNA. One or a combination of two or more.
  • the interfering RNA is siRNA.
  • the length of the interfering RNA is 17-25nt, preferably 19-21nt, such as 17, 18, 19, 20, 21, 22, 23, 24, 25nt.
  • the interfering RNA also includes dangling bases to increase interfering RNA stability and activity.
  • the interfering RNA contains 1-10 dangling bases. 2-4 dangling bases are further preferred. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 dangling bases.
  • the hanging base is located at the 3' end of the sense strand and/or antisense strand of the interfering RNA.
  • the dangling bases are deoxynucleosides; preferably, the dangling bases can be n identical or different deoxynucleosides (such as deoxythymidine (dT), deoxycytidine (dC), deoxyuridine (dU) etc.), n is an integer from 1 to 10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).
  • the ends of the sense strand and/or the antisense strand of the interference RNA are provided with two identical dangling bases.
  • the ends of the sense strand and/or the antisense strand of the interference RNA are provided with two different dangling bases.
  • the dangling base is dTdT, dTdC or dUdU.
  • the interfering RNA includes a sense strand and/or an antisense strand.
  • the sense strand and the antisense strand are complementary to each other.
  • the sense strand includes any one or two or more nucleotide sequences shown in SEQ ID NO: 14-26. Further preferably, the sense strand is as shown in any one of the nucleotide sequences in SEQ ID NO: 14-26. Further preferably, the sense strand includes any one or two or more nucleotide sequences shown in SEQ ID NO: 14-23 or 25. More preferably, the sense strand includes any one or two or more nucleotide sequences shown in SEQ ID NO: 14-16 or 18-19 or 21-23.
  • the sense strand includes SEQ ID NO: 15, 21 and /23.
  • the antisense strand includes any one or two or more nucleotide sequences shown in SEQ ID NO: 27-39. Further preferably, the antisense strand is represented by any one of the nucleotide sequences in SEQ ID NO: 27-39. Further preferably, the antisense strand includes any one or two or more nucleotide sequences shown in SEQ ID NO: 27-36 or 38. More preferably, the antisense strand includes SEQ ID NO: 27-29 Or any one or two or more of the nucleotide sequences shown in 31-32 or 34-36.
  • the antisense strand includes SEQ ID NO: 28, 34 and/or 36.
  • the interfering RNA may further include at least one modification.
  • Modified interfering RNA has better properties than the corresponding unmodified interfering RNA, such as higher stability, lower immunostimulation, etc.
  • the modifications include modifications to the chemical structures of bases, sugar rings and/or phosphates.
  • base modifications include but are not limited to 5-position pyrimidine modification, 8-position purine modification and/or 5-bromouracil substitution.
  • the modification of the sugar ring includes but is not limited to substitution of 2'-OH by groups such as H, OZ, Z, halo, SH, SZ, NH2, NHZ, NZ2 or CN, where Z is an alkyl group .
  • the alkyl group represents a linear or branched hydrocarbon group without unsaturated bonds, and the hydrocarbon group is connected to other parts of the molecule with a single bond.
  • Typical alkyl groups contain 1 to 20 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert.
  • the phosphate backbone modification includes but is not limited to phosphorothioate modification.
  • the modifications also include inosine, braidin, xanthine, 2'-methylribose, non-natural phosphodiester bonds (such as methylphosphonate, thiophosphonate) and/or peptides of nucleotides.
  • the modification can be methylation, fluorination, phosphorothioate, pseudouracil, etc.
  • the modification may occur at any position in the sequence, for example, it may be a partial modification or a complete modification.
  • the same sequence can be modified at some positions of the same or different modification types, or at all positions of the same or different modification types.
  • the interfering RNA includes one or a combination of two or more of the following target site sequences, sense strand and antisense strand sequence combinations:
  • SEQ ID NO: 1 SEQ ID NO: 14, SEQ ID NO: 27;
  • SEQ ID NO: 8 SEQ ID NO: 56, SEQ ID NO: 60;
  • the interfering RNA includes group B), group F) and/or group H).
  • the interfering RNA contains one or more than two of the nucleic acid sequences shown in Table 1 or Table 15.
  • the interfering RNA can be prepared by any method in the existing technology, such as chemical synthesis, etc.
  • a second aspect of the present invention provides a delivery system, said delivery system comprising the above-mentioned interfering RNA.
  • the delivery system further includes a carrier.
  • the carrier can be any carrier suitable for delivering the above-mentioned interfering RNA of the present invention to target tissues or target cells, etc., such as existing technologies (such as Chen Zhonghua, Zhu Desheng, Li Jun, Huang Zhanqin. "Non-viral siRNA” "Progress in Carrier Research”. Chinese Pharmacological Bulletin. 2015, 31(7): 910-4; Wang Rui, Qu Bingnan, Yang Jing. "Research Progress in Nanopreparations Carrying siRNA”. Chinese Pharmacy. 2017, 28(31) :4452-4455).
  • existing technologies such as Chen Zhonghua, Zhu Desheng, Li Jun, Huang Zhanqin. "Non-viral siRNA” "Progress in Carrier Research". Chinese Pharmacological Bulletin. 2015, 31(7): 910-4; Wang Rui, Qu Bingnan, Yang Jing. "Research Progress in Nanopreparations Carrying siRNA”. Chinese Pharmacy. 2017, 28(31) :4452-4455).
  • the vector is a viral vector.
  • the viral vector includes but is not limited to lentiviral vector, retroviral vector, adenoviral vector, adeno-associated virus vector, poxvirus One or more of vectors or herpes virus vectors.
  • the vector is a non-viral vector.
  • the non-viral vector includes but is not limited to liposomes, lipid nanoparticles (LNP), polymers, polypeptides, and antibodies. , aptamer or N-acetylgalactosamine (GalNAc), any one or a combination of two or more; further preferably, the non-viral vector includes lipid nanoparticles (LNP).
  • the weight ratio of the interfering RNA and the non-viral vector can be any value from 1:1 to 50 (for example, 1:1, 1:5, 1:6, 1:7, 1:8, 1: 9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50), preferably any one of 1:1-10 numerical value.
  • the above-mentioned lipid nanoparticles/liposomes include: one or a combination of two or more of cationic lipids, neutral lipids, polyethylene glycol lipids, steroidal lipids or anionic lipids.
  • the cationic lipid includes: stearamide (SA), lauryltrimethylammonium bromide, cetyltrimethylammonium bromide, myristyltrimethylammonium bromide, Dimethyldioctadecylamine (DDAB), [(4-hydroxybutyl)azadialkyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC- 0315), 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP), 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane and 1,2-di-hexadecanoyl-3-trimethylammonium-propane, 3 ⁇ -[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), Dimethyloctadecyl ammoni
  • the cationic lipid is a steroid-cationic lipid compound, and the structure of the compound is: one or more than two of them.
  • the neutral lipid includes: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine Base (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-di Myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC) ), one or a combination of two or more of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) or distearoylphosphatidylethanolamine (DSPE).
  • DSPC 1,2-distearoyl-s
  • the polyethylene glycol lipid includes: 2-[(polyethylene glycol)-2000]-N,N-tetradecyl acetamide (ALC-0159), 1,2-dimethylaminoglycan Myristoyl-sn-glycerylmethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[amino(polyethylene glycol)]( PEG-DSPE), PEG-disterol Glycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG- DPPE) or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA), One or a combination of two or more of the above
  • n is selected from an integer from 20 to 300, such as 20, 30, 50, 80, 100, 150, 200, 250, 300, etc.
  • the polyethylene glycol lipid is a polyethylene glycol lipid of a single molecular weight.
  • the polyethylene glycol lipid includes:
  • the anionic liposome includes: dioleoylphosphatidylglycerol and/or dioleoylphosphatidylethanolamine, etc.
  • the steroidal lipids include: avenasterol, ⁇ -sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, blackseasterol, epicholesterol, ergosterol, fuccasterol, hexahydroluminosterol, hydroxycholesterol, lanosterol, luminosterol, alginasterol, sitostanol, sitosterol, stigmasterol, stigmasterol, bile acid, glycocholic acid, taurocholic acid, deoxycholic acid or lithocholic acid, or a combination of two or more thereof.
  • the above-mentioned polymer can be a synthetic polymer (such as polyethylenimine, cyclodextrin, etc.) or a natural polymer (such as chitosan, telocollagen, etc.) or a mixture thereof.
  • a synthetic polymer such as polyethylenimine, cyclodextrin, etc.
  • a natural polymer such as chitosan, telocollagen, etc.
  • the above-mentioned polypeptide can be a cell-penetrating peptide (CPP) (such as protamine, Tat peptide, transportan peptide, penetratin peptide, oligoarginine peptide, etc.).
  • CPP cell-penetrating peptide
  • the above-mentioned antibody can be a single-chain antibody (such as scFv-tp, scFv-9R, etc.).
  • the delivery system of this application can also encapsulate various ingredients that are beneficial to the human body and deliver them directly into cells to produce the desired effects faster and better.
  • a third aspect of the present invention provides a cell containing the above-mentioned interfering RNA or the above-mentioned delivery system.
  • the cells may be tumor cells.
  • a fourth aspect of the present invention provides a method for preparing cells.
  • the preparation method includes introducing the above-mentioned interfering RNA or the above-mentioned delivery system into cells.
  • a fifth aspect of the present invention provides a lipid nanoparticle, which contains the above-mentioned interfering RNA.
  • the lipid nanoparticles further comprise one or more of polyethylene glycol lipid compounds, cationic lipids, steroidal lipids or neutral lipids.
  • polyethylene glycol lipid compounds cationic lipids, steroidal lipids and neutral lipids are the same as in the second aspect of this application.
  • the lipid nanoparticles contain the above-mentioned interfering RNA as well as polyethylene glycol lipid compounds, cationic lipids (excluding steroid-cationic lipid compounds), steroid lipids and neutral lipids.
  • the molar ratio of polyethylene glycol lipid compounds, cationic lipids, steroidal lipids and neutral lipids is (0.5-5):(30-55):( 30-55):(5-20), for example (0.5, 1, 2, 3, 4, 5): (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): (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):(5,6,7,8,9,10,11,12 ,13,14,15,16,17,18,19,20).
  • the molar ratio of polyethylene glycol lipid compound, cationic lipid, steroidal lipid and neutral lipid is (1-5):(
  • the lipid nanoparticles comprise the above-mentioned interfering RNA and a steroid-cationic lipid compound, a second lipid and a polyethylene glycol lipid.
  • the second lipid is selected from the group consisting of neutral lipids, zwitterionic lipids or anionic lipids.
  • the molar ratio of the steroid-cationic lipid: second lipid: polyethylene glycol lipid is (10-30): (60-80): (10-25 ), such as (10, 15, 20, 25, 30): (60, 65, 70, 75, 80): (10, 15, 20, 25), preferably (20-30): (60-70): (10-20).
  • the lipid nanoparticles of the present application can also encapsulate various components that are beneficial to the human body and deliver them directly into cells to produce the desired effects faster and better.
  • the lipid nanoparticles can be prepared by conventional lipid nanoparticle preparation methods in the field, such as high-pressure homogenization method, emulsification precipitation method, ultrasonic dispersion method, etc.
  • a sixth aspect of the present invention provides a medicine or kit, which contains the above-mentioned interfering RNA, the above-mentioned delivery system, the above-mentioned lipid nanoparticles or the above-mentioned cells.
  • the medicine also includes pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable excipients include but are not limited to carriers, diluents, adhesives, lubricants, wetting agents and the like.
  • the administration methods of the drug include but are not limited to oral, enteral, subcutaneous, intramuscular, intravenous, nasal, transdermal, subconjunctival, intraocular, orbital, retro-ocular, retinal, choroidal, intrathecal, and the like.
  • the dosage forms of the drug include but are not limited to tablets, capsules, pills, injections, inhalants, lozenges, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, and suspoemulsions. , cream, Jelly, spray, etc.
  • Various dosage forms of the drug can be prepared according to conventional production methods in the pharmaceutical field.
  • the mass content of the above-mentioned interfering RNA, the above-mentioned delivery system or the above-mentioned cells in the drug can be 1%-100%, such as 1%, 2%, 3%, 4%, 5%, 6% ,7%,8%,9%,10%,15%,20%,25%,30%,35%,40%,45%,50%,55%,60%,65%,70%,75 %, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100%.
  • the seventh aspect of the present invention provides an antibody-nucleic acid conjugated drug, which contains the above-mentioned interfering RNA or the above-mentioned delivery system or the above-mentioned lipid nanoparticles.
  • the antibody-nucleic acid conjugate drug contains one or more interfering RNAs and antibodies.
  • the antibody and the nucleic acid can be directly connected or connected through a linking group or linking peptide.
  • the general formula (I) of the antibody-nucleic acid conjugated drug is:
  • R is the above-mentioned interfering RNA
  • x1 is an integer from 1 to 144; preferably, x1 is an integer from 1 to 9; more preferably, x1 is an integer from 1 to 3;
  • x2 is an integer from 1 to 8; preferably, x2 is an integer from 1 to 2;
  • Ab is an antibody, protein, or peptide
  • L is the connecting unit connecting Ab and R.
  • the L part has the structure of general formula (II):
  • x3 is selected from an integer of 1-12; preferably 1-3;
  • x4 is selected from an integer of 1-12; preferably 1-3;
  • P 1 and P 2 are the same or different polyethylene glycol residues
  • L1 is a linker unit connecting Ab and P1 ;
  • L 2 is the connecting unit connecting P 2 and R;
  • a 1 is the connection unit connecting P 1 and P 2 ;
  • the P 1 and P 2 are independently selected from linear, Y-shaped, multi-branched polyethylene glycol residues;
  • the molecular weight is 88-4400 Da, and more preferably, the molecular weight of P 1 and P 2 is 176-1056 Da;
  • P 1 and P 2 are non-single molecular weight polyethylene glycol
  • the molecular weight is 1000Da-40kDa
  • the molecular weight of P 1 and P 2 is 2000Da-10kDa.
  • the L 1 is a connecting group selected from linear or branched C 1-12 alkylene, C 6-12 arylene, C 3-12 cycloalkylene, -S-, One or a combination of two or more groups;
  • Any H atom on the linear or branched C 1-12 chain alkylene group, C 6-12 arylene group or C 3-12 cycloalkylene group is replaced by -H, -F, -Cl, -Br, -I, -O-, -S-, -SO 2 , -NO 2 , C 1-12 alkyl, C 3-12 cycloalkyl, C 6-12 aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, Substituted by one type or a group consisting of two or more groups;
  • the L 2 is a connecting group selected from linear or branched C 1-12 alkylene, C 6-12 arylene, C 3-12 cycloalkylene, -S-, One or a combination of two or more groups;
  • Any H atom on the linear or branched C 1-12 chain alkylene group, C 6-12 arylene group or C 3-12 cycloalkylene group is replaced by -H, -F, -Cl, -Br, -I, -O-, -S-, -SO 2 , -NO 2 , C 1-12 alkyl, C 3-12 cycloalkyl, C 6-12 aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, Substituted by a group consisting of one or more groups;
  • the L 1 is an amide bond, a hydrazone bond and a thiol-maleimide bond
  • the L 1 is an amide bond
  • the L 2 is a disulfide bond and a thiol-maleimide bond
  • the L 2 is a disulfide bond.
  • the A 1 is a connecting group connecting P 1 and P 2 , which is selected from linear or branched C 1-12 alkylene, C 6-12 arylene, and C 3-12 ring. Alkylene, -S-, One or a combination of two or more groups;
  • Any H atom on the linear or branched C 1-12 chain alkylene group, C 6-12 arylene group or C 3-12 cycloalkylene group is replaced by -H, -F, -Cl, -Br, -I, -O-, -S-, -SO 2 , -NO 2 , C 1-12 alkyl, C 3-12 cycloalkyl, C 6-12 aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, Substitute with one or more groups consisting of two or more groups.
  • the general formula (IV) of the antibody-nucleic acid conjugate drug is:
  • the L 1 is a connecting group, selected from linear or branched C 1-12 alkylene, C 6-12 arylene, C 3-12 cycloalkylene, -S-, One or a combination of two or more groups;
  • P 1 is the same or different polyethylene glycol residue
  • the molecular weight is 88-4400 Da, and more preferably, the molecular weight of P 1 is 176-1056 Da;
  • the molecular weight is 1000Da-40kDa; more preferably, the molecular weight of the P1 is 2000Da-10kDa.
  • the L 2 is a connecting group, selected from linear or branched C 1-12 alkylene, C 6-12 arylene, C 3-12 cycloalkylene, -S-, One or a combination of two or more groups;
  • x1 is an integer from 1 to 144; preferably, x1 is an integer from 1 to 9; more preferably, x1 is an integer from 1 to 3;
  • x2 is an integer from 1 to 8; preferably, x2 is an integer from 1 to 2.
  • the antibody-nucleic acid conjugated drug has the following structure:
  • n 1 is selected from an integer from 4 to 100, preferably 4 to 24; n 1 can be a fixed value or an average value.
  • the antibody-nucleic acid conjugated drug has the following structure:
  • n 1 and n 2 are independently selected from integers of 4-100, preferably 4-24.
  • n1 and n2 can be fixed values or mean values.
  • the Ab is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antibody fragments, and antibody fusion fragments.
  • the antibody can be a single domain antibody or a single chain antibody.
  • the Ab is a monoclonal antibody; more preferably, the monoclonal antibody is reactive to antigens or epitopes related to cancer, malignant cells, infectious organisms or autoimmune diseases.
  • the Ab is selected from: anti-HER2 antibody, anti-EGFR antibody, anti-PMSA antibody, anti-VEGFR antibody, anti-CD30 antibody, anti-CD22 antibody, anti-CD56 antibody, anti-CD29 antibody, anti-GPNMB Antibody, anti-CD138 antibody, anti-CD74 antibody, anti-ENPP3 antibody, anti-Nectin-4 antibody, anti-EGFRVIII antibody, anti-SLC44A4 antibody, anti-mesothelin antibody, anti-ET8R antibody, anti-CD37 antibody, anti-CEACAM5 antibody, anti-CD70 antibody, Anti-MUC16 antibody, anti-CD79b antibody, anti-MUC16 antibody, anti-Muc1 antibody, anti-CD3 antibody, anti-CD28 antibody, anti-CD38 antibody, anti-CD19 antibody, anti-PD-L1 antibody, anti-4-1BB antibody, etc.
  • the eighth aspect of the present invention provides an application of the above-mentioned interfering RNA, the above-mentioned delivery system, the above-mentioned cell, the above-mentioned medicine or kit, the above-mentioned lipid nanoparticle or the above-mentioned antibody-nucleic acid conjugated drug, Applications described include:
  • the working concentration of the interfering RNA used in the treatment, the above-mentioned delivery system, the above-mentioned cells, the above-mentioned drugs or kits, the above-mentioned lipid nanoparticles or the above-mentioned antibody-nucleic acid conjugated drugs is 0.01-1000nM, For example, 0.01, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000nM.
  • diseases related to increased expression of TUBB2 or excessive cell proliferation include tumors.
  • the tumors include digestive tract tumors (oral cancer, tongue cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, etc.), nervous system tumors (glioma (such as glioma), Neuroepithelioma, schwannoma, astrocytoma, neurofibroma (such as neurofibrosarcoma), ependymoma, medulloblastoma, meningioma, brain metastases, etc.), respiratory tract tumors (nasopharyngeal carcinoma, Laryngeal cancer, bronchial cancer, lung cancer, etc.), genitourinary system tumors (prostate cancer, kidney tumors, bladder cancer, breast cancer, cervical cancer, ovarian cancer, placental choriocarcinoma, urinary tract system tumors, seminal vesicle gland tumors, test
  • the tumor is selected from genitourinary system tumors and/or skin system tumors.
  • the tumor is selected from prostate cancer, breast cancer or melanoma.
  • the ninth aspect of the present invention provides a method for inhibiting TUBB2 expression, which method includes adding the above-mentioned interfering RNA, the above-mentioned delivery system, the above-mentioned cells, the above-mentioned lipid nanoparticles, the above-mentioned medicine or kit Or the above-mentioned antibody-nucleic acid conjugate drugs.
  • the method inhibits TUBB2 expression in cells, and the cells are tumor cells.
  • the method includes delivering the above-mentioned interfering RNA into cells.
  • Said delivery uses the delivery system described above.
  • a tenth aspect of the present invention provides a method for treating diseases related to increased expression of TUBB2 or excessive cell proliferation.
  • the method includes applying to a subject a therapeutically effective amount of the above-mentioned interfering RNA, the above-mentioned delivery system, the above-mentioned Lipid nanoparticles, the above-mentioned cells, the above-mentioned drugs or kits, or the above-mentioned antibody-nucleic acid conjugated drugs.
  • diseases related to increased expression of TUBB2 or excessive cell proliferation include tumors. Further preferably,
  • the tumors include digestive tract tumors (oral cancer, tongue cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, etc.), nervous system tumors (glioma (such as glioma), neuroepithelial tumors, Schwannoma, astrocytoma, neurofibroma (such as neurofibrosarcoma), ependymoma, medulloblastoma, meningioma, brain metastases, etc.), respiratory tract tumors (nasopharyngeal cancer, laryngeal cancer, bronchial carcinoma, etc.) cancer, lung cancer, etc.), genitourinary system tumors (prostate cancer, kidney tumors, bladder cancer, breast cancer, cervical cancer, ovarian cancer, placental choriocarcinoma, urinary tract system tumors, seminal vesicle tumors, testicular and paratesticular tissue tumors, penis Tumors, vulvar cancer,
  • An eleventh aspect of the present invention provides a method for treating tumors, which method includes applying to a subject a therapeutically effective amount of the above-mentioned interfering RNA, the above-mentioned delivery system, the above-mentioned cells, and the above-mentioned lipid nanoparticles. particles, the above-mentioned drugs or kits, or the above-mentioned antibody-nucleic acid conjugated drugs.
  • the tumors include digestive tract tumors (oral cancer, tongue cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, etc.), nervous system tumors (glioma (such as glioma), nerve Epithelioma, schwannoma, astrocytoma, neurofibroma (such as neurofibrosarcoma), ependymoma, medulloblastoma, meningioma, brain metastases, etc.), respiratory tract tumors (nasopharyngeal carcinoma, laryngeal carcinoma, etc.) cancer, bronchial cancer, lung cancer, etc.), genitourinary system tumors (prostate cancer, kidney tumors, bladder cancer, breast cancer, cervical cancer, ovarian cancer, placental choriocarcinoma, urinary tract system tumors, seminal vesicle gland tumors, testis and paratesticular tissue Tumors, penile tumors, etc.
  • the tumor is selected from urogenital system tumors and/or skin system tumors.
  • the tumor is selected from prostate cancer, breast cancer or melanoma.
  • the twelfth aspect of the present invention provides a method for inhibiting excessive cell proliferation, which comprises applying a therapeutically effective amount of the above-mentioned interfering RNA, the above-mentioned delivery system, the above-mentioned cells, the above-mentioned lipid nanoparticles, the above-mentioned drug or kit or the above-mentioned antibody-nucleic acid conjugate drug to a subject.
  • a thirteenth aspect of the present invention provides an anti-mitosis method, which method includes applying to a subject a therapeutically effective amount of the above-mentioned interfering RNA, the above-mentioned delivery system, the above-mentioned cells, and the above-mentioned lipid nanoparticles. particles, the above-mentioned drugs or kits, or the above-mentioned antibody-nucleic acid conjugated drugs.
  • the TUBB2 siRNA sequence screened out in this application can be used to treat diseases related to vigorous cell proliferation (such as various tumors, etc.) and has strong development potential. Experiments have confirmed that it can significantly inhibit the mRNA expression level of TUBB2 in a variety of cells (such as A375, MDA-MB-231-GFP, PC-3, SKOV-3, HCT116 and other cells). Therefore, it is expected to be used in diseases related to increased tubulin expression, such as various malignant tumors and other disease states. By inhibiting the expression of tubulin ⁇ 2, blocking or reducing cell proliferation, it can slow down disease progression and improve disease outcomes. the goal of.
  • the "interfering RNA” described in the present invention includes single-stranded RNA (e.g., mature miRNA) or double-stranded RNA (e.g., siRNA, shRNA, aiRNA or precursor miRNA).
  • single-stranded RNA e.g., mature miRNA
  • double-stranded RNA e.g., siRNA, shRNA, aiRNA or precursor miRNA.
