WO2024061354A1 - 一种抑制top1基因表达的干扰rna及其应用 - Google Patents
一种抑制top1基因表达的干扰rna及其应用 Download PDFInfo
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- WO2024061354A1 WO2024061354A1 PCT/CN2023/120758 CN2023120758W WO2024061354A1 WO 2024061354 A1 WO2024061354 A1 WO 2024061354A1 CN 2023120758 W CN2023120758 W CN 2023120758W WO 2024061354 A1 WO2024061354 A1 WO 2024061354A1
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- cancer
- antibody
- interfering rna
- tumors
- top1
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Classifications
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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
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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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
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
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- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
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- A61P35/02—Antineoplastic agents specific for leukemia
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
Definitions
- the invention relates to the technical fields of molecular biology and biomedicine, and specifically relates to an interfering RNA that inhibits TOP1 expression, especially siRNA, and its application.
- Topoisomerases are essential enzymes in the cells of higher eukaryotic organisms.
- Topoisomerases I (TOP1) belong to type I topoisomerases and can form a TOP1-DNA cleavage complex (TOP1cc) with DNA to catalyze the formation of instantaneous single-strand breaks in DNA, and the process does not require ATP hydrolysis to provide energy.
- TOP1cc TOP1-DNA cleavage complex
- the shearing and reconnection of TOP1 on the phosphate backbone can help change the topological structure of DNA and maintain topological homeostasis in multiple processes such as DNA replication, transcription, repair, and recombination.
- TOP1 is one of the drug targets for treating various tumors.
- camptothecin drugs such as topotecan, irinotecan, berotecan, etc.
- ADC drug carrying camptothecin-like small molecules The mechanism of action is basically the same: during DNA replication or transcription, camptothecin can reversibly bind to TOP1cc, making the replication or transcription process unable to proceed smoothly, thereby causing DNA damage.
- camptothecin drugs have their own limitations: 1) Camptothecin needs to be combined with TOP1cc for a long time to form DNA damage; 2) Camptothecin has certain side effects, such as leukopenia, diarrhea, etc., which makes its dosage has been restricted; 3) The structural change of camptothecin will make it unable to target TOP1, but it will bind to serum albumin. In order to solve these problems, the development of camptothecin derivatives and non-camptothecin drugs is of great significance.
- Small interfering RNA is an RNA fragment about 20 nt in length. In the body, small interfering RNA can participate in the formation of the RISC complex (RNA-induced silencing complex) and target the target through complementary base pairing. specific mRNA and degrade it. Therefore, this application uses small interfering RNA to silence the expression level of TOP1.
- RISC complex RNA-induced silencing complex
- a first aspect of the invention provides an interfering RNA targeting the TOP1 gene.
- the interfering RNA inhibits the expression of TOP1.
- the target site sequence of the interfering RNA includes any one or more than two nucleotide sequences shown in SEQ ID NO: 1-15.
- the target site sequence of the interfering RNA is as shown in any one of the nucleotide sequences in SEQ ID NO: 1-15.
- the target site sequence of the interfering RNA includes any one or two or more nucleotide sequences shown in SEQ ID NO: 1, 9 or 10.
- the interfering RNA comprises one or a combination of two or more of siRNA, dsRNA, shRNA, aiRNA or miRNA.
- 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 dangling 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 antisense strand are complementary pairs.
- the sense strand contains any one or two or more cores shown in SEQ ID NO: 16-30 nucleotide sequence. Further preferably, the sense strand is as shown in any one of the nucleotide sequences in SEQ ID NO: 16-30. Further preferably, the sense strand includes any one or two or more nucleotide sequences shown in SEQ ID NO: 16, 24 or 25.
- the antisense strand includes any one or more than two nucleotide sequences shown in SEQ ID NO: 31-45. Further preferably, the antisense strand is as shown in any nucleotide sequence of SEQ ID NO: 31-45. Further preferably, the antisense strand includes any one or two or more nucleotide sequences shown in SEQ ID NO: 31, 39 or 40.
- 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 on the chemical structure 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, NH 2 , NHZ, NZ 2 or CN, where Z is an alkyl group 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 may be methylation, fluorination, thiophosphorylation, 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: 16 SEQ ID NO: 31;
- the interfering RNA includes group A), group I) and/or group J).
- the interfering RNA contains one or a combination of two or more nucleic acid sequences shown in Table 1 or Table 13.
- the interfering RNA can be prepared by any method in the prior art, such as chemical synthesis.
- 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, herpes virus vectors, etc.
- 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, preferably any value from 1:1 to 10 (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 ).
- 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
- JK-0315-CA (JK-0315-CA) or a combination of two or more.
- 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-glycerol-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl -sn-glycerol-3-phosphate-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), or distearyl One or a combination of two or more of acylphosphatidylethanolamine (DSPE), etc.
- DPPC 1,2-dipalmitoy
- 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-disterylglycerol (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 of 20-300, for example, 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 Lipids include:
- the anionic liposome includes: dioleoylphosphatidylglycerol and/or dioleoylphosphatidylethanolamine, etc.
- the steroidal lipids include: avenasterol, ⁇ -sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, chain sterol, di- Hydroergocalciferol, dihydrocholesterol, dihydroergosterol, acerol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, lanosterol, photosterol, algasterol, sitostanol , one or a combination of two or more of sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid or lithocholic acid.
- 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 may 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.
- the third aspect of the present invention provides a cell, wherein the cell comprises 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 include the above-mentioned interfering RNA and polyethylene glycol lipid compounds, cationic lipids (excluding steroid-cationic lipid compounds), steroidal lipids and Neutral lipids.
- the molar ratio of polyethylene glycol lipid compounds, cationic lipids, steroidal lipids and neutral lipids in the lipid nanoparticles 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): (35-50): (40-50): (8-15 ).
- 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 steroid-cationic lipid: second lipid: polyethylene glycol lipid in the lipid nanoparticle is (10-30): (60-80): (10-25) , for example (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, etc. agents, wetting agents, etc.
- the drug administration methods include but are not limited to oral administration, enteral administration, subcutaneous injection, intramuscular injection, intravenous injection, nasal administration, transdermal administration, subconjunctival administration, intraocular administration, orbital administration.
- 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. , creams, jelly, sprays, 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 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
- L 1 is the connecting unit connecting Ab and P 1 ;
- 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, and 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 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 one or more groups consisting of two 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 L2 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 conjugated 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 P 1 is 2000Da-10kDa.
- the L2 is a linking group selected from a linear or branched C1-12 alkylene group, a C6-12 arylene group, a C3-12 cycloalkylene group, -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.
- n1 is selected from an integer of 4-100, preferably 4-24; n1 can be a fixed value or an average value.
- 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.
- 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.
- 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.
- the TOP1 expression-related diseases are tumors with TOP1 expression or high expression.
- the diseases related to TOP1 expression are tumors marked by TOP1 expression.
- the diseases related to TOP1 expression are tumors that need to be treated by inhibiting TOP1 expression.
- the diseases related to TOP1 expression include tumors.
- the tumors include digestive system tumors (oral cancer, tongue cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, etc.), nervous system tumors (glioma (such as glioma), neuroepithelioma, neurothecoma, astrocytoma, neurofibroma (such as neurofibrosarcoma), ependymoma, medulloblastoma, meningioma, brain metastasis, etc.), respiratory tract tumors (nasopharyngeal carcinoma, laryngeal cancer, bronchial cancer, lung cancer, etc.), urinary system tumors (such as prostate cancer, renal cell carcinoma, bladder cancer, etc.) , reproductive system tumors (breast cancer, cervical cancer, ovarian cancer, placental choriocarcinoma, etc.), blood and lymphatic system tumors (multiple myelo
- the tumor is selected from digestive system tumors, skin system tumors or urinary system tumors.
