WO2016129531A1 - 非小細胞肺がん細胞(h1975)に結合するdnaアプタマー - Google Patents
非小細胞肺がん細胞(h1975)に結合するdnaアプタマー Download PDFInfo
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Definitions
- the present invention relates to a DNA aptamer that can specifically bind to cancer cells, particularly non-small cell lung cancer cells (H1975), and a composition containing the DNA aptamer.
- DNA is a biopolymer composed of four nucleobases: guanine (G), cytosine (C), adenine (A), and thymine (T), and plays a role mainly in the preservation, expression and propagation of genes in vivo. Yes.
- G guanine
- C cytosine
- A adenine
- T thymine
- Nucleotide sequences that have high binding affinity for low molecular weight compounds such as drugs, DNA, RNA, peptides, and proteins have been discovered and selected so far, and they have selective recognition ability for specific molecules.
- DNA is called “DNA aptamer”.
- the selection of nucleic acid aptamers such as DNA aptamers is generally based on the in vitro selection method, in particular, the method based on the in vitro evolution method (Systematic Evolution of Ligands by EXPERIMENTAL ENRICHEMENT: SELEX method).
- SELEX method is a nucleic acid molecule (single-stranded DNA, RNA) having affinity for a target substance by selecting a nucleic acid ligand (aptamer) that binds to the target substance and repeating exponential amplification by PCR multiple times. Is to get
- various improvements have been made in recent years, and aptamers can be recovered with fewer cycles.
- the method is excellent in efficiency and selectivity, and not only small molecules and proteins but also cells and tissues (exactly on the surface)
- a method for obtaining an aptamer that binds to a molecule (Cell-SELEX method; for example, Non-patent Document 3) has been reported.
- Such a DNA aptamer is similar to an antibody in that it is a biopolymer with molecular recognition ability, but is easier to synthesize and modify than an antibody; it has excellent stability against environmental changes such as heat and pH.
- aptamers can be obtained for any substance, and the target substance to be targeted is not limited; it can be amplified quickly and inexpensively by PCR and other techniques, and the cost is excellent. It has the following advantages.
- lung cancer is roughly divided into non-small cell lung cancer and small cell lung cancer, and the former accounts for 80 to 85% of all lung cancers.
- Non-small cell lung cancer is classified into squamous cell carcinoma, adenocarcinoma, large cell carcinoma, etc. according to its histological type, 2/3 of which are already unresectable at the time of discovery, and drug therapy is the center of treatment.
- Increasing the therapeutic results of advanced lung cancer that is, improving the results of chemotherapy, is essential for improving the therapeutic results of non-small cell lung cancer.
- a biomolecule that distinguishes normal cells from cancer cells and specifically binds to cancer cells is required. Therefore, there is a need for the development of new lung cancer biomarkers useful for earlier diagnosis and development of effective therapeutic agents.
- an object of the present invention is to provide a novel DNA aptamer useful for cancer basic research such as diagnosis and treatment of lung cancer, prevention of metastasis, and cancer metastasis research. .
- the present inventors have identified a nucleotide sequence having a specific binding ability for non-small cell lung cancer cells by using the Cell-SELEX method.
- the inventors have newly found that a DNA having the sequence can function as a specific aptamer for non-small cell lung cancer cells, and have completed the present invention.
- the present invention relates to detection of lung cancer cells by the above DNA aptamer, and in particular, (9) A composition for detecting lung cancer cells, comprising the DNA aptamer according to any one of (1) to (8) above; (10) A kit for detecting lung cancer cells, comprising the DNA aptamer according to any one of (1) to (8) above; (11) A method for detecting lung cancer, characterized by using the DNA aptamer according to any one of (1) to (8) above; and (12) lung DNA, lung tissue, blood, serum, wherein the DNA aptamer is used.
- the present invention relates to the use of the above DNA aptamer in pharmaceutical compositions and drug delivery systems, in particular, (14) A pharmaceutical composition for preventing or treating metastasis of lung cancer, comprising the DA aptamer according to any one of (1) to (8) above; (15) Use of the DNA aptamer according to any one of (1) to (8) above for the manufacture of a pharmaceutical composition for preventing or treating lung cancer metastasis; (16) A drug delivery system for preventing or treating metastasis of lung cancer, comprising the DNA aptamer according to any one of (1) to (8) above.
- a novel DNA aptamer that specifically binds to non-small cell lung cancer cells such lung cancer cells can be efficiently detected.
- a kit containing a DNA aptamer provided with a detection site such as a fluorescent label simple and high-throughput detection or imaging using a lung cell or tissue collected from a living body as a measurement target can be performed. Detection of such lung cancer cells makes it possible to diagnose the onset of cancer, the presence or absence of metastasis, the prognosis of cancer, and the grade of malignancy.
- the DNA aptamer of the present invention has the property of being able to specifically bind to non-small cell lung cancer cells
- the above-mentioned DNA aptamer can be used by conjugating a drug such as an anticancer agent to the DNA aptamer. Since the drug can act on the target site reliably, it can be expected to be useful as a pharmaceutical composition for preventing or treating lung cancer metastasis or a drug delivery system for such a pharmaceutical composition.
- the consensus sequence in the DNA aptamer of the present invention is a relatively short region of only about 30 bases, it is possible to suppress the labor and cost for production, and to perform desired chemical modification or the like depending on various applications. There is also an advantage that it is easy to add a further function.
- FIG. 1 is a schematic diagram showing the formation of single-stranded double-stranded DNA using magnetic particles.