  • the interfering RNA and the target gene or sequence are in the same cell, they can reduce or inhibit the expression of the target gene or sequence (e.g., by mediating degradation and/or inhibiting the translation of mRNA complementary to the interfering RNA sequence).
  • siRNA is a small interfering RNA, and each strand of its molecule contains nucleotides with a length of about 15nt to about 60nt (for example, a length of about 15-60, 15-50, 15-40, 15-30, 15- 25, or 19-25 nt nucleotides, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nt in length).
  • siRNA can be chemically synthesized.
  • the siRNA molecules of the invention are capable of silencing the expression of target sequences in vitro and/or in vivo.
  • siRNA may contain no modified nucleotides; in other embodiments, siRNA may contain at least one modified nucleotide, for example, siRNA may contain one, two, three, or four modified nucleotides in the double-stranded region. , five, six, seven, eight, nine, ten or more modified nucleotides.
  • shRNA is small hairpin RNA or short Hairpin RNA, which includes short RNA sequences that create tight hairpin turns that can be used to silence gene expression through RNA interference, shRNA hairpin structures that can be cleaved into siRNA by cellular machinery.
  • miRNA miRNA is a single-stranded RNA molecule about 21-23 nucleotides in length that regulates gene expression.
  • “Inhibiting the expression of a target gene” in the present invention refers to the ability of the interfering RNA (for example, siRNA) of the present invention to silence, reduce or inhibit the expression of a target gene (for example, the TUBB2 gene).
  • interfering RNA for example, siRNA
  • the term “comprises” or “comprises” used in the present invention is an open-ended expression, containing the specified components or steps described, as well as other specified components or steps that will not substantially affect the process.
  • the 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 same Identical or similar activity to the original sequence.
  • a "tumor” as used herein may be any undesirable cell proliferation (or any disease manifesting itself as undesirable cell proliferation), neoplasia, or a predisposition or increased risk of undesirable cell proliferation, neoplasia, or neoplasia. It can be benign or malignant, primary or secondary (metastatic). A neoplasm can be any abnormal growth or proliferation of cells and can be located in any tissue.
  • tissues include adrenal glands, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, Endometrium, gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, Oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary glands, colon, skin, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsils, trachea, uterus, vulva, rectum.
  • central nervous system including or excluding the brain
  • cerebellum including or
  • the tumors include digestive tract tumors (oral cancer, tongue cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, etc.), nervous system tumors (glioma (such as glioma), Neuroepithelioma, schwannoma, astrocytoma, neurofibroma (such as neurofibrosarcoma), ependymoma, medulloblastoma, meningioma, brain metastases, etc.), respiratory tract tumors (nasopharyngeal carcinoma, Laryngeal cancer, bronchial cancer, lung cancer, etc.), urinary system tumors (such as renal cell carcinoma, bladder cancer, etc.), reproductive system tumors (prostate cancer, testicular cancer, breast cancer, cervical cancer, ovarian cancer, placental choriocarcinoma, etc.), blood Lymphoid system tumors (multiple myeloma, mesothelioma,
  • the "subject" of the present invention can be a human or a non-human mammal, and the non-human mammal can be a wild animal, a zoo animal, an economic animal, a pet, an experimental animal, etc.
  • the non-human mammals include, but are not limited to, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, mink, camels, dogs, cats, rabbits, mice (such as rats, mice, guinea pigs, hamsters , gerbils, chinchillas, squirrels) or monkeys, etc.
  • Treatment as used herein means to slow, interrupt, arrest, control, stop, alleviate, or reverse the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but not necessarily Involves the complete elimination of all disease-related signs, symptoms, conditions, or disorders.
  • Prevention as used in the present invention means a method implemented to prevent or delay the occurrence of a disease, illness or symptom in the body.
  • the "effective amount” of the present invention refers to the amount of the product of the present invention that provides the desired effect (such as treatment, prevention or inhibition of TUBB2 expression) after administration to a subject or its cells or organs in single or multiple doses. or dose.
  • Lipids refer to a group of organic compounds, including but not limited to esters of fatty acids, and are usually characterized by being poorly soluble in water but soluble in many organic solvents.
  • the "cationic lipid” mentioned in the present invention refers to a lipid molecule capable of being positively charged.
  • neutral lipid refers to uncharged, non-phosphoglyceride lipid molecules.
  • polyethylene glycol lipid described in the present invention refers to a molecule comprising a lipid portion and a polyethylene glycol portion.
  • Lipid nanoparticles refer to particles with at least one nanometer scale size, which contain at least one lipid.
  • the "delivery system” mentioned in the present invention refers to a preparation or composition that regulates the distribution of biologically active ingredients in space, time and dosage within the body.
  • Figure 1 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-112.
  • Figure 2 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-176.
  • Figure 3 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-184.
  • Figure 4 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-236.
  • Figure 5 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-333.
  • Figure 6 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-358.
  • Figure 7 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-373.
  • Figure 8 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-457.
  • Figure 9 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-460.
  • Figure 10 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-1092.
  • Figure 11 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-1093.
  • Figure 12 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-1342.
  • FIG. 13 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-1345.
  • Figure 14 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-598.
  • Figure 15 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-982.
  • Figure 16 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-984.
  • Figure 17 Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-985.
  • Figure 18 Effects of different siRNA on the relative expression of TUBB2b mRNA in A375 cells.
  • Figure 19 Effects of different siRNA on the relative expression of TUBB2 mRNA in A375 cells.
  • Figure 20 Effects of different siRNA on the relative expression of TUBB2 mRNA in PC-3 cells.
  • Figure 21 Experimental results of inhibition of PC-3 cell proliferation by siRNA at different concentrations.
  • Figure 22 Experimental results of inhibition of A375 cell proliferation by different siRNA.
  • Figure 23 Effect of LNP-siTUBB2 on the relative expression level of TUBB2 mRNA in MDA-MB-231-GFP cells Influence.
  • Figure 24 Effects of different concentrations of LNP-siTUBB2 on the relative expression of TUBB2 mRNA in PC-3 cells.
  • FIG. 25 Time trend of the inhibitory effect of LNP-siTUBB2 on PC-3 cell proliferation.
  • Figure 26 Effect of LNP-siTUBB2 on the relative expression of TUBB2 mRNA in MDA-MB-231-GFP cells.
  • Figure 28 Grouping and administration time and frequency.
  • Figure 29 Changes in animal body weight under different dosage regimens.
  • Figure 30 Tumor size with different dosing regimens.
  • FIG. 31 Tumor inhibition rates of different dosage regimens.
  • siRNAs in order to detect the inhibitory effect of siRNA corresponding to tubulin ⁇ 2 (TUBB2) mentioned in the present invention, a variety of siRNAs were prepared for TUBB2.
  • the prepared siRNA is first delivered to cultured cells. Cell samples are collected at a predetermined time after transfection and RNA is extracted. After the RNA is reverse transcribed into cDNA, the Ct value is obtained using real-time quantitative PCR. By normalizing each group of data, the relative expression of TUBB2mRNA was obtained, so as to compare the inhibitory effect of each siRNA on TUBB2mRNA expression.
  • cytotoxicity experiments were conducted to verify cell phenotypic changes.
  • various siRNAs designed above were prepared into lipid nanoparticles and delivered into the body, which also achieved the effect of treating tumors.
  • Example 1 siRNA design and synthesis
  • siRNA sequences based on the TUBB2b sequence (GenBank: NM_178012.5). The sequence information is as follows: As shown in Table 1, the mass spectrometry results are shown in Figure 1-13. In actual use, 2 dangling bases dTdT are added to the 3' end of the 19bp siRNA sense strand and antisense strand, and the siRNA exceeding 19bp remains unchanged.
  • siRNA sequences similar to the sequences 45402, 165110/165111, and 165148/165149 (1618322/1618323) in the patent WO2012177639A3 were synthesized (corresponding to positions 598, 982, 984, and 985 of the TUBB2b sequence respectively, and the mass spectrum is shown in Figure 14-17). For comparison, see Table 2.
  • A375 cells human melanoma cells
  • PC-3 human prostate cancer cells
  • A375 cells were cultured in DMEM containing 10% FBS, 100 U/mL Penicillin, and 100 ⁇ g/ml Streptomycin
  • PC-3 cells were cultured in F12K containing 10% FBS, 100 U/mL Penicillin, and 100 ⁇ g/ml Streptomycin at 37°C with 5% CO 2.
  • RNA quality inspection Nanodrop detects RNA content and purity, and 1% agarose gel electrophoresis detects RNA integrity.
  • RNA reverse transcription Use the total RNA extracted from the sample as a template and use the Lamblid Reverse Transcription Kit to establish RNA reverse transcription in Table 3 system.
  • the TUBB2-1F and TUBB2-1R primer sequences can be amplified using TUBB2a and TUBB2b as templates at the same time, while the TUBB2-2F and TUBB2-2R primer sequences can only be amplified using TUBB2b as the template.
  • the relative expression of TUBB2 mRNA or TUBB2b mRNA was calculated by the ⁇ Ct method; with the expression level of the blank group as 100%, the mRNA expression levels of each group were standardized.
  • TUBB2b or TUBB2 in cells treated with the siRNA designed in Table 1 in Example 1 was inhibited.
  • siTUBB2-176, siTUBB2-457 and siTUBB2-1092 significantly inhibit the expression of TUBB2 mRNA in cells, where siTUBB2-457 and siTUBB2-1092 can inhibit about 90% of TUBB2 mRNA expression.
  • A375 cells use DMEM medium containing 7.5% FBS (containing 100U/mL Penicillin, 100 ⁇ g/mL Streptomycin), PC-3 cells use F12K medium containing 10% FBS (containing 100U/mL Penicillin, 100 ⁇ g/ml Streptomycin), cultured in a 37°C 5% CO2 saturated humidity incubator. Culture in a 37°C 5% CO2 saturated humidity incubator. 24 hours before the experiment, 2.5 ⁇ 10 3 cells (A375) or 1 ⁇ 10 4 cells (PC-3) per well were seeded in a 96-well plate and cultured overnight.
  • siTUBB2 designed in Table 1 of Example 1 can kill tumor cells.
  • the cell survival rate is only 16%-18%.
  • the cell survival rate is only 16%-18%.
  • the survival rate is only about 40%, and the cell survival rate in the siTUBB2-176 treatment group is only 50%-60%. Therefore, it can be considered that siTUBB2-176, siTUBB2-457 and siTUBB2-1092 can significantly inhibit cell proliferation (see Tables 9 and 10 and Figures 22 and 21).
  • Example 4 LNP preparation, biological activity and cell proliferation inhibitory activity evaluation
  • LNP/EtOH solution uses channel 1, solution volume 2mL, flow rate 5mL/min;
  • siRNA/buffer solution uses channel 2, solution volume 6mL, flow rate 15mL/min;
  • the particle size and zeta potential of LNP-siTUBB2 were detected using NanoBrook (Brookhaven, USA) nanoparticle size analyzer.
  • the prepared LNP-siTUBB2 100 ⁇ L was diluted to 2mL with DEPC water and tested on the machine. Each sample was tested three times, and the particle size was expressed as the mean of the effective particle size (D50).
  • D50 effective particle size
  • the method is the same as in Example 2, using human breast cancer MDA-MB-231-GFP cells (the medium is 1640 medium + 10% FBS + 1% penicillin) or PC-3 (the medium is F12K medium + 10% FBS + 1% penicillin-streptomycin).
  • the results show that LNP can effectively deliver siTUBB2 into MDA-MB-231-GFP cells or PC-3 cells and significantly inhibit the expression of TUBB2 mRNA (see Tables 12 and 13 and Figures 23 and 24).
  • Example 3 The method is the same as Example 3, using PC-3 cells for testing.
  • the results showed that LNP-siTUBB2 could significantly inhibit the proliferation of PC-3 cells, and the cell survival rate was significantly reduced, which could last for a long time (see Table 14 and Figure 25).
  • Example 5 Chemical modification of siTUBB2, evaluation of biological effects, and evaluation of cell proliferation inhibition
  • siTUBB2-457 and siTUBB2-1092 were selected from Table 1 of Example 1 and chemically modified, including methylation, fluorination, thiophosphorylation, pseudouracil, etc. (see Table 15).
  • the designed modified type of siTUBB2 was synthesized by Shanghai Sangon Biotechnology Co., Ltd.
  • modified siTUBB2 The experimental method for evaluating the biological effects of modified siTUBB2 is the same as in Example 2.
  • siTUBB2-457-4 and siTUBB2-1092-4 have comparable biological effects to unmodified siTUBB2 (Table 16, Figure 26).
  • siTUBB2-1092 could cause significant cell cycle arrest in A375 cells.
  • the proportion of cells in the G0G1% phase decreased and the cells were arrested in the G2M phase (Figure 27).
  • the human prostate cancer cell line PC-3 was cultured in F-12K culture medium containing 10% fetal bovine serum in a 37°C, 5% CO 2 cell culture incubator. The cells were divided into bottles and passaged every 3 to 4 days after they were full. Tumor cells in the logarithmic growth phase are used for in vivo tumor inoculation. Resuspend PC-3 tumor cells at a concentration of 5 ⁇ 10 7 cells/mL and inoculate them subcutaneously into the right flank of Balb/c nude mice at 100 ⁇ L/mouse until the tumor grows to an average volume of 100-150 mm 3 Give medicine in groups when left and right, There are 7 groups in total, with 6 animals in each group. The grouping, dosage and frequency are as shown in Table 17 and Figure 28. Weigh regularly (Figure 29), detect tumor size (Figure 30), and calculate the tumor inhibition rate (TGI) ( Figure 31).
  • mice in the 457 intravenous group exceeded that of the paclitaxel group.
  • the weight loss of mice in other groups was greater than that of the Vehicle group, but lower than that of the Paclitaxel group.
  • tumor volume the 1092 intravenous group and the 457 intratumoral group were lower than the paclitaxel group and other groups.
  • tumor inhibition index TGI
  • the 1092 intravenous group and the 457 intratumoral group were still higher than the paclitaxel group 20 days after stopping administration, reaching 42.28 and 39.13 respectively at the end of the experiment (64 days after vaccination, 27 days after stopping administration).
  • the paclitaxel group dropped to 33.03. This shows that LNP-siTUBB2-457 and 1092 can exert a longer-lasting effect than paclitaxel and inhibit tumor growth.

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Abstract

The present invention provides an interfering RNA targeting microtubule protein β2 (TUBB2). The interfering RNA can reduce the expression of TUBB2, thereby treating a disease related to the increase of TUBB2 expression or excessive proliferation of cells. The present invention further provides a lipid nanoparticle drug comprising the interfering RNA. The lipid nanoparticle drug is used for treating a disease related to the increase of TUBB2 expression or excessive proliferation of cells. The present invention further provides an antibody-nucleic acid conjugate drug, which comprises the interfering RNA and an antibody and is used for treating a disease related to the increase of TUBB2 expression or excessive proliferation of cells.

Description

抑制TUBB2基因表达的干扰RNA及其应用Interfering RNA that inhibits TUBB2 gene expression and its application 技术领域Technical field
本发明涉及分子生物学与生物医药技术领域，具体涉及一种有效抑制TUBB2表达的干扰RNA，特别是siRNA，及其应用。The present invention relates to the technical fields of molecular biology and biomedicine, and specifically relates to an interfering RNA that effectively inhibits TUBB2 expression, especially siRNA, and its application.
背景技术Background technique
微管蛋白是构成真核细胞骨架的重要部分，广泛存在于活细胞的微管中。微管是组成真核细胞骨架的基本元素，主要由微管蛋白α和β聚合而成。在正常情况下，微管和微管蛋白二聚体之间存在动态平衡。微管在细胞支持和细胞运动中起着至关重要的作用，可以帮助细胞抵抗压缩力，使其能够保持其预期的形状和结构；此外，微管对于细胞运动、有丝分裂期间的染色体分离以及细胞器的细胞内运输等过程同样至关重要。Tubulin is an important part of the eukaryotic cytoskeleton and is widely present in microtubules of living cells. Microtubules are the basic elements of the eukaryotic cytoskeleton and are mainly composed of tubulin α and β polymers. Under normal circumstances, there is a dynamic balance between microtubules and tubulin dimers. Microtubules play a vital role in cell support and cell movement, helping cells resist compression forces so that they can maintain their intended shape and structure; in addition, microtubules are also essential for processes such as cell movement, chromosome segregation during mitosis, and intracellular transport of organelles.
在人类中，微管蛋白有五个亚组：α-微管蛋白、β-微管蛋白、γ-微管蛋白、δ和ε-微管蛋白以及ζ-微管蛋白。β-微管蛋白特别令人感兴趣，因为它形成了一种仅在神经元中表达的微管。此外，所有与人微管蛋白结合的药物也与β-微管蛋白结合，使其成为一种治疗靶点。In humans, there are five subgroups of tubulin: α-tubulin, β-tubulin, γ-tubulin, δ and ε-tubulin, and ζ-tubulin. β-Tubulin is of particular interest because it forms a type of microtubule that is expressed only in neurons. Additionally, all drugs that bind to human tubulin also bind to beta-tubulin, making it a therapeutic target.
抗有丝分裂化疗药物以微管蛋白为靶点，微管蛋白是有丝分裂纺锤体中的主要蛋白质，微管蛋白亚型组成被认为是肿瘤进展诊断和细胞对化疗反应的决定因素。这意味着正常组织和肿瘤组织的亚型组成是不同的，为肿瘤的化疗药物的开发提供了理论基础。目前常见的抗微管蛋白药物有长春碱、紫杉醇等。其中，紫杉醇可使微管与微管蛋白二聚体之间失去动态平衡，诱导和促进微管蛋白聚合，防止解聚，稳定微管。长春碱主要抑制微管蛋白的聚合，而妨碍纺锤体微管的形成，使核分裂停止于中期；也可引起核崩溃，呈空泡式固缩；作用于细胞膜，干扰细胞膜对氨基酸的运转，使蛋白质的合成受抑制；可通过抑制RNA聚合酶的活力而抑制RNA的合成，将细胞杀灭于G1期。这些作用导致细胞在进行有丝分裂时不能形成纺锤体和纺锤丝，抑制了细胞分裂和增殖，从而发挥抗肿瘤作用。因此，通过抑制或促进微管蛋白聚合，干扰微管的平衡，从而影响细胞的有丝分裂，抑制细 胞增殖。Antimitotic chemotherapy drugs target tubulin, the major protein in the mitotic spindle, and tubulin isoform composition is considered a determinant of tumor progression diagnosis and cellular response to chemotherapy. This means that the subtype composition of normal tissue and tumor tissue is different, which provides a theoretical basis for the development of chemotherapy drugs for tumors. Common anti-tubulin drugs currently include vinblastine, paclitaxel, etc. Among them, paclitaxel can cause the dynamic balance between microtubules and tubulin dimers to lose, induce and promote tubulin polymerization, prevent depolymerization, and stabilize microtubules. Vinblastine mainly inhibits the polymerization of tubulin, hinders the formation of spindle microtubules, and stops nuclear division in metaphase; it can also cause nuclear collapse and vacuolar pyknosis; it acts on the cell membrane, interfering with the movement of amino acids in the cell membrane, causing Protein synthesis is inhibited; RNA synthesis can be inhibited by inhibiting the activity of RNA polymerase, killing cells in the G1 phase. These effects prevent cells from forming spindles and spindle fibers during mitosis, inhibiting cell division and proliferation, thus exerting anti-tumor effects. Therefore, by inhibiting or promoting tubulin polymerization, it interferes with the balance of microtubules, thereby affecting cell mitosis and inhibiting cell mitosis. cell proliferation.
β2微管蛋白存在于细胞核，也可以非微管形式存在，可以显著影响细胞的行为。β2微管蛋白参与了几种类型肿瘤的发生发展，如神经上皮瘤和脑肿瘤、结肠癌和前列腺癌。在侵袭性转移瘤中，发现了位于细胞核的β2微管蛋白，暗示其可以与抗微管药紫杉醇和长春碱相互作用。细胞质中没有被装配到微管中的β2微管蛋白可以与电压依赖性阴离子通道(VDAC)相互作用，为肌肉细胞提供能量。研究发现，β2微管蛋白的过表达与结肠直肠癌患者较短的预期寿命相关。β2微管蛋白定位于细胞核的肿瘤患者的预期寿命甚至更短。β2 tubulin exists in the nucleus and can also exist in non-microtubule forms and can significantly affect cell behavior. β2-tubulin is involved in the development of several types of tumors, such as neuroepithelial tumors and brain tumors, colon cancer, and prostate cancer. In invasive metastases, β2-tubulin was found localized in the nucleus, suggesting that it could interact with the antimicrotubule drugs paclitaxel and vinblastine. β2-tubulin in the cytoplasm that is not assembled into microtubules can interact with voltage-dependent anion channels (VDAC) to provide energy to muscle cells. Studies have found that overexpression of β2-tubulin is associated with shorter life expectancy in patients with colorectal cancer. Patients with tumors whose β2-tubulin localizes to the nucleus have even shorter life expectancies.
因此，本申请筛选出抑制活性较高的干扰RNA序列，有望制备成RNA药物，用于治疗肿瘤。另外，siRNA分子较大，带有大量负电荷，很难通过同样带负电荷的细胞膜。因此寻找合适的递送系统是开发siRNA药物过程中重要的一部分，本申请提供了脂质纳米颗粒(lipid nanoparticles，LNP)和抗体偶联siRNA(Antibody–siRNA conjugates，ARC)两种可行的递送方式。Therefore, this application screens out interfering RNA sequences with high inhibitory activity, which are expected to be prepared into RNA drugs for the treatment of tumors. In addition, siRNA molecules are larger and carry a large number of negative charges, making it difficult for them to pass through the similarly negatively charged cell membrane. Therefore, finding a suitable delivery system is an important part of the process of developing siRNA drugs. This application provides two feasible delivery methods: lipid nanoparticles (LNP) and antibody-conjugated siRNA (Antibody–siRNA conjugates, ARC).
发明内容Contents of the invention
本发明的第一方面，提供了一种靶向微管蛋白β2(TUBB2)基因的干扰RNA。A first aspect of the invention provides an interfering RNA targeting the tubulin β2 (TUBB2) gene.
所述的干扰RNA抑制TUBB2的表达。The interfering RNA inhibits the expression of TUBB2.
所述干扰RNA的靶位点序列包含SEQ ID NO：1-13中的任一个或两个以上所示的核苷酸序列。优选的，所述干扰RNA的靶位点序列如SEQ ID NO：1-13中的任一个核苷酸序列所示。The target site sequence of the interfering RNA includes any one or more than two nucleotide sequences shown in SEQ ID NO: 1-13. Preferably, the target site sequence of the interfering RNA is as shown in any one of the nucleotide sequences in SEQ ID NO: 1-13.
优选的，所述干扰RNA的靶位点序列包含SEQ ID NO：1-10或12中的任一个或两个以上所示的核苷酸序列。优选的，所述干扰RNA的靶位点序列如SEQ ID NO：1-10或12中的任一个核苷酸序列所示。Preferably, the target site sequence of the interfering RNA includes any one or more of the nucleotide sequences shown in SEQ ID NO: 1-10 or 12. Preferably, the target site sequence of the interfering RNA is as shown in any one of the nucleotide sequences in SEQ ID NO: 1-10 or 12.
进一步优选的，所述干扰RNA的靶位点序列包含SEQ ID NO：1-3或5-6或8-10中的任一个或两个以上所示的核苷酸序列。优选的，所述干扰RNA的靶位点序列如SEQ ID NO：1-3或5-6或8-10中的任一个核苷酸序列所示。Further preferably, the target site sequence of the interference RNA includes any one or two or more nucleotide sequences shown in SEQ ID NO: 1-3 or 5-6 or 8-10. Preferably, the target site sequence of the interfering RNA is as shown in any one of the nucleotide sequences in SEQ ID NO: 1-3 or 5-6 or 8-10.
在本发明的一个具体实施方式中，所述干扰RNA的靶位点序列包含SEQ ID NO：2、8和/或10。所述的干扰RNA包含siRNA、dsRNA、shRNA、aiRNA或miRNA中 一种或两种以上的组合。In a specific embodiment of the present invention, the target site sequence of the interfering RNA includes SEQ ID NO: 2, 8 and/or 10. The interfering RNA includes siRNA, dsRNA, shRNA, aiRNA or miRNA. One or a combination of two or more.
在本发明的一个具体实施方式中，所述的干扰RNA为siRNA。In a specific embodiment of the present invention, the interfering RNA is siRNA.