- the tumor is selected from colon cancer, liver cancer, prostate cancer or melanoma.
- a ninth aspect of the present invention provides a method for inhibiting TOP1 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 TOP1 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 TOP1 expression, 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 lipid nanoparticles, The above-mentioned cells, the above-mentioned drugs or kits, or the above-mentioned antibody-nucleic acid conjugate drugs.
- diseases associated with TOP1 expression include tumors.
- the tumors include digestive system 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 tumors (such as neurofibrosarcoma), ependymoma, medulloblastoma, meningioma, brain metastases, etc.), respiratory tract tumors (nasopharyngeal cancer, laryngeal cancer, bronchial cancer, lung cancer, etc.), urinary system tumors (such as Prostate cancer, renal cell carcinoma, bladder cancer, etc.), reproductive system tumors (breast cancer, cervical cancer, ovarian cancer, placental choriocarcinoma, etc.), blood and lymphatic system tumors (multiple myelo
- 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 TOP1 expression-related diseases are tumors with TOP1 expression or high expression.
- the diseases related to TOP1 expression are tumors marked by TOP1 expression.
- the diseases related to TOP1 expression are tumors that need to be treated by inhibiting TOP1 expression.
- the tumors include digestive system 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.), urinary system tumors (such as prostate cancer, renal cell carcinoma, bladder cancer, etc.), reproductive system tumors (breast cancer, cervical cancer, ovarian cancer, placental choriocarcinoma, etc.), blood and lymphatic system tumors (Multiple myeloma, mesothelioma,
- the tumor is selected from digestive system tumors, skin system tumors or urinary system tumors.
- the tumor is selected from colon cancer, liver cancer, prostate cancer or melanoma.
- Interfering RNA includes single-stranded RNA (ssRNA, for example, mature miRNA) or double-stranded RNA.
- RNA e.g., siRNA, shRNA, aiRNA, or pre-miRNA
- dsRNA e.g., siRNA, shRNA, aiRNA, or pre-miRNA
- dsRNA e.g., siRNA, shRNA, aiRNA, or pre-miRNA
- 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 15-60nt, 15-50nt, 15-40nt, 15-30nt, 15- 25nt or 19-25nt nucleotides, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25nt 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.
- a dsRNA or precursor RNA molecule includes any precursor molecule that is processed in vivo by an endonuclease to produce active siRNA.
- shRNA is small hairpin RNA or short hairpin RNA, including short RNA sequences that create tight hairpin turns that can be used to silence gene expression through RNA interference.
- shRNA hairpin structures can be used in cells is processed into siRNA.
- miRNA miRNA (microRNA) 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 TOP1 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 Fluid, bone, bone marrow, brain, mammary gland, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gallbladder, esophagus , glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary , pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsils
- the tumors include digestive system 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 prostate cancer, renal cell carcinoma, bladder cancer, etc.), reproductive system tumors (breast cancer, cervical cancer, ovarian cancer, placental choriocarcinoma, etc.), blood and lymphatic system Tumors (multiple myeloma, mesothelioma, myelo
- 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” used in the present invention refers to the present invention that provides the desired effect (such as treatment, prevention of TOP1 expression-related diseases or inhibition of TOP1 expression) after administration to a subject or its cells or organs in single or multiple doses.
- the amount or dosage of the product refers to the present invention that provides the desired effect (such as treatment, prevention of TOP1 expression-related diseases or inhibition of TOP1 expression) after administration to a subject or its cells or organs in single or multiple doses.
- Lipids refer to a group of organic compounds, including but not limited to esters of fatty acids, and are generally characterized by being poorly soluble in water but soluble in many organic solvents.
- the "cationic lipid” described in the present invention refers to a lipid molecule that can carry a positive charge.
- neutral lipid refers to uncharged, non-phosphoglyceride lipid molecules.
- Polyethylene glycol lipid refers to a molecule containing a lipid moiety and a polyethylene glycol moiety.
- 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 siTOP1-267 sense strand (ss) and antisense strand (as).
- Figure 2 Mass spectra of siTOP1-628 sense strand (ss) and antisense strand (as).
- Figure 3 Mass spectra of siTOP1-896 sense strand (ss) and antisense strand (as).
- Figure 4 Mass spectra of siTOP1-1261 sense strand (ss) and antisense strand (as).
- Figure 5 Mass spectra of the sense strand (ss) and antisense strand (as) of siTOP1-1282.
- Figure 7 Mass spectra of siTOP1-1459 sense strand (ss) and antisense strand (as).
- Figure 8 Mass spectra of siTOP1-1530 sense strand (ss) and antisense strand (as).
- Figure 9 Mass spectra of siTOP1-1604 sense strand (ss) and antisense strand (as).
- Figure 10 Mass spectra of siTOP1-1630 sense strand (ss) and antisense strand (as).
- Figure 11 Mass spectra of siTOP1-1679 sense strand (ss) and antisense strand (as).
- Figure 12 Mass spectra of the sense strand (ss) and antisense strand (as) of siTOP1-1776.
- Figure 13 Mass spectra of siTOP1-2273 sense strand (ss) and antisense strand (as).
- Figure 14 Mass spectra of siTOP1-2585 sense strand (ss) and antisense strand (as).
- Figure 15 Mass spectra of siTOP1-2898 sense strand (ss) and antisense strand (as).
- Figure 16 Testing the inhibitory effect of siTOP1 on TOP1 mRNA in A375 cells.
- Figure 17 Inhibitory effect of siTOP1 on A375 cell proliferation.
- Figure 18A Dose dependence of siTOP1-1604 in human prostate cancer cells.
- Figure 18B Dose dependence of siTOP1-1604 in human colon cancer cells.
- FIG 19 Multimodal particle size distribution of LNP-siTOP1-1604.
- Figure 20 Testing the inhibitory effect of LNP-siTOP1-1604 on TOP1 mRNA in various tumor cells.
- Figure 21 Inhibitory effect of single transfection of LNP-siTOP1-1604 on PC-3 cell proliferation.
- Figure 22A Inhibitory effect of LNP-siTOP1-1604 on tumor volume in mice.
- Figure 22B Tumor inhibition rate at different days after LNP-siTOP1-1604 treatment.
- Figure 23A Inhibitory effect of LNP-siTOP1-1604 on HCT116 colony formation.
- FIG. 23B Inhibitory effect of LNP-siTOP1-1604 on HCT116 colony area and colony number.
- Figure 24 Inhibitory effect of siTOP1 modified sequence on TOP1mRNA.
- Example 1 siRNA design and synthesis
- TOP1 siRNA targeting topoisomerase I
- Table 1 TOP1 target sequence and siRNA sequence
- Example 2 Inhibitory effect of siTOP1 on TOP1 mRNA in human melanoma cells
- melanoma A375 cells were plated in a 24-well plate at 6 ⁇ 10 4 -8 ⁇ 10 4 cells/well in 500 ⁇ L DMEM containing 10% FBS and 1% Penicillin-Streptomycin, and placed in a 37°C, 5% CO 2 incubator for 24 hours to allow the cells to adhere. Three replicate wells were set up for each group.
- Opti-MEM medium Take 25 ⁇ L Opti-MEM medium and dilute siRNA.
