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WO2023280001A1 - Application de mir-31-5p dans le traitement de la leucémie myéloïde aiguë - Google Patents

Application de mir-31-5p dans le traitement de la leucémie myéloïde aiguë Download PDF

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WO2023280001A1
WO2023280001A1 PCT/CN2022/101571 CN2022101571W WO2023280001A1 WO 2023280001 A1 WO2023280001 A1 WO 2023280001A1 CN 2022101571 W CN2022101571 W CN 2022101571W WO 2023280001 A1 WO2023280001 A1 WO 2023280001A1
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mir
aml
cells
pri
vectors
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Chinese (zh)
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闫道广
钟文彬
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Jinan University
University of Jinan
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Jinan University
University of Jinan
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the disclosure belongs to the field of biomedicine, relates to the diagnosis and treatment of tumors, and specifically relates to the application of miR-31-5p in the diagnosis and treatment of acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • Leukemia is a kind of clonal malignant disease originating from hematopoietic stem cells. According to statistics, about 350,000 people worldwide die of leukemia every year. Therefore, leukemia has become the main malignant tumor that threatens human health.
  • Leukemia stem cells are a very small group of cells in leukemia patients, accounting for about 0.1% to 1% of all leukemia cells. LSC was first discovered in acute myeloid leukemia (AML) and has been widely recognized.
  • AML acute myeloid leukemia
  • LSCs can cause and maintain leukemia; at the same time, unlike leukemia cells, more than 95% of LSCs are in the "dormant" state of G0 phase, and these cells are not effective for traditional cell cycle chemotherapy. Drug insensitivity, the retained LSC increases the relapse rate of leukemia patients through self-replication (Renewal). Therefore, finding the unique self-protection mechanism of LSC and targeting to kill LSC is an important way to completely cure leukemia.
  • MicroRNAs are a class of non-coding small RNA molecules, about 19-25bp in length, including a 6-8nt seed sequence and a 12-17nt supplementary sequence.
  • the initial miRNA is produced, which forms a hairpin structure under the processing of Drosha enzyme. According to the order of transcription, it is divided into 5' end arm and 3' end arm, also known as 5p and 3p.
  • the partial sequences of the 5' end arm and the 3' end arm are complementary to form a double-stranded RNA, which becomes a mature miRNA precursor (pre-miRNA) and is transported to the cytoplasm to play a role in post-transcriptional control of gene expression.
  • the pre-miRNA Once the pre-miRNA enters the cytoplasm from the nucleus, it will be further processed by Dicer enzyme to become a mature miRNA. These miRNAs play an important role in the regulation of gene expression, and are involved in cell differentiation, proliferation, and maintenance of cell homeostasis.
  • Chinese patent CN106636308B discloses a probe combination and kit for detecting skin cancer-related markers, wherein hsa-miR-31-5p is used as a detection of skin cancer-related markers.
  • the purpose of the present disclosure is to screen out acute myeloid leukemia as a molecular diagnostic biomarker, and develop a corresponding diagnostic kit to diagnose and treat acute myeloid leukemia or provide its prognosis.
  • the present disclosure provides a product for detecting miR-31-5p, pri-miR-31 and/or pre-miR-31 in preparation for assisting in diagnosing subjects with acute myeloid leukemia (AML) And/or use in a reagent, chip or kit for prognosing the survival period of a subject.
  • AML acute myeloid leukemia
  • the present disclosure provides a use of miR-31-5p, pri-miR-31 and/or pre-miR-31 in the preparation of a drug for preventing and/or treating acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • the present disclosure provides a drug for treating acute myeloid leukemia (AML), the drug comprising miR-31 naive miRNA, miR-31 precursor miRNA, and/or mature miR-31-5p.
  • AML acute myeloid leukemia
  • the present disclosure provides a method of treating acute myeloid leukemia (AML), comprising administering to a subject a therapeutically effective amount of miR-31-5p, pri-miR-31 and/or pre-miR- 31 or a pharmaceutical composition comprising it.
  • AML acute myeloid leukemia
  • Figure 1 shows the principle of miRNA tailing reverse transcription.
  • Figure 2 is the QPCR detection of miR-31-5p expression in bone marrow leukemia stem cells (LSCs) of AML patients and normal human bone marrow hematopoietic stem cells (HSCs).
  • LSCs bone marrow leukemia stem cells
  • HSCs normal human bone marrow hematopoietic stem cells
  • Figure 3 shows the expression of miR-31-5p detected in AML patient bone marrow cells (AML) and normal human bone marrow cells (BM) by QPCR.
  • Fig. 4 is a graph showing the relationship between the expression level of miR-31-5p and the prognosis and survival period of AML patients.