- FIG. 2 is a fluorescence imaging diagram of H1975 cells by a DNA aptamer having SEQ ID NO: 1.
- FIG. 3 is a fluorescence imaging diagram of H1975 cells by a DNA aptamer having SEQ ID NO: 2.
- DNA aptamer means a single-stranded oligo DNA that can specifically recognize a target molecule or substance, and the DNA aptamer according to the present invention is specific to non-small cell lung cancer cells. It is a single-stranded oligo DNA having a function of binding to.
- non-small cell lung cancer is classified into squamous cell carcinoma (ASC), adenocarcinoma (ADC), and large cell carcinoma (LCC) mainly depending on the tissue type.
- non-limiting examples of “non-small cell lung cancer cells” herein include squamous cell carcinoma cells NCI-H226, NCI-H647; lung adenocarcinoma cells NCI-H1975, A549, LC319. , PC-3, PC-9, PC-14, A427, NCI-H1373, and LX1, which is a large cell lung cancer cell.
- the binding target of the DNA aptamer according to the present invention is preferably a lung adenocarcinoma cell, and more preferably an H1975 cell (for details of the H1975 cell, research subject name: Obioid cancer cell proliferation and stem cell differentiation) Influence, issue number 23781731, scientific research fund grant project (scientific research grant fund grant) research result report, described on May 15, 2013, etc.).
- the DNA aptamer according to the present invention has a nucleotide sequence represented by any of SEQ ID NOs: 1 and 2 shown below.
- the nucleotide sequence is written from left to right in the direction from the 5 ′ end to the 3 ′ end.
- the DNA aptamer according to the present invention has a function of specifically binding to non-small cell lung cancer cells, one or a plurality of nucleotides in SEQ ID NOs: 1 and 2 are substituted, deleted, or added. It may be an array.
- the number of nucleotides to be substituted, deleted or added is 1 to 3, more preferably 1 or 2, and still more preferably 1.
- the DNA aptamer sequence according to the present invention is 90% or more, preferably 93% or more, more preferably 96, with each of SEQ ID NOs: 1 and 2. % Or more homologous sequences (hereinafter sometimes referred to as “homologues”).
- the term “homology” refers to the generally accepted meaning in the art.
- the term is typically referred to when examined by sequence analysis programs (eg, Karlin and Altschul, 1990, PNAS 87: 2264-2268; Karlin and Altschul, 1993, PNAS 90: 5873-5877) or by visual inspection.
- sequence analysis programs eg, Karlin and Altschul, 1990, PNAS 87: 2264-2268; Karlin and Altschul, 1993, PNAS 90: 5873-5877
- universal base indicates its commonly accepted meaning in the art.
- the term generally refers to nucleotide base analogs that form base pairs with each base of standard DNA / RNA almost indistinguishably and are recognized by intracellular enzymes (see, eg, Loakes et al., 1997, J. Mol. Bio.270: 426-435).
- Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carbozamide, and nitroazole derivatives (3′-nitropyrrole, 4-nitro Indole, 5-nitroindole, and 6-nitroindole) (Loakes, 2001, Nucleic Acids Res. 29: 2437).
- the length of the DNA aptamer according to the present invention is not limited as long as it has a function of specifically binding to non-small cell lung cancer cells.
- the upper limit of the length of the DNA aptamer in the present embodiment is, for example, 200 bases or less, preferably 150 bases or less, more preferably 100 bases or less. .
- the lower limit is the number of bases in SEQ ID NOS: 1 and 2 or more, that is, 34 bases or 35 bases or more.
- the DNA aptamer is preferably single-stranded (ssDNA), but even when a double-stranded structure is partially formed by taking a hairpin loop structure, the length of the DNA aptamer is one. Calculate as the length of the chain.
- the DNA aptamer according to the present invention may be a nucleotide sequence consisting of the sequence of any of SEQ ID NOs: 1 and 2 and a primer / primer recognition sequence on the 5 ′ and 3 ′ terminal sides thereof. That is, in this case, the DNA aptamer is 5'-P 1 -XP 2 -3 ' It has the nucleotide sequence represented by these.
- X is a nucleotide sequence selected from the sequences shown in SEQ ID NOs: 1 and 2, or a sequence comprising 1 to 3 nucleotide substitutions, deletions or additions in these sequences.
- P 1 and P 2 are the first and second primer recognition sequence introduced for the PCR amplification.
- P 1 is GCC TGT TGT GAG CCT CCT (SEQ ID NO: 3) and P 2 is CGC TTA TTC TTG TCT CCC (SEQ ID NO: 4).
- the DNA aptamer according to the present invention may be chemically modified in order to increase stability in vivo.
- Non-limiting examples of such chemical modifications include chemical substitution at the sugar chain moiety (eg, 2′-0 methylation), chemical substitution at the phosphate ester moiety (eg, phosphorothioate, amino group) , Lower alkylamine groups, acetyl groups, and the like), and chemical substitution at the base moiety.
- it can have additional bases at the 5 'or 3' end. The length of the additional base is usually 5 bases or less.
- the additional base may be DNA or RNA, but if DNA is used, the stability of the aptamer may be improved in some cases.
- Examples of such an additional base sequence include ug-3 ′, uu-3 ′, tg-3 ′, tt-3 ′, ggg-3 ′, guuu-3 ′, gttt-3 ′, and ttttt-3.
- Examples of such sequences include, but are not limited to, ', uuuu-3'.
- the DNA aptamer according to the present invention can have a detection label linked to, for example, the 5 'end or the 3' end for use in the detection method of lung cancer cells described later or the detection kit.