- the negative control group is siNC of the same concentration (use the siNC in the reference (O Sordet et al., 2008), and the target sequence is 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 46)), take another 25 ⁇ L Opti-MEM culture medium to dilute Lipofectamine TM RNAiMAX transfection reagent (Lipo), and let it stand at room temperature for 5 minutes; mix the two and let it stand at room temperature for 5 minutes.
- Lipofectamine TM RNAiMAX transfection reagent Lipo
- RNA template 1) Use NanoDrop TM to detect the quality and concentration of RNA and configure RNA template: take 1 ⁇ g RNA, add RNase-free H 2 O to a volume of 10 ⁇ L; 70°C for 5 minutes, then ice bath for 5 minutes before use;
- the primer sequences used for detection are as follows:
- GAPDH-F TCTGACTTCAACAGCGACAC (SEQ ID NO: 47);
- GAPDH-R GCCAAATTCGTTGTCATACC (SEQ ID NO: 48);
- TOP1-F CTGTAGCCCTGTACTTCATCG (SEQ ID NO: 49);
- siRNA targeting TOP1 designed in Example 1 can inhibit the expression of TOP1.
- siTOP1-1282 inhibited 93.4%
- siTOP1-1459 90.1% inhibition
- siTOP1-1530 90.6% inhibition
- siTOP1-1604 94.0% inhibition
- siTOP1-1679 91.1% inhibition
- siTOP1-1776 91.1% inhibition
- siTOP1-2585 91.2% inhibition
- siTOP1-2898 93.0% inhibition
- Example 3 Inhibitory effect of siTOP1 on human melanoma cell proliferation
- melanoma cells A375 were plated in a 96-well plate at 2500-3000 cells/well, with a total volume of 200 ⁇ L of DMEM medium containing 7.5% FBS and 1% Penicillin-Streptomycin, and placed at 37°C. Wait for cells to adhere to the wall in a 5% CO2 incubator for 24 hours, and set 5 multiple wells in each group.
- Opti-MEM medium Take an appropriate amount of Opti-MEM medium to dilute siRNA, and another appropriate amount of Opti-MEM medium to dilute it Lipofectamine TM RNAiMAX transfection reagent (Lipo), let stand at room temperature for 5 minutes; mix the two and let stand at room temperature for 5 minutes.
- Lipofectamine TM RNAiMAX transfection reagent Lipo
- siRNA designed in Example 1 can inhibit the proliferation of A375 cells.
- exemplary results are shown in Table 5 and Figure 17.
- siTOP1-267 inhibited by approximately 42%
- siTOP1-1604 inhibited by 58%
- siTOP1 -1630 suppressed 56%.
- Example 4 Inhibitory effect of siTOP1 on TOP1 mRNA in human prostate cancer cells and human colon cancer cells
- Opti-MEM medium On the day of transfection, configure the transfection system: take an appropriate amount of Opti-MEM medium to dilute siTOP1-1604, and another appropriate amount of Opti-MEM medium to dilute Lipofectamine TM RNAiMAX transfection reagent (Lipo), and let stand at room temperature for 5 minutes; mix the two. , let stand at room temperature for 5 minutes, and then add 50 ⁇ L to each well in a 24-well cell culture plate.
- Human prostate cancer cell PC-3 was added into a 24-well cell culture plate at 4 ⁇ 10 5 cells/well, and the medium was 450 ⁇ L F12K containing 10% FBS; human colon cancer cell HCT116 was added at 3.5 ⁇ 10 5 cells/well.
- the wells were added to a 24-well cell culture plate, and the culture medium was 450 ⁇ L of IMDM containing 10% FBS, and mixed with siRNA.
- the final transfection volume was 500 ⁇ L, and the working concentrations of siRNA were 10, 1.67, 0.27, 0.046, 0.0077, 0.0013, and 0.00021nM.
- the control is Blank (equal volume Opti-MEM), transfection reagent Lipofectamine TM RNAiMAX (Lipo) and 10 nM siNC.
- the final concentration of siTOP1 is 0.1 mg/ml.
- the effective particle size of LNP-siTOP1-1604 is 102.42nm and the polydispersity coefficient is 0.064 ( Figure 19).
- human colon cancer cell HCT116, human liver cancer cell HepG2 and human prostate cancer cell PC-3 were plated in a 24-well plate at 1.25 ⁇ 10 5 -1.5 ⁇ 10 5 cells/well, and the culture medium was 500 ⁇ L.
- IMDM, MEM and F12K containing 10% FBS and 1% Penicillin-Streptomycin
- human melanoma A375 cells were plated in a 24-well plate at 6 ⁇ 10 4 -8 ⁇ 10 4 cells/well, and the culture medium was 500 ⁇ L containing 10 %FBS and 1% Penicillin-Streptomycin in DMEM.
- TOP1 in A375 was reduced by 84.7%
- TOP1 in HCT116 was reduced by 95.0%
- TOP1 in HepG2 was reduced by 93.2%
- TOP1 in PC-3 was reduced by 92.4%.
- Example 7 Inhibitory effect of LNP-siTOP1 on proliferation of human prostate cancer cells
- human prostate cancer cells PC-3 were plated in a 96-well cell culture plate at 1 ⁇ 10 4 cells/well.
- the medium was F12K containing 10% FBS and 1% Penicillin-Streptomycin. After plating, The cell culture plate was placed in a 37°C, 5% CO 2 incubator for 24 hours until the cells adhered to the wall. Each group of 96-well plate was set with 4 multiple wells.
- change to F12K medium without antibiotics containing 10% FBS
- add LNP-siTOP1-1604 diluted with Opti-MEM medium, and the siRNA working concentration is 200 nM.
- Controls were Blank (equal volume of Opti-MEM) and LNP-mock without siRNA diluted with Opti-MEM. Place the cell culture plate in a 37°C, 5% CO 2 incubator to continue culturing, and detect the cell survival rate every 72 hours according to the method in Example 3.
- the results show that LNP-siTOP1-1604 can inhibit the proliferation of PC-3.
- the number of viable cells in the LNP-siTOP1-1604 group was the lowest on the 9th day after transfection, which was reduced by 71.2% compared with the Blank group (Table 9, Figure 21) .
- Example 9 Toxicity of LNP-siTOP1 and irinotecan to human colon cancer cells
- human colon cancer cells HCT116 were plated in a 96-well cell culture plate at 5000 cells/well.
- the medium was IMDM containing 10% FBS and 1% Penicillin-Streptomycin. After plating, the cell culture plate was placed on Wait for cells to adhere to the wall in a 37°C, 5% CO 2 incubator for 24 hours, and set 3 duplicate wells in each group.
- change to antibiotic-free IMDM medium containing 10% FBS
- add LNP-siTOP1-1604 diluted with Opti-MEM medium, and siRNA working concentrations are 500, 200, 100, 50, 20, and 5nM.
- the control is Blank (equal volume of Opti-MEM), and the concentrations of positive control drug irinotecan are 500, 200, 100, 50, 20, 5, and 1 ⁇ M.
- the cell culture plate was placed in a 37°C, 5% CO 2 incubator to continue culturing for 72 hours, and the cell survival rate was detected using the method in Example 3 (Table 10).
- the results showed that the IC50 of LNP-siTOP1-1604 was 25.90nM, which was significantly lower than the IC50 of irinotecan (Table 11).
- Example 10 LNP-siTOP1 inhibits colony formation of human colon cancer cell HCT116
- human colon cancer cells HCT116 were plated in a 6-well cell culture plate at 5 ⁇ 10 5 cells/well.