所述干扰RNA的长度为17-25nt，优选19-21nt，例如17、18、19、20、21、22、23、24、25nt。The length of the interfering RNA is 17-25nt, preferably 19-21nt, such as 17, 18, 19, 20, 21, 22, 23, 24, 25nt.
所述干扰RNA还包括悬挂碱基，以增加干扰RNA稳定性和活性。The interfering RNA also includes dangling bases to increase interfering RNA stability and activity.
优选的，所述的干扰RNA包含1-10个悬挂碱基。进一步优选2-4个悬挂碱基。例如1、2、3、4、5、6、7、8、9、10个悬挂碱基。Preferably, the interfering RNA contains 1-10 dangling bases. 2-4 dangling bases are further preferred. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 dangling bases.
所述的悬挂碱基位于所述的干扰RNA的正义链和/或反义链的3’末端。The hanging base is located at the 3' end of the sense strand and/or antisense strand of the interfering RNA.
所述的悬挂碱基为脱氧核苷；优选的，悬挂碱基可以为n个相同或不同的脱氧核苷(例如脱氧胸苷(dT)、脱氧胞苷(dC)、脱氧尿苷(dU)等)，n为1-10的整数(例如1、2、3、4、5、6、7、8、9、10)。The dangling bases are deoxynucleosides; preferably, the dangling bases can be n identical or different deoxynucleosides (such as deoxythymidine (dT), deoxycytidine (dC), deoxyuridine (dU) etc.), n is an integer from 1 to 10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).
在本发明的一个具体实施方式中，所述的干扰RNA的正义链和/或反义链的末端设有2个相同的悬挂碱基。In a specific embodiment of the present invention, the ends of the sense strand and/or the antisense strand of the interference RNA are provided with two identical dangling bases.
在本发明的一个具体实施方式中，所述的干扰RNA的正义链和/或反义链的末端设有2个不同的悬挂碱基。In a specific embodiment of the present invention, the ends of the sense strand and/or the antisense strand of the interference RNA are provided with two different dangling bases.
在本发明的一个具体实施方式中，所述的悬挂碱基为dTdT、dTdC或dUdU。In a specific embodiment of the present invention, the dangling base is dTdT, dTdC or dUdU.
在本发明的一个具体实施方式中，所述的干扰RNA包含正义链和/或反义链。In a specific embodiment of the present invention, the interfering RNA includes a sense strand and/or an antisense strand.
优选的，所述的正义链和反义链互补配对。Preferably, the sense strand and the antisense strand are complementary to each other.
优选的，所述的正义链包含SEQ ID NO：14-26中的任一个或两个以上所示的核苷酸序列。进一步优选的，所述的正义链如SEQ ID NO：14-26中的任一个核苷酸序列所示。进一步优选的，所述的正义链包含SEQ ID NO：14-23或25中的任一个或两个以上所示的核苷酸序列。更进一步优选的，所述的正义链包含SEQ ID NO：14-16或18-19或21-23中的任一个或两个以上所示的核苷酸序列。Preferably, the sense strand includes any one or two or more nucleotide sequences shown in SEQ ID NO: 14-26. Further preferably, the sense strand is as shown in any one of the nucleotide sequences in SEQ ID NO: 14-26. Further preferably, the sense strand includes any one or two or more nucleotide sequences shown in SEQ ID NO: 14-23 or 25. More preferably, the sense strand includes any one or two or more nucleotide sequences shown in SEQ ID NO: 14-16 or 18-19 or 21-23.
在本发明的一个具体实施方式中，所述的正义链包含SEQ ID NO：15、21和/23。In a specific embodiment of the invention, the sense strand includes SEQ ID NO: 15, 21 and /23.
优选的，所述的反义链包含SEQ ID NO：27-39中的任一个或两个以上所示的核苷酸序列。进一步优选的，所述的反义链如SEQ ID NO：27-39中的任一个核苷酸序列所示。进一步优选的，所述的反义链包含SEQ ID NO：27-36或38中的任一个或两个以上所示的核苷酸序列。更进一步优选的，所述的反义链包含SEQ ID NO：27-29 或31-32或34-36中的任一个或两个以上所示的核苷酸序列。Preferably, the antisense strand includes any one or two or more nucleotide sequences shown in SEQ ID NO: 27-39. Further preferably, the antisense strand is represented by any one of the nucleotide sequences in SEQ ID NO: 27-39. Further preferably, the antisense strand includes any one or two or more nucleotide sequences shown in SEQ ID NO: 27-36 or 38. More preferably, the antisense strand includes SEQ ID NO: 27-29 Or any one or two or more of the nucleotide sequences shown in 31-32 or 34-36.
在本发明的一个具体实施方式中，所述的反义链包含SEQ ID NO：28、34和/或36。In a specific embodiment of the invention, the antisense strand includes SEQ ID NO: 28, 34 and/or 36.
在本发明的一个具体实施方式中，所述的干扰RNA中还可以包含至少一个修饰。修饰的干扰RNA具有比相应未修饰的干扰RNA更佳的性质，如更高的稳定性，更低的免疫刺激性等。In a specific embodiment of the present invention, the interfering RNA may further include at least one modification. Modified interfering RNA has better properties than the corresponding unmodified interfering RNA, such as higher stability, lower immunostimulation, etc.
所述的修饰包括在碱基、糖环和/或磷酸盐的化学结构上进行的修饰。The modifications include modifications to the chemical structures of bases, sugar rings and/or phosphates.
优选的，碱基的修饰包括但不限于5位嘧啶修饰、8位嘌呤修饰和/或5-溴尿嘧啶取代。Preferably, base modifications include but are not limited to 5-position pyrimidine modification, 8-position purine modification and/or 5-bromouracil substitution.
优选的，所述的糖环的修饰包括但不限于2'-OH被H、OZ、Z、halo、SH、SZ、NH2、NHZ、NZ2或CN等基团取代，其中Z为烷基基团。Preferably, the modification of the sugar ring includes but is not limited to substitution of 2'-OH by groups such as H, OZ, Z, halo, SH, SZ, NH2, NHZ, NZ2 or CN, where Z is an alkyl group .
所述的烷基基团代表直链或支链的且不含不饱和键的烃基，且该烃基以单键与分子其它部分连接。典型的烷基基团含有1至20(例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20)个碳原子，例如甲基、乙基、正丙基、异丙基、正丁基、仲丁基、异丁基、叔丁基、正戊基、异戊基、新戊基、叔戊基、正己基、异己基、正庚基、异庚基、正辛基、壬基、癸基、十一烷基、1-甲基十一烷基、十二烷基、十三烷基、十四烷基、十五烷基、十六烷基、十七烷基、十八烷基、十九烷基及二十烷基等。The alkyl group represents a linear or branched hydrocarbon group without unsaturated bonds, and the hydrocarbon group is connected to other parts of the molecule with a single bond. Typical alkyl groups contain 1 to 20 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert. Pentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, nonyl, decyl, undecyl, 1-methylundecyl, dodecyl, tridecyl , Tetradecyl, Pentadecyl, Hexadecyl, Heptadecyl, Octadecyl, Nonadecyl and Eicosyl, etc.
优选的，所述的磷酸骨架修饰包括但不限于硫代磷酸修饰。Preferably, the phosphate backbone modification includes but is not limited to phosphorothioate modification.
优选的，所述的修饰还包括具有肌苷、辫苷、黄嘌呤、2'-甲基核糖、非天然磷酸二酯键(如甲基膦酸酯、硫代膦酸酯)和/或肽的核苷酸。Preferably, the modifications also include inosine, braidin, xanthine, 2'-methylribose, non-natural phosphodiester bonds (such as methylphosphonate, thiophosphonate) and/or peptides of nucleotides.
在本发明的一个具体实施方式中，所述的修饰可以为甲基化、氟化、硫代磷酸化、假尿嘧啶等。In a specific embodiment of the present invention, the modification can be methylation, fluorination, phosphorothioate, pseudouracil, etc.
所述的修饰可以发生在序列的任一位置，例如可以为部分位置修饰或全部修饰。The modification may occur at any position in the sequence, for example, it may be a partial modification or a complete modification.
同一条序列可以为相同或不同修饰类型的部分位置修饰，也可以相同或不同修饰类型的全部位置修饰。The same sequence can be modified at some positions of the same or different modification types, or at all positions of the same or different modification types.
在本发明的一个具体实施方式中，所述的干扰RNA包含如下靶位点序列、正义链和反义链序列组合中的一种或两种以上的组合：In a specific embodiment of the present invention, the interfering RNA includes one or a combination of two or more of the following target site sequences, sense strand and antisense strand sequence combinations:
A)SEQ ID NO：1，SEQ ID NO：14，SEQ ID NO：27； A) SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 27;
B)SEQ ID NO：2，SEQ ID NO：15，SEQ ID NO：28；B) SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 28;
C)SEQ ID NO：3，SEQ ID NO：16，SEQ ID NO：29；C) SEQ ID NO: 3, SEQ ID NO: 16, SEQ ID NO: 29;
D)SEQ ID NO：5，SEQ ID NO：18，SEQ ID NO：31；D)SEQ ID NO: 5, SEQ ID NO: 18, SEQ ID NO: 31;
E)SEQ ID NO：6，SEQ ID NO：19，SEQ ID NO：32；E) SEQ ID NO: 6, SEQ ID NO: 19, SEQ ID NO: 32;
F)SEQ ID NO：8，SEQ ID NO：21，SEQ ID NO：34；F) SEQ ID NO: 8, SEQ ID NO: 21, SEQ ID NO: 34;
G)SEQ ID NO：9，SEQ ID NO：22，SEQ ID NO：35；G) SEQ ID NO: 9, SEQ ID NO: 22, SEQ ID NO: 35;
H)SEQ ID NO：10，SEQ ID NO：23，SEQ ID NO：36；H)SEQ ID NO: 10, SEQ ID NO: 23, SEQ ID NO: 36;
I)SEQ ID NO：8，SEQ ID NO：56，SEQ ID NO：60；I) SEQ ID NO: 8, SEQ ID NO: 56, SEQ ID NO: 60;
J)SEQ ID NO：8，SEQ ID NO：57，SEQ ID NO：61；J) SEQ ID NO: 8, SEQ ID NO: 57, SEQ ID NO: 61;
K)SEQ ID NO：10，SEQ ID NO：58，SEQ ID NO：62；K)SEQ ID NO: 10, SEQ ID NO: 58, SEQ ID NO: 62;
L)SEQ ID NO：10，SEQ ID NO：59，SEQ ID NO：63。L)SEQ ID NO: 10, SEQ ID NO: 59, SEQ ID NO: 63.
优选的，所述的干扰RNA包含B)组、F)组和/或H)组。Preferably, the interfering RNA includes group B), group F) and/or group H).
在本发明的一个具体实施方式中，所述的干扰RNA包含表1或表15所示的核酸序列中的一种或两种以上。In a specific embodiment of the present invention, the interfering RNA contains one or more than two of the nucleic acid sequences shown in Table 1 or Table 15.
所述的干扰RNA可以采用现有技术任意方法制备获得，例如化学合成等。The interfering RNA can be prepared by any method in the existing technology, such as chemical synthesis, etc.
本发明的第二方面，提供了一种递送系统，所述的递送系统包含上述的干扰RNA。A second aspect of the present invention provides a delivery system, said delivery system comprising the above-mentioned interfering RNA.
优选的，所述的递交系统还包括载体。Preferably, the delivery system further includes a carrier.
优选的，所述的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)中公开的载体。Preferably, the carrier can be any carrier suitable for delivering the above-mentioned interfering RNA of the present invention to target tissues or target cells, etc., such as existing technologies (such as Chen Zhonghua, Zhu Desheng, Li Jun, Huang Zhanqin. "Non-viral siRNA" "Progress in Carrier Research". Chinese Pharmacological Bulletin. 2015, 31(7): 910-4; Wang Rui, Qu Bingnan, Yang Jing. "Research Progress in Nanopreparations Carrying siRNA". Chinese Pharmacy. 2017, 28(31) :4452-4455).
在本发明的一个具体实施方式中，所述的载体为病毒载体，优选的，所述的病毒载体包括但不限于慢病毒载体、逆转录病毒载体、腺病毒载体、腺相关病毒载体、痘病毒载体或疱疹病毒载体等中的一种或两种以上。In a specific embodiment of the present invention, the vector is a viral vector. Preferably, the viral vector includes but is not limited to lentiviral vector, retroviral vector, adenoviral vector, adeno-associated virus vector, poxvirus One or more of vectors or herpes virus vectors.
在本发明的一个具体实施方式中，所述的载体为非病毒载体，优选的，所述的非病毒载体包括但不限于脂质体、脂质纳米颗粒(LNP)、聚合物、多肽、抗体、适配体或N-乙酰半乳糖胺(GalNAc)中的任一种或两种以上的组合；进一步优选的，所述的非病毒载体包括脂质纳米颗粒(LNP)。其中，所述的干扰RNA与非病毒载体的重量比可以为1：1-50中的任一数值(例如1:1、1:5、1:6、1:7、1:8、1:9、1:10、1:15、1:20、1:25、1:30、1:35、1:40、1:45、1:50)，优选为1：1-10中的任一数值。 In a specific embodiment of the present invention, the vector is a non-viral vector. Preferably, the non-viral vector includes but is not limited to liposomes, lipid nanoparticles (LNP), polymers, polypeptides, and antibodies. , aptamer or N-acetylgalactosamine (GalNAc), any one or a combination of two or more; further preferably, the non-viral vector includes lipid nanoparticles (LNP). Wherein, the weight ratio of the interfering RNA and the non-viral vector can be any value from 1:1 to 50 (for example, 1:1, 1:5, 1:6, 1:7, 1:8, 1: 9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50), preferably any one of 1:1-10 numerical value.
优选的，上述脂质纳米颗粒/脂质体包括：阳离子脂质、中性脂质、聚乙二醇脂质、甾族脂质或阴离子脂质中的一种或两种以上组合。Preferably, the above-mentioned lipid nanoparticles/liposomes include: one or a combination of two or more of cationic lipids, neutral lipids, polyethylene glycol lipids, steroidal lipids or anionic lipids.
进一步优选的，所述阳离子脂质包括：十八酰胺(SA)、溴化月桂基三甲基铵、溴化十六烷基三甲基铵、溴化肉豆蔻基三甲基铵、溴化二甲基二-十八铵(DDAB)、[(4-羟基丁基)氮杂二烷基]双(己烷-6，1-二基)双(2-己基癸酸酯)(ALC-0315)、1,2-二油酰基氧基-3-(三甲基铵基)丙烷(DOTAP)、1,2-二-(9Z-十八烯酰基)-3-三甲基铵-丙烷和1,2-二-十六酰基-3-三甲基铵-丙烷、3β-[N-(N',N'-二甲基氨基乙烷)-氨基甲酰基]胆固醇(DC胆固醇)、二甲基二十八烷基铵(DDA)、1,2-二肉豆蔻酰基-3-三甲基铵丙烷(DMTAP)、二棕榈酰(C16:0)三甲基铵丙烷(DPTAP)、二硬脂酰基三甲基铵丙烷(DSTAP)、N-[1-(2,3-二烯丙氧基)丙基]-N,N,N-三甲基氯化铵(DOTMA)、N,N-二油酰基-N,N-二甲基氯化铵(DODAC)、1,2-二油酰基-sn-丙三氧基-3-乙基磷酸胆碱(DOEPC)、1,2-二油酰基-3-二甲基铵丙烷(DODAP)、1,2-二亚油基氧基-3-二甲基氨基丙烷(DLinDMA)、1,2-二-十四酰基-3-二甲基铵-丙烷、1,2-二-十六酰基-3-二甲基铵-丙烷和1,2-二-十八酰基-3-二甲基铵-丙烷、1,2-二油酰基-c-(4'-三甲基铵)-丁酰基-sn-甘油(DOTB)、二-十八酰胺-丙氨酰基精胺、SAINT-2、聚阳离子脂质2,3-二油酰基氧基-N-[2(精胺-羧酰氨基)乙基]-N,N-二甲基-1-丙铵三氟乙酸盐(DOSPA)、 (JK-0315-CA)中的一种或两种以上的组合。Further preferably, the cationic lipid includes: stearamide (SA), lauryltrimethylammonium bromide, cetyltrimethylammonium bromide, myristyltrimethylammonium bromide, Dimethyldioctadecylamine (DDAB), [(4-hydroxybutyl)azadialkyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC- 0315), 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP), 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane and 1,2-di-hexadecanoyl-3-trimethylammonium-propane, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), Dimethyloctadecyl ammonium (DDA), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), dipalmitoyl (C16:0) trimethylammonium propane (DPTAP), Distearoyltrimethylammonium propane (DSTAP), N-[1-(2,3-diallyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), N ,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-propanetrioxy-3-ethylphosphocholine (DOEPC), 1,2 -Dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 1,2-ditetradecanoyl-3- Dimethylammonium-propane, 1,2-di-hexadecanoyl-3-dimethylammonium-propane and 1,2-di-octadecanoyl-3-dimethylammonium-propane, 1,2-dimethylammonium-propane Oleoyl-c-(4'-trimethylammonium)-butyryl-sn-glycerol (DOTB), di-octadecanamide-alanylspermine, SAINT-2, polycationic lipid 2,3-di Oleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propylammonium trifluoroacetate (DOSPA), (JK-0315-CA) or a combination of two or more.
优选的，所述阳离子脂质为类固醇-阳离子脂质化合物，所述化合物的结构为： 中的一种或两种以上。Preferably, the cationic lipid is a steroid-cationic lipid compound, and the structure of the compound is: one or more than two of them.
进一步优选的，所述中性脂质包括：1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)(DOPG)、油酰磷脂酰胆碱(POPC)、1-棕榈酰基-2-油酰基磷脂酰乙醇胺(POPE)或二硬脂酰磷脂酰乙醇胺(DSPE)中的一种或两种以上的组合。Further preferably, the neutral lipid includes: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine Base (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-di Myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC) ), one or a combination of two or more of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) or distearoylphosphatidylethanolamine (DSPE).
进一步优选的，所述聚乙二醇脂质包括：2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺(ALC-0159)、1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)](PEG-DSPE)、PEG-二甾醇基 甘油(PEG-DSG)、PEG-二棕榈油基、PEG-二油基、PEG-二硬脂基、PEG-二酰基甘油酰胺(PEG-DAG)、PEG-二棕榈酰基磷脂酰乙醇胺(PEG-DPPE)或PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺(PEG-c-DMA)、等中的一种或两种以上的组合；Further preferably, the polyethylene glycol lipid includes: 2-[(polyethylene glycol)-2000]-N,N-tetradecyl acetamide (ALC-0159), 1,2-dimethylaminoglycan Myristoyl-sn-glycerylmethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[amino(polyethylene glycol)]( PEG-DSPE), PEG-disterol Glycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG- DPPE) or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA), One or a combination of two or more of the above;
其中，n选自20-300的整数，例如20、30、50、80、100、150、200、250、300等。Wherein, n is selected from an integer from 20 to 300, such as 20, 30, 50, 80, 100, 150, 200, 250, 300, etc.
优选的，所述聚乙二醇脂质为单一分子量的聚乙二醇脂质，优选的，所述的聚乙二醇脂质包括：

Preferably, the polyethylene glycol lipid is a polyethylene glycol lipid of a single molecular weight. Preferably, the polyethylene glycol lipid includes:

进一步优选的，所述阴离子脂质体包括：二油酰磷脂酰甘油和/或二油酰磷脂酰乙醇胺等。Further preferably, the anionic liposome includes: dioleoylphosphatidylglycerol and/or dioleoylphosphatidylethanolamine, etc.
进一步优选的，所述的甾族脂质包括：燕麦甾醇、β-谷甾醇、菜子甾醇、麦角骨化醇、菜油甾醇、胆甾烷醇、胆固醇、粪甾醇、脱氢胆固醇、链甾醇、二氢麦角骨化醇、二氢胆固醇、二氢麦角甾醇、黑海甾醇、表胆甾醇、麦角甾醇、岩藻甾醇、六氢光甾醇、羟基胆固醇、羊毛甾醇、光甾醇、海藻甾醇、谷甾烷醇、谷甾醇、豆甾烷醇、豆甾醇、胆酸、甘氨胆酸、牛磺胆酸、脱氧胆酸或石胆酸中的一种或两种以上的组合。Further preferably, the steroidal lipids include: avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, blackseasterol, epicholesterol, ergosterol, fuccasterol, hexahydroluminosterol, hydroxycholesterol, lanosterol, luminosterol, alginasterol, sitostanol, sitosterol, stigmasterol, stigmasterol, bile acid, glycocholic acid, taurocholic acid, deoxycholic acid or lithocholic acid, or a combination of two or more thereof.
优选的，上述聚合物可以为合成型聚合物(如聚乙烯亚胺、环糊精等)或天然型聚合物(如壳聚糖、端胶原等)或其混合物。Preferably, the above-mentioned polymer can be a synthetic polymer (such as polyethylenimine, cyclodextrin, etc.) or a natural polymer (such as chitosan, telocollagen, etc.) or a mixture thereof.
优选的，上述多肽可以为细胞穿透肽(CPP)(如鱼精蛋白、Tat肽、transportan肽、penetratin肽、寡聚精氨酸肽等)。Preferably, the above-mentioned polypeptide can be a cell-penetrating peptide (CPP) (such as protamine, Tat peptide, transportan peptide, penetratin peptide, oligoarginine peptide, etc.).
优选的，上述抗体可以为单链抗体(如scFv-tp、scFv-9R等)。Preferably, the above-mentioned antibody can be a single-chain antibody (such as scFv-tp, scFv-9R, etc.).
本申请的递送系统还可以包裹各种对人体有益的成分，将其直接递送到细胞内发挥作用，可以更快更好地产生预期效果。The delivery system of this application can also encapsulate various ingredients that are beneficial to the human body and deliver them directly into cells to produce the desired effects faster and better.
本发明的第三方面，提供了一种细胞，所述的细胞中包含上述的干扰RNA或上述的递送系统。A third aspect of the present invention provides a cell containing the above-mentioned interfering RNA or the above-mentioned delivery system.
所述的细胞中TUBB2的表达量被抑制。The expression of TUBB2 in the cells was inhibited.
所述的细胞可以为肿瘤细胞。The cells may be tumor cells.
本发明的第四方面，提供了一种细胞的制备方法，所述的制备方法包括将上述的干扰RNA或上述的递送系统导入到细胞中。A fourth aspect of the present invention provides a method for preparing cells. The preparation method includes introducing the above-mentioned interfering RNA or the above-mentioned delivery system into cells.
本发明的第五方面，提供了一种脂质纳米颗粒，所述的脂质纳米颗粒包含上述的干扰RNA。A fifth aspect of the present invention provides a lipid nanoparticle, which contains the above-mentioned interfering RNA.
优选的，所述的脂质纳米颗粒还包含聚乙二醇脂质化合物、阳离子脂质、甾族脂质或中性脂质中的一种或两种以上。Preferably, the lipid nanoparticles further comprise one or more of polyethylene glycol lipid compounds, cationic lipids, steroidal lipids or neutral lipids.
其中，聚乙二醇脂质化合物、阳离子脂质、甾族脂质及中性脂质的限定同本申请第二方面。 Among them, the definitions of polyethylene glycol lipid compounds, cationic lipids, steroidal lipids and neutral lipids are the same as in the second aspect of this application.
在本发明的一个具体实施方式中，所述的脂质纳米颗粒包含上述的干扰RNA以及聚乙二醇脂质化合物、阳离子脂质(不包括类固醇-阳离子脂质化合物)、甾族脂质和中性脂质。In a specific embodiment of the present invention, the lipid nanoparticles contain the above-mentioned interfering RNA as well as polyethylene glycol lipid compounds, cationic lipids (excluding steroid-cationic lipid compounds), steroid lipids and neutral lipids.
优选的，所述的脂质纳米颗粒中，聚乙二醇脂质化合物、阳离子脂质、甾族脂质和中性脂质的摩尔比为(0.5-5):(30-55):(30-55):(5-20)，例如(0.5、1、2、3、4、5):(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)：(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):(5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20)。优选的，聚乙二醇脂质化合物、阳离子脂质、甾族脂质和中性脂质的摩尔比为(1-5):(35-50):(40-50):(8-15)。Preferably, in the lipid nanoparticles, the molar ratio of polyethylene glycol lipid compounds, cationic lipids, steroidal lipids and neutral lipids is (0.5-5):(30-55):( 30-55):(5-20), for example (0.5, 1, 2, 3, 4, 5): (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): (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):(5,6,7,8,9,10,11,12 ,13,14,15,16,17,18,19,20). Preferably, the molar ratio of polyethylene glycol lipid compound, cationic lipid, steroidal lipid and neutral lipid is (1-5):(35-50):(40-50):(8-15 ).