- a detection label is preferably a fluorescent label, but a Raman label, an enzyme label or an infrared label may be used.
- a fluorescent label a fluorescent labeling agent commonly used in the art can be used.
- 6-carboxytetramethylrhodamine TAMRA TM
- fluorescein isothiocyanate FITC
- 6-carboxyfluorocein- Examples thereof include fluorophores that can be introduced by commercially available oligonucleotide solid phase synthesis services such as aminohexyl (FAM) and cyanine fluorescent dyes (Cy3, Cy5).
- FAM aminohexyl
- Cy3, Cy5 cyanine fluorescent dyes
- a quencher quenching substance
- the fluorescence is detected by separating the fluorescent substance and the quencher during the detection reaction.
- an enhancement is made by using an enhanced electric field formed by a surface plasma wave of a nanoparticle by adsorbing an organic substance, particularly a substance whose absorption band is slightly affected by the laser wavelength, to a nanometer-sized gold particle.
- Raman scattering can also be used as a labeling agent.
- a fluorescent dye is frequently used as the organic substance, but it is not particularly necessary to be particular about this, and a sufficient Raman signal can be obtained even with a dye such as crystal violet.
- enzyme labels include ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like.
- luminol, luminol derivatives, luciferin, lucigenin, etc. may be used as a labeling agent.
- DNA aptamer of the present invention can be selected and obtained using in vitro selection methods well known in the art.
- an in vitro evolution method System Evolution of Ligands by EXPERIMENTAL ENRICHEMENT: SELEX method
- SELEX method single-stranded DNA, RNA
- nucleic acid ligand (aptamer) that binds to the target substance and exponential amplification by PCR multiple times.
- a Cell-SELEX method as described in, for example, Guo, et al., Int. J. Mol. Sci., 9 (4): 668, 2008. Since this method can use the cell itself as a target, it does not require analysis of membrane proteins on the cell surface and can simultaneously select multiple aptamers that can bind to the cell surface, compared to the conventional SELEX method. And an aptamer that more specifically binds to the target cell can be selected.
- the DNA aptamer of the present invention can be selected and obtained using a method well known in the art.
- the “in vitro selection method” selects an aptamer molecule having affinity for a target molecule or cell from a pool of nucleic acid molecules (so-called DNA pool) containing a random nucleotide sequence, and determines the affinity. It is a method of eliminating molecules that do not have. By repeating the cycle of amplifying only the selected aptamer molecule by PCR or the like, and further selecting by affinity, aptamer molecules having strong binding ability can be concentrated.
- a single-stranded nucleic acid molecule containing a random nucleotide sequence (base sequence) region of about 20 to 300 bases, preferably 30 to 150, more preferably about 30 to 100 bases, for example, an oligo DNA is prepared.
- the primer recognition sequence portion may have an appropriate restriction enzyme site so that the primer portion can be excised with a restriction enzyme after PCR amplification.
- the length of the primer recognition sequence portion to be used is not particularly limited, but is about 20 to 50, preferably about 20 to 30 bases.
- the 5 ′ end may be labeled with a radiolabel, a fluorescent label, or the like.
- the nucleic acid molecule (library pool) having the random nucleotide sequence obtained above and the target cell are mixed at an appropriate concentration ratio and incubated under an appropriate condition. After incubation, the mixture is centrifuged to separate the nucleic acid molecule-target cell complex and free nucleic acid molecule. The supernatant portion of the separation solution is removed, and a cell-bound nucleic acid sequence is amplified by performing a PCR reaction using the obtained nucleic acid molecule-target cell complex. Thereafter, the nucleic acid molecule forming a complex with the target cell is made into a single strand according to a technique well known in the art.
- ssDNA having cell binding ability can be separated from the amplified nucleic acid duplex, and unnecessary coexisting substances contained in the PCR reaction solution such as DNA polymerase can be removed. Thereafter, the same operation is performed using the recovered ssDNA as a library pool.
- the obtained nucleic acid molecule can be subjected to sequence analysis by a technique well known in the art.
- DNA aptamers according to the present invention has a function to specifically bind to non-small cell lung cancer cells, of the lung cancer cells
- the composition for detection that can be suitably used for detection and contains the DNA aptamer of the present invention can also be used as a tumor marker for non-small cell lung cancer.
- the detection composition comprising the DNA aptamer of the present invention is brought into contact with a sample collected from a living body selected from the group consisting of lung cells, lung tissue, blood, serum, plasma, saliva, and sputum, Thereafter, the presence of lung cancer cells is detected by observing a response (the presence or absence of a signal) due to the binding between the sample and the DNA aptamer.
- a sample collected from a living body is a sample collected from an animal, preferably a human, and is in a form particularly if it is a sample or secretory fluid that can be secured with minimal invasiveness, an in vitro cell culture fluid component sample, or the like. It is not limited.
- the “response” for detecting the presence of lung cancer cells is preferably a fluorescence response. Therefore, as described above, the fluorescence of TAMRA TM FITC or the like is present at the 5 ′ end or 3 ′ end of the DNA aptamer. It is preferable to link a labeling agent.
- the composition for detecting lung cancer cells of the present invention can also be provided as a kit containing a DNA aptamer in order to improve its convenience and portability.
- the DNA aptamer can be provided usually in the form of an aqueous solution dissolved at an appropriate concentration, or in the form of a DNA array in which the DNA aptamer is immobilized on a solid phase carrier.