- the culture medium was 2 mL of IMDM containing 10% FBS and 1% Penicillin-Streptomycin. After plating, the cells were The culture plate was placed in a 37°C, 5% CO2 incubator for 24 hours until the cells adhered.
- replace it with 1.8 mL of antibiotic-free medium (containing 10% FBS), and then add 200 ⁇ L of LNP-siTOP1-1604 diluted with Opti-MEM.
- the final transfection volume is 2 mL, and the working concentration of siRNA is 50 nM.
- control groups were Opti-MEM and LNP-mock without siRNA diluted with Opti-MEM. After continuing to culture for 24 hours, the cells were digested with 0.25% trypsin-EDTA, and re-plated on a 6-well cell culture plate at 1,000 cells/well. Each group was set up with two duplicate wells and placed in a 37°C, 5% CO2 incubator. Continue culturing for 2 weeks, changing the culture medium regularly.
- the siTOP1-1604 sequence was chemically modified, and the modified sequence is as shown in Table 13.
- the modified siTOP1 was transfected into human prostate cancer cell PC-3.
- the working concentration of siRNA was 50 nM, and the TOP1 mRNA level was detected 48 hours after transfection.
- the control was Blank (equal volume Opti-MEM) and transfection reagent Lipofectamine TM RNAiMAX (Lipo), and the positive control was unmodified siTOP1-1604.
- Experimental results showed that compared with the control group, pseudouracil-modified siTOP1 inhibited the expression of TOP1 mRNA to 4.3% (Table 14, Figure 24).
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Abstract
本发明提供了一种靶向TOP1基因的干扰RNA,该干扰RNA可以降低TOP1的表达,从而达到治疗TOP1表达相关的疾病。本发明还提供了一种包含干扰RNA的脂质纳米颗粒药物,用于治疗TOP1表达相关的疾病。本发明还提供了一种抗体-核酸偶联药物,其中包含干扰RNA,用于治疗TOP1表达相关的疾病。
Description
本发明涉及分子生物学与生物医药技术领域,具体涉及一种抑制TOP1表达的干扰RNA,特别是siRNA,及其应用。
拓扑异构酶(Topoisomerases)是高等真核生物细胞中必不可少的一种酶。拓朴异构酶I(TOP1)属于I型拓朴异构酶,可以与DNA形成TOP1-DNA切割复合体(TOP1-DNA cleavage complex,TOP1cc),催化DNA形成瞬时的单链断裂,且作用过程无需ATP水解提供能量。TOP1对磷酸骨架的剪切、重新连接作用可以在DNA复制、转录、修复、重组等多个过程中帮助改变DNA的拓朴结构,维持拓朴稳态。
TOP1是治疗多种肿瘤的药物靶点之一,目前靶向TOP1的药物分为两种:一种是喜树碱类药物,如托泊替康、伊立替康、贝罗替康等;另一种是携带喜树碱类小分子的ADC药物。其作用机制基本是相同的:在DNA复制或转录时,喜树碱可以与TOP1cc可逆地结合,使得复制或转录过程无法顺利进行下去,进而造成DNA损伤。但喜树碱类药物有其自身的局限性:1)喜树碱需要和TOP1cc结合较长时间才能形成DNA损伤;2)喜树碱具有一定的副作用,如白细胞减少、腹泻等,使得其用量受到了限制;3)喜树碱结构的变化会使其无法靶向TOP1,但会与血清白蛋白结合。为了解决这些问题,喜树碱衍生物及非喜树碱类药物的研发就具有重要意义。
有研究表明,在稳定敲低的TOP1结肠癌和乳腺癌细胞系中,TOP1的表达水平为正常的10-20%,而这些细胞均表现出染色体畸变、复制缺陷、基因表达水平改变等现象。小干扰RNA(small interfering RNA,siRNA)是一段长度约20nt左右的RNA片段,在体内,小干扰RNA可以参与形成RISC复合体(RNA-induced silencing complex),通过碱基互补配对的方式靶向到特定的mRNA并使其降解。因此,本申请采用小干扰RNA沉默TOP1的表达水平。
发明内容
本发明的第一方面,提供了一种靶向TOP1基因的干扰RNA。
所述的干扰RNA抑制TOP1的表达。
所述干扰RNA的靶位点序列包含SEQ ID NO:1-15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:1-15中的任一个核苷酸序列所示。进一步优选的,所述干扰RNA的靶位点序列包含SEQ ID NO:1、9或10中的任一个或两个以上所示的核苷酸序列。
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。
在本发明的一个具体实施方式中,所述的干扰RNA为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。
所述干扰RNA还包括悬挂碱基,以增加干扰RNA稳定性和活性。
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。
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。
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)。
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。
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。
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。
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。
优选的,所述的正义链和反义链互补配对。
优选的,所述的正义链包含SEQ ID NO:16-30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:16-30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:16、24或25中的任一个或两个以上所示的核苷酸序列。
优选的,所述的反义链包含SEQ ID NO:31-45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:31-45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:31、39或40中的任一个或两个以上所示的核苷酸序列。
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。
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。
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。
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。
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。
优选的,所述的磷酸骨架修饰包括但不限于硫代磷酸修饰。
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。
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。
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。
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。
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:
A)SEQ ID NO:1,SEQ ID NO:16,SEQ ID NO:31;
B)SEQ ID NO:2,SEQ ID NO:17,SEQ ID NO:32;
C)SEQ ID NO:3,SEQ ID NO:18,SEQ ID NO:33;
D)SEQ ID NO:4,SEQ ID NO:19,SEQ ID NO:34;
E)SEQ ID NO:5,SEQ ID NO:20,SEQ ID NO:35;
F)SEQ ID NO:6,SEQ ID NO:21,SEQ ID NO:36;
G)SEQ ID NO:7,SEQ ID NO:22,SEQ ID NO:37;
H)SEQ ID NO:8,SEQ ID NO:23,SEQ ID NO:38;
I)SEQ ID NO:9,SEQ ID NO:24,SEQ ID NO:39;
J)SEQ ID NO:10,SEQ ID NO:25,SEQ ID NO:40;
K)SEQ ID NO:11,SEQ ID NO:26,SEQ ID NO:41;
L)SEQ ID NO:12,SEQ ID NO:27,SEQ ID NO:42;
M)SEQ ID NO:13,SEQ ID NO:28,SEQ ID NO:43;
N)SEQ ID NO:14,SEQ ID NO:29,SEQ ID NO:44;
O)SEQ ID NO:15,SEQ ID NO:30,SEQ ID NO:45;
P)SEQ ID NO:9,SEQ ID NO:51,SEQ ID NO:52;
Q)SEQ ID NO:9,SEQ ID NO:53,SEQ ID NO:54;
R)SEQ ID NO:9,SEQ ID NO:55,SEQ ID NO:56。
优选的,所述的干扰RNA包含A)组、I)组和/或J)组。
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13所示的核酸序列的一种或两种以上的组合。