在本发明的一个具体实施方式中，所述的脂质纳米颗粒包含上述的干扰RNA以及类固醇-阳离子脂质化合物、第二脂质和聚乙二醇脂质。其中，所述的第二脂质选自：中性脂质、两性离子脂质或阴离子脂质。In a specific embodiment of the present invention, the lipid nanoparticles comprise the above-mentioned interfering RNA and a steroid-cationic lipid compound, a second lipid and a polyethylene glycol lipid. Wherein, the second lipid is selected from the group consisting of neutral lipids, zwitterionic lipids or anionic lipids.
优选的，所述的脂质纳米颗粒中，所述类固醇-阳离子脂质:第二脂质:聚乙二醇脂质摩尔比为(10-30):(60-80):(10-25)，例如(10、15、20、25、30)：(60、65、70、75、80)：(10、15、20、25)，优选(20-30):(60-70):(10-20)。Preferably, in the lipid nanoparticles, the molar ratio of the steroid-cationic lipid: second lipid: polyethylene glycol lipid is (10-30): (60-80): (10-25 ), such as (10, 15, 20, 25, 30): (60, 65, 70, 75, 80): (10, 15, 20, 25), preferably (20-30): (60-70): (10-20).
本申请的脂质纳米颗粒还可以包裹各种对人体有益的成分，将其直接递送到细胞内发挥作用，可以更快更好地产生预期效果。The lipid nanoparticles of the present application can also encapsulate various components that are beneficial to the human body and deliver them directly into cells to produce the desired effects faster and better.
所述的脂质纳米颗粒可以采用本领域常规的脂质纳米颗粒制备方法制备得到，例如高压乳匀法、乳化沉淀法、超声分散法等。The lipid nanoparticles can be prepared by conventional lipid nanoparticle preparation methods in the field, such as high-pressure homogenization method, emulsification precipitation method, ultrasonic dispersion method, etc.
本发明的第六方面，提供了一种药物或试剂盒，所述的药物或试剂盒包含上述的干扰RNA、上述的递送系统、上述的脂质纳米颗粒或上述的细胞。A sixth aspect of the present invention provides a medicine or kit, which contains the above-mentioned interfering RNA, the above-mentioned delivery system, the above-mentioned lipid nanoparticles or the above-mentioned cells.
所述的药物还包括药学上可接受的辅料。The medicine also includes pharmaceutically acceptable excipients.
优选的，所述的药学上可接受的辅料包括但不限于载体、稀释剂、粘合剂、润滑剂、润湿剂等等。Preferably, the pharmaceutically acceptable excipients include but are not limited to carriers, diluents, adhesives, lubricants, wetting agents and the like.
优选的，所述的药物的给药方式包括但不限于口服、肠给药、皮下注射、肌肉注射、静脉注射、鼻腔给药、透皮给药、结膜下给药、眼球内给药、眼眶给药、眼球后给药、视网膜给药、脉络膜给药、鞘内注射等等。Preferably, the administration methods of the drug include but are not limited to oral, enteral, subcutaneous, intramuscular, intravenous, nasal, transdermal, subconjunctival, intraocular, orbital, retro-ocular, retinal, choroidal, intrathecal, and the like.
优选的，所述的药物的剂型包括但不限于片剂、胶囊、丸剂、注射剂、吸入剂、含片、栓剂、乳剂、微乳剂、亚微乳剂、纳米颗粒、凝胶剂、粉剂、悬乳液、乳膏剂、 胶冻剂、喷雾剂等等。所述药物的各种剂型可以按照药学领域的常规生产方法制备。Preferably, the dosage forms of the drug include but are not limited to tablets, capsules, pills, injections, inhalants, lozenges, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, and suspoemulsions. , cream, Jelly, spray, etc. Various dosage forms of the drug can be prepared according to conventional production methods in the pharmaceutical field.
优选的，所述的药物中上述的干扰RNA、上述的递送系统或上述的细胞的质量含量可以为1％-100％，例如1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、99.5％、100％。Preferably, the mass content of the above-mentioned interfering RNA, the above-mentioned delivery system or the above-mentioned cells in the drug can be 1%-100%, such as 1%, 2%, 3%, 4%, 5%, 6% ,7%,8%,9%,10%,15%,20%,25%,30%,35%,40%,45%,50%,55%,60%,65%,70%,75 %, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100%.
本发明的第七方面，提供了一种抗体-核酸偶联药物，所述的抗体-核酸偶联药物中包含上述的干扰RNA或上述的递送系统或上述的脂质纳米颗粒。The seventh aspect of the present invention provides an antibody-nucleic acid conjugated drug, which contains the above-mentioned interfering RNA or the above-mentioned delivery system or the above-mentioned lipid nanoparticles.
所述的抗体-核酸偶联药物中包含一个或多个干扰RNA，以及抗体。The antibody-nucleic acid conjugate drug contains one or more interfering RNAs and antibodies.
所述的抗体-核酸偶联药物中抗体与核酸可以直接相连或通过连接基团或连接肽相连。In the antibody-nucleic acid conjugated drug, the antibody and the nucleic acid can be directly connected or connected through a linking group or linking peptide.
所述的抗体-核酸偶联药物的通式(I)为：
The general formula (I) of the antibody-nucleic acid conjugated drug is:
其中，R为上述的干扰RNA；Among them, R is the above-mentioned interfering RNA;
x1为1-144的整数；优选的，x1为1-9的整数；更优选的，x1为1-3的整数；x1 is an integer from 1 to 144; preferably, x1 is an integer from 1 to 9; more preferably, x1 is an integer from 1 to 3;
x2为1-8的整数；优选的，x2为1-2的整数；x2 is an integer from 1 to 8; preferably, x2 is an integer from 1 to 2;
Ab为抗体、蛋白质、多肽；Ab is an antibody, protein, or peptide;
L为连接Ab与R之间的连接单元。L is the connecting unit connecting Ab and R.
优选的，所述L部分具有通式(Ⅱ)结构：
Preferably, the L part has the structure of general formula (II):
其中，in,
式Ⅱ中x3选自1-12的整数；优选为1-3；In formula II, x3 is selected from an integer of 1-12; preferably 1-3;
式Ⅱ中x4选自1-12的整数；优选为1-3；In formula II, x4 is selected from an integer of 1-12; preferably 1-3;
P1、P2相同或不同的聚乙二醇残基； P 1 and P 2 are the same or different polyethylene glycol residues;
L1为连接Ab与P1之间的连接单元； L1 is a linker unit connecting Ab and P1 ;
L2为连接P2与R之间的连接单元；L 2 is the connecting unit connecting P 2 and R;
A1为连接P1与P2之间的连接单元；A 1 is the connection unit connecting P 1 and P 2 ;
优选的，所述P1、P2独立地选自直链、Y型、多分支的聚乙二醇残基；Preferably, the P 1 and P 2 are independently selected from linear, Y-shaped, multi-branched polyethylene glycol residues;
优选的，当所述P1、P2为单一分子量聚乙二醇，分子量为88-4400Da，更优选的，所述P1、P2分子量为176-1056Da；Preferably, when P 1 and P 2 are single molecular weight polyethylene glycol, the molecular weight is 88-4400 Da, and more preferably, the molecular weight of P 1 and P 2 is 176-1056 Da;
优选的，当所述P1、P2为非单一分子量聚乙二醇，分子量为1000Da-40kDa；Preferably, when P 1 and P 2 are non-single molecular weight polyethylene glycol, the molecular weight is 1000Da-40kDa;
更优选的，所述P1、P2分子量为2000Da-10kDa。More preferably, the molecular weight of P 1 and P 2 is 2000Da-10kDa.
优选的，所述L1为连接基团，选自直链或支链的C1-12亚烷基、C6-12亚芳基、C3-12环亚烷基、-S-、 中的一种或者两种以上基团的组合；Preferably, the L 1 is a connecting group selected from linear or branched C 1-12 alkylene, C 6-12 arylene, C 3-12 cycloalkylene, -S-, One or a combination of two or more groups;
所述直链或支链的C1-12链亚烷基、C6-12的亚芳基或C3-12环亚烷基上的任意的H原子被-H、-F、-Cl、-Br、-I、-O-、-S-、-SO2、-NO2、C1-12链烷基、C3-12环烷基、C6-12芳烷基、取代或未取代的杂环基或取代或未取代的杂环基烷基、 中的一种或者两种以上基团组成的组取代；Any H atom on the linear or branched C 1-12 chain alkylene group, C 6-12 arylene group or C 3-12 cycloalkylene group is replaced by -H, -F, -Cl, -Br, -I, -O-, -S-, -SO 2 , -NO 2 , C 1-12 alkyl, C 3-12 cycloalkyl, C 6-12 aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, Substituted by one type or a group consisting of two or more groups;
优选的，所述L2为连接基团，选自直链或支链的C1-12亚烷基、C6-12亚芳基、C3-12环亚烷基、-S-、 中的一种或者两种以上基团的组合；Preferably, the L 2 is a connecting group selected from linear or branched C 1-12 alkylene, C 6-12 arylene, C 3-12 cycloalkylene, -S-, One or a combination of two or more groups;
所述直链或支链的C1-12链亚烷基、C6-12的亚芳基或C3-12环亚烷基上的任意的H原子被-H、-F、-Cl、-Br、-I、-O-、-S-、-SO2、-NO2、C1-12链烷基、C3-12环烷基、C6-12芳烷基、取代或未取代的杂环基或取代或未取代的杂环基烷基、 中一种或者以上基团组成的组取代；Any H atom on the linear or branched C 1-12 chain alkylene group, C 6-12 arylene group or C 3-12 cycloalkylene group is replaced by -H, -F, -Cl, -Br, -I, -O-, -S-, -SO 2 , -NO 2 , C 1-12 alkyl, C 3-12 cycloalkyl, C 6-12 aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, Substituted by a group consisting of one or more groups;
优选的，所述L1为酰胺键、腙键以及巯基-马来酰亚胺键；Preferably, the L 1 is an amide bond, a hydrazone bond and a thiol-maleimide bond;
更优选的，所述L1为酰胺键；More preferably, the L 1 is an amide bond;
优选的，所述L2为二硫键以及巯基-马来酰亚胺键；Preferably, the L 2 is a disulfide bond and a thiol-maleimide bond;
更优选的，所述L2为二硫键。More preferably, the L 2 is a disulfide bond.
优选的，所述A1为连接P1与P2之间的连接基团，选自直链或支链的C1-12亚烷基、C6-12亚芳基、C3-12环亚烷基、-S-、 中的一种或者两种以上基团的组合；Preferably, the A 1 is a connecting group connecting P 1 and P 2 , which is selected from linear or branched C 1-12 alkylene, C 6-12 arylene, and C 3-12 ring. Alkylene, -S-, One or a combination of two or more groups;
所述直链或支链的C1-12链亚烷基、C6-12的亚芳基或C3-12环亚烷基上的任意的H原子被-H、-F、-Cl、-Br、-I、-O-、-S-、-SO2、-NO2、C1-12链烷基、C3-12环烷基、C6-12芳烷基、取代或未取代的杂环基或取代或未取代的杂环基烷基、 中一种或者两种以上基团组成的组取代。Any H atom on the linear or branched C 1-12 chain alkylene group, C 6-12 arylene group or C 3-12 cycloalkylene group is replaced by -H, -F, -Cl, -Br, -I, -O-, -S-, -SO 2 , -NO 2 , C 1-12 alkyl, C 3-12 cycloalkyl, C 6-12 aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, Substitute with one or more groups consisting of two or more groups.
进一步优选的，所述的抗体-核酸偶联药物的通式(IV)：
Further preferably, the general formula (IV) of the antibody-nucleic acid conjugate drug is:
所述L1为连接基团，选自直链或支链的C1-12亚烷基、C6-12亚芳基、C3-12环亚烷基、-S-、 中的一种或者两种以上基团的组合；The L 1 is a connecting group, selected from linear or branched C 1-12 alkylene, C 6-12 arylene, C 3-12 cycloalkylene, -S-, One or a combination of two or more groups;
优选的，P1为相同或不同的聚乙二醇残基； Preferably, P 1 is the same or different polyethylene glycol residue;
优选的，当所述P1为单一分子量聚乙二醇，分子量为88-4400Da，更优选的，所述P1分子量为176-1056Da；Preferably, when P 1 is a single molecular weight polyethylene glycol, the molecular weight is 88-4400 Da, and more preferably, the molecular weight of P 1 is 176-1056 Da;
优选的，当所述P1为非单一分子量聚乙二醇，分子量为1000Da-40kDa；更优选的，所述P1分子量为2000Da-10kDa。Preferably, when the P1 is a non-single molecular weight polyethylene glycol, the molecular weight is 1000Da-40kDa; more preferably, the molecular weight of the P1 is 2000Da-10kDa.
所述L2为连接基团，选自直链或支链的C1-12亚烷基、C6-12亚芳基、C3-12环亚烷基、-S-、 中的一种或者两种以上基团的组合；The L 2 is a connecting group, selected from linear or branched C 1-12 alkylene, C 6-12 arylene, C 3-12 cycloalkylene, -S-, One or a combination of two or more groups;
x1为1-144的整数；优选的，x1为1-9的整数；更优选的，x1为1-3的整数；x1 is an integer from 1 to 144; preferably, x1 is an integer from 1 to 9; more preferably, x1 is an integer from 1 to 3;
x2为1-8的整数；优选的，x2为1-2的整数。x2 is an integer from 1 to 8; preferably, x2 is an integer from 1 to 2.
在本发明的一个具体实施方式中，所述的抗体-核酸偶联药物具有如下结构：
In a specific embodiment of the invention, the antibody-nucleic acid conjugated drug has the following structure:
所述n1选自4-100的整数，优选为4-24；n1可以为定值也可以为均值。The n 1 is selected from an integer from 4 to 100, preferably 4 to 24; n 1 can be a fixed value or an average value.
在本发明的一个具体实施方式中，所述的抗体-核酸偶联药物具有如下结构：
In a specific embodiment of the invention, the antibody-nucleic acid conjugated drug has the following structure:
所述n1、n2独立地选自4-100的整数，优选为4-24。n1、n2可以为定值也可以为均值。Said n 1 and n 2 are independently selected from integers of 4-100, preferably 4-24. n1 and n2 can be fixed values or mean values.
优选的，所述的Ab选自单克隆抗体、多克隆抗体、抗体片段、抗体融合片段。Preferably, the Ab is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antibody fragments, and antibody fusion fragments.
所述的抗体可以为单域抗体或单链抗体。The antibody can be a single domain antibody or a single chain antibody.
进一步优选的，Ab为单克隆抗体；更优选的，所述单克隆抗体对癌症、恶性细胞、感染性生物或自身免疫性疾病相关的抗原或其表位是反应性的。Further preferably, the Ab is a monoclonal antibody; more preferably, the monoclonal antibody is reactive to antigens or epitopes related to cancer, malignant cells, infectious organisms or autoimmune diseases.
在本发明的一个具体实施方式中，所述Ab选自：抗HER2抗体、抗EGFR抗体、抗PMSA抗体、抗VEGFR抗体、抗CD30抗体、抗CD22抗体、抗CD56抗体、抗CD29抗体、抗GPNMB抗体、抗CD138抗体、抗CD74抗体、抗ENPP3抗体、抗Nectin-4抗体、抗EGFRⅧ抗体、抗SLC44A4抗体、抗间皮素抗体、抗ET8R抗体、抗CD37抗体、抗CEACAM5抗体、抗CD70抗体、抗MUC16抗体、抗CD79b抗体、抗MUC16抗体、抗Muc1抗体、抗CD3抗体、抗CD28抗体、抗CD38抗体、抗CD19抗体、抗PD-L1抗体、抗4-1BB抗体等。In a specific embodiment of the invention, the Ab is selected from: anti-HER2 antibody, anti-EGFR antibody, anti-PMSA antibody, anti-VEGFR antibody, anti-CD30 antibody, anti-CD22 antibody, anti-CD56 antibody, anti-CD29 antibody, anti-GPNMB Antibody, anti-CD138 antibody, anti-CD74 antibody, anti-ENPP3 antibody, anti-Nectin-4 antibody, anti-EGFRVIII antibody, anti-SLC44A4 antibody, anti-mesothelin antibody, anti-ET8R antibody, anti-CD37 antibody, anti-CEACAM5 antibody, anti-CD70 antibody, Anti-MUC16 antibody, anti-CD79b antibody, anti-MUC16 antibody, anti-Muc1 antibody, anti-CD3 antibody, anti-CD28 antibody, anti-CD38 antibody, anti-CD19 antibody, anti-PD-L1 antibody, anti-4-1BB antibody, etc.
本发明的第八方面，提供了一种上述的干扰RNA、上述的递送系统、上述的细胞、上述的药物或试剂盒、上述的脂质纳米颗粒或上述的抗体-核酸偶联药物的应用，所述的应用包括：The eighth aspect of the present invention provides an application of the above-mentioned interfering RNA, the above-mentioned delivery system, the above-mentioned cell, the above-mentioned medicine or kit, the above-mentioned lipid nanoparticle or the above-mentioned antibody-nucleic acid conjugated drug, Applications described include:
A)在制备治疗和/或预防TUBB2表达增加或细胞过度增殖相关疾病的药物中的应用；A) Application in the preparation of drugs for the treatment and/or prevention of diseases related to increased expression of TUBB2 or excessive cell proliferation;
B)在抑制TUBB2表达中的应用；B) Application in inhibiting TUBB2 expression;
C)在治疗和/或预防TUBB2表达增加或细胞过度增殖相关疾病中的应用。C) Use in treating and/or preventing diseases associated with increased TUBB2 expression or excessive cell proliferation.
优选的，所述治疗使用的干扰RNA、上述的递送系统、上述的细胞、上述的药物或试剂盒、上述的脂质纳米颗粒或上述的抗体-核酸偶联药物的工作浓度为0.01-1000nM，例如0.01、1、2、3、4、5、6、7、8、9、10、15、20、25、30、35、 40、45、50、55、60、65、70、75、80、85、90、95、100、200、300、400、500、600、700、800、900或1000nM。Preferably, the working concentration of the interfering RNA used in the treatment, the above-mentioned delivery system, the above-mentioned cells, the above-mentioned drugs or kits, the above-mentioned lipid nanoparticles or the above-mentioned antibody-nucleic acid conjugated drugs is 0.01-1000nM, For example, 0.01, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000nM.
优选的，TUBB2表达增加或细胞过度增殖相关疾病包括肿瘤。进一步优选的，所述的肿瘤包括消化道肿瘤(口腔癌、舌癌、食管癌、胃癌、肝癌、胰腺癌、结肠直肠癌等)、神经系统肿瘤(胶质瘤(例如神经胶质瘤)、神经上皮瘤、神经鞘瘤、星形细胞瘤、神经纤维瘤(例如神经纤维肉瘤)、室管膜瘤、成神经管细胞瘤、脑膜瘤、脑转移瘤等)、呼吸道肿瘤(鼻咽癌、喉癌、支气管癌、肺癌等)、泌尿生殖系统肿瘤(前列腺癌、肾脏肿瘤、膀胱癌、乳腺癌、宫颈癌、卵巢癌、胎盘绒毛癌、尿路系统肿瘤、精囊腺肿瘤、睾丸及睾丸旁组织肿瘤、阴茎肿瘤、阴道癌、外阴癌、子宫癌、子宫内膜癌等)、血液淋巴系统肿瘤(白血病(例如慢性粒细胞白血病、急性髓系白血病、慢性淋巴细胞白血病、骨髓增生异常综合征、慢性骨髓细胞性白血病、急性淋巴细胞白血病等)、多发性骨髓瘤、间皮瘤、骨髓瘤、淋巴瘤(例如非霍奇金淋巴瘤、霍奇金淋巴瘤、皮肤T细胞淋巴瘤)、皮肤系统肿瘤(如皮肤癌、表皮样癌、黑色素瘤、基底细胞癌、鳞状细胞癌、Paget病、卡波西肉瘤、Merkel细胞癌、不典型纤维黄瘤、附属器肿瘤、皮肤T细胞淋巴瘤(蕈状肉芽肿)等)等以及横纹肌肉瘤、头颈部癌等。Preferably, diseases related to increased expression of TUBB2 or excessive cell proliferation include tumors. Further preferably, the tumors include digestive tract tumors (oral cancer, tongue cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, etc.), nervous system tumors (glioma (such as glioma), Neuroepithelioma, schwannoma, astrocytoma, neurofibroma (such as neurofibrosarcoma), ependymoma, medulloblastoma, meningioma, brain metastases, etc.), respiratory tract tumors (nasopharyngeal carcinoma, Laryngeal cancer, bronchial cancer, lung cancer, etc.), genitourinary system tumors (prostate cancer, kidney tumors, bladder cancer, breast cancer, cervical cancer, ovarian cancer, placental choriocarcinoma, urinary tract system tumors, seminal vesicle gland tumors, testicles and paratesticular tumors Tissue tumors, penile tumors, vaginal cancer, vulvar cancer, uterine cancer, endometrial cancer, etc.), hematological lymphoid system tumors (leukemias (such as chronic myelogenous leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome) , chronic myeloid leukemia, acute lymphoblastic leukemia, etc.), multiple myeloma, mesothelioma, myeloma, lymphoma (such as non-Hodgkin lymphoma, Hodgkin lymphoma, cutaneous T-cell lymphoma), Cutaneous system tumors (such as skin cancer, epidermoid carcinoma, melanoma, basal cell carcinoma, squamous cell carcinoma, Paget's disease, Kaposi's sarcoma, Merkel cell carcinoma, atypical fibroxanthoma, adnexal tumors, cutaneous T-cell lymphoma tumors (mycosis fungoides, etc.), as well as rhabdomyosarcoma, head and neck cancer, etc.
进一步优选的，所述的肿瘤选自泌尿生殖系统肿瘤和/或皮肤系统肿瘤。Further preferably, the tumor is selected from genitourinary system tumors and/or skin system tumors.
在本发明的一个具体实施方式中，所述的肿瘤选自前列腺癌、乳腺癌或黑色素瘤。In a specific embodiment of the invention, the tumor is selected from prostate cancer, breast cancer or melanoma.
本发明的第九方面，提供了一种抑制TUBB2表达的方法，所述的方法包括加入上述的干扰RNA、上述的递送系统、上述的细胞、上述的脂质纳米颗粒、上述的药物或试剂盒或上述的抗体-核酸偶联药物。The ninth aspect of the present invention provides a method for inhibiting TUBB2 expression, which method includes adding the above-mentioned interfering RNA, the above-mentioned delivery system, the above-mentioned cells, the above-mentioned lipid nanoparticles, the above-mentioned medicine or kit Or the above-mentioned antibody-nucleic acid conjugate drugs.
优选的，所述的方法抑制细胞中TUBB2表达，所述的细胞为肿瘤细胞。Preferably, the method inhibits TUBB2 expression in cells, and the cells are tumor cells.
优选的，所述的方法包括将上述干扰RNA递送入细胞中。所述的递送使用上述的递送系统。Preferably, the method includes delivering the above-mentioned interfering RNA into cells. Said delivery uses the delivery system described above.
本发明的第十方面，提供了一种治疗TUBB2表达增加或细胞过度增殖相关疾病的方法，所述的方法包括向受试者施加治疗有效量的上述的干扰RNA、上述的递送系统、上述的脂质纳米颗粒、上述的细胞、上述的药物或试剂盒或上述的抗体-核酸偶联药物。A tenth aspect of the present invention provides a method for treating diseases related to increased expression of TUBB2 or excessive cell proliferation. The method includes applying to a subject a therapeutically effective amount of the above-mentioned interfering RNA, the above-mentioned delivery system, the above-mentioned Lipid nanoparticles, the above-mentioned cells, the above-mentioned drugs or kits, or the above-mentioned antibody-nucleic acid conjugated drugs.