- biotin is bound to the end of a DNA aptamer to form a complex
- streptavidin is immobilized on the surface of the solid support
- the DNA aptamer is immobilized on the solid support by the interaction of biotin and streptavidin. can do.
- the kit may appropriately contain other reagents as necessary.
- additives such as a solubilizing agent, a pH adjusting agent, a buffering agent, and a tonicity agent may be used as an additive. These blending amounts can be appropriately selected by those skilled in the art.
- the present invention provides a pharmaceutical composition for preventing or treating lung cancer metastasis comprising the above DNA aptamer.
- the pharmaceutical composition can contain an effective amount of a pharmaceutical compound (active ingredient) for preventing or treating metastasis of lung cancer, and a pharmaceutically acceptable carrier.
- a pharmaceutical compound active ingredient
- this aptamer bound to gold nanoparticles can be used as a reagent for cancer thermotherapy.
- lung cancer is a non-small cell lung cancer including squamous cell carcinoma (ASC), adenocarcinoma (ADC), and large cell carcinoma (LCC), and preferably an adenocarcinomas associated with H1975 cells.
- ASC squamous cell carcinoma
- ADC adenocarcinoma
- LCC large cell carcinoma
- Methodastasis prevention or treatment can include cancer cell release, migration, metastasis, suppression of invasion or proliferation, induction of apoptosis. Suppression of metastasis means that cancer cells reach a different site from the primary lesion and prevent secondary cancer from occurring at the site.
- the active ingredient contained in the pharmaceutical composition of the present invention is not particularly limited as long as it is effective for preventing or treating lung cancer metastasis, but is preferably an anticancer agent.
- anticancer agents include alkylating agents, antimetabolites, antitumor antibiotics, chemotherapeutic agents, and other anticancer agents.
- alkylating agent include nitrogen mustard, chlorambutyl, dibromodalcitol, thiotepa, carmustine, and busulfan.
- the antimetabolite include 6-mercaptopurine, fluorouracil, tegafur, doxyfluridine, cytarabine, enocitabine, methotrexate, and the like.
- Antibiotics such as mitomycin C, bleomycin, peplomycin, doxorubicin, THP-adriamycin, actinomycin D and other anticancer agents include Amrubicin hydrochloride, irinotecan hydrochloride, ifosfamide, etoposidrastat, gefitinib, cyclophosphamide, cisplatin, trastuzumab, fluorouracil, imanitib mesylate, methotrexate, rituxan, adriamycin, carboplatin, tamoxifen, camptothecin, melphalan cyclatanase Etc.
- Amrubicin hydrochloride irinotecan hydrochloride, ifosfamide, etoposidrastat, gefitinib, cyclophosphamide, cisplatin, trastuzumab, fluorouracil, imanitib mes
- Examples of the pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention include excipients such as sucrose and starch, binders such as cellulose and methylcellulose, disintegrants such as starch and carboxymethylcellulose, and stearic acid.
- Lubricants such as magnesium and aerosil, fragrances such as citric acid and menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinylpyrrolidone, and interfaces
- examples include, but are not limited to, dispersants such as active agents, diluents such as water and physiological saline, and base waxes.
- the composition may further contain a reagent for introducing a nucleic acid.
- a reagent for introducing nucleic acid cationic lipids such as atelocollagen, liposome, nanoparticle, lipofectin, lipfectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDAB, HDAB, polybrene, or polyethyleneimine are used. I can do it.
- the pharmaceutical composition of the present invention can be administered to, for example, mammals (eg, humans, rats, mice, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.).
- mammals eg, humans, rats, mice, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.
- the pharmaceutical composition of the present invention can take various dosage forms, for example, capsules, tablets, liquids and the like, but is not limited, but more generally, it is liquefied and made into injections. Or an oral agent or a sustained-release agent.
- the injection can be prepared by a well-known method in the technical field. For example, it can be prepared by dissolving in an appropriate solvent such as sterilized water, buffer solution, physiological saline, etc., sterilizing by filtration with a filter or the like, and then filling in an aseptic container.
- Oral preparations are formulated into dosage forms such as tablets, granules, fine granules, powders, soft or hard capsules, solutions, emulsions, suspensions, syrups and the like.
- sustained release agent As a sustained release agent, it is formulated into dosage forms such as tablets, granules, fine granules, powders, soft or hard capsules, microcapsules and the like.
- a stabilizer such as albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol or the like can be preferably added.
- necessary auxiliary additives such as excipients, solubilizers, antioxidants, soothing agents, tonicity agents and the like may be included.
- a liquid preparation it is desirable to store it after removing moisture by freeze storage or freeze drying.
- sustained release carriers include soluble collagen or soluble collagen derivatives, proteins such as gelatin, ceramic porous bodies, polyamino acids, polylactic acid, chitin or chitin derivatives, water-swellable polymer gels, etc. Can be used.
- the pharmaceutical composition of the present invention can be administered by an appropriate administration route according to the form. It can be administered orally or parenterally, but is preferably administered parenterally. For example, it can be administered into a vein, artery, subcutaneous, intramuscular, etc. in the form of an injection. Moreover, it can administer by implanting in the living body, for example, an affected part, subcutaneous, intramuscular, etc. in the form of a sustained release agent.
- the dose and frequency of administration vary depending on the purpose of administration, administration method, type and size of cancer, and the situation (gender, age, body weight, etc.) of the subject of administration, but basically a desirable dosage form for the above active ingredients follows.
- the present invention provides a drug delivery system for preventing or treating lung cancer metastasis comprising the above DNA aptamer.