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。
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。
优选的,所述的递送系统还包括载体。
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。
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、疱疹病毒载体等等中的一种或两种以上。
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,优选为1:1-10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。
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)、
(SM-102)、(JK-102-CA)、
(JK-0315-CA)中的一种或两种以上的组合。
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。
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)等中的一种或两种以上的组合。
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;
其中,n选自20-300的整数,例如20、30、50、80、100、150、200、250、300等。
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:
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。
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。
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。
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)。
优选的,上述抗体可以为单链抗体(如scFv-tp、scFv-9R等)。
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。
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。
所述的细胞中TOP1的表达量被抑制。
所述的细胞可以为肿瘤细胞。
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。
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。
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。
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。
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。
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)。
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。
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。
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。
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。
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。
所述的药物还包括药学上可接受的辅料。
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。
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。
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。
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%。
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。
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。
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。
所述的抗体-核酸偶联药物的通式(I)为:
其中,R为上述的干扰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;
x2为1-8的整数;优选的,x2为1-2的整数;
Ab为抗体、蛋白质、多肽;
L为连接Ab与R之间的连接单元。
所述L部分具有通式(Ⅱ)结构:
其中,
式Ⅱ中x3选自1-12的整数;优选为1-3;
式Ⅱ中x4选自1-12的整数;优选为1-3;
P1、P2相同或不同的聚乙二醇残基;
L1为连接Ab与P1之间的连接单元;
L2为连接P2与R之间的连接单元;
A1为连接P1与P2之间的连接单元;
优选的,所述P1、P2独立的选自直链、Y型、多分支的聚乙二醇残基;
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;
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;
更优选的,所述P1、P2分子量为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;
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;
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;
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;
优选的,所述L1为酰胺键、腙键以及巯基-马来酰亚胺键;
更优选的,所述L1为酰胺键;
优选的,所述L2为二硫键以及巯基-马来酰亚胺键;
更优选的,所述L2为二硫键。
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;
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。
优选的,所述的抗体-核酸偶联药物的通式(IV):
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;
优选的,P1为相同或不同的聚乙二醇残基;
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;
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。
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;
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;
x2为1-8的整数;优选的,x2为1-2的整数。
优选的,其具有如下结构:
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。
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。
所述的抗体可以为单域抗体或单链抗体。
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。
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。
在本发明的一个具体实施方式中,其具有如下结构:
所述n1、n2独立的选自4-100的整数,优选为4-24;n1、n2可以为定值也可以为均值。
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:
A)在制备治疗和/或预防TOP1表达相关疾病的药物中的应用;
B)在抑制TOP1表达中的应用;
C)在治疗和/或预防TOP1表达相关疾病中的应用。
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。
所述的TOP1表达相关疾病为TOP1表达或高表达的肿瘤。
所述的TOP1表达相关疾病为以TOP1表达为标志的肿瘤。
所述的TOP1表达相关疾病为需要抑制TOP1表达进行治疗的肿瘤。
优选的,TOP1表达相关疾病包括肿瘤。进一步优选的,所述的肿瘤包括消化系统肿瘤(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(如前列腺癌、肾细胞癌、膀胱癌等)、生殖系统肿瘤(乳腺癌、宫颈癌、卵巢癌、胎盘绒毛癌等)、血液和淋巴系统肿瘤(多发性骨髓瘤、间皮瘤、骨髓瘤、骨髓增生异常综合征、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、急性髓性白血病、慢性淋巴细胞白血病等)、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。
进一步优选的,所述的肿瘤选自消化系统肿瘤、皮肤系统肿瘤或泌尿系统肿瘤。
在本发明的一个具体实施方式中,所述的肿瘤选自结肠癌、肝癌、前列腺癌或黑色素瘤。
本发明的第九方面,提供了一种抑制TOP1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。
优选的,所述的方法抑制细胞中TOP1表达,所述的细胞为肿瘤细胞。
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。
本发明的第十方面,提供了一种治疗TOP1表达相关疾病的方法,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。
优选的,TOP1表达相关疾病包括肿瘤。进一步优选的,所述的肿瘤包括消化系统肿瘤(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(如前列腺癌、肾细胞癌、膀胱癌等)、生殖系统肿瘤(乳腺癌、宫颈癌、卵巢癌、胎盘绒毛癌等)、血液和淋巴系统肿瘤(多发性骨髓瘤、间皮瘤、骨髓瘤、骨髓增生异常综合征、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、急性髓性白血病、慢性淋巴细胞白血病等)、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。
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。
所述的TOP1表达相关疾病为TOP1表达或高表达的肿瘤。
所述的TOP1表达相关疾病为以TOP1表达为标志的肿瘤。