优选的，TUBB2表达增加或细胞过度增殖相关疾病包括肿瘤。进一步优选的， Preferably, diseases related to increased expression of TUBB2 or excessive cell proliferation include tumors. Further preferably,
所述的肿瘤包括消化道肿瘤(口腔癌、舌癌、食管癌、胃癌、肝癌、胰腺癌、结肠直肠癌等)、神经系统肿瘤(胶质瘤(例如神经胶质瘤)、神经上皮瘤、神经鞘瘤、星形细胞瘤、神经纤维瘤(例如神经纤维肉瘤)、室管膜瘤、成神经管细胞瘤、脑膜瘤、脑转移瘤等)、呼吸道肿瘤(鼻咽癌、喉癌、支气管癌、肺癌等)、泌尿生殖系统肿瘤(前列腺癌、肾脏肿瘤、膀胱癌、乳腺癌、宫颈癌、卵巢癌、胎盘绒毛癌、尿路系统肿瘤、精囊腺肿瘤、睾丸及睾丸旁组织肿瘤、阴茎肿瘤、外阴癌、阴道癌、子宫癌、子宫内膜癌等)、血液淋巴系统肿瘤(白血病(例如慢性粒细胞白血病、急性髓系白血病、慢性淋巴细胞白血病、骨髓增生异常综合征、慢性骨髓细胞性白血病、急性淋巴细胞白血病等)、多发性骨髓瘤、间皮瘤、骨髓瘤、淋巴瘤(例如非霍奇金淋巴瘤、霍奇金淋巴瘤、皮肤T细胞淋巴瘤)、皮肤系统肿瘤(如皮肤癌、表皮样癌、黑色素瘤、基底细胞癌、鳞状细胞癌、Paget病、卡波西肉瘤、Merkel细胞癌、不典型纤维黄瘤、附属器肿瘤、皮肤T细胞淋巴瘤(蕈状肉芽肿)等)等以及横纹肌肉瘤、头颈部癌等。The tumors include digestive tract tumors (oral cancer, tongue cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, etc.), nervous system tumors (glioma (such as glioma), neuroepithelial tumors, Schwannoma, astrocytoma, neurofibroma (such as neurofibrosarcoma), ependymoma, medulloblastoma, meningioma, brain metastases, etc.), respiratory tract tumors (nasopharyngeal cancer, laryngeal cancer, bronchial carcinoma, etc.) cancer, lung cancer, etc.), genitourinary system tumors (prostate cancer, kidney tumors, bladder cancer, breast cancer, cervical cancer, ovarian cancer, placental choriocarcinoma, urinary tract system tumors, seminal vesicle tumors, testicular and paratesticular tissue tumors, penis Tumors, vulvar cancer, vaginal cancer, uterine cancer, endometrial cancer, etc.), hematolymphatic system tumors (leukemias (such as chronic myelogenous leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, chronic myeloid leukemia) leukemia, acute lymphoblastic leukemia, etc.), multiple myeloma, mesothelioma, myeloma, lymphoma (such as non-Hodgkin lymphoma, Hodgkin lymphoma, cutaneous T-cell lymphoma), cutaneous system tumors ( Such as skin cancer, epidermoid carcinoma, melanoma, basal cell carcinoma, squamous cell carcinoma, Paget's disease, Kaposi's sarcoma, Merkel cell carcinoma, atypical fibroxanthoma, adnexal tumors, cutaneous T-cell lymphoma (mycosis fungoides) Granuloma), etc.) as well as rhabdomyosarcoma, head and neck cancer, etc.
本发明的第十一方面，提供了一种治疗肿瘤的方法，所述的方法包括向受试者施加治疗有效量的上述的干扰RNA、上述的递送系统、上述的细胞、上述的脂质纳米颗粒、上述的药物或试剂盒或上述的抗体-核酸偶联药物。An eleventh aspect of the present invention provides a method for treating tumors, which method includes applying to a subject a therapeutically effective amount of the above-mentioned interfering RNA, the above-mentioned delivery system, the above-mentioned cells, and the above-mentioned lipid nanoparticles. particles, the above-mentioned drugs or kits, or the above-mentioned antibody-nucleic acid conjugated drugs.
优选的，所述的肿瘤包括消化道肿瘤(口腔癌、舌癌、食管癌、胃癌、肝癌、胰腺癌、结肠直肠癌等)、神经系统肿瘤(胶质瘤(例如神经胶质瘤)、神经上皮瘤、神经鞘瘤、星形细胞瘤、神经纤维瘤(例如神经纤维肉瘤)、室管膜瘤、成神经管细胞瘤、脑膜瘤、脑转移瘤等)、呼吸道肿瘤(鼻咽癌、喉癌、支气管癌、肺癌等)、泌尿生殖系统肿瘤(前列腺癌、肾脏肿瘤、膀胱癌、乳腺癌、宫颈癌、卵巢癌、胎盘绒毛癌、尿路系统肿瘤、精囊腺肿瘤、睾丸及睾丸旁组织肿瘤、阴茎肿瘤、外阴癌、阴道癌、子宫癌、子宫内膜癌等)、血液淋巴系统肿瘤(白血病(例如慢性粒细胞白血病、急性髓系白血病、慢性淋巴细胞白血病、骨髓增生异常综合征、慢性骨髓细胞性白血病、急性淋巴细胞白血病等)、多发性骨髓瘤、间皮瘤、骨髓瘤、淋巴瘤(例如非霍奇金淋巴瘤、霍奇金淋巴瘤、皮肤T细胞淋巴瘤)、皮肤系统肿瘤(如皮肤癌、表皮样癌、黑色素瘤、基底细胞癌、鳞状细胞癌、Paget病、卡波西肉瘤、Merkel细胞癌、不典型纤维黄瘤、附属器肿瘤、皮肤T细胞淋巴瘤(蕈状肉芽肿)等)等以及横纹肌肉瘤、头颈部癌等。 Preferably, the tumors include digestive tract tumors (oral cancer, tongue cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, etc.), nervous system tumors (glioma (such as glioma), nerve Epithelioma, schwannoma, astrocytoma, neurofibroma (such as neurofibrosarcoma), ependymoma, medulloblastoma, meningioma, brain metastases, etc.), respiratory tract tumors (nasopharyngeal carcinoma, laryngeal carcinoma, etc.) cancer, bronchial cancer, lung cancer, etc.), genitourinary system tumors (prostate cancer, kidney tumors, bladder cancer, breast cancer, cervical cancer, ovarian cancer, placental choriocarcinoma, urinary tract system tumors, seminal vesicle gland tumors, testis and paratesticular tissue Tumors, penile tumors, vulvar cancer, vaginal cancer, uterine cancer, endometrial cancer, etc.), hematological and lymphatic system tumors (leukemias (such as chronic myelogenous leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, Chronic myeloid leukemia, acute lymphoblastic leukemia, etc.), multiple myeloma, mesothelioma, myeloma, lymphoma (such as non-Hodgkin lymphoma, Hodgkin lymphoma, cutaneous T-cell lymphoma), skin Systemic tumors (such as skin cancer, epidermoid carcinoma, melanoma, basal cell carcinoma, squamous cell carcinoma, Paget's disease, Kaposi's sarcoma, Merkel cell carcinoma, atypical fibroxanthoma, adnexal tumors, cutaneous T-cell lymphoma (Mycosis fungoides), etc.), as well as rhabdomyosarcoma, head and neck cancer, etc.
进一步优选的，所述的肿瘤选自泌尿生殖系统肿瘤和/或皮肤系统肿瘤。Further preferably, the tumor is selected from urogenital system tumors and/or skin system tumors.
在本发明的一个具体实施方式中，所述的肿瘤选自前列腺癌、乳腺癌或黑色素瘤。In a specific embodiment of the invention, the tumor is selected from prostate cancer, breast cancer or melanoma.
本发明的第十二方面，提供了一种抑制细胞过度增殖的方法，所述的方法包括向受试者施加治疗有效量的上述的干扰RNA、上述的递送系统、上述的细胞、上述的脂质纳米颗粒、上述的药物或试剂盒或上述的抗体-核酸偶联药物。The twelfth aspect of the present invention provides a method for inhibiting excessive cell proliferation, which comprises applying a therapeutically effective amount of the above-mentioned interfering RNA, the above-mentioned delivery system, the above-mentioned cells, the above-mentioned lipid nanoparticles, the above-mentioned drug or kit or the above-mentioned antibody-nucleic acid conjugate drug to a subject.
本发明的第十三方面，提供了一种抗有丝分裂的方法，所述的方法包括向受试者施加治疗有效量的上述的干扰RNA、上述的递送系统、上述的细胞、上述的脂质纳米颗粒、上述的药物或试剂盒或上述的抗体-核酸偶联药物。A thirteenth aspect of the present invention provides an anti-mitosis method, which method includes applying to a subject a therapeutically effective amount of the above-mentioned interfering RNA, the above-mentioned delivery system, the above-mentioned cells, and the above-mentioned lipid nanoparticles. particles, the above-mentioned drugs or kits, or the above-mentioned antibody-nucleic acid conjugated drugs.
本申请筛选出的TUBB2siRNA序列，可以用于细胞增殖旺盛相关疾病(如各种肿瘤等)的治疗，有强大的开发潜力。并经实验证实，可显著抑制多种细胞(如A375、MDA-MB-231-GFP、PC-3、SKOV-3、HCT116等细胞)中TUBB2的mRNA表达水平。因此，有望用于与微管蛋白表达增加相关的疾病，如各种恶性肿瘤等疾病状态，通过抑制微管蛋白β2的表达，阻断或降低细胞的增殖，实现减缓疾病进展和改善疾病转归的目的。The TUBB2 siRNA sequence screened out in this application can be used to treat diseases related to vigorous cell proliferation (such as various tumors, etc.) and has strong development potential. Experiments have confirmed that it can significantly inhibit the mRNA expression level of TUBB2 in a variety of cells (such as A375, MDA-MB-231-GFP, PC-3, SKOV-3, HCT116 and other cells). Therefore, it is expected to be used in diseases related to increased tubulin expression, such as various malignant tumors and other disease states. By inhibiting the expression of tubulin β2, blocking or reducing cell proliferation, it can slow down disease progression and improve disease outcomes. the goal of.
本发明所述的“干扰RNA”，包括单链RNA(例如，成熟miRNA)或双链RNA(例如，siRNA、shRNA、aiRNA或前体miRNA)，当干扰RNA与靶基因或序列处于同一细胞中时，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/或抑制与干扰RNA序列互补的mRNA的翻译)。The "interfering RNA" described in the present invention includes single-stranded RNA (e.g., mature miRNA) or double-stranded RNA (e.g., siRNA, shRNA, aiRNA or precursor miRNA). When the interfering RNA and the target gene or sequence are in the same cell, they can reduce or inhibit the expression of the target gene or sequence (e.g., by mediating degradation and/or inhibiting the translation of mRNA complementary to the interfering RNA sequence).
其中，siRNA为小干扰RNA，其分子的每条链包含长度为约15nt至约60nt的核苷酸(例如，长度为约15-60、15-50、15-40、15-30、15-25、或19-25nt的核苷酸，或者长度为15、16、17、18、19、20、21、22、23、24、或25nt的核苷酸)。在一个具体实施方式中，siRNA可以是化学合成的。本发明的siRNA分子能够在体外和/或体内沉默靶序列的表达。在一些实施方式中，siRNA可以不含有修饰的核苷酸；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。shRNA为小发夹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。miRNA(微RNA)是长度约21-23个核苷酸的调节基因表达的单链RNA分子。Wherein, siRNA is a small interfering RNA, and each strand of its molecule contains nucleotides with a length of about 15nt to about 60nt (for example, a length of about 15-60, 15-50, 15-40, 15-30, 15- 25, or 19-25 nt nucleotides, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nt in length). In a specific embodiment, siRNA can be chemically synthesized. The siRNA molecules of the invention are capable of silencing the expression of target sequences in vitro and/or in vivo. In some embodiments, siRNA may contain no modified nucleotides; in other embodiments, siRNA may contain at least one modified nucleotide, for example, siRNA may contain one, two, three, or four modified nucleotides in the double-stranded region. , five, six, seven, eight, nine, ten or more modified nucleotides. shRNA is small hairpin RNA or short Hairpin RNA, which includes short RNA sequences that create tight hairpin turns that can be used to silence gene expression through RNA interference, shRNA hairpin structures that can be cleaved into siRNA by cellular machinery. miRNA (microRNA) is a single-stranded RNA molecule about 21-23 nucleotides in length that regulates gene expression.
本发明所述的“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TUBB2基因)表达的能力。"Inhibiting the expression of a target gene" in the present invention refers to the ability of the interfering RNA (for example, siRNA) of the present invention to silence, reduce or inhibit the expression of a target gene (for example, the TUBB2 gene).
本发明所述的“包含”或“包括”为开放式写法，含有所描述的指定成分或步骤，以及不会实质上影响的其他指定成分或步骤。当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，但仍然具有与原序列相同或相似的活性。The term "comprises" or "comprises" used in the present invention is an open-ended expression, containing the specified components or steps described, as well as other specified components or steps that will not substantially affect the process. When used to describe the sequence of a protein or nucleic acid, the 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 same Identical or similar activity to the original sequence.
本发明所述的“肿瘤”可以是任何不良的细胞增殖(或本身表现为不良细胞增殖的任何疾病)、赘生物，或不良细胞增殖、赘生物或肿瘤的倾向性或风险增加。其可以是良性或恶性的，也可以是原发性或继发性(转移性)。赘生物可以是细胞的任何异常生长或增殖，并且可以位于任何组织中。组织的实例包括肾上腺、肾上腺髓质、肛门、阑尾、膀胱、血液、骨、骨髓、脑、乳腺、盲肠、中枢神经系统(包括或排除大脑)、小脑、子宫颈、结肠、十二指肠、子宫内膜、胆囊、食道、神经胶质细胞、心脏、回肠、空肠、肾、泪腺、喉、肝、肺、淋巴、淋巴结、上颌骨、纵隔、肠系膜、子宫肌层、鼻咽、网膜、口腔、卵巢、胰腺、腮腺、周围神经系统、腹膜、胸膜、前列腺、唾液腺、结肠、皮肤、软组织、脾、胃、睾丸、胸腺、甲状腺、舌、扁桃体、气管、子宫、外阴、直肠。进一步优选的，所述的肿瘤包括消化道肿瘤(口腔癌、舌癌、食管癌、胃癌、肝癌、胰腺癌、结肠直肠癌等)、神经系统肿瘤(胶质瘤(例如神经胶质瘤)、神经上皮瘤、神经鞘瘤、星形细胞瘤、神经纤维瘤(例如神经纤维肉瘤)、室管膜瘤、成神经管细胞瘤、脑膜瘤、脑转移瘤等)、呼吸道肿瘤(鼻咽癌、喉癌、支气管癌、肺癌等)、泌尿系统肿瘤(如肾细胞癌、膀胱癌等)、生殖系统肿瘤(前列腺癌、睾丸癌、乳腺癌、宫颈癌、卵巢癌、胎盘绒毛癌等)、血液淋巴系统肿瘤(多发性骨髓瘤、间皮瘤、骨髓瘤、淋巴瘤(例如非霍奇金淋巴瘤、霍奇 金淋巴瘤、皮肤T细胞淋巴瘤)、白血病(例如慢性粒细胞白血病、急性髓系白血病、慢性淋巴细胞白血病、骨髓增生异常综合征等)、胸腺癌等)、皮肤系统肿瘤(如皮肤癌、表皮样癌、黑色素瘤等)等以及横纹肌肉瘤、头颈部癌等。A "tumor" as used herein may be any undesirable cell proliferation (or any disease manifesting itself as undesirable cell proliferation), neoplasia, or a predisposition or increased risk of undesirable cell proliferation, neoplasia, or neoplasia. It can be benign or malignant, primary or secondary (metastatic). A neoplasm can be any abnormal growth or proliferation of cells and can be located in any tissue. Examples of tissues include adrenal glands, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, Endometrium, gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, Oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary glands, colon, skin, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsils, trachea, uterus, vulva, rectum. Further preferably, the tumors include digestive tract tumors (oral cancer, tongue cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, etc.), nervous system tumors (glioma (such as glioma), Neuroepithelioma, schwannoma, astrocytoma, neurofibroma (such as neurofibrosarcoma), ependymoma, medulloblastoma, meningioma, brain metastases, etc.), respiratory tract tumors (nasopharyngeal carcinoma, Laryngeal cancer, bronchial cancer, lung cancer, etc.), urinary system tumors (such as renal cell carcinoma, bladder cancer, etc.), reproductive system tumors (prostate cancer, testicular cancer, breast cancer, cervical cancer, ovarian cancer, placental choriocarcinoma, etc.), blood Lymphoid system tumors (multiple myeloma, mesothelioma, myeloma, lymphoma (such as non-Hodgkin lymphoma, Hodgkin lymphoma) Golden lymphoma, cutaneous T-cell lymphoma), leukemias (such as chronic myelogenous leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, etc.), thymic cancer, etc.), cutaneous system tumors (such as skin cancer, Epidermoid carcinoma, melanoma, etc.) as well as rhabdomyosarcoma, head and neck cancer, etc.
本发明所述的“受试者”可以为人或非人哺乳动物，所述的非人哺乳动物可以为野生动物、动物园动物、经济动物、宠物、实验动物等等。优选的，所述的非人哺乳动物包括但不限于猪、牛、羊、马、驴、狐、貉、貂、骆驼、狗、猫、兔、鼠(例如大鼠、小鼠、豚鼠、仓鼠、沙鼠、南美洲栗鼠、松鼠)或猴等等。The "subject" of the present invention can be a human or a non-human mammal, and the non-human mammal can be a wild animal, a zoo animal, an economic animal, a pet, an experimental animal, etc. Preferably, the non-human mammals include, but are not limited to, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, mink, camels, dogs, cats, rabbits, mice (such as rats, mice, guinea pigs, hamsters , gerbils, chinchillas, squirrels) or monkeys, etc.
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。"Treat" as used herein means to slow, interrupt, arrest, control, stop, alleviate, or reverse the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but not necessarily Involves the complete elimination of all disease-related signs, symptoms, conditions, or disorders.
本发明所述的“预防”表示为了阻止或延迟疾病或病症或症状在机体内的发生而实施的方式。"Prevention" as used in the present invention means a method implemented to prevent or delay the occurrence of a disease, illness or symptom in the body.
本发明所述“有效量”是指在以单个或多个剂量给予至受试者或其细胞或器官之后提供所希望的效果(例如治疗、预防或抑制TUBB2表达)的本发明的产品的量或剂量。The "effective amount" of the present invention refers to the amount of the product of the present invention that provides the desired effect (such as treatment, prevention or inhibition of TUBB2 expression) after administration to a subject or its cells or organs in single or multiple doses. or dose.
本发明所述“脂质”是指一组有机化合物，其包括但不限于脂肪酸的酯，并且通常以难溶于水但可溶于许多有机溶剂为特征。"Lipids" as used herein refer to a group of organic compounds, including but not limited to esters of fatty acids, and are usually characterized by being poorly soluble in water but soluble in many organic solvents.
本发明所述“阳离子脂质”是指能够带正电的脂质分子。The "cationic lipid" mentioned in the present invention refers to a lipid molecule capable of being positively charged.
本发明所述“中性脂质”术语是指不带电荷的、非磷酸甘油酯的脂质分子。The term "neutral lipid" as used herein refers to uncharged, non-phosphoglyceride lipid molecules.
本发明所述“聚乙二醇脂质”是指包含脂质部分和聚乙二醇部分的分子。The "polyethylene glycol lipid" described in the present invention refers to a molecule comprising a lipid portion and a polyethylene glycol portion.
本发明所述“脂质纳米颗粒”是指具有至少一个纳米量级尺寸的颗粒，其包含至少一种脂质。"Lipid nanoparticles" as used in the present invention refer to particles with at least one nanometer scale size, which contain at least one lipid.
本发明所述“递送系统”是指调控生物活性成分在空间、时间及剂量在生物体内分布的制剂或组合物。The "delivery system" mentioned in the present invention refers to a preparation or composition that regulates the distribution of biologically active ingredients in space, time and dosage within the body.
附图说明 Description of the drawings
以下，结合附图来详细说明本发明的实施例，其中：Below, the embodiments of the present invention are described in detail with reference to the accompanying drawings, wherein:
图1：siTUBB2-112的正义链(左)和反义链(右)的质谱图。Figure 1: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-112.
图2：siTUBB2-176的正义链(左)和反义链(右)的质谱图。Figure 2: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-176.
图3：siTUBB2-184的正义链(左)和反义链(右)的质谱图。Figure 3: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-184.
图4：siTUBB2-236的正义链(左)和反义链(右)的质谱图。Figure 4: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-236.
图5：siTUBB2-333的正义链(左)和反义链(右)的质谱图。Figure 5: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-333.
图6：siTUBB2-358的正义链(左)和反义链(右)的质谱图。Figure 6: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-358.
图7：siTUBB2-373的正义链(左)和反义链(右)的质谱图。Figure 7: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-373.
图8：siTUBB2-457的正义链(左)和反义链(右)的质谱图。Figure 8: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-457.
图9：siTUBB2-460的正义链(左)和反义链(右)的质谱图。Figure 9: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-460.
图10：siTUBB2-1092的正义链(左)和反义链(右)的质谱图。Figure 10: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-1092.
图11：siTUBB2-1093的正义链(左)和反义链(右)的质谱图。Figure 11: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-1093.
图12：siTUBB2-1342的正义链(左)和反义链(右)的质谱图。Figure 12: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-1342.
图13：siTUBB2-1345的正义链(左)和反义链(右)的质谱图。FIG. 13 : Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-1345.
图14：siTUBB2-598的正义链(左)和反义链(右)的质谱图。Figure 14: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-598.
图15：siTUBB2-982的正义链(左)和反义链(右)的质谱图。Figure 15: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-982.
图16：siTUBB2-984的正义链(左)和反义链(右)的质谱图。Figure 16: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-984.
图17：siTUBB2-985的正义链(左)和反义链(右)的质谱图。Figure 17: Mass spectra of the sense strand (left) and antisense strand (right) of siTUBB2-985.
图18：不同siRNA对A375细胞中TUBB2b mRNA相对表达的影响。Figure 18: Effects of different siRNA on the relative expression of TUBB2b mRNA in A375 cells.
图19：不同siRNA对A375细胞中TUBB2mRNA相对表达的影响。Figure 19: Effects of different siRNA on the relative expression of TUBB2 mRNA in A375 cells.
图20：不同siRNA对PC-3细胞中TUBB2mRNA相对表达的影响。Figure 20: Effects of different siRNA on the relative expression of TUBB2 mRNA in PC-3 cells.
图21：不同浓度siRNA对PC-3细胞增殖抑制实验结果。Figure 21: Experimental results of inhibition of PC-3 cell proliferation by siRNA at different concentrations.
图22：不同siRNA对A375细胞增殖抑制实验结果。Figure 22: Experimental results of inhibition of A375 cell proliferation by different siRNA.
图23：LNP-siTUBB2对MDA-MB-231-GFP细胞中TUBB2mRNA相对表达水平的 影响。Figure 23: Effect of LNP-siTUBB2 on the relative expression level of TUBB2 mRNA in MDA-MB-231-GFP cells Influence.
图24：不同浓度LNP-siTUBB2对PC-3细胞中TUBB2mRNA相对表达的影响。Figure 24: Effects of different concentrations of LNP-siTUBB2 on the relative expression of TUBB2 mRNA in PC-3 cells.
图25：LNP-siTUBB2对PC-3细胞的增殖抑制作用的时间趋势。FIG. 25 : Time trend of the inhibitory effect of LNP-siTUBB2 on PC-3 cell proliferation.
图26：LNP-siTUBB2对MDA-MB-231-GFP细胞中TUBB2mRNA相对表达的影响。Figure 26: Effect of LNP-siTUBB2 on the relative expression of TUBB2 mRNA in MDA-MB-231-GFP cells.
图27：siTUBB2对细胞周期的阻滞作用。Figure 27: Cell cycle arresting effect of siTUBB2.
图28：分组及给药时间、频次。Figure 28: Grouping and administration time and frequency.
图29：不同给药方案的动物体重变化。Figure 29: Changes in animal body weight under different dosage regimens.
图30：不同给药方案的肿瘤大小。Figure 30: Tumor size with different dosing regimens.
图31：不同给药方案的抑瘤率。Figure 31: Tumor inhibition rates of different dosage regimens.
具体实施方式Detailed ways
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。The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.