- the pharmaceutical ingredients that can be transported by the drug delivery system are typically the above-mentioned anticancer agents, but as long as they are substances useful for the prevention or treatment of lung cancer metastasis, there are other toxins and cancer growth inhibitors. It can also be a siRNA (small interfering RNA) that inhibits the expression of a gene, a suicide gene, or a gene that plays an important role in the growth and metastasis of lung cancer.
- Example 1 Selection of Aptamers Aptamers that specifically bind to non-small cell lung cancer cells, H1975 cells, were selected from DNA pools having random sequences using the Cell-SELEX method. Each step in the Cell-SELEX method is as follows. 1) Preparation of DNA pool (solution preparation of DNA aptamer candidate group) 2) Mixing with target substance-H1975 cells 3) Separation of target-binding DNA and non-binding DNA 4) Replication of target-binding DNA (amplification of DNA aptamer bound to target substance) 5) Purification of target-binding DNA (step of purifying amplified DNA aptamer into single-stranded DNA) 6) Cloning of target-binding nucleic acid (pretreatment for sequence analysis of the obtained DNA aptamer) 7) Perform 8 rounds of steps 1) to 6) 8) Sequence analysis of target-binding nucleic acid (analysis of nucleotide sequence of DNA aptamer using sequencer)
- the DNA pool used was an oligo DNA having the following sequence with a total length of 70 bases and a random sequence part (N) of 34 bases.
- DNA pool Random 34 (Tsukuba Oligo Service Co., Ltd.) Sequence: 5′-GCC TGT TGT GAG CCT CCT (N 34 ) CGC TTA TTC TTG TCT CCC-3 ′ ⁇ Length: 70 bases (random sequence is the center 34 bases) ⁇ Molecular weight: 21391.3 g / mol Molar extinction coefficient: 630475 L / mol ⁇ cm
- a 1 ⁇ M DNA pool was prepared by using the random DNA as a solvent in a cell culture medium (Wako Pure Chemical Industries, Ltd .: RPM1-1640, Whith Glucose + calf serum + antibiotics) buffer.
- H1975 cells were cultured in a culture dish and cultured until the number of cells reached 10 6 to 10 7 .
- the medium was removed, and 2 mL of phosphate buffered saline (hereinafter referred to as PBS) was added to the petri dish to wash the cells.
- 1 mL of 0.05% trypsin solution containing EDTA was added to the petri dish, and left in a 37 ° C. incubator for 2 minutes.
- 4 mL of PBS was added, and this was collected in a 15 mL centrifuge tube and centrifuged at 200 g for 3 minutes. Thereafter, the supernatant was removed with an aspirator.
- the obtained mixed solution was allowed to stand in a 37 ° C. incubator for 1 hour. Thereafter, centrifugation was performed at 400 g for 4 minutes using the same centrifuge tube. After removing the supernatant, 500 ⁇ L of PBS was added and centrifuged at 400 g for 4 minutes. This washing operation was performed three times. After removing the supernatant, 200 ⁇ L of PBS was added, and the mixture was heated at 95 ° C. for 10 minutes. Centrifugation was performed at 13000 g for 5 minutes, and the supernatant was collected.
- the separated H1975 cell binding DNA was amplified by PCR.
- the apparatus used was a Thermal Cycler (TAKARA-TP600).
- As the primer an 18-base primer corresponding to the common sequence of the random DNA was used (manufactured by Tsukuba Oligo Service Co., Ltd.).
- the 5 'terminal side of the primer is modified with biotin to enable separation of single-stranded DNA as described later.
- Streptavidin is added to the purified DNA and adsorbed on the magnetic particles. After collecting the magnetic particles with a magnet, the supernatant is removed, and then single-stranded DNA not bound to the magnetic particles is recovered in the supernatant by denaturation with alkaline buffer ( FIG. 1). Thereafter, the alkaline buffer was replaced with a PBS buffer, and the single-stranded DNA, which was the target compound bound to the magnetic particles, was recovered. This was defined as one round, and this operation was performed 8 times.
- PCR amplification was performed using an 18-base primer not modified with biotin, and the PCR product was subjected to sequencer analysis. Analysis is performed by a sequencer analyzer Ion PGMTM (registered trademark) manufactured by Life Technologies Japan, and further, DNA aptamers targeting mouse whole blood and mouse mononuclear cells using the same method as described above by the Cell-SELEX method, respectively.
- sequencer analyzer Ion PGMTM registered trademark
- DNA aptamers targeting mouse whole blood and mouse mononuclear cells using the same method as described above by the Cell-SELEX method, respectively.
- the mouse aptamer group for mouse H1975 cells that has already been found and the common aptamer sequences targeting mouse whole blood and mouse mononuclear cells were pointed out. Sequences 1 and 2 were found as sequences that do not bind to mononuclear cells but bind only to H1975 cells.
- Example 2 Staining of non-small cell lung cancer cells with a fluorescently labeled DNA aptamer H1975 cells are cultured in a culture dish and cultured until the number of cells reaches 10 6 to 10 7 .
- 30 ⁇ L of the aptamer solution was taken and added while being dispersed in a culture dish (medium RPMI-1640; 2 mL). This was left still in an incubator at 37 ° C. for 1 hour.