  • Figure 5 shows the effect of miR-31-5p on the ability of AML-LSC colony formation.
  • Fig. 6 is a graph showing the results of miR-31-5p-induced death of AML-LSC and bone marrow AML cells.
  • Fig. 7 is a graph showing the results of miR-31-5p enhancing the chemotherapy drug cytarabine (Ara-C) to induce AML-LSC and bone marrow AML cell death.
  • Figure 8 is a verification of the therapeutic effect of miR-31-5p on AML disease in B-NDG mice.
  • primary miR-31 or “pri-miR-31” refers to miRNA obtained after transcription of miR-31 gene by RNA polymerase.
  • pre-miR-31 refers to the original miR-31 (pri-miR-31) under the processing of Drosha enzyme to form a hairpin structure sequence with 71 nucleosides Acid (71nt), its sequence is as follows: 5'-ggagaggaggcaagaugcuggcauagcuguugaacugggaaccugcuaugccaacauauugccaucuuucc-3'(SEQ ID NO3).
  • mature miR-31-5p or “miR-31-5p” refers to the sequence formed by the processing of the precursor miR-31 (pre-miR-31) by Dicer enzyme, having 21 nucleosides Acid (21nt), its sequence is as follows: 5'-aggcaagaugcuggcauagcu-3' (SEQ ID NO 1).
  • vector refers to a construct capable of delivering and optionally expressing one or more polynucleotides of interest into a host cell.
  • examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes , and certain eukaryotic cells, such as producer cells.
  • Vectors can be stable and self-replicating. There is no limitation regarding the types of vectors that can be used.
  • a vector may be a cloning vector, a polynucleotide suitable for propagation and obtaining incorporation with various foreign organisms, a genetic construct or an expression vector.
  • Suitable vectors include prokaryotic expression vectors (such as pUC18, pUC19, Bluescript and their derivatives), mpl8, mpl9, pBR322, pMB9, CoIE1, pCR1, RP4, phage and shuttle vectors (such as pSA3 and pAT28), and viral-based vectors ( eukaryotic expression vectors such as adenovirus, adeno-associated virus, and retroviruses and lentiviruses), and non-viral vectors such as pSilencer 4.1-CMV (LifeTechnologies Corp., Carslbad, CA, USA), pcDNA3, pcDNA3.1/hyg pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HC
  • plasmid refers to a small, circular, double-stranded, self-replicating DNA molecule obtained by genetic engineering techniques capable of transferring genetic material of interest to cells, which results in the production of The product encoded by the genetic material (such as protein polypeptide, peptide or functional RNA) in.
  • recombinant plasmid or “plasmid” also refers to a small, circular, double-stranded, self-replicating DNA molecule obtained by genetic engineering techniques used during the preparation of viral vectors as vectors for recombinant vector genomes.
  • viral vector refers to an agent obtained from a naturally occurring virus by genetic engineering techniques capable of transferring genetic material of interest (such as DNA or RNA) to a cell, resulting in the production of The product encoded by a substance such as a protein polypeptide, peptide or functional RNA.
  • composition refers to a formulation of various preparations.
  • Formulations containing therapeutically effective amounts of miR-31-5p, pri-miR-31 and/or pre-miR-31 are in sterile liquid solution, liquid suspension or lyophilized form, optionally comprising stabilizers or excipients .
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation, which is not an active ingredient, and which is nontoxic to a subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • treating an individual suffering from a disease or condition means that the individual's symptoms are partially or fully alleviated, or remain unchanged after treatment.
  • treatment includes prophylaxis, treatment and/or cure.
  • Prevention refers to preventing an underlying disease and/or preventing worsening of symptoms or development of a disease.
  • therapeutic effect means the effect resulting from treatment of an individual that alters, usually ameliorates or ameliorate the symptoms of, or cures a disease or condition.
  • terapéuticaally effective amount or “therapeutically effective dose” refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect when administered to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest or partially arrest the symptoms of a disease or disorder.
  • prophylactically effective amount or “prophylactically effective dose” refers to the amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the intended prophylactic effect, e.g., prevent or delay a disease or symptom occurrence or recurrence of the disease or symptoms, and to reduce the likelihood of occurrence or recurrence of the disease or symptoms.
  • a full prophylactically effective dose does not have to occur by administering one dose, and can only occur after administering a series of doses.
  • a prophylactically effective amount can be administered in one or more administrations.