本申请为了检测本发明提及的微管蛋白β2(TUBB2)对应的siRNA的抑制效果，针对TUBB2先后制备了多种siRNA。在细胞实验中，首先将制备的siRNA递送至培养的细胞中，在转染后预定时间收集细胞样品、提取RNA；将RNA反转录成cDNA后，利用实时定量PCR方法获得Ct值。通过对各组数据进行归一化处理，获得TUBB2mRNA的相对表达情况，从而比较各条siRNA对TUBB2mRNA表达的抑制效果。此外针对微管蛋白在细胞增殖生长过程中的作用机制，进行了细胞毒性实验验证细胞表型变化。进一步的，将上述设计的多种siRNA制备为脂质纳米颗粒，递送至体内，也获得了治疗肿瘤的效果。In this application, in order to detect the inhibitory effect of siRNA corresponding to tubulin β2 (TUBB2) mentioned in the present invention, a variety of siRNAs were prepared for TUBB2. In cell experiments, the prepared siRNA is first delivered to cultured cells. Cell samples are collected at a predetermined time after transfection and RNA is extracted. After the RNA is reverse transcribed into cDNA, the Ct value is obtained using real-time quantitative PCR. By normalizing each group of data, the relative expression of TUBB2mRNA was obtained, so as to compare the inhibitory effect of each siRNA on TUBB2mRNA expression. In addition, based on the mechanism of tubulin's role in cell proliferation and growth, cytotoxicity experiments were conducted to verify cell phenotypic changes. Furthermore, various siRNAs designed above were prepared into lipid nanoparticles and delivered into the body, which also achieved the effect of treating tumors.
实施例1：siRNA设计和合成Example 1: siRNA design and synthesis
针对TUBB2b序列(GenBank:NM_178012.5)设计13条siRNA序列，序列信息如 表1所示，质谱结果见图1-13。实际使用时，在19bp siRNA正义链和反义链的3’端加入2个悬挂碱基dTdT，超过19bp的siRNA保持不变。Design 13 siRNA sequences based on the TUBB2b sequence (GenBank: NM_178012.5). The sequence information is as follows: As shown in Table 1, the mass spectrometry results are shown in Figure 1-13. In actual use, 2 dangling bases dTdT are added to the 3' end of the 19bp siRNA sense strand and antisense strand, and the siRNA exceeding 19bp remains unchanged.
表1：siRNA序列

Table 1: siRNA sequences

上述13条序列由北京擎科生物科技有限公司合成，1条无关序列(siNC，正义链：5’-UUC UCC GAA CGU GUC ACG UTT(SEQ ID NO：40)，反义链：5’-ACG UGA CAC GUU CGG AGA ATT(SEQ ID NO：41))由生工生物工程(上海)股份有限公司设计合成。另外合成四条与专利WO2012177639A3中序列45402、165110/165111、165148/165149(1618322/1618323)相似的siRNA序列(分别对应TUBB2b序列的598、982、984、985位，质谱图见图14-17)用作对照，见表2。The above 13 sequences were synthesized by Beijing Qingke Biotechnology Co., Ltd., 1 irrelevant sequence (siNC, sense strand: 5'-UUC UCC GAA CGU GUC ACG UTT (SEQ ID NO: 40), antisense strand: 5'-ACG UGA CAC GUU CGG AGA ATT (SEQ ID NO: 41)) was designed and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. In addition, four siRNA sequences similar to the sequences 45402, 165110/165111, and 165148/165149 (1618322/1618323) in the patent WO2012177639A3 were synthesized (corresponding to positions 598, 982, 984, and 985 of the TUBB2b sequence respectively, and the mass spectrum is shown in Figure 14-17). For comparison, see Table 2.
表2：专利WO2012177639A3中序列
Table 2: Sequence in patent WO2012177639A3
实施例2：TUBB2基因表达抑制检测Example 2: TUBB2 gene expression inhibition detection
(1)细胞培养和转染 (1) Cell culture and transfection
采用A375细胞(人黑色素瘤细胞)和PC-3(人前列腺癌细胞)验证siRNA的抑制效果。A375细胞使用含10％FBS、100U/mL Penicillin、100μg/ml Streptomycin的DMEM培养基，PC-3细胞使用含10％FBS、100U/mL Penicillin、100μg/ml Streptomycin的F12K培养基于37℃5％CO2饱和湿度培养箱中培养。转染实验前24h，以每孔8×104细胞(A375)或1.5×105细胞(PC-3)接种于24孔板中培养过夜。A375 cells (human melanoma cells) and PC-3 (human prostate cancer cells) were used to verify the inhibitory effect of siRNA. A375 cells were cultured in DMEM containing 10% FBS, 100 U/mL Penicillin, and 100 μg/ml Streptomycin, and PC-3 cells were cultured in F12K containing 10% FBS, 100 U/mL Penicillin, and 100 μg/ml Streptomycin at 37°C with 5% CO 2. Culture in a saturated humidity incubator. 24 hours before the transfection experiment, 8×10 4 cells (A375) or 1.5×10 5 cells (PC-3) per well were seeded in a 24-well plate and cultured overnight.
(2)取22.5μL OPTI-MEM培养基稀释2.5μL siTUBB2或siNC(母液浓度为10μM)，取23.5μL OPTI-MEM培养基稀释1.5μL Lipofectamine RNAiMAX转染试剂，二者混合，轻轻摇匀，静置5min。此外，设置Blank组、NC组。(2) Take 22.5μL OPTI-MEM medium and dilute 2.5μL siTUBB2 or siNC (the mother solution concentration is 10μM), take 23.5μL OPTI-MEM medium and dilute 1.5μL Lipofectamine RNAiMAX transfection reagent, mix the two and shake gently. Let stand for 5 minutes. In addition, set the Blank group and NC group.
(3)细胞板中各孔细胞进行换液，添加无抗生素培养基，每孔体积450μL，然后各孔加入上述混合液50μL，最终每孔总体积500μL。siTUBB2(或siNC)转染浓度均为50nM。(3) Change the medium of the cells in each well of the cell plate, add antibiotic-free medium, and add a volume of 450 μL to each well. Then add 50 μL of the above mixed solution to each well, and the final total volume of each well is 500 μL. The transfection concentration of siTUBB2 (or siNC) was 50nM.
(4)转染48h后将24孔板从37℃，5％CO2的培养箱中取出，收集细胞用于提取RNA进行后续检测。(4) 48 hours after transfection, take the 24-well plate out of the incubator at 37°C and 5% CO2 , and collect the cells for RNA extraction for subsequent detection.
(5)RNA提取：采用Promega公司RNA提取试剂盒提取RNA。简言之，在PBS洗涤细胞后，加入300μL裂解液，用移液器吹打细胞，充分裂解后加入300μL稀释液，然后70℃水浴3min。14000rpm离心10min，将上清液转移至新的1.5mL Ep管中，加入300μL无水乙醇，充分混匀后，加入吸附柱中。14000rpm离心1min，弃去滤液，加入600μL洗涤液，再次离心1min。弃去滤液，每孔中加入现配的50μL DNA酶反应液，室温静置15min；加入600μL洗涤液，离心1min；弃去滤液后再加入600μL洗涤液再次离心1min；弃去滤液，离心2min，每孔中加入50μL无核酸酶水，室温静置5min，然后离心2min收集洗脱的RNA。(5) RNA extraction: RNA was extracted using Promega company's RNA extraction kit. Briefly, after washing the cells with PBS, add 300 μL of lysis solution, pipette the cells, add 300 μL of diluent after full lysis, and then incubate in a 70°C water bath for 3 min. Centrifuge at 14000 rpm for 10 min, transfer the supernatant to a new 1.5 mL Ep tube, add 300 μL of absolute ethanol, mix thoroughly, and add to the adsorption column. Centrifuge at 14000 rpm for 1 min, discard the filtrate, add 600 μL washing solution, and centrifuge again for 1 min. Discard the filtrate, add 50 μL of freshly prepared DNase reaction solution to each well, and let stand at room temperature for 15 min; add 600 μL of washing solution, and centrifuge for 1 min; discard the filtrate, then add 600 μL of washing solution and centrifuge again for 1 min; discard the filtrate, and centrifuge for 2 min. Add 50 μL nuclease-free water to each well, let stand at room temperature for 5 minutes, and then centrifuge for 2 minutes to collect the eluted RNA.
RNA质检，Nanodrop检测RNA含量和纯度，1％琼脂糖凝胶电泳检测RNA的完整性。RNA quality inspection, Nanodrop detects RNA content and purity, and 1% agarose gel electrophoresis detects RNA integrity.
(6)RNA反转录(6)RNA reverse transcription
以样品提取的总RNA作为模板，使用兰博利德反转录试剂盒，建立表3RNA反转录 体系。Use the total RNA extracted from the sample as a template and use the Lamblid Reverse Transcription Kit to establish RNA reverse transcription in Table 3 system.
表3：RNA反转录体系
Table 3: RNA reverse transcription system
以上体系混匀，离心收集液体至管底，37℃2min，55℃15min，85℃5min；产物即为cDNA模板，冰上保存备用。Mix the above system, centrifuge to collect the liquid to the bottom of the tube, 37°C for 2 minutes, 55°C for 15 minutes, and 85°C for 5 minutes; the product is the cDNA template and is stored on ice for later use.
(7)荧光定量PCR检测(7) Fluorescent quantitative PCR detection
使用TB green Premix Ex Taq II(Tli RNaseH Plus)(Takara)试剂(或其他同类试剂)，建立表4所示反应体系。Use TB green Premix Ex Taq II (Tli RNaseH Plus) (Takara) reagent (or other similar reagents) to establish the reaction system shown in Table 4.
表4：荧光定量PCR反应体系
Table 4: Fluorescence quantitative PCR reaction system
按如下程序进行PCR扩增，引物序列见表5。Perform PCR amplification according to the following procedure. The primer sequences are shown in Table 5.
95℃预变性10min，然后进入如下循环Pre-denature at 95°C for 10 minutes, then enter the following cycle
*95℃10s*95℃10s
60℃20s60℃20s
70℃10s70℃10s
读板Plate reading
返回*共进行40个循环。Return* for a total of 40 cycles.
制作熔解曲线：65℃至95℃之间，每0.5℃读板一次并停5s。Make a melting curve: between 65°C and 95°C, read the plate every 0.5°C and stop for 5 seconds.
表5：引物序列
Table 5: Primer sequences
注：TUBB2-1F和TUBB2-1R引物序列可同时以TUBB2a和TUBB2b为模板扩增，TUBB2-2F和TUBB2-2R引物序列仅可以TUBB2b为模板扩增。Note: The TUBB2-1F and TUBB2-1R primer sequences can be amplified using TUBB2a and TUBB2b as templates at the same time, while the TUBB2-2F and TUBB2-2R primer sequences can only be amplified using TUBB2b as the template.
(8)抑制效果(8) Inhibitory effect
以GAPDH为内标基因，通过ΔΔCt法计算TUBB2mRNA或TUBB2b mRNA的相对表达量；以blank组表达水平为100％，对各组mRNA表达水平进行标准化。Using GAPDH as the internal standard gene, the relative expression of TUBB2 mRNA or TUBB2b mRNA was calculated by the ΔΔCt method; with the expression level of the blank group as 100%, the mRNA expression levels of each group were standardized.
各组细胞mRNA的相对表达水平见下表6、7、8以及图18、19、20。The relative expression levels of cell mRNA in each group are shown in Tables 6, 7, and 8 below and Figures 18, 19, and 20.
结果表明，A375细胞中，实施例1中表1设计的siRNA，除siTUBB2-1093或siTUBB2-1345外，其他siRNA处理的细胞TUBB2b或TUBB2的相对表达均受到抑制。而且，除siTUBB2-236、siTUBB2-373、siTUBB2-1342外，其他均较对照组序列具有更好的抑制作用。尤其是TUBB2b(siTUBB2-112、siTUBB2-184、siTUBB2-176、siTUBB2-333)或TUBB2(siTUBB2-457、siTUBB2-1092)mRNA相对于blank组、siNC组的相对表达水平均受到显著的抑制。The results showed that in A375 cells, except for siTUBB2-1093 or siTUBB2-1345, the relative expression of TUBB2b or TUBB2 in cells treated with other siRNAs was inhibited by the siRNA designed in Table 1 in Example 1. Moreover, except for siTUBB2-236, siTUBB2-373, and siTUBB2-1342, all others had better inhibitory effects than the control sequence. In particular, the relative expression levels of TUBB2b (siTUBB2-112, siTUBB2-184, siTUBB2-176, siTUBB2-333) or TUBB2 (siTUBB2-457, siTUBB2-1092) mRNA were significantly inhibited compared to the blank group and siNC group.
表6：使用TUBB2F和TUBB2R引物对扩增结果

Table 6: Amplification results using TUBB2F and TUBB2R primer pairs

表7：使用TUBB1F和TUBB1R引物对扩增结果
Table 7: Amplification results using TUBB1F and TUBB1R primer pairs
在PC-3细胞中，实施例1中表1设计的siRNA处理的细胞中TUBB2b或TUBB2的相对表达均受到抑制。示例性的，siTUBB2-176、siTUBB2-457和siTUBB2-1092显著抑制细胞中TUBB2mRNA的表达，其中siTUBB2-457和siTUBB2-1092可抑制约90％的TUBB2mRNA表达。In PC-3 cells, the relative expression of TUBB2b or TUBB2 in cells treated with the siRNA designed in Table 1 in Example 1 was inhibited. Exemplarily, siTUBB2-176, siTUBB2-457 and siTUBB2-1092 significantly inhibit the expression of TUBB2 mRNA in cells, where siTUBB2-457 and siTUBB2-1092 can inhibit about 90% of TUBB2 mRNA expression.
表8：部分序列在PC-3细胞中对TUBB2mRNA表达的抑制效果

Table 8: Inhibitory effect of some sequences on TUBB2mRNA expression in PC-3 cells

实施例3：细胞增殖抑制实验Example 3: Cell proliferation inhibition experiment
(1)A375细胞使用含7.5％FBS的DMEM培养基(含100U/mL Penicillin，100μg/mL Streptomycin)，PC-3细胞使用含10％FBS的F12K培养基(含100U/mL Penicillin、100μg/ml Streptomycin)，于37℃5％CO2饱和湿度培养箱中培养。37℃5％CO2饱和湿度培养箱中培养。实验前24h以每孔2.5×103细胞(A375)或1×104细胞(PC-3)接种于96孔板中培养过夜。(1) A375 cells use DMEM medium containing 7.5% FBS (containing 100U/mL Penicillin, 100μg/mL Streptomycin), PC-3 cells use F12K medium containing 10% FBS (containing 100U/mL Penicillin, 100μg/ml Streptomycin), cultured in a 37°C 5% CO2 saturated humidity incubator. Culture in a 37°C 5% CO2 saturated humidity incubator. 24 hours before the experiment, 2.5×10 3 cells (A375) or 1×10 4 cells (PC-3) per well were seeded in a 96-well plate and cultured overnight.
(2)取4.5μL OPTI-MEM培养基稀释0.5μL siTUBB2或siNC(母液浓度为10μM)，取4.7μL OPTI-MEM培养基稀释0.3μL Lipofectamine RNAiMAX转染试剂，二者混合，轻轻摇匀，静置5min。测试不同浓度siTUBB2的细胞增殖抑制效果时，按终浓度计算相应用量。此外，设置Blank组、lipofectamine RNAiMAX组、lipofectamine RNAiMAX+NC组。(2) Take 4.5μL OPTI-MEM medium and dilute 0.5μL siTUBB2 or siNC (the mother solution concentration is 10μM), take 4.7μL OPTI-MEM medium and dilute 0.3μL Lipofectamine RNAiMAX transfection reagent, mix the two and shake gently. Let stand for 5 minutes. When testing the cell proliferation inhibitory effect of siTUBB2 at different concentrations, calculate the corresponding dosage based on the final concentration. In addition, set the Blank group, lipofectamine RNAiMAX group, and lipofectamine RNAiMAX+NC group.
(3)细胞板中各孔细胞进行换液，添加无抗生素培养基，每孔体积90μL，然后各孔加入上述混合液10μL，最终每孔总体积100μL。siTUBB2(或siNC)转染浓度均为50nM。(3) Change the medium of the cells in each well of the cell plate, add antibiotic-free medium, and add 90 μL of volume to each well. Then add 10 μL of the above mixed solution to each well, and the final total volume of each well is 100 μL. The transfection concentration of siTUBB2 (or siNC) was 50nM.
(4)转染后48h或72h将96孔板从37℃，5％CO2的培养箱中取出，每孔加入10μl CCK-8。继续在37℃5％CO2培养箱中培养约4h，用酶标仪检测450nm处吸光度。
细胞存活率(Viability％)＝100*A实验/A对照 (4) 48h or 72h after transfection, remove the 96-well plate from the incubator at 37°C and 5% CO2 , and add 10 μl CCK-8 to each well. Continue to culture in a 37°C 5% CO2 incubator for about 4 hours, and use a microplate reader to detect the absorbance at 450 nm.
Cell viability (Viability%) = 100*A experiment /A control
结果显示，实施例1表1中设计的siTUBB2均可以杀伤肿瘤细胞，示例性的，siTUBB2-1092处理的细胞中，细胞存活率仅为16％-18％，siTUBB2-457处理的细胞中，细胞存活率仅为40％左右，siTUBB2-176处理组的细胞存活率仅为50％-60％。因此，可以认为siTUBB2-176、siTUBB2-457和siTUBB2-1092可以明显抑制细胞增殖(见表9、10及图22、图21)。The results show that the siTUBB2 designed in Table 1 of Example 1 can kill tumor cells. For example, in the cells treated with siTUBB2-1092, the cell survival rate is only 16%-18%. In the cells treated with siTUBB2-457, the cell survival rate is only 16%-18%. The survival rate is only about 40%, and the cell survival rate in the siTUBB2-176 treatment group is only 50%-60%. Therefore, it can be considered that siTUBB2-176, siTUBB2-457 and siTUBB2-1092 can significantly inhibit cell proliferation (see Tables 9 and 10 and Figures 22 and 21).
表9：A375细胞增殖抑制实验
Table 9: A375 cell proliferation inhibition experiment
表10：PC-3细胞增殖抑制实验
Table 10: PC-3 cell proliferation inhibition experiment
实施例4：LNP制备、生物学活性和细胞增殖抑制活性评价Example 4: LNP preparation, biological activity and cell proliferation inhibitory activity evaluation
(1)LNP-siTUBB2制备(1) Preparation of LNP-siTUBB2
称取SM-102 71.02mg，加入EtOH10mL，溶解得SM-102母液； Weigh 71.02 mg of SM-102, add 10 mL of EtOH, and dissolve to obtain SM-102 mother liquor;
称取M-DMG-2000 249.5mg，无水EtOH 10mL，溶解得PEG组分母液；Weigh 249.5mg of M-DMG-2000, 10mL of anhydrous EtOH, and dissolve to obtain the PEG component mother liquor;
称取DSPC 79.02mg，无水EtOH 10mL，溶解得DSPC母液；Weigh 79.02mg of DSPC, 10mL of anhydrous EtOH, and dissolve to obtain the DSPC mother liquor;
称取胆固醇38.66mg，无水EtOH 10mL，溶解得胆固醇母液。Weigh 38.66 mg of cholesterol and dissolve it in 10 mL of anhydrous EtOH to obtain a cholesterol mother solution.
分别取SM-102母液2160μL、PEG组分母液64.8μL、DSPC母液432μL、胆固醇母液1664μL，混合均匀得LNP/EtOH溶液；Take 2160 μL of SM-102 mother liquor, 64.8 μL of PEG component mother liquor, 432 μL of DSPC mother liquor, and 1664 μL of cholesterol mother liquor respectively, and mix them evenly to obtain an LNP/EtOH solution;
取实施例1表1中的siTUBB2 150nmol，加入50mM柠檬酸缓冲液(pH＝4.0)6.5mL，得siRNA/buffer溶液；Take 150nmol of siTUBB2 in Table 1 of Example 1, add 6.5mL of 50mM citric acid buffer (pH=4.0) to obtain a siRNA/buffer solution;
微流控制备：LNP/EtOH溶液使用通路1，溶液体积2mL，流速5mL/min；siRNA/buffer溶液使用通路2，溶液体积6mL，流速15mL/min；Microfluidic preparation: LNP/EtOH solution uses channel 1, solution volume 2mL, flow rate 5mL/min; siRNA/buffer solution uses channel 2, solution volume 6mL, flow rate 15mL/min;
取微流控制备溶液4mL，使用PBS(pH＝7.4)透析16h，得到LNP-siTUBB2，siTUBB2终浓度0.2mg/mL。Take 4 mL of the microfluidic preparation solution and dialyze it with PBS (pH=7.4) for 16 hours to obtain LNP-siTUBB2. The final concentration of siTUBB2 is 0.2 mg/mL.
(2)纳米粒径仪检测LNP-siTUBB2的粒径和zeta电位(2) Detection of particle size and zeta potential of LNP-siTUBB2 using a nanoparticle size analyzer
使用NanoBrook(美国布鲁克海文公司)纳米粒径仪检测LNP-siTUBB2的粒径和zeta电位。制备好的LNP-siTUBB2 100μL使用DEPC水稀释到2mL，上机检测。每个样品检测三次，以有效粒径(D50)的均值表示粒径，示例性的结果见表11。The particle size and zeta potential of LNP-siTUBB2 were detected using NanoBrook (Brookhaven, USA) nanoparticle size analyzer. The prepared LNP-siTUBB2 100μL was diluted to 2mL with DEPC water and tested on the machine. Each sample was tested three times, and the particle size was expressed as the mean of the effective particle size (D50). The exemplary results are shown in Table 11.
表11：LNP-siTUBB2-457和LNP-siTUBBb-1092的有效粒径和zeta电位
Table 11: Effective particle size and zeta potential of LNP-siTUBB2-457 and LNP-siTUBBb-1092
结果显示，两种LNP的有效粒径都在100nm左右，zeta电位均为正电。The results show that the effective particle size of both LNPs is around 100nm, and the zeta potential is positive.
(3)LNP-siTUBB2生物学活性评价(3) Evaluation of biological activity of LNP-siTUBB2
方法同实施例2，使用人乳腺癌细胞MDA-MB-231-GFP细胞(培养基为1640培养基+10％FBS+1％青链霉素)或PC-3(培养基为F12K培养基+10％FBS+1％青链霉素)。结果表明，LNP可以将siTUBB2有效地递送到MDA-MB-231-GFP细胞或PC-3细胞中，并显著抑制TUBB2mRNA的表达(见表12、13以及图23、图24)。 The method is the same as in Example 2, using human breast cancer MDA-MB-231-GFP cells (the medium is 1640 medium + 10% FBS + 1% penicillin) or PC-3 (the medium is F12K medium + 10% FBS + 1% penicillin-streptomycin). The results show that LNP can effectively deliver siTUBB2 into MDA-MB-231-GFP cells or PC-3 cells and significantly inhibit the expression of TUBB2 mRNA (see Tables 12 and 13 and Figures 23 and 24).
表12：LNP-siTUBB2对MDA-MB-231-GFP细胞中TUBB2mRNA的表达影响
Table 12: Effect of LNP-siTUBB2 on the expression of TUBB2 mRNA in MDA-MB-231-GFP cells
表13：LNP-siTUBB2对PC-3细胞中TUBB2mRNA的表达影响
Table 13: Effect of LNP-siTUBB2 on the expression of TUBB2 mRNA in PC-3 cells
(4)LNP-siTUBB2细胞增殖抑制活性评价(4) Evaluation of LNP-siTUBB2 cell proliferation inhibitory activity
方法同实施例3，使用PC-3细胞测试。结果表明，LNP-siTUBB2可以显著抑制PC-3细胞的增殖，细胞存活率显著下降，并且可持续较长时间(见表14及图25)。 The method is the same as Example 3, using PC-3 cells for testing. The results showed that LNP-siTUBB2 could significantly inhibit the proliferation of PC-3 cells, and the cell survival rate was significantly reduced, which could last for a long time (see Table 14 and Figure 25).