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Abstract
Description
(1)配列番号1および2で示される配列から選択される少なくとも1つのヌクレオチド配列を有し、非小細胞肺がん細胞に対して特異的に結合することを特徴とするDNAアプタマー;
(2)配列番号1および2で示される配列から選択される少なくとも1つのヌクレオチド配列において、1~3個のヌクレオチドの置換、欠失、又は付加を含む配列を有し、非小細胞肺がん細胞に対して特異的に結合することを特徴とするDNAアプタマー;
(3)非小細胞肺がん細胞に対して特異的に結合するDNAアプタマーであって、
5’-P1-X-P2-3’
で表されるヌクレオチド配列を有し、
ここで、Xは、1)配列番号1および2で示される配列から選択されるヌクレオチド配列、又は2)配列番号1および2で示される配列から選択されるヌクレオチド配列において1~3個のヌクレオチドの置換、欠失、又は付加を含む配列であり、
P1及びP2は、PCR増幅のために導入された第1及び第2プライマー認識配列である、
該DNAアプタマー;
(4)P1は、配列番号3で示される第1プライマー認識配列であり、及びP2は、配列番号4で示される第2プライマー認識配列である、上記(3)に記載のDNAアプタマー。
(5)前記非小細胞肺がん細胞が、H1975細胞である、上記(1)~(4)のいずれか1に記載のDNAアプタマー;
(6)糖鎖部分での化学的置換、リン酸エステル部分での化学的置換及び核酸塩基部分での化学的置換から成る群より選択される、少なくとも1つの化学修飾を含む、上記(1)~(5)のいずれか1に記載のDNAアプタマー;
(7)5’末端又は3’末端に蛍光標識を有する、上記(1)~(6)のいずれか1に記載のDNAアプタマー;及び
(8)前記蛍光標識が、6-カルボキシテトラメチルローダミン、フルオレセインイソチオシアネート、6-カルボキシフルオロセイン-アミノヘキシル、又はシアニン系蛍光色素である、上記(7)に記載のDNAアプタマー
に関する。
(9)上記(1)~(8)のいずれか1に記載のDNAアプタマーを含む、肺がん細胞の検出用組成物;
(10)上記(1)~(8)のいずれか1に記載のDNAアプタマーを含む、肺がん細胞の検出用キット;
(11)上記(1)~(8)のいずれか1に記載のDNAアプタマーを用いることを特徴とする、肺がんの検出方法;及び
(12)前記DNAアプタマーを肺細胞、肺組織、血液、血清、血漿、唾液、及び喀痰よりなる群から選択される生体から採取された試料と接触させる工程、及び当該試料とDNAアプタマーとの結合による応答を観測することによって肺がん細胞の存在を検出する工程を含む、上記(11)に記載の検出方法;
(13)前記応答が、蛍光応答もしくはラマン散乱応答である、上記(12)に記載の検出方法
に関する。
(14)上記(1)~(8)のいずれか1に記載のDAアプタマーを含有する、肺がんの転移予防又は治療用医薬組成物;
(15)肺がんの転移予防又は治療用医薬組成物の製造のための上記(1)~(8)のいずれか1に記載のDNAアプタマーの使用;
(16)上記(1)~(8)のいずれか1に記載のDNAアプタマーを含有する、肺がんの転移予防又は治療用ドラッグデリバリーシステム
に関する。
本願において「DNAアプタマー」とは、ターゲットとなる分子や物質を特異的に認識できる一本鎖オリゴDNAを意味し、本発明に係るDNAアプタマーは、非小細胞肺がん細胞に対して特異的に結合する機能を有する一本鎖オリゴDNAである。
<配列番号1>ACA GTT CGT CAG TGT TTG GGG TTC AGC TTA GGT G (34 mer)
<配列番号2>
TGC GCG TGG GTG GTT TTT GTC TGT CAG CTT GGG TC (35 mer)
5’-P1-X-P2-3’
で表されるヌクレオチド配列を有する。ここで、Xは、配列番号1および2で示される配列から選択されるヌクレオチド配列、又はそれらの配列において1~3個のヌクレオチドの置換、欠失、又は付加を含む配列である。P1及びP2は、PCR増幅のために導入された第1及び第2プライマー認識配列である。好ましくは、P1は、GCC TGT TGT GAG CCT CCT(配列番号3)であり、及びP2は、CGC TTA TTC TTG TCT CCC(配列番号4)である。
酵素標識の例としては、β-ガラクトシダーゼ、β-グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素等が挙げられる。また、発光基質として、ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニン等)などを標識剤として用いてもよい。
本発明のDNAアプタマーは、当該技術分野において周知のインビトロセレクション法を用いて選別及び取得することができる。そのような手法の好ましい例として、試験管内進化法(Systematic Evolution of Ligands by EXponential enrichment:SELEX法)が用いられる。当該SELEX法は、ターゲット物質に結合する核酸リガンド(アプタマー)の選別と、PCRによる指数関数的な増幅を複数回繰り返すことにより、ターゲット物質に親和性を有する核酸分子(一本鎖DNA、RNA)を得るというものである。また、その改良手法として、例えばGuo,et al.,Int.J.Mol.Sci.,9(4):668,2008に記載されているようなCell-SELEX法を用いることが好ましい。この手法は、ターゲットとして細胞自体を用いることができるため、往来のSELEX法と比較して、細胞表面に膜タンパク質の解析が不要であること、細胞表面に結合し得る複数のアプタマーを同時に選抜できること、及びターゲット細胞に対してより特異的に結合するアプタマーを選抜できるといった利点を有するものである。なお、これら以外にも当該技術分野において周知の方法を用いて本発明のDNAアプタマーを選別及び取得することもできる。
上述のように、本発明に係るDNAアプタマーは、非小細胞肺がん細胞に対して特異的に結合する機能を有することから、当該肺がん細胞の検出において好適に用いることができ、本発明のDNAアプタマーを含む検出用組成物は、非小細胞肺がんに対する腫瘍マーカとしても用いることができる。