  • the present disclosure provides a product for detecting miR-31-5p, pri-miR-31 and/or pre-miR-31 in preparation for assisting in diagnosing subjects with acute myeloid leukemia (AML) And/or the purposes in the reagent, chip or test kit that are used for prognostic experimenter's survival period, wherein, the sequence of miR-31-5p is: 5'-aggcaagaugcuggcauagcu-3'(SEQ ID NO1), pre-miR- The sequence of 31 is: 5'-ggagaggaggcaagaugcuggcauagcuguugaacugggaaccugcuaugccaacauauugccaucuuucc-3' (SEQ ID NO 3).
  • the reagent is capable of detecting the level of miR-31-5p, pri-miR-31 and/or pre-miR-31 in a biological sample.
  • the level of miR-31-5p, pri-miR-31 and/or pre-miR-31 in the biological sample is lower than the corresponding miR-31-5p, pri-miR in the normal control sample
  • a level of -31 and/or pre-miR-31 indicates that the subject has acute myeloid leukemia (AML).
  • the biological sample is selected from one or more of peripheral blood, bone marrow, and tissue suspected of having leukemia cells.
  • the subject can be a human or other mammal.
  • the level of miR-31-5p, pri-miR-31 and/or pre-miR-31 adopts high-throughput sequencing method, miRNA expression profiling chip, quantitative PCR method and/or probe hybridization method for detection.
  • the reagent comprises a forward primer for amplifying miR-31-5p; preferably, the sequence of the forward primer is as follows: 5'-aggcaagatgctggcatagct-3' (SEQ ID NO 2) .
  • miR-31-5p, pri-miR-31 and/or pre-miR-31 in the biological sample are relative to the corresponding miR-31-5p, pri-miR-31 and And/or the transcript level of the pre-miR-31 gene is low, the subjects have a shorter survival period.
  • the chip comprises a solid support and oligonucleotide probes immobilized on the solid support.
  • the present disclosure provides a use of miR-31-5p, pri-miR-31 and/or pre-miR-31 in the preparation of a drug for preventing and/or treating acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • the disclosed experiment proves that the increased level of miR-31-5p in cells can directly induce the death of AML and AML-LSC cells, and enhance the cytotoxicity of the chemotherapeutic drug cytarabine.
  • the miR-31-5p gene is transcribed by RNA polymerase to obtain the initial miR-31 (pri-miR-31), and the initial miR-31 (pri-miR-31) is processed by Drosha enzyme to form a hairpin
  • the structure of the precursor miR-31 (pre-miR-31), the precursor miR-31 (pre-miR-31) is processed by Dicer enzyme to form miR-31-5p.
  • pri-miR-31 and/or pre-miR-31 increases, and pri-miR-31 and/or pre-miR-31 will be processed in the cell to form miR-31-5p, which plays a role in increasing miR-31-5p had the same effect.
  • miR-31-5p, pri-miR-31 and/or pre-miR-31 are natural, artificially synthesized, or can be expressed using miR-31-5p, pri-miR-31 and/or pre-miR-31 DNA fragment expression vector transfected cells, wherein the gene sequence of miR-31-5p is: 5'-aggcaagatgctggcatagct-3'(SEQ ID NO 2); pre-miR- The gene sequence of 31-5p is: 5'-ggagaggaggcaagatgctggcatagctgttgaactgggaacctgctatgccaacatattgccatctttcc-3' (SEQ ID NO 4).
  • the expression vector is selected from vectors selected from plasmids, phages, phagemids, cosmids, viral vectors, virions, prokaryotic expression vectors and eukaryotic expression vectors.
  • the viral vector is selected from the group consisting of retroviral vectors, lentiviruses, adenoviral vectors, adeno-associated viral vectors, herpesvirus vectors, alphavirus vectors, baculoviruses, and vaccinia viruses.
  • the prokaryotic expression vector is selected from an E. coli expression vector and a Bacillus subtilis expression vector.
  • the eukaryotic expression vector is selected from a yeast expression vector, an insect expression vector, or a mammalian expression vector.
  • the drug may be administered alone or in combination with other drugs capable of inhibiting AML.
  • other drugs capable of inhibiting AML are selected from daunorubicin, cytarabine, thioguanine, etoposide, harringtonine, vincristine, prednisone, Mitoxantrone, doxorubicin, cyclophosphamide, carboplatin, decitabine, methotrexate, etoposide, doxorubicin (doxorubicin), cisplatin, dexamethasone, sacola Neo, methylnitrosourea, fluorouracil, 5-fluorouracil, vinblastine, camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide, oxaliplatin, irinotecan, topotecan
  • Kang Kang, leucovorin, carmustine, streptozocin, paclitaxel, tamoxifen, dacarbazine, imatinib, aza
  • the medicament further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is selected from lactose, dextrose, sucrose, polyvinylpyrrolidone, alginate, gelatin, cellulose, syrup, sorbitol, mannitol, starch, arabic Rubber, Talc, Magnesium Stearate, Calcium Phosphate, Calcium Silicate, Microcrystalline Cellulose, Methyl Cellulose, Methyl Hydroxybenzoate, Propyl Hydroxybenzoate, Mineral Oil, Microcapsules & Microspheres, Nano One or more of particles and liposomes.
  • the dosage form of the drug is solution, injection, oral liquid, suspension, emulsion, extract, powder, granule, suppository, aerosol, granule, tablet or capsule .
  • injections include sterile or sterile solutions, aqueous injections, oil injections, powder injections, etc.; oral liquid dosage forms include solutions, syrups, emulsions, suspensions, etc.; tablets include ordinary Compressed tablets, sugar-coated tablets, effervescent tablets, chewable tablets, multilayer tablets, implanted tablets, sustained-release tablets, controlled-release tablets, etc.
  • the medicament further comprises a dispersant or stabilizer.
  • the present disclosure provides a reagent, a chip or a kit for assisting in diagnosing that a subject has acute myeloid leukemia (AML) and/or for prognosing the survival of a subject;
  • the reagent, chip or kit comprises Assays for products that detect miR-31-5p and/or its precursors in biological samples.
  • the present disclosure provides a drug for treating acute myeloid leukemia (AML), the drug comprising miR-31-5p, pri-miR-31 and/or pre-miR-31.
  • AML acute myeloid leukemia
  • the present disclosure provides a pharmaceutical composition for treating acute myeloid leukemia (AML), the medicine comprising miR-31-5p, pri-miR-31 and/or pre-miR-31, and a pharmaceutically acceptable Carrier.
  • AML acute myeloid leukemia
  • the present disclosure provides a method of treating acute myeloid leukemia (AML), comprising administering to a subject a therapeutically effective amount of miR-31-5p, pri-miR-31 and/or pre-miR- 31 or a pharmaceutical composition comprising it.
  • AML acute myeloid leukemia
  • a medicament or pharmaceutical composition of an embodiment is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal administration.
  • Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: sterile diluents for injection such as water, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; Antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphate, and agents to adjust osmotic pressure, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable pharmaceutically acceptable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and a suitable mixture thereof.
  • Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, the maintenance of the desired particle size in the case of dispersions, and the use of surfactants.
  • Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent delaying absorption, for example, aluminum monostearate and gelatin.
  • the methods of preparation are vacuum-drying and freeze-drying to obtain a powder containing the active ingredient plus any additional desired ingredient from a sterile-filtered solution of these ingredients as previously described .
  • Dosage unit form refers to physically separable units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of a drug calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. miR-31-5p, pri-miR-31 and/or pre-miR-31.
  • the pharmaceutical composition can be presented in a container, pack, or dispenser together with instructions for administration.
  • one or more of miR-31-5p, pri-miR-31 and/or pre-miR-31 may be administered in combination therapy, ie in combination with other drugs capable of inhibiting AML.
  • the term "in conjunction” herein means that the agents are administered substantially simultaneously, simultaneously or sequentially. If administered sequentially, the first of the two compounds is still preferably detectable at the site of treatment at an effective concentration when administration of the second compound is initiated.
  • “combination” can also include miR-31-5p, pri-miR-31 and/or pre-miR-31 and other therapeutic agents in the kit at the same time.
  • combination therapy may comprise miR-31-5p, pri-miR-31 and/or pre-miR-31 herein with one or more additional therapeutic agents (e.g., one or more cytokines and growth factor inhibitory agents, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and/or cytotoxic or cytostatic agents, as described in more detail below) are co-formulated and/or co-administered.
  • additional therapeutic agents e.g., one or more cytokines and growth factor inhibitory agents, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and/or cytotoxic or cytostatic agents, as described in more detail below.
  • additional therapeutic agents e.g., one or more cytokines and growth factor inhibitory agents, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and/or cytotoxic or cytostatic agents, as described in more detail below.
  • Such combination therapy may advantageously utilize lower doses of the therapeutic agents administered
  • AML acute myeloid leukemia
  • the processing methods for collecting bone marrow samples from AML patients and normal people are as follows:
  • Bone marrow sample collection requirements heparin anticoagulation, no blood clots, bone marrow volume greater than 2mL, cell sorting within 24 hours after sample collection.
  • Mononuclear cells were separated from the AML patient bone marrow samples and normal human bone marrow samples collected in step 1 using Dayou's human lymphocyte separation medium (product number 711101X), including the following steps:
  • PBS phosphate buffered saline
  • step B Add a certain volume of separation liquid into a 15mL centrifuge tube, spread the diluted bone marrow sample obtained in step A above the liquid surface of the separation liquid, keep the interface between the two liquid surfaces clear, and obtain a mixture of bone marrow and separation liquid. Ensure that the volume ratio of the lymphocyte separation medium to the diluted bone marrow sample in PBS is 1:2.
  • step C At room temperature, adjust the centrifugal force of the horizontal rotor of the centrifuge to 700-800g (or 2000-2500rpm/min), and centrifuge the mixture of bone marrow and separation solution obtained in step B for 20-30min.
  • step E Dilute the mononuclear cells collected in step D with 5 mL of PBS, and mix upside down to obtain diluted mononuclear cells.
  • step G Resuspend the mononuclear cell pellet obtained in step F with PBS or a suitable medium for use.
  • LSC leukemia stem cells
  • HSC hematopoietic stem cells
  • step B Add 200 ⁇ L FcR blocking solution and 200 ⁇ L magnetic bead-coupled anti-CD34 antibody to the resuspended mononuclear cells obtained in step A, and incubate at 4°C for 30 min to obtain blocked and coupled mononuclear cells.
  • step C Add 50 ⁇ L of FITC-labeled anti-CD38 antibody to the blocked and conjugated mononuclear cells obtained in step B, and incubate at 4°C for 10 min to obtain FITC-labeled mononuclear cells.
  • step D Add the FITC-labeled mononuclear cells obtained in step D to the magnetic bead sorter, let the cells not bound to the CD34 antibody pass through the magnetic field, and retain the cells bound to the CD34 antibody.
  • step G Add 20 ⁇ L of enzymatic hydrolysis solution to the CD34-positive cells collected in step F, and incubate at 4°C for 10 minutes. Centrifuge at 250g (or 1000rpm/min) for 10min, discard the supernatant, and collect the cell pellet.
  • step G Suspend the cell pellet collected in step G with 20 ⁇ L PBS, add 60 ⁇ L enzymatic hydrolysis stop solution, add 100 ⁇ L anti-FITC antibody, and incubate at 4°C for 30 min.
  • step H Add the cells incubated in step H to the magnetic bead sorter, and collect the cells not bound to the FITC antibody, which are CD34 + CD38 - stem cells.
  • Embodiment 2 detection of miR-31-5p gene expression
  • Example 1 The mononuclear cells or CD34 + CD38 - stem cells sorted in Example 1 were washed twice with PBS.
  • miRNA is different from mRNA.
  • the length of mature miRNA is only about 20nt, which is very short.
  • the forward primer is enough to cover its full length or even more than that, and the reverse primer has nowhere to be placed.
  • the solution is to try to increase the length of the reverse transcription product during reverse transcription.
  • the most direct way to increase the length of the miRNA reverse transcription product is to increase the length of the miRNA, that is, to add a sequence behind the 3' end of the miRNA, and then perform the reverse transcription reaction.
  • the length of the obtained cDNA is increased from the original 20nt to more than 80nt, so that the qPCR amplification of the miRNA can be realized.
  • the tailing method is completed by the joint action of two enzymes, which are PolyA polymerase and reverse transcriptase.
  • PolyA polymerase is responsible for adding PolyA tails to miRNAs, increasing their length.
  • the reverse transcription primer is bound to the PolyA sequence, and the synthesis of the extended version of cDNA is completed by reverse transcriptase.
  • the process is shown in Figure 1.
  • reaction system volume RNA (0.5-8 ⁇ g) 3.75 ⁇ L mRQ buffer (2 ⁇ ) 5 ⁇ L mRQ enzyme 1.25 ⁇ L Total volume after adding DEPC water 10 ⁇ L
  • step temperature time 1 37°C 60min 2 85°C 5s 3 4°C termination
  • reaction product was stored in a -20°C refrigerator for subsequent experiments.
  • the forward primer is a forward primer for miR-31-5p or internal reference U6, wherein:
  • the nucleotide sequence of the miR-31-5p forward primer is: 5'-aggcaagatgctggcatagct-3' (SEQ ID NO 2).
  • the nucleotide sequence of U6 forward primer is: 5'-ggaacgatacagagaagattagc-3' (SEQ ID NO 5).
  • the reverse primer is the mRQ3' primer provided by the kit, wherein:
  • nucleotide sequence of mRQ3' primer is: 5'-ctcaactggtgtcgtgga-3' (SEQ ID NO 6).
  • the software configured with fluorescent quantitative PCR was used to process the data of quantitative PCR, and the threshold and baseline of each gene were established, and the melting amplification curve was automatically generated by the software. Obtain the CT value corresponding to