所述的TOP1表达相关疾病为需要抑制TOP1表达进行治疗的肿瘤。
优选的,所述的肿瘤包括消化系统肿瘤(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(如前列腺癌、肾细胞癌、膀胱癌等)、生殖系统肿瘤(乳腺癌、宫颈癌、卵巢癌、胎盘绒毛癌等)、血液和淋巴系统肿瘤(多发性骨髓瘤、间皮瘤、骨髓瘤、骨髓增生异常综合征、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、急性髓性白血病、慢性淋巴细胞白血病等)、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。
进一步优选的,所述的肿瘤选自消化系统肿瘤、皮肤系统肿瘤或泌尿系统肿瘤。
在本发明的一个具体实施方式中,所述的肿瘤选自结肠癌、肝癌、前列腺癌或黑色素瘤。
本发明所述的“干扰RNA”,包括单链RNA(ssRNA,例如,成熟miRNA)或双链
RNA(dsRNA,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,影响mRNA的翻译)。
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15-60nt、15-50nt、15-40nt、15-30nt、15-25nt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。dsRNA或前体RNA分子包括在体内由核酸内切酶加工以产生活性siRNA的任何前体分子。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发夹结构可以在细胞内被加工为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。
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TOP1基因)表达的能力。
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。
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、上皮细胞(例如肾上皮细胞)、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、淋巴母细胞、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、乙状结肠、皮肤、小肠、软组织、脾、胃、睾丸、胸腺、甲状腺、舌、扁桃体、气管、子宫、外阴、白细胞。进一步优选的,所述的肿瘤包括消化系统肿瘤(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(如前列腺癌、肾细胞癌、膀胱癌等)、生殖系统肿瘤(乳腺癌、宫颈癌、卵巢癌、胎盘绒毛癌等)、血液和淋巴系统肿瘤(多发性骨髓瘤、间皮瘤、骨髓瘤、骨髓增生异常综合征、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、急性髓性白血病、慢性淋巴细胞白血病等)、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。
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、龙猫、松鼠)或猴等等。
本发明所述的“治疗”表示在疾病已开始发展后减缓、中断、阻止、控制、停止、减轻、或逆转一种体征、症状、失调、病症、或疾病的进展或严重性,但不一定涉及所有疾病相关体征、症状、病症、或失调的完全消除。
本发明所述的“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。
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TOP1表达相关疾病或抑制TOP1表达)的本发明的产品的量或剂量。
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。
本发明所述“阳离子脂质”是指能够带正电的脂质分子。
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。
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。
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。
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。
以下,结合附图来详细说明本发明的实施例,其中:
图1:siTOP1-267正义链(ss)和反义链(as)质谱图。
图2:siTOP1-628正义链(ss)和反义链(as)质谱图。
图3:siTOP1-896正义链(ss)和反义链(as)质谱图。
图4:siTOP1-1261正义链(ss)和反义链(as)质谱图。
图5:siTOP1-1282正义链(ss)和反义链(as)质谱图。
图6:siTOP1-1357正义链(ss)和反义链(as)质谱图。
图7:siTOP1-1459正义链(ss)和反义链(as)质谱图。
图8:siTOP1-1530正义链(ss)和反义链(as)质谱图。
图9:siTOP1-1604正义链(ss)和反义链(as)质谱图。
图10:siTOP1-1630正义链(ss)和反义链(as)质谱图。
图11:siTOP1-1679正义链(ss)和反义链(as)质谱图。
图12:siTOP1-1776正义链(ss)和反义链(as)质谱图。
图13:siTOP1-2273正义链(ss)和反义链(as)质谱图。
图14:siTOP1-2585正义链(ss)和反义链(as)质谱图。
图15:siTOP1-2898正义链(ss)和反义链(as)质谱图。
图16:A375细胞中测试siTOP1对TOP1mRNA的抑制效果。
图17:siTOP1对A375细胞增殖的抑制效果。
图18A:siTOP1-1604在人前列腺癌细胞中的剂量依赖性。
图18B:siTOP1-1604在人结肠癌细胞中的剂量依赖性。
图19:LNP-siTOP1-1604的多峰粒径分布图。
图20:多种肿瘤细胞中测试LNP-siTOP1-1604对TOP1mRNA的抑制效果。
图21:单次转染LNP-siTOP1-1604对PC-3细胞增殖的抑制效果。
图22A:LNP-siTOP1-1604对小鼠体内肿瘤体积的抑制作用。
图22B:LNP-siTOP1-1604处理后不同天数的肿瘤抑制率。
图23A:LNP-siTOP1-1604对HCT116集落形成的抑制作用。
图23B:LNP-siTOP1-1604对HCT116集落面积和集落数量的抑制作用。
图24:siTOP1修饰序列对TOP1mRNA的抑制作用。
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明的部分实施例,而不是全部。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1:siRNA设计和合成
设计合成靶向拓朴异构酶I(TOP1)的siRNA,用于降低TOP1的表达水平,治疗
TOP1表达相关疾病,并经过筛选获得表1所示序列,质谱结果见图1-15。
表1:TOP1靶序列和siRNA序列
实施例2:siTOP1对人黑色素瘤细胞中TOP1mRNA的抑制作用
1、细胞转染
1)转染前一天,将黑色素瘤细胞A375按照6×104-8×104个细胞/孔铺于24孔板中,培养基为500μL含10%FBS和1%Penicillin-Streptomycin的DMEM,置于37℃、5%CO2培养箱中24小时待细胞贴壁。每组设置3复孔。
2)转染当天更换为450μL不含抗生素的DMEM培养基(含10%FBS)。
3)取25μL Opti-MEM培养基稀释siRNA,阴性对照组为同浓度的siNC(使用参考文献(O Sordet et al.,2008)中的siNC,靶序列为5’-TTCTCCGAACGTGTCACGT-3’(SEQ ID NO:46)),另取25μL Opti-MEM培养基稀释LipofectamineTMRNAiMAX转染试剂(Lipo),室温静置5min;将二者混匀,室温静置5min。
4)每孔加入上述混合液50μL,最终转染体积为500μL,siRNA的工作浓度为50nM。
5)转染后培养48小时后,收集细胞。
2、提取RNA
1)使用EastepTM Super总RNA提取试剂盒提取RNA,加入300μL RNA裂解液和300μL RNA稀释液,混匀后转移至1.5mL离心管中。
2)14000g离心5min,小心吸取上清至新的1.5mL离心管中。
3)加入0.5倍体积的无水乙醇,颠倒混匀15-20次后转移至离心柱中,12000g离心1min,弃滤液。
4)加入600μL RNA洗液,12000g离心1min,弃滤液。
5)加入50μL DNA酶I孵育液,室温消化DNA 15min。
6)加入600μL RNA洗液,12000g离心1min,弃滤液;重复1次;
7)14000g离心2min去除残余RNA洗液,将离心柱将置于收集管中;
8)加入适量RNase-free H2O,室温静置12min,14000g离心1min,-70℃保存备用。
3、检测基因表达水平
1)使用NanoDropTM检测RNA的质量和浓度,配置RNA template:取1μg RNA,加
RNase-free H2O至体积为10μL;70℃5min,冰浴5min备用;
2)按照表2建立反应体系:
表2:反应体系
3)将以上反应体系混匀,离心收集液体至管底,25℃ 5min,42℃ 60min,72℃ 15min,产物即为cDNA;
4)按照表3配置Q-PCR体系,检测TOP1基因的水平:
表3:反应体系
以GAPDH为内参,检测所用引物序列如下:
GAPDH-F:TCTGACTTCAACAGCGACAC(SEQ ID NO:47);
GAPDH-R:GCCAAATTCGTTGTCATACC(SEQ ID NO:48);
TOP1-F:CTGTAGCCCTGTACTTCATCG(SEQ ID NO:49);
TOP1-R:CTACCACATATTCCTGACCATCC(SEQ ID NO:50)。
结果表明,在转染siRNA培养48小时后的A375细胞中,与Mock和siNC相比,实施例1设计的靶向TOP1的siRNA均可以抑制TOP1的表达。其中siTOP1-1282(抑制了
93.4%)、siTOP1-1459(抑制了90.1%)、siTOP1-1530(抑制了90.6%)、siTOP1-1604(抑制了94.0%)、siTOP1-1679(抑制了91.1%)、siTOP1-1776(抑制了92.6%)、siTOP1-2585(抑制了91.2%)和siTOP1-2898(抑制了93.0%)的抑制效果较好(表4、图16)。
表4:细胞中测试siTOP1对基因的抑制效果
实施例3:siTOP1对人黑色素瘤细胞增殖的抑制作用
1、细胞转染
1)转染前一天,将黑色素瘤细胞A375按照2500-3000个细胞/孔铺于96孔板中,总体积200μL含7.5%FBS和1%Penicillin-Streptomycin的DMEM培养基,置于37℃、5%CO2培养箱中24小时待细胞贴壁,每组设置5复孔。
2)转染当天更换为90μL不含抗生素的减血清培养基,FBS含量为7.5%。