別の態様において、本発明は、上記DNAアプタマーを含有する、肺がんの転移予防又は治療用医薬組成物を提供するものである。好ましくは、当該医薬組成物は、DNAアプタマーに加えて、有効量の肺がんの転移予防又は治療のための医薬化合物(有効成分)、及び医薬上許容される担体を含むことができる。例えば、本アプタマーを金ナノ粒子に結合したものを用いて、癌の温熱療法用の試薬として利用できる可能性がある。
塩酸アムルビシン、塩酸イリノテカン、イホスファミド、エトポシドラステット、ゲフィニチブ、シクロホスファミド、シスプラチン、トラスツズマブ、フルオロウラシル、メシル酸イマニチブ、メソトレキサート、リツキサン、アドリアマイシン、カルボプラチン、タモキシフェン、カンプトテシン、メルファラン、L-アスパラギナーゼ、アセクラトン、シゾフィランなどが挙げられる。
Cell-SELEX法を用いて、ランダムな配列を有するDNAプールから非小細胞肺がん細胞であるH1975細胞に特異的に結合するアプタマーの選別を行った。当該Cell-SELEX法における各工程は以下のとおりである。
1) DNAプールの調製(DNAアプタマー候補群の溶液調製)
2) ターゲット物質-H1975細胞-との混合
3) ターゲット結合性DNAと非結合性DNAの分離
4) ターゲット結合性DNAの複製(ターゲット物質と結合したDNAアプタマーを増幅する工程)
5) ターゲット結合性DNAの精製(増幅したDNAアプタマーを1本鎖DNAに精製する工程)
6) ターゲット結合性核酸のクローンニング(得られたDNAアプタマーの配列解析の前処理)
7) これら1)~6)の工程を8ラウンド実行
8) ターゲット結合性核酸の配列解析(シーケンサーによるDNAアプタマーのヌクレオチド配列の解析)
DNA pool:Random34(つくばオリゴサービス株式会社製)
・配列: 5’-GCC TGT TGT GAG CCT CCT(N34)CGC TTA TTC TTG TCT CCC-3’
・長さ: 70塩基(ランダム配列は中央の34塩基)
・分子量: 21391.3 g/mol
・モル吸光係数: 630475 L/mol・cm
H1975細胞を培養シャーレで培養し、細胞数が106~107になるまで培養する。これにシークエンサー解析により見出されたH1975細胞に対して親和性のあるヌクレオチド配列からなるDNAアプタマーの5‘末端をCy3.5(商標)で修飾し、このDNAを超純水により100μMに調製したアプタマー溶液を30μLとり、培養シャーレ(培地RPMI-1640;2mL)に分散させながら添加した。これを37℃1時間インキュベーターに静置した。その後、PBSで細胞を3回洗浄した後、さらにPBSを2mL添加した状態でオリンパス製の倒立型蛍光顕微鏡IX51により観察した。配列番号1および2を有するDNAアプタマーによる結果を、それぞれ図2および図3に示す。これらの蛍光標識DNAアプタマーがH1975細胞に特異的に結合し、それによってH1975細胞に対する良好な蛍光イメージング画像が得られることが明らかとなった。
Claims (16)
- 配列番号1および2で示される配列から選択される少なくとも1つのヌクレオチド配列を有し、非小細胞肺がん細胞に対して特異的に結合することを特徴とするDNAアプタマー。
- 配列番号1および2で示される配列から選択される少なくとも1つのヌクレオチド配列において、1~3個のヌクレオチドの置換、欠失、又は付加を含む配列を有し、非小細胞肺がん細胞に対して特異的に結合することを特徴とするDNAアプタマー。
- 非小細胞肺がん細胞に対して特異的に結合するDNAアプタマーであって、
5’-P1-X-P2-3’
で表されるヌクレオチド配列を有し、ここで、
Xは、1)配列番号1および2で示される配列から選択されるヌクレオチド配列、又は2)配列番号1および2で示される配列から選択されるヌクレオチド配列において1~3個のヌクレオチドの置換、欠失、又は付加を含む配列であり、
P1及びP2は、PCR増幅のために導入された第1及び第2プライマー認識配列である
該DNAアプタマー。 - P1は、配列番号3で示される第1プライマー認識配列であり、及び
P2は、配列番号4で示される第2プライマー認識配列である、請求項3に記載のDNAアプタマー。 - 前記非小細胞肺がん細胞が、H1975細胞である、請求項1~4のいずれか1に記載のDNAアプタマー。
- 糖鎖部分での化学的置換、リン酸エステル部分での化学的置換及び核酸塩基部分での化学的置換から成る群より選択される、少なくとも1つの化学修飾を含む、請求項1~5のいずれか1に記載のDNAアプタマー。
- 5’末端又は3’末端に蛍光標識を有する、請求項1~6のいずれか1に記載のDNAアプタマー。
- 前記蛍光標識が、6-カルボキシテトラメチルローダミン、フルオレセインイソチオシアネート、6-カルボキシフルオロセイン-アミノヘキシル、又はシアニン系蛍光色素である、請求項7に記載のDNAアプタマー。
- 請求項1~8のいずれか1に記載のDNAアプタマーを含む、肺がん細胞の検出用組成物。
- 請求項1~8のいずれか1に記載のDNAアプタマーを含む、肺がん細胞の検出用キット。
- 請求項1~8のいずれか1に記載のDNAアプタマーを用いることを特徴とする、肺がんの検出方法。
- 前記DNAアプタマーを肺細胞、肺組織、血液、血清、血漿、唾液、及び喀痰よりなる群から選択される生体から採取された試料と接触させる工程、及び当該試料とDNAアプタマーとの結合による応答を観測することによって肺がん細胞の存在を検出する工程を含む、請求項11に記載の検出方法。
- 前記応答が、蛍光応答もしくはラマン散乱応答である、請求項12に記載の検出方法。
- 請求項1~8のいずれか1に記載のDNAアプタマーを含有する、肺がんの転移予防又は治療用医薬組成物。
- 肺がんの転移予防又は治療用医薬組成物の製造のための請求項1~8のいずれか1に記載のDNAアプタマーの使用。
- 請求項1~8のいずれか1に記載のDNAアプタマーを含有する、肺がんの転移予防又は治療用ドラッグデリバリーシステム。
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KR1020177025022A KR20170109674A (ko) | 2015-02-10 | 2016-02-05 | 비소세포 폐암 세포(h1975)에 결합하는 dna 앱타머 |
US15/549,843 US20180016582A1 (en) | 2015-02-10 | 2016-02-05 | Dna aptamer binding to non-small cell lung cancer cells (h1975) |