表14：LNP-siTUBB2对PC-3细胞的增殖抑制作用
Table 14: Inhibitory effect of LNP-siTUBB2 on the proliferation of PC-3 cells
实施例5：siTUBB2化学修饰、生物学效果评价、细胞增殖抑制评价Example 5: Chemical modification of siTUBB2, evaluation of biological effects, and evaluation of cell proliferation inhibition
从实施例1表1中选取siTUBB2-457和siTUBB2-1092，进行化学修饰，包括甲基化、氟代、硫代磷酸化、假尿嘧啶等(见表15)。设计好修饰类型的siTUBB2由上海生工生物科技有限公司合成。siTUBB2-457 and siTUBB2-1092 were selected from Table 1 of Example 1 and chemically modified, including methylation, fluorination, thiophosphorylation, pseudouracil, etc. (see Table 15). The designed modified type of siTUBB2 was synthesized by Shanghai Sangon Biotechnology Co., Ltd.
表15：siTUBB2修饰序列修饰
Table 15: siTUBB2 modified sequence modifications
其中，m为甲基化修饰(2’-O-methyl)；f为氟代修饰(2’-deoxy-2’-fluoro)；-为硫代磷酸化修饰(Phosphorothioate，p)；Ψ为假尿嘧啶修饰(pseudouridine)。Among them, m is methylation modification (2'-O-methyl); f is fluorination modification (2'-deoxy-2'-fluoro); - is phosphorothioate modification (Phosphorothioate, p); Ψ is false Uracil modification (pseudouridine).
修饰的siTUBB2的生物学效果评价实验方法同实施例2。修饰的siTUBB2序列中，siTUBB2-457-4和siTUBB2-1092-4具有与未修饰siTUBB2相当的生物学效果(表16，图26)。 The experimental method for evaluating the biological effects of modified siTUBB2 is the same as in Example 2. Among the modified siTUBB2 sequences, siTUBB2-457-4 and siTUBB2-1092-4 have comparable biological effects to unmodified siTUBB2 (Table 16, Figure 26).
表16：siTUBB2修饰序列在MDA-MB-231-GFP中TUBB2mRNA相对表达的影响
Table 16: Effect of siTUBB2 modification sequence on relative expression of TUBB2 mRNA in MDA-MB-231-GFP
实施例6：细胞周期阻滞实验Example 6: Cell cycle arrest experiment
按实施例2中所述方法将siTUBB2-1092转染到A375细胞中后72h，用PBS洗涤细胞，加胰酶将细胞消化下来，用带血清培养基终止消化后，300×g离心，弃上清；用PBS再洗涤2次，弃上清；用4℃预冷的70％冰乙醇重悬分散细胞，轻轻吹打混匀后，封口膜封口，4℃过夜。次日，将细胞500×g离心5min，去上清液，2ml PBS洗两次，500×g离心5min，去上清液。每管加入100μL 100μg/mL RNase A和400μl 50μg/mL PI溶液，37℃避光孵育30min；加入2ml PBS洗涤细胞，500×g离心5min弃上清；加入0.5ml PBS重悬细胞。然后进行流式细胞仪检测，计数106个细胞，在激发波长488nm出检测红色荧光，用FlowJo软件分析细胞周期时相分布。72 hours after transfecting siTUBB2-1092 into A375 cells according to the method described in Example 2, wash the cells with PBS, add trypsin to digest the cells, stop the digestion with serum-containing medium, centrifuge at 300×g, and discard the cells. Clear; wash twice with PBS, discard the supernatant; resuspend and disperse the cells in 70% ice-cold ethanol pre-cooled at 4°C, mix gently by pipetting, seal with parafilm, and store overnight at 4°C. The next day, centrifuge the cells at 500×g for 5 min, remove the supernatant, wash twice with 2 ml of PBS, centrifuge at 500×g for 5 min, and remove the supernatant. Add 100 μL of 100 μg/mL RNase A and 400 μL of 50 μg/mL PI solution to each tube, and incubate in the dark at 37°C for 30 min; add 2 ml of PBS to wash the cells, centrifuge at 500 × g for 5 min and discard the supernatant; add 0.5 ml of PBS to resuspend the cells. Then perform flow cytometry detection, count 10 6 cells, detect red fluorescence at the excitation wavelength of 488 nm, and use FlowJo software to analyze the cell cycle phase distribution.
结果显示，siTUBB2-1092可以使A375细胞发生明显的细胞周期阻滞，G0G1％期细胞比例下降，细胞被阻滞在G2M期(图27)。The results showed that siTUBB2-1092 could cause significant cell cycle arrest in A375 cells. The proportion of cells in the G0G1% phase decreased and the cells were arrested in the G2M phase (Figure 27).
实施例7：肿瘤抑制实验Example 7: Tumor Inhibition Experiment
人前列腺癌细胞系PC-3培养在含10％胎牛血清的F-12K培养液，37℃、5％CO2细胞培养箱中，每隔3至4天待细胞长满后分瓶传代，将处于对数生长期的肿瘤细胞用于体内肿瘤的接种。重悬PC-3肿瘤细胞，浓度为5×107个细胞/mL，接种于Balb/c nude小鼠的右侧胁肋部皮下，100μL/只，在肿瘤生长至平均体积为100-150mm3左右时分组给药， 共7组，每组6只，分组和给药剂量、频次按表17、图28。定期称重(图29)，检测肿瘤大小(图30)，并计算抑瘤率(TGI)(图31)。The human prostate cancer cell line PC-3 was cultured in F-12K culture medium containing 10% fetal bovine serum in a 37°C, 5% CO 2 cell culture incubator. The cells were divided into bottles and passaged every 3 to 4 days after they were full. Tumor cells in the logarithmic growth phase are used for in vivo tumor inoculation. Resuspend PC-3 tumor cells at a concentration of 5×10 7 cells/mL and inoculate them subcutaneously into the right flank of Balb/c nude mice at 100 μL/mouse until the tumor grows to an average volume of 100-150 mm 3 Give medicine in groups when left and right, There are 7 groups in total, with 6 animals in each group. The grouping, dosage and frequency are as shown in Table 17 and Figure 28. Weigh regularly (Figure 29), detect tumor size (Figure 30), and calculate the tumor inhibition rate (TGI) (Figure 31).
表17：动物分组及给药方案
Table 17: Animal grouping and dosage regimen
结果显示，截至接种后64d，仅457静脉组小鼠体重下降超过了紫杉醇组。其他组别小鼠体重下降幅度大于Vehicle组，但低于紫杉醇组。在肿瘤体积方面，1092静脉组和457瘤内组低于紫杉醇组和其他组。就肿瘤抑制指数(TGI)来说，1092静脉组和457瘤内组在停止给药20天后仍高于紫杉醇组，至实验终点(接种后64d，停止给药后27d)时分别达到42.28和39.13，紫杉醇组则降到了33.03。说明LNP-siTUBB2-457和1092可以比紫杉醇发挥更长时间的作用，可以抑制肿瘤生长。The results showed that as of 64 days after vaccination, only the weight loss of mice in the 457 intravenous group exceeded that of the paclitaxel group. The weight loss of mice in other groups was greater than that of the Vehicle group, but lower than that of the Paclitaxel group. In terms of tumor volume, the 1092 intravenous group and the 457 intratumoral group were lower than the paclitaxel group and other groups. In terms of tumor inhibition index (TGI), the 1092 intravenous group and the 457 intratumoral group were still higher than the paclitaxel group 20 days after stopping administration, reaching 42.28 and 39.13 respectively at the end of the experiment (64 days after vaccination, 27 days after stopping administration). , the paclitaxel group dropped to 33.03. This shows that LNP-siTUBB2-457 and 1092 can exert a longer-lasting effect than paclitaxel and inhibit tumor growth.
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。The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, a variety of simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.
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。 In addition, it should be noted that each of the specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner without conflict. In order to avoid unnecessary repetition, the present invention combines various possible combinations. The combination method will not be further explained.

Claims (37)

  1. 一种靶向TUBB2基因的干扰RNA，其特征在于，所述干扰RNA的靶位点序列包含SEQ ID NO：1-13中的任一个或两个以上所示的核苷酸序列。An interfering RNA targeting the TUBB2 gene, characterized in that the target site sequence of the interfering RNA includes any one or more than two nucleotide sequences shown in SEQ ID NO: 1-13.
  2. 根据权利要求1所述的干扰RNA，其特征在于，所述的干扰RNA包含siRNA、dsRNA、shRNA、aiRNA或miRNA中一种或两种以上的组合；The interfering RNA according to claim 1, characterized in that the interfering RNA includes one or a combination of two or more of siRNA, dsRNA, shRNA, aiRNA or miRNA;
    优选的，所述的干扰RNA为siRNA。Preferably, the interfering RNA is siRNA.
  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还包括悬挂碱基；优选的，所述的干扰RNA包含1-10个悬挂碱基。The interfering RNA according to claim 1 or 2, characterized in that the interfering RNA further includes dangling bases; preferably, the interfering RNA contains 1-10 dangling bases.
  4. 根据权利要求3所述的干扰RNA，其特征在于，所述的悬挂碱基位于所述的干扰RNA的正义链和/或反义链的3’末端。The interfering RNA according to claim 3, wherein the dangling base is located at the 3' end of the sense strand and/or the antisense strand of the interfering RNA.
  5. 根据权利要求3或4所述的干扰RNA，其特征在于，所述的悬挂碱基为脱氧核苷；The interfering RNA according to claim 3 or 4, characterized in that the dangling base is a deoxynucleoside;
    优选的，所述的悬挂碱基为dTdT、dTdC或dUdU。Preferably, the dangling base is dTdT, dTdC or dUdU.
  6. 根据权利要求1-5任一所述的干扰RNA，其特征在于，所述的干扰RNA包含正义链和/或反义链；The interfering RNA according to any one of claims 1 to 5, characterized in that the interfering RNA contains a sense strand and/or an antisense strand;
    优选的，所述的正义链包含SEQ ID NO：14-26中的任一个或两个以上所示的核苷酸序列；Preferably, the sense strand includes any one or more than two nucleotide sequences shown in SEQ ID NO: 14-26;
    优选的，所述的反义链包含SEQ ID NO：27-39中的任一个或两个以上所示的核苷酸序列。Preferably, the antisense strand includes any one or more than two nucleotide sequences shown in SEQ ID NO: 27-39.
  7. 根据权利要求1-6任一所述的干扰RNA，其特征在于，所述干扰RNA还包括至少一种修饰；所述的修饰包括在碱基、糖环和/或磷酸骨架上进行的修饰；The interfering RNA according to any one of claims 1 to 6, characterized in that the interfering RNA further includes at least one modification; the modification includes modifications on bases, sugar rings and/or phosphate backbones;
    优选的，碱基的修饰包括但不限于5位嘧啶修饰、8位嘌呤修饰、假尿嘧啶修饰和/或5-溴尿嘧啶取代；Preferably, base modifications include but are not limited to 5-position pyrimidine modification, 8-position purine modification, pseudouracil modification and/or 5-bromouracil substitution;
    优选的，所述的糖环的修饰包括但不限于2'-OH被H、OZ、Z、halo、SH、SZ、NH2、NHZ、NZ2或CN基团取代，其中Z为烷基基团；Preferably, the modification of the sugar ring includes but is not limited to substitution of 2'-OH by H, OZ, Z, halo, SH, SZ, NH 2 , NHZ, NZ 2 or CN group, where Z is an alkyl group group;
    优选的，所述的磷酸骨架修饰包括但不限于硫代磷酸修饰。Preferably, the phosphate backbone modification includes but is not limited to phosphorothioate modification.
  8. 根据权利要求7所述的干扰RNA，其特征在于，所述的修饰还包括具有肌苷、辫苷、黄嘌呤、2'-甲基核糖、非天然磷酸二酯键(如甲基膦酸酯、硫代膦酸酯)和/或肽的 核苷酸。The interfering RNA according to claim 7, characterized in that the modification also includes inosine, braidin, xanthine, 2'-methylribose, non-natural phosphodiester bonds (such as methylphosphonate , thiophosphonates) and/or peptides Nucleotides.
  9. 一种权利要求1-8任一所述的干扰RNA的递送系统，其特征在于，所述的递送系统包含权利要求1-8任一所述的干扰RNA和载体。A delivery system for interfering RNA according to any one of claims 1 to 8, characterized in that the delivery system contains the interfering RNA according to any one of claims 1 to 8 and a carrier.
  10. 根据权利要求9所述的递送系统，其特征在于，所述的载体为病毒载体或非病毒载体；The delivery system according to claim 9, wherein the vector is a viral vector or a non-viral vector;
    优选的，所述病毒载体包括：慢病毒载体、逆转录病毒载体、腺病毒载体、腺相关病毒载体、痘病毒载体或疱疹病毒载体中的一种或两种及以上的组合；Preferably, the viral vector includes: one or a combination of two or more of lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated virus vectors, poxvirus vectors or herpes virus vectors;
    优选的，所述非病毒载体包括：脂质体、脂质纳米颗粒、聚合物、多肽、抗体、适配体或N-乙酰半乳糖胺(GalNAc)中任一种或两种及以上的组合。Preferably, the non-viral vector includes: any one or a combination of two or more of liposomes, lipid nanoparticles, polymers, polypeptides, antibodies, aptamers or N-acetylgalactosamine (GalNAc). .
  11. 根据权利要求10所述的递送系统，其特征在于，所述脂质纳米颗粒/脂质体包括：阳离子脂质、中性脂质、聚乙二醇脂质、甾族脂质或阴离子脂质中的一种或两种以上组合。The delivery system according to claim 10, wherein the lipid nanoparticles/liposomes include: cationic lipids, neutral lipids, polyethylene glycol lipids, steroidal lipids or anionic lipids One or a combination of two or more of them.
  12. 根据权利要求11所述的递送系统，其特征在于，所述阳离子脂质包括：十八酰胺(SA)、溴化月桂基三甲基铵、溴化十六烷基三甲基铵、溴化肉豆蔻基三甲基铵、溴化二甲基二-十八铵(DDAB)、[(4-羟基丁基)氮杂二烷基]双(己烷-6，1-二基)双(2-己基癸酸酯)(ALC-0315)、1,2-二油酰基氧基-3-(三甲基铵基)丙烷(DOTAP)、1,2-二-(9Z-十八烯酰基)-3-三甲基铵-丙烷和1,2-二-十六酰基-3-三甲基铵-丙烷、3β-[N-(N',N'-二甲基氨基乙烷)-氨基甲酰基]胆固醇(DC胆固醇)、二甲基二十八烷基铵(DDA)、1,2-二肉豆蔻酰基-3-三甲基铵丙烷(DMTAP)、二棕榈酰(C16:0)三甲基铵丙烷(DPTAP)、二硬脂酰基三甲基铵丙烷(DSTAP)、N-[1-(2,3-二烯丙氧基)丙基]-N,N,N-三甲基氯化铵(DOTMA)、N,N-二油酰基-N,N-二甲基氯化铵(DODAC)、1,2-二油酰基-sn-丙三氧基-3-乙基磷酸胆碱(DOEPC)、1,2-二油酰基-3-二甲基铵丙烷(DODAP)、1,2-二亚油基氧基-3-二甲基氨基丙烷(DLinDMA)、1,2-二-十四酰基-3-二甲基铵-丙烷、1,2-二-十六酰基-3-二甲基铵-丙烷和1,2-二-十八酰基-3-二甲基铵-丙烷、1,2-二油酰基-c-(4'-三甲基铵)-丁酰基-sn-甘油(DOTB)、二-十八酰胺-丙氨酰基精胺、SAINT-2、聚阳离子脂质2,3-二油酰基氧基-N-[2(精胺-羧酰氨基)乙基]-N,N- 二甲基-1-丙铵三氟乙酸盐(DOSPA)、 (JK-0315-CA)中的一种或两种以上的组合。The delivery system according to claim 11, wherein the cationic lipid includes: stearamide (SA), lauryltrimethylammonium bromide, cetyltrimethylammonium bromide, Myristyl trimethyl ammonium, dimethyl dioctadecyl ammonium bromide (DDAB), [(4-hydroxybutyl) azadialkyl] bis (hexane-6,1-diyl) bis( 2-Hexyldecanoate) (ALC-0315), 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP), 1,2-di-(9Z-octadecenoyl )-3-trimethylammonium-propane and 1,2-di-hexadecanoyl-3-trimethylammonium-propane, 3β-[N-(N',N'-dimethylaminoethane)- Carbamoyl]cholesterol (DC cholesterol), dimethyloctadecyl ammonium (DDA), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), dipalmitoyl (C16:0 )trimethylammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP), N-[1-(2,3-diallyloxy)propyl]-N,N,N-tri Methyl ammonium chloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-propanetrioxy-3-ethyl Phosphocholine (DOEPC), 1,2-dioleyl-3-dimethylammonium propane (DODAP), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 1, 2-Ditetradecanoyl-3-dimethylammonium-propane, 1,2-di-tetradecanoyl-3-dimethylammonium-propane and 1,2-di-octadecanoyl-3-dimethyl ammonium-propane, 1,2-dioleoyl-c-(4'-trimethylammonium)-butyryl-sn-glycerol (DOTB), di-octadecanamide-alanylspermine, SAINT-2 , polycationic lipid 2,3-dioleoyloxy-N-[2(spermine-carboxamido)ethyl]-N,N- Dimethyl-1-propylammonium trifluoroacetate (DOSPA), (JK-0315-CA) or a combination of two or more.
  13. 根据权利要求11所述的递送系统，其特征在于，所述中性脂质包括：1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)(DOPG)、油酰磷脂酰胆碱(POPC)或1-棕榈酰基-2-油酰基磷脂酰乙醇胺(POPE)、二硬脂酰磷脂酰乙醇胺(DSPE)中的一种或两种以上的组合。The delivery system according to claim 11, wherein the neutral lipid includes: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl Acyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphate Ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DOPG ), one or a combination of two or more of oleoylphosphatidylcholine (POPC) or 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), distearoylphosphatidylethanolamine (DSPE).
  14. 根据权利要求11所述的递送系统，其特征在于，所述聚乙二醇脂质包括：2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺(ALC-0159)1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)](PEG-DSPE)、PEG-二甾醇基甘油(PEG-DSG)、PEG-二棕榈油基、PEG-二油基、PEG-二硬脂基、PEG-二酰基甘油酰胺(PEG-DAG)、PEG-二棕榈酰基磷脂酰乙醇胺(PEG-DPPE)或PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺(PEG-c-DMA)、 中的一种或两种以上的组合；The delivery system according to claim 11, wherein the polyethylene glycol lipid includes: 2-[(polyethylene glycol)-2000]-N,N-tetradecyl acetamide (ALC -0159) 1,2-dimyristoyl-sn-glycerylmethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[ Amino (polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacyl Glyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE) or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA), One or a combination of two or more of them;
    其中，n选自20-300的整数。Wherein, n is selected from an integer of 20-300.
  15. 根据权利要求11所述的递送系统，其特征在于，所述聚乙二醇脂质为单一分子量的聚乙二醇脂质，优选的，所述的聚乙二醇脂质包括：
    The delivery system according to claim 11, wherein the polyethylene glycol lipid is a polyethylene glycol lipid of a single molecular weight. Preferably, the polyethylene glycol lipid includes:
  16. 根据权利要求11所述的递送系统，其特征在于，所述阳离子脂质为类固醇-阳离子脂质化合物，The delivery system of claim 11, wherein the cationic lipid is a steroid-cationic lipid compound,
    所述化合物的结构为： The structure of the compound is:
    中的一种或两种以上。 one or more than two of them.
  17. 根据权利要求11所述的递送系统，其特征在于，所述阴离子脂质体包括：二油酰磷脂酰甘油或二油酰磷脂酰乙醇胺中的一种或两种以上的组合。The delivery system according to claim 11, wherein the anionic liposome includes: one or a combination of two or more of dioleoylphosphatidylglycerol or dioleoylphosphatidylethanolamine.
  18. 根据权利要求11所述的递送系统，其特征在于，所述的甾族脂质包括：燕麦甾醇、β-谷甾醇、菜子甾醇、麦角骨化醇、菜油甾醇、胆甾烷醇、胆固醇、粪甾醇、脱氢胆固醇、链甾醇、二氢麦角骨化醇、二氢胆固醇、二氢麦角甾醇、黑海甾醇、表胆甾醇、麦角甾醇、岩藻甾醇、六氢光甾醇、羟基胆固醇、羊毛甾醇、光甾醇、海藻甾醇、谷甾烷醇、谷甾醇、豆甾烷醇、豆甾醇、胆酸、甘氨胆酸、牛磺胆酸、脱氧胆酸或石胆酸中的一种或两种以上的组合。The delivery system according to claim 11, wherein the steroidal lipids include: avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, feces Sterols, dehydrocholesterol, chain sterols, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, melanosterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, lanosterol, One or more of photosterol, algasterol, sitosteranol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid or lithocholic acid The combination.
  19. 一种细胞，其特征在于，所述的细胞中包含权利要求1-8任一所述的干扰RNA或权利要求9-18任一所述的递送系统。 A cell, characterized in that the cell contains the interfering RNA described in any one of claims 1-8 or the delivery system described in any one of claims 9-18.
  20. 一种细胞的制备方法，其特征在于，所述的制备方法包括将权利要求1-8任一所述的干扰RNA或权利要求9-18任一所述的递送系统导入到细胞中。A method for preparing cells, characterized in that the preparation method comprises introducing the interfering RNA described in any one of claims 1-8 or the delivery system described in any one of claims 9-18 into cells.
  21. 一种药物或试剂盒，其特征在于，所述的药物或试剂盒包含权利要求1-8任一所述的干扰RNA、权利要求9-18任一所述的递送系统或权利要求19所述的细胞。A medicine or kit, characterized in that the medicine or kit contains the interfering RNA described in any one of claims 1-8, the delivery system described in any one of claims 9-18, or the delivery system described in claim 19 Cell.
  22. 一种抗体-核酸偶联药物，其特征在于，所述的抗体-核酸偶联药物中包含权利要求1-8任一所述的干扰RNA或权利要求9-18任一所述的递送系统。An antibody-nucleic acid conjugated drug, characterized in that the antibody-nucleic acid conjugated drug contains the interfering RNA described in any one of claims 1-8 or the delivery system described in any one of claims 9-18.
  23. 根据权利要求22所述的抗体-核酸偶联药物，其特征在于，所述的抗体-核酸偶联药物的通式(I)为：
    The antibody-nucleic acid conjugated drug according to claim 22, wherein the general formula (I) of the antibody-nucleic acid conjugated drug is:
    其中，R为权利要求1-8任一所述的干扰RNA；Wherein, R is the interfering RNA described in any one of claims 1-8;
    x1为1-144的整数；优选的，x1为1-9的整数；更优选的，x1为1-3的整数；x1 is an integer from 1 to 144; preferably, x1 is an integer from 1 to 9; more preferably, x1 is an integer from 1 to 3;
    x2为1-8的整数；优选的，x2为1-2的整数；x2 is an integer from 1 to 8; preferably, x2 is an integer from 1 to 2;
    Ab为抗体、蛋白质、多肽；Ab is an antibody, protein, or peptide;
    L为连接Ab与R之间的连接单元。L is the connecting unit connecting Ab and R.
  24. 根据权利要求23所述的抗体-核酸偶联药物，其特征在于，所述L部分具有通式(Ⅱ)结构：
    The antibody-nucleic acid conjugate drug according to claim 23, characterized in that the L portion has a general formula (II):
    其中，in,
    式Ⅱ中x3选自1-12的整数；优选为1-3；In formula II, x3 is selected from an integer of 1-12; preferably 1-3;
    式Ⅱ中x4选自1-12的整数；优选为1-3；In formula II, x4 is selected from an integer of 1-12; preferably 1-3;
    P1、P2相同或不同的聚乙二醇残基；P 1 and P 2 are the same or different polyethylene glycol residues;
    L1为连接Ab与P1之间的连接单元； L 1 is the connecting unit connecting Ab and P 1 ;
    L2为连接P2与R之间的连接单元；L 2 is the connecting unit connecting P 2 and R;
    A1为连接P1与P2之间的连接单元；A 1 is the connection unit connecting P 1 and P 2 ;
    优选的，所述P1、P2独立地选自直链、Y型、多分支的聚乙二醇残基；Preferably, the P 1 and P 2 are independently selected from linear, Y-shaped, multi-branched polyethylene glycol residues;
    优选的，当所述P1、P2为单一分子量聚乙二醇，分子量为88-4400Da，更优选的，所述P1、P2分子量为176-1056Da；Preferably, when P 1 and P 2 are single molecular weight polyethylene glycol, the molecular weight is 88-4400 Da, and more preferably, the molecular weight of P 1 and P 2 is 176-1056 Da;
    优选的，当所述P1、P2为非单一分子量聚乙二醇，分子量为1000Da-40kDa；Preferably, when P 1 and P 2 are non-single molecular weight polyethylene glycol, the molecular weight is 1000Da-40kDa;
    更优选的，所述P1、P2分子量为2000Da-10kDa。More preferably, the molecular weight of P 1 and P 2 is 2000Da-10kDa.