3)取适量Opti-MEM培养基稀释siRNA,另取适量Opti-MEM培养基稀释
LipofectamineTMRNAiMAX转染试剂(Lipo),室温静置5min;将二者混匀,室温静置5min。
4)每孔加入上述混合液10μL,最终转染体积为100μL,siRNA的工作浓度为50nM。
5)在37℃、5%CO2培养箱中培养72h。
2、细胞增殖抑制实验
转染72h后,每孔加入10μL CCK-8溶液,于37℃、5%CO2培养箱中继续培养4h,使用酶标仪读取450nm处的吸光值。按照以下公式计算细胞存活率:
细胞存活率(%)=100*OD450实验组/OD450Blank
结果表明,实施例1设计的siRNA可以抑制A375细胞的增殖,与Blank相比,示例性的结果见表5和图17,siTOP1-267抑制了约42%,siTOP1-1604抑制了58%,siTOP1-1630抑制了56%。
表5:siTOP1对A375细胞增殖的抑制效果
实施例4:siTOP1对人前列腺癌细胞和人结肠癌细胞中TOP1mRNA的抑制作用
转染当天,配置转染体系:取适量Opti-MEM培养基稀释siTOP1-1604,另取适量Opti-MEM培养基稀释LipofectamineTMRNAiMAX转染试剂(Lipo),室温静置5min;将二者混匀,室温静置5min,然后加入24孔细胞培养板中,每孔50μL。将人前列腺癌细胞PC-3按照4×105个细胞/孔加入24孔细胞培养板中,培养基为450μL含10%FBS的F12K;将人结肠癌细胞HCT116按照3.5×105个细胞/孔加入24孔细胞培养板中,培养基为450μL含10%FBS的IMDM,与siRNA混匀。最终转染体积为500μL,siRNA的工作浓度为10、1.67、0.27、0.046、0.0077、0.0013、0.00021nM。对照为Blank(等体积
Opti-MEM)、转染试剂LipofectamineTMRNAiMAX(Lipo)以及10nM siNC。
转染24小时后收集细胞,按照实施例2中的方法提取细胞总RNA并检测TOP1mRNA的水平。结果表明,两种肿瘤细胞中TOP1的相对表达水平与siTOP1-1604的浓度具有剂量依赖性(表6)。使用Graphpad软件对qPCR结果进行非线性拟合,计算siTOP1的IC50,结果表明,以siNC为参照,siTOP1-1604在人前列腺癌细胞中的IC50是0.075nM,在人结肠癌细胞中的IC50是0.006nM(表7、图18A和图18B)。
表6:不同浓度siTOP1处理后TOP1的相对表达水平
表7:siTOP1-1604在PC-3和HCT116中的IC50(24h)
实施例5:LNP-siTOP1的制备
1)配制各组分母液:
称取SM-102(Jenkem)71.02mg,加入无水EtOH 10ml,溶解得SM-102母液;
称取M-DMG-2000(Jenkem)249.5mg,加入无水EtOH 10ml,溶解得PEG母液;
称取DSPC(Sigma)79.02mg,加入无水EtOH 10ml,溶解得DSPC母液;
称取胆固醇(Sigma)38.66mg,加入无水EtOH 10ml,溶解得胆固醇母液。
2)分别取SM-102母液2160μl、PEG母液64.8μl、DSPC母液432μl、胆固醇母液1664μl,混合均匀得LNP/EtOH溶液;取siTOP1(实施例1中筛选的siTOP1)100nmol,加入50mM柠檬酸缓冲液(pH=4.0)6.5ml,得siTOP1/buffer溶液;
3)微流控制备:LNP/EtOH溶液使用通路1,溶液体积2ml,流速5ml/min;siTOP1/buffer溶液使用通路2,溶液体积6ml,流速15ml/min;
取微流控制备溶液4ml,使用PBS(PH=7.4)透析16h,得到LNP-siTOP1,siTOP1终浓度0.1mg/ml。使用NanoBrook 90Plus PALS粒度仪检测有效粒径,示例性的,LNP-siTOP1-1604的有效粒径为102.42nm,多分散系数为0.064(图19)。
实施例6:体外细胞实验中测试LNP-siTOP1对基因表达的抑制
转染前一天,将人结肠癌细胞HCT116、人肝癌细胞HepG2和人前列腺癌细胞PC-3按照1.25×105-1.5×105个细胞/孔铺于24孔板中,培养基分别为500μL含10%FBS和1%Penicillin-Streptomycin的IMDM、MEM和F12K;将人黑色素瘤细胞A375按照6×104-8×104个细胞/孔铺于24孔板中,培养基为500μL含10%FBS和1%Penicillin-Streptomycin的DMEM。铺板后将细胞培养板置于37℃、5%CO2培养箱中24小时待细胞贴壁。每组设置3复孔。转染当天更换为450μL不含抗生素的培养基(含10%FBS),然后加入50μL用Opti-MEM稀释的LNP-siTOP1-1604,最终转染体积为500μL,siRNA的工作浓度为50nM。对照组为Blank(Opti-MEM)和用Opti-MEM稀释的不含siRNA的LNP-mock。转染后继续培养48小时,收集细胞,按照实施例2中的方法提取细胞总RNA并检测TOP1mRNA的水平。结果表明,实施例5制备的LNP-siTOP1-1604可以在体外实验中抑制多种肿瘤细胞中TOP1mRNA的表达水平。具体的,转染48h后,与Blank相比,A375中的TOP1降低了84.7%,HCT116中的TOP1降低了95.0%,HepG2中的TOP1降低了93.2%,PC-3中的TOP1降低了92.4%(表8、图20)。
表8:肿瘤细胞中测试LNP-siTOP1-1604对基因的抑制效果
实施例7:LNP-siTOP1对人前列腺癌细胞增殖的抑制作用
转染前一天,将人前列腺癌细胞PC-3按照1×104个细胞/孔铺板于96孔细胞培养板中,培养基为含10%FBS和1%Penicillin-Streptomycin的F12K,铺板后将细胞培养板置于37℃、5%CO2培养箱中24小时待细胞贴壁,96孔板每组设置4复孔。转染当天更换为不含抗生素的F12K培养基(含10%FBS),加入用Opti-MEM培养基稀释的LNP-siTOP1-1604,siRNA工作浓度为200nM。对照为Blank(等体积的Opti-MEM)和用Opti-MEM稀释的不含siRNA的LNP-mock。将细胞培养板置于37℃、5%CO2培养箱中继续培养,每72h按照实施例3中的方法检测一次细胞存活率。结果表明,LNP-siTOP1-1604可以抑制PC-3的增殖,转染后第9天时LNP-siTOP1-1604组的活细胞数量最低,与Blank组相比降低了71.2%(表9、图21)。
表9:单次转染LNP-siTOP1-1604对PC-3细胞增殖的抑制效果
实施例8:前列腺癌小鼠模型中LNP-siTOP1的药效学研究
体内药效学试验使用6-8周NCG雄性小鼠为研究对象,将处于对数生长期的人前列腺癌细胞PC-3接种于试验动物的右侧胁肋部皮下,在肿瘤生长至平均体积为100-150mm3
时进行分组,每组6只,分组当天记为D0。试验分组、给药剂量及频次如下:①溶媒组(Vehicle);②伊立替康组(Irinotecan),D0和D14各给药一次,30mg/kg;③LNP-siTOP1-1604组,D0、D15和D23各瘤内注射一次,3mg/kg。定期测量并记录肿瘤体积和小鼠体重。动物试验结果表明,与溶媒对照相比,LNP-siTOP1-1604对肿瘤的生长具有一定的抑制作用,D28天时TGI(肿瘤抑制率)达到28(图22A和图22B)。
实施例9:LNP-siTOP1与伊立替康对人结肠癌细胞的毒性
转染前一天,将人结肠癌细胞HCT116按照5000个细胞/孔铺板于96孔细胞培养板中,培养基为含10%FBS和1%Penicillin-Streptomycin的IMDM,铺板后将细胞培养板置于37℃、5%CO2培养箱中24小时待细胞贴壁,每组设置3复孔。转染当天更换为不含抗生素的IMDM培养基(含10%FBS),加入用Opti-MEM培养基稀释的LNP-siTOP1-1604,siRNA工作浓度为500、200、100、50、20、5nM。对照为Blank(等体积的Opti-MEM),阳性对照药伊立替康的浓度为500、200、100、50、20、5、1μM。将细胞培养板置于37℃、5%CO2培养箱中继续培养72h,用实施例3中的方法检测细胞存活率(表10)。使用Graphpad软件进行拟合,结果表明,LNP-siTOP1-1604的IC50为25.90nM,显著低于伊立替康的IC50(表11)。
表10:不同浓度的LNP-siTOP1和伊立替康对HCT116增殖的抑制效果
表11:LNP-siTOP1-1604和伊立替康在HCT116中的IC50(72h)
实施例10:LNP-siTOP1抑制人结肠癌细胞HCT116的集落形成
转染前一天,将人结肠癌细胞HCT116按照5×105个细胞/孔铺于6孔细胞培养板中,培养基为2mL含10%FBS和1%Penicillin-Streptomycin的IMDM,铺板后将细胞培养板置于37℃、5%CO2培养箱中24小时待细胞贴壁。转染当天更换为1.8mL不含抗生素的培养基(含10%FBS),然后加入200μL用Opti-MEM稀释的LNP-siTOP1-1604,最终转染体积为2mL,siRNA的工作浓度为50nM。对照组为Opti-MEM和用Opti-MEM稀释的不含siRNA的LNP-mock。继续培养24小时后,用0.25%胰蛋白酶-EDTA消化细胞,按照1000个细胞/孔重新铺于6孔细胞培养板,每组设置两复孔,置于37℃、5%CO2培养箱中继续培养2周,定期更换培养基。
实验结束后,去除培养基并用PBS清洗,加入1ml 0.1%的结晶紫染色液室温染色10min,PBS洗去浮色后用相机和成像仪拍照,使用ImageJ软件对各组集落数量及面积进行统计。结果表明,LNP-siTOP1-1604组的集落数量和集落面积明显少于对照组(表12、图23A和图23B),说明LNP-siTOP1-1604可以抑制人结肠癌细胞HCT116的生长。
表12:LNP-siTOP1-1604对HCT116的抑制作用
实施例11:siTOP1-1604修饰序列对基因的抑制作用
对siTOP1-1604序列进行化学修饰,修饰序列如下表13。
表13:siTOP1-1604修饰序列
其中,m为甲基化修饰(2’-O-methyl);f为氟化修饰(2’-deoxy-2’-fluoro);-为硫代磷酸化修饰(Phosphorothioate,p);Ψ为假尿嘧啶修饰(pseudouridine)。
按照实施例2中的方法,将修饰过的siTOP1转染入人前列腺癌细胞PC-3中,siRNA工作浓度为50nM,检测转染48h的TOP1mRNA水平。对照为Blank(等体积Opti-MEM)和转染试剂LipofectamineTMRNAiMAX(Lipo),阳性对照为未修饰的siTOP1-1604。实验结果表明,与对照组相比,假尿嘧啶修饰的siTOP1将TOP1mRNA的表达抑制到了4.3%(表14、图24)。
表14:siTOP1-1604修饰序列对基因的抑制效果
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
Claims (37)
- 一种靶向TOP1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:1-15中的任一个或两个以上所示的核苷酸序列。
- 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;优选的,所述的干扰RNA为siRNA。