EP16749175.2A EP3257940A4 (en) | 2015-02-10 | 2016-02-05 | Dna aptamer capable of binding to non-small cell lung cancer cell (h1975) |
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CN107488663A (zh) * | 2017-07-19 | 2017-12-19 | 中国科学院化学研究所 | 核酸、dna g‑四链体、试剂、其制备方法和用途 |
JP2020519615A (ja) * | 2017-05-08 | 2020-07-02 | オーグマニティ ナノ リミテッド | 急速に進化する生物学的実体の処置 |
JP2023543335A (ja) * | 2020-10-30 | 2023-10-13 | スピア、トッド | オリゴヌクレオチドベースの治療剤およびその使用 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101914542A (zh) * | 2009-04-28 | 2010-12-15 | 中国科学院化学研究所 | 用于不同亚型非小细胞肺癌分型的一种核酸适体及其筛选方法 |
CN101914543A (zh) * | 2009-04-28 | 2010-12-15 | 中国科学院化学研究所 | 一种用于不同亚型非小细胞肺癌分型的核酸适体及其筛选方法 |
CN101955939A (zh) * | 2009-04-28 | 2011-01-26 | 中国科学院化学研究所 | 用于不同亚型非小细胞肺癌分型的核酸适体及其筛选方法 |
JP2014217311A (ja) * | 2013-05-08 | 2014-11-20 | 日産化学工業株式会社 | がん細胞に結合するdnaアプタマー |
JP2015019606A (ja) * | 2013-07-17 | 2015-02-02 | 日産化学工業株式会社 | 非小細胞肺癌に特異的に結合する核酸 |
-
2016
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- 2016-02-05 JP JP2016574783A patent/JPWO2016129531A1/ja active Pending
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Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101914542A (zh) * | 2009-04-28 | 2010-12-15 | 中国科学院化学研究所 | 用于不同亚型非小细胞肺癌分型的一种核酸适体及其筛选方法 |
CN101914543A (zh) * | 2009-04-28 | 2010-12-15 | 中国科学院化学研究所 | 一种用于不同亚型非小细胞肺癌分型的核酸适体及其筛选方法 |
CN101955939A (zh) * | 2009-04-28 | 2011-01-26 | 中国科学院化学研究所 | 用于不同亚型非小细胞肺癌分型的核酸适体及其筛选方法 |
JP2014217311A (ja) * | 2013-05-08 | 2014-11-20 | 日産化学工業株式会社 | がん細胞に結合するdnaアプタマー |
JP2015019606A (ja) * | 2013-07-17 | 2015-02-02 | 日産化学工業株式会社 | 非小細胞肺癌に特異的に結合する核酸 |
Non-Patent Citations (4)
Title |
---|
KAUR J ET AL.: "Ets1 identified as a novel molecular target of RNA aptamer selected against metastatic cells for targeted delivery of nano-formulation", ONCOGENE, vol. 34, no. 41, 2015, pages 5216 - 5228, XP055457073, ISSN: 0950-9232 * |
See also references of EP3257940A4 * |
XU, LI ET AL.: "Cellular internalization and cytotoxicity of aptamers selected from lung cancer cell", AM. J. BIOMED. SCI., vol. 5, no. 1, 2013, pages 47 - 58, XP055457072, ISSN: 1937-9080 * |
ZHAO, ZILONG ET AL.: "Recognition of subtype non-small cell lung cancer by DNA aptamers selected from living cells", ANALYST, vol. 134, no. 9, 2009, pages 1808 - 1814, XP009503956, ISSN: 0003-2654 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2020519615A (ja) * | 2017-05-08 | 2020-07-02 | オーグマニティ ナノ リミテッド | 急速に進化する生物学的実体の処置 |
CN107488663A (zh) * | 2017-07-19 | 2017-12-19 | 中国科学院化学研究所 | 核酸、dna g‑四链体、试剂、其制备方法和用途 |
JP2023543335A (ja) * | 2020-10-30 | 2023-10-13 | スピア、トッド | オリゴヌクレオチドベースの治療剤およびその使用 |
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US20180016582A1 (en) | 2018-01-18 |
TW201643249A (zh) | 2016-12-16 |
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