  25. 根据权利要求24所述的抗体-核酸偶联药物，其特征在于，所述L1为连接基团，选自直链或支链的C1-12亚烷基、C6-12亚芳基、C3-12环亚烷基、-S-、 中的一种或者两种以上基团的组合；The antibody-nucleic acid conjugated drug according to claim 24, characterized in that said L 1 is a connecting group selected from linear or branched C 1-12 alkylene and C 6-12 arylene. , C 3-12 cycloalkylene, -S-, One or a combination of two or more groups;
    所述直链或支链的C1-12链亚烷基、C6-12的亚芳基或C3-12环亚烷基上的任意的H原子被-H、-F、-Cl、-Br、-I、-O-、-S-、-SO2、-NO2、C1-12链烷基、C3-12环烷基、C6-12芳烷基、取代或未取代的杂环基或取代或未取代的杂环基烷基、 中的一种或者两种以上基团组成的组取代；Any H atom on the linear or branched C 1-12 chain alkylene group, C 6-12 arylene group or C 3-12 cycloalkylene group is replaced by -H, -F, -Cl, -Br, -I, -O-, -S-, -SO 2 , -NO 2 , C 1-12 alkyl, C 3-12 cycloalkyl, C 6-12 aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, Substituted by one type or a group consisting of two or more groups;
    所述L2为连接基团，选自直链或支链的C1-12亚烷基、C6-12亚芳基、C3-12环亚烷基、-S-、 中的一种或者两种以上基团的组合；The L 2 is a connecting group, selected from linear or branched C 1-12 alkylene, C 6-12 arylene, C 3-12 cycloalkylene, -S-, One or a combination of two or more groups;
    所述直链或支链的C1-12链亚烷基、C6-12的亚芳基或C3-12环亚烷基上的任意的H原子被-H、-F、-Cl、-Br、-I、-O-、-S-、-SO2、-NO2、C1-12链烷基、C3-12环烷基、C6-12芳烷基、取代或未取代的杂环基或取代或未取代的杂环基烷基、 中一种或者两种以上基团组成的组取代；Any H atom on the linear or branched C 1-12 chain alkylene group, C 6-12 arylene group or C 3-12 cycloalkylene group is replaced by -H, -F, -Cl, -Br, -I, -O-, -S-, -SO 2 , -NO 2 , C 1-12 alkyl, C 3-12 cycloalkyl, C 6-12 aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, Substituted by one or more groups consisting of two or more groups;
    优选的，所述L1为酰胺键、腙键以及巯基-马来酰亚胺键；Preferably, the L 1 is an amide bond, a hydrazone bond and a thiol-maleimide bond;
    更优选的，所述L1为酰胺键；More preferably, the L 1 is an amide bond;
    优选的，所述L2为二硫键以及巯基-马来酰亚胺键；Preferably, the L 2 is a disulfide bond and a thiol-maleimide bond;
    更优选的，所述L2为二硫键。More preferably, the L 2 is a disulfide bond.
  26. 根据权利要求24或25所述的抗体-核酸偶联药物，其特征在于，所述A1为连接P1与P2之间的连接基团，选自直链或支链的C1-12亚烷基、C6-12亚芳基、C3-12环亚烷基、-S-、 中的一种或者两种以上基团的组合； The antibody-nucleic acid conjugated drug according to claim 24 or 25, characterized in that said A 1 is a connecting group between P 1 and P 2 , selected from linear or branched C 1-12 Alkylene, C 6-12 arylene, C 3-12 cycloalkylene, -S-, One or a combination of two or more groups;
    所述直链或支链的C1-12链亚烷基、C6-12的亚芳基或C3-12环亚烷基上的任意的H原子被-H、-F、-Cl、-Br、-I、-O-、-S-、-SO2、-NO2、C1-12链烷基、C3-12环烷基、C6-12芳烷基、取代或未取代的杂环基或取代或未取代的杂环基烷基、 中一种或者两种以上基团组成的组取代。Any H atom on the linear or branched C 1-12 chain alkylene group, C 6-12 arylene group or C 3-12 cycloalkylene group is replaced by -H, -F, -Cl, -Br, -I, -O-, -S-, -SO 2 , -NO 2 , C 1-12 alkyl, C 3-12 cycloalkyl, C 6-12 aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, Substitute with one or more groups consisting of two or more groups.
  27. 根据权利要求23-26任一所述的抗体-核酸偶联药物，其特征在于，所述的抗体-核酸偶联药物的通式(IV)：
    The antibody-nucleic acid conjugated drug according to any one of claims 23 to 26, characterized in that the general formula (IV) of the antibody-nucleic acid conjugated drug is:
    所述L1为连接基团，选自直链或支链的C1-12亚烷基、C6-12亚芳基、C3-12环亚烷基、-S-、 中的一种或者两种以上基团的组合；The L 1 is a connecting group, selected from linear or branched C 1-12 alkylene, C 6-12 arylene, C 3-12 cycloalkylene, -S-, One or a combination of two or more groups;
    优选的，P1为相同或不同的聚乙二醇残基；Preferably, P 1 is the same or different polyethylene glycol residue;
    优选的，当所述P1为单一分子量聚乙二醇，分子量为88-4400Da，更优选的，所述P1分子量为176-1056Da；Preferably, when P 1 is a single molecular weight polyethylene glycol, the molecular weight is 88-4400 Da, and more preferably, the molecular weight of P 1 is 176-1056 Da;
    优选的，当所述P1为非单一分子量聚乙二醇，分子量为1000Da-40kDa；更优选的，所述P1分子量为2000Da-10kDa； Preferably, when P 1 is non-single molecular weight polyethylene glycol, the molecular weight is 1000Da-40kDa; more preferably, the molecular weight of P 1 is 2000Da-10kDa;
    所述L2为连接基团，选自直链或支链的C1-12亚烷基、C6-12亚芳基、C3-12环亚烷基、-S-、 中的一种或者两种以上基团的组合；The L 2 is a connecting group, selected from linear or branched C 1-12 alkylene, C 6-12 arylene, C 3-12 cycloalkylene, -S-, One or a combination of two or more groups;
    x1为1-144的整数；优选的，x1为1-9的整数；更优选的，x1为1-3的整数；x1 is an integer from 1 to 144; preferably, x1 is an integer from 1 to 9; more preferably, x1 is an integer from 1 to 3;
    x2为1-8的整数；优选的，x2为1-2的整数。x2 is an integer from 1 to 8; preferably, x2 is an integer from 1 to 2.
  28. 根据权利要求23-27任一所述的抗体-核酸偶联药物，其特征在于，其具有如下结构：
    The antibody-nucleic acid conjugate drug according to any one of claims 23 to 27, characterized in that it has the following structure:
    所述n1选自4-100的整数，优选为4-24；n1可以为定值也可以为均值。The n1 is selected from an integer of 4-100, preferably 4-24; n1 can be a fixed value or an average value.
  29. 根据权利要求23-28任一所述的抗体-核酸偶联药物，其特征在于，所述的Ab选自单克隆抗体、多克隆抗体、抗体片段、抗体融合片段；优选的，Ab为单克隆抗体；The antibody-nucleic acid coupling drug according to any one of claims 23 to 28, characterized in that the Ab is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antibody fragments, and antibody fusion fragments; preferably, the Ab is a monoclonal antibody Antibody;
    更优选的，所述Ab选自：抗HER2抗体、抗EGFR抗体、抗PMSA抗体、抗VEGFR抗体、抗CD30抗体、抗CD22抗体、抗CD56抗体、抗CD29抗体、抗GPNMB抗体、抗CD138抗体、抗CD74抗体、抗ENPP3抗体、抗Nectin-4抗体、抗EGFRⅧ抗体、抗SLC44A4抗体、抗间皮素抗体、抗ET8R抗体、抗CD37抗体、抗CEACAM5抗体、抗CD70抗体、抗MUC16抗体、抗CD79b抗体、抗MUC16抗体、抗Muc1抗体、抗CD3抗体、抗CD28抗体、抗CD38抗体、抗CD19抗体、抗PD-L1抗体或抗4-1BB抗体中的一种或两种以上。 More preferably, the Ab is selected from: anti-HER2 antibody, anti-EGFR antibody, anti-PMSA antibody, anti-VEGFR antibody, anti-CD30 antibody, anti-CD22 antibody, anti-CD56 antibody, anti-CD29 antibody, anti-GPNMB antibody, anti-CD138 antibody, Anti-CD74 antibody, anti-ENPP3 antibody, anti-Nectin-4 antibody, anti-EGFRVIII antibody, anti-SLC44A4 antibody, anti-mesothelin antibody, anti-ET8R antibody, anti-CD37 antibody, anti-CEACAM5 antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-CD79b One or more of antibodies, anti-MUC16 antibodies, anti-Muc1 antibodies, anti-CD3 antibodies, anti-CD28 antibodies, anti-CD38 antibodies, anti-CD19 antibodies, anti-PD-L1 antibodies or anti-4-1BB antibodies.
  30. 根据权利要求23-29任一所述的抗体-核酸偶联药物，其特征在于，其具有如下结构：
    The antibody-nucleic acid conjugate drug according to any one of claims 23 to 29, characterized in that it has the following structure:
    所述n1、n2独立地选自4-100的整数，优选为4-24；n1、n2可以为定值也可以为均值。The n 1 and n 2 are independently selected from integers from 4 to 100, preferably 4 to 24; n 1 and n 2 can be fixed values or average values.
  31. 一种权利要求1-8任一所述的干扰RNA、权利要求9-18任一所述的递送系统、权利要求19所述的细胞、权利要求21所述的药物或试剂盒或权利要求22-30任一所述的抗体-核酸偶联药物的应用，其特征在于，所述的应用包括：An interfering RNA according to any one of claims 1 to 8, a delivery system according to any one of claims 9 to 18, a cell according to claim 19, a medicine or kit according to claim 21, or claim 22 -30 The application of any of the antibody-nucleic acid conjugated drugs, characterized in that the application includes:
    A)在制备治疗和/或预防TUBB2表达增加或细胞过度增殖相关疾病的药物中的应用；A) Application in the preparation of drugs for the treatment and/or prevention of diseases related to increased expression of TUBB2 or excessive cell proliferation;
    B)在抑制TUBB2表达中的应用；或，B) Application in inhibiting TUBB2 expression; or,
    C)在治疗和/或预防TUBB2表达增加或细胞过度增殖相关疾病中的应用；C) Application in the treatment and/or prevention of diseases related to increased expression of TUBB2 or excessive cell proliferation;
    优选的，TUBB2表达增加或细胞过度增殖相关疾病包括肿瘤。Preferably, diseases related to increased expression of TUBB2 or excessive cell proliferation include tumors.
  32. 根据权利要求31所述的应用，其特征在于，所述的肿瘤包括消化道肿瘤、神经系统肿瘤、呼吸道肿瘤、泌尿生殖系统肿瘤、血液淋巴系统肿瘤、皮肤系统肿瘤、横纹肌肉瘤或头颈部癌；The application according to claim 31, characterized in that the tumors include digestive tract tumors, nervous system tumors, respiratory tract tumors, genitourinary system tumors, hemolymphatic system tumors, skin system tumors, rhabdomyosarcoma or head and neck cancer. ;
    优选的，消化道肿瘤包括口腔癌、舌癌、食管癌、胃癌、肝癌、胰腺癌或结肠直肠癌中的一种或两种以上；Preferably, the digestive tract tumor includes one or more of oral cancer, tongue cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer or colorectal cancer;
    优选的，神经系统肿瘤包括胶质瘤(例如神经胶质瘤)、神经上皮瘤、神经鞘瘤、星形细胞瘤、神经纤维瘤(例如神经纤维肉瘤)、室管膜瘤、成神经管细胞瘤、脑膜瘤或脑转移瘤中的一种或两种以上；Preferably, nervous system tumors include gliomas (eg, gliomas), neuroepithelialoma, schwannoma, astrocytoma, neurofibroma (eg, neurofibrosarcoma), ependymoma, medulloblastoma One or more of tumor, meningioma or brain metastasis;
    优选的，呼吸道肿瘤包括鼻咽癌、喉癌、支气管癌或肺癌中的一种或两种以上；Preferably, the respiratory tract tumors include one or more of nasopharyngeal cancer, laryngeal cancer, bronchial cancer or lung cancer;
    优选的，泌尿生殖系统肿瘤包括肾脏肿瘤、尿路系统肿瘤、前列腺癌、精囊腺肿瘤、睾丸及睾丸旁组织肿瘤、阴茎肿瘤、乳腺癌、外阴癌、阴道癌、宫颈癌、子宫癌、子宫内膜癌、卵巢癌中的一种或两种以上； Preferably, genitourinary system tumors include kidney tumors, urinary tract system tumors, prostate cancer, seminal vesicle tumors, testicular and paratesticular tissue tumors, penile tumors, breast cancer, vulvar cancer, vaginal cancer, cervical cancer, uterine cancer, intrauterine cancer One or more types of pancreatic cancer and ovarian cancer;
    优选的，血液淋巴系统肿瘤包括白血病、多发性骨髓瘤、间皮瘤、骨髓瘤或淋巴瘤(例如非霍奇金淋巴瘤、霍奇金淋巴瘤、皮肤T细胞淋巴瘤)中的一种或两种以上；Preferably, the blood lymphatic system tumor includes one of leukemia, multiple myeloma, mesothelioma, myeloma or lymphoma (such as non-Hodgkin lymphoma, Hodgkin lymphoma, cutaneous T-cell lymphoma) or Two or more types;
    更优选的，白血病包括急性淋巴细胞白血病、急性髓系白血病、慢性骨髓细胞性白血病、慢性淋巴细胞白血病或骨髓增生异常综合征中的一种或两种以上；More preferably, the leukemia includes one or more of acute lymphoblastic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia or myelodysplastic syndrome;
    优选的，皮肤系统肿瘤包括基底细胞癌、鳞状细胞癌、黑色素瘤、Paget病、卡波西肉瘤、Merkel细胞癌、不典型纤维黄瘤、附属器肿瘤或皮肤T细胞淋巴瘤(蕈状肉芽肿)中的一种或两种以上。Preferably, cutaneous system tumors include basal cell carcinoma, squamous cell carcinoma, melanoma, Paget's disease, Kaposi's sarcoma, Merkel cell carcinoma, atypical fibroxanthoma, adnexal tumors or cutaneous T-cell lymphoma (mycosis fungoides). Swelling) one or more than two.
  33. 一种抑制TUBB2表达的方法，其特征在于，所述的方法包括加入权利要求1-8任一所述的干扰RNA、权利要求9-18任一所述的递送系统、权利要求19所述的细胞、权利要求21所述的药物或试剂盒或权利要求22-30任一所述的抗体-核酸偶联药物。A method for inhibiting TUBB2 expression, characterized in that the method includes adding the interfering RNA described in any one of claims 1-8, the delivery system described in any one of claims 9-18, and the cells, the drug or kit according to claim 21, or the antibody-nucleic acid conjugated drug according to any one of claims 22-30.
  34. 一种治疗TUBB2表达增加或细胞过度增殖相关疾病的方法，其特征在于，所述的方法包括向受试者施加治疗有效量的权利要求1-8任一所述的干扰RNA、权利要求9-18任一所述的递送系统、权利要求19所述的细胞、权利要求21所述的药物或试剂盒或权利要求22-30任一所述的抗体-核酸偶联药物。A method for treating diseases related to increased expression of TUBB2 or excessive cell proliferation, characterized in that the method includes applying to the subject a therapeutically effective amount of the interfering RNA described in any one of claims 1-8, claim 9- The delivery system of any one of 18, the cell of claim 19, the drug or kit of claim 21, or the antibody-nucleic acid conjugated drug of any one of claims 22-30.
  35. 根据权利要求34所述的方法，其特征在于，所述的TUBB2表达增加或细胞过度增殖相关疾病包括肿瘤；优选的，所述的肿瘤包括消化道肿瘤、神经系统肿瘤、呼吸道肿瘤、泌尿生殖系统肿瘤、血液淋巴系统肿瘤、皮肤系统肿瘤、横纹肌肉瘤或头颈部癌；The method according to claim 34, wherein the diseases related to increased TUBB2 expression or excessive cell proliferation include tumors; preferably, the tumors include digestive tract tumors, nervous system tumors, respiratory tract tumors, and genitourinary system tumors. Tumors, tumors of the blood lymphatic system, tumors of the cutaneous system, rhabdomyosarcoma, or head and neck cancer;
    优选的，消化道肿瘤包括口腔癌、舌癌、食管癌、胃癌、肝癌、胰腺癌或结肠直肠癌中的一种或两种以上；Preferably, the digestive tract tumors include one or more of oral cancer, tongue cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer or colorectal cancer;
    优选的，神经系统肿瘤包括胶质瘤(例如神经胶质瘤)、神经上皮瘤、神经鞘瘤、星形细胞瘤、神经纤维瘤(例如神经纤维肉瘤)、室管膜瘤、成神经管细胞瘤、脑膜瘤或脑转移瘤中的一种或两种以上；Preferably, nervous system tumors include gliomas (eg, gliomas), neuroepithelialoma, schwannoma, astrocytoma, neurofibroma (eg, neurofibrosarcoma), ependymoma, medulloblastoma One or more of tumor, meningioma or brain metastasis;
    优选的，呼吸道肿瘤包括鼻咽癌、喉癌、支气管癌或肺癌中的一种或两种以上；Preferably, the respiratory tract tumors include one or more of nasopharyngeal cancer, laryngeal cancer, bronchial cancer or lung cancer;
    优选的，泌尿生殖系统肿瘤包括肾脏肿瘤、尿路系统肿瘤、前列腺癌、精囊腺肿瘤、睾丸及睾丸旁组织肿瘤、阴茎肿瘤、乳腺癌、外阴癌、阴道癌、宫颈癌、子宫癌、子宫内膜癌、卵巢癌中的一种或两种以上；Preferably, genitourinary system tumors include kidney tumors, urinary tract system tumors, prostate cancer, seminal vesicle tumors, testicular and paratesticular tissue tumors, penile tumors, breast cancer, vulvar cancer, vaginal cancer, cervical cancer, uterine cancer, intrauterine cancer One or more types of pancreatic cancer and ovarian cancer;
    优选的，血液淋巴系统肿瘤包括白血病、多发性骨髓瘤、间皮瘤、骨髓瘤或淋巴瘤(例如非霍奇金淋巴瘤、霍奇金淋巴瘤、皮肤T细胞淋巴瘤)中的一种或两种以上； Preferably, the blood lymphatic system tumor includes one of leukemia, multiple myeloma, mesothelioma, myeloma or lymphoma (such as non-Hodgkin lymphoma, Hodgkin lymphoma, cutaneous T-cell lymphoma) or Two or more types;
    更优选的，白血病包括急性淋巴细胞白血病、急性髓系白血病、慢性骨髓细胞性白血病、慢性淋巴细胞白血病或骨髓增生异常综合征中的一种或两种以上；More preferably, the leukemia includes one or more of acute lymphoblastic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia or myelodysplastic syndrome;
    优选的，皮肤系统肿瘤包括基底细胞癌、鳞状细胞癌、黑色素瘤、Paget病、卡波西肉瘤、Merkel细胞癌、不典型纤维黄瘤、附属器肿瘤或皮肤T细胞淋巴瘤(蕈状肉芽肿)中的一种或两种以上。Preferably, cutaneous system tumors include basal cell carcinoma, squamous cell carcinoma, melanoma, Paget's disease, Kaposi's sarcoma, Merkel cell carcinoma, atypical fibroxanthoma, adnexal tumors or cutaneous T-cell lymphoma (mycosis fungoides). Swelling) one or more than two.
  36. 一种治疗肿瘤的方法，其特征在于，所述的方法包括向受试者施加治疗有效量的权利要求1-8任一所述的干扰RNA、权利要求9-18任一所述的递送系统、权利要求19所述的细胞、权利要求21所述的药物或试剂盒或权利要求22-30任一所述的抗体-核酸偶联药物。A method of treating tumors, characterized in that the method includes applying to a subject a therapeutically effective amount of the interfering RNA of any one of claims 1-8 and the delivery system of any one of claims 9-18 The cell of claim 19, the drug or kit of claim 21, or the antibody-nucleic acid conjugated drug of any one of claims 22-30.
  37. 根据权利要求36所述的方法，其特征在于，所述的肿瘤包括消化道肿瘤、神经系统肿瘤、呼吸道肿瘤、泌尿生殖系统肿瘤、血液淋巴系统肿瘤、皮肤系统肿瘤、横纹肌肉瘤或头颈部癌；The method according to claim 36, wherein the tumor includes a digestive tract tumor, a nervous system tumor, a respiratory tract tumor, a genitourinary system tumor, a hemolymphatic system tumor, a cutaneous system tumor, rhabdomyosarcoma or head and neck cancer. ;
    优选的，消化道肿瘤包括口腔癌、舌癌、食管癌、胃癌、肝癌、胰腺癌或结肠直肠癌中的一种或两种以上；Preferably, the digestive tract tumors include one or more of oral cancer, tongue cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer or colorectal cancer;
    优选的，神经系统肿瘤包括胶质瘤(例如神经胶质瘤)、神经上皮瘤、神经鞘瘤、星形细胞瘤、神经纤维瘤(例如神经纤维肉瘤)、室管膜瘤、成神经管细胞瘤、脑膜瘤或脑转移瘤中的一种或两种以上；Preferably, nervous system tumors include gliomas (eg, gliomas), neuroepithelialoma, schwannoma, astrocytoma, neurofibroma (eg, neurofibrosarcoma), ependymoma, medulloblastoma One or more of tumor, meningioma or brain metastasis;
    优选的，呼吸道肿瘤包括鼻咽癌、喉癌、支气管癌或肺癌中的一种或两种以上；Preferably, the respiratory tract tumors include one or more of nasopharyngeal cancer, laryngeal cancer, bronchial cancer or lung cancer;
    优选的，泌尿生殖系统肿瘤包括肾脏肿瘤、尿路系统肿瘤、前列腺癌、精囊腺肿瘤、睾丸及睾丸旁组织肿瘤、阴茎肿瘤、乳腺癌、外阴癌、阴道癌、宫颈癌、子宫癌、子宫内膜癌、卵巢癌中的一种或两种以上；Preferably, genitourinary system tumors include kidney tumors, urinary tract system tumors, prostate cancer, seminal vesicle tumors, testicular and paratesticular tissue tumors, penile tumors, breast cancer, vulvar cancer, vaginal cancer, cervical cancer, uterine cancer, intrauterine cancer One or more types of pancreatic cancer and ovarian cancer;
    优选的，血液淋巴系统肿瘤包括白血病、多发性骨髓瘤、间皮瘤、骨髓瘤或淋巴瘤(例如非霍奇金淋巴瘤、霍奇金淋巴瘤、皮肤T细胞淋巴瘤)中的一种或两种以上；Preferably, the blood lymphatic system tumor includes one of leukemia, multiple myeloma, mesothelioma, myeloma or lymphoma (such as non-Hodgkin lymphoma, Hodgkin lymphoma, cutaneous T-cell lymphoma) or Two or more types;
    更优选的，白血病包括急性淋巴细胞白血病、急性髓系白血病、慢性骨髓细胞性白血病、慢性淋巴细胞白血病或骨髓增生异常综合征中的一种或两种以上；More preferably, the leukemia includes one or more of acute lymphoblastic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia or myelodysplastic syndrome;
    优选的，皮肤系统肿瘤包括基底细胞癌、鳞状细胞癌、黑色素瘤、Paget病、卡波西肉瘤、Merkel细胞癌、不典型纤维黄瘤、附属器肿瘤或皮肤T细胞淋巴瘤(蕈状肉芽肿)中的一种或两种以上。 Preferably, cutaneous system tumors include basal cell carcinoma, squamous cell carcinoma, melanoma, Paget's disease, Kaposi's sarcoma, Merkel cell carcinoma, atypical fibroxanthoma, adnexal tumors or cutaneous T-cell lymphoma (mycosis fungoides). Swelling) one or more than two.
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