- 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。
- 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。
- 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;优选的,所述的悬挂碱基为dTdT、dTdC或dUdU。
- 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;优选的,所述的正义链包含SEQ ID NO:16-30中的任一个或两个以上所示的核苷酸序列;优选的,所述的反义链包含SEQ ID NO:31-45中的任一个或两个以上所示的核苷酸序列。
- 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还包括至少一个修饰;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;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;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;优选的,所述的磷酸骨架修饰包括但不限于硫代磷酸修饰。
- 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。
- 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。
- 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;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一种或两种及以上的组合;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。
- 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。
- 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)、 (SM-102)、(JK-102-CA)、(JK-0315-CA)中的一种或两种以上的组合。
- 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)、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。
- 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;其中,n选自20-300的整数。
- 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:
- 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,所述化合物的结构为: 中的一种或两种以上。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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:
其中,R为权利要求1-8任一所述的干扰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;x2为1-8的整数;优选的,x2为1-2的整数;Ab为抗体、蛋白质、多肽;L为连接Ab与R之间的连接单元。 - 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:
其中,式Ⅱ中x3选自1-12的整数;优选为1-3;式Ⅱ中x4选自1-12的整数;优选为1-3;P1、P2相同或不同的聚乙二醇残基;L1为连接Ab与P1之间的连接单元;L2为连接P2与R之间的连接单元;A1为连接P1与P2之间的连接单元;优选的,所述P1、P2独立的选自直链、Y型、多分支的聚乙二醇残基;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;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;更优选的,所述P1、P2分子量为2000Da-10kDa。 - 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;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;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;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;优选的,所述L1为酰胺键、腙键以及巯基-马来酰亚胺键;更优选的,所述L1为酰胺键;优选的,所述L2为二硫键以及巯基-马来酰亚胺键;更优选的,所述L2为二硫键。
- 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;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。
- 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):
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;优选的,P1为相同或不同的聚乙二醇残基;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;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;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;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;x2为1-8的整数;优选的,x2为1-2的整数。 - 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:
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。 - 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;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。
- 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:
所述n1、n2独立的选自4-100的整数,优选为4-24;n1、n2可以为定值也可以为均值。 - 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:A)在制备治疗和/或预防TOP1表达相关疾病的药物中的应用;B)在抑制TOP1表达中的应用;或,C)在治疗和/或预防TOP1表达相关疾病中的应用;优选的,TOP1表达相关疾病包括肿瘤。
- 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系统肿瘤、神经系统肿瘤、呼吸道肿瘤、泌尿系统肿瘤、生殖系统肿瘤、血液和淋巴系统肿瘤、皮肤系统肿瘤、横纹肌肉瘤或头颈部癌;优选的,消化系统肿瘤包括口腔癌、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;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;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;优选的,泌尿系统肿瘤包括前列腺癌、肾细胞癌或膀胱癌中的一种或两种以上;优选的,生殖系统肿瘤包括乳腺癌、宫颈癌、卵巢癌或胎盘绒毛癌中的一种或两种以上;优选的,血液和淋巴系统肿瘤包括多发性骨髓瘤、间皮瘤、骨髓瘤、骨髓增生异常综合征、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、急性髓性白血病或慢性淋巴细胞白血病)中的一种或两种以上;优选的,皮肤系统肿瘤包括皮肤癌、表皮样癌或黑色素瘤中的一种或两种以上。
- 一种抑制TOP1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。
- 一种治疗TOP1表达相关疾病的方法,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。
- 根据权利要求34所述的方法,其特征在于,所述的TOP1表达相关疾病包括肿瘤;优选的,所述的肿瘤包括消化系统肿瘤、神经系统肿瘤、呼吸道肿瘤、泌尿系统肿瘤、生殖系统肿瘤、血液和淋巴系统肿瘤、皮肤系统肿瘤、横纹肌肉瘤或头颈部癌;优选的,消化系统肿瘤包括口腔癌、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;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;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;优选的,泌尿系统肿瘤包括前列腺癌、肾细胞癌或膀胱癌中的一种或两种以上;优选的,生殖系统肿瘤包括乳腺癌、宫颈癌、卵巢癌或胎盘绒毛癌中的一种或两种以上;优选的,血液和淋巴系统肿瘤包括多发性骨髓瘤、间皮瘤、骨髓瘤、骨髓增生异常综合征、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、急性髓性白血病或慢性淋巴细胞白血病)中的一种或两种以上;优选的,皮肤系统肿瘤包括皮肤癌、表皮样癌或黑色素瘤中的一种或两种以上。
- 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。
- 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系统肿瘤、神经系统肿瘤、呼吸道肿瘤、泌尿系统肿瘤、生殖系统肿瘤、血液和淋巴系统肿瘤、皮肤系统肿瘤、横纹肌肉瘤或头颈部癌;优选的,消化系统肿瘤包括口腔癌、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;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;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;优选的,泌尿系统肿瘤包括前列腺癌、肾细胞癌或膀胱癌中的一种或两种以上;优选的,生殖系统肿瘤包括乳腺癌、宫颈癌、卵巢癌或胎盘绒毛癌中的一种或两种以上;优选的,血液和淋巴系统肿瘤包括多发性骨髓瘤、间皮瘤、骨髓瘤、骨髓增生异常综合征、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、急性髓性白血病或慢性淋巴细胞白血病)中的一种或两种以上;优选的,皮肤系统肿瘤包括皮肤癌、表皮样癌或黑色素瘤中的一种或两种以上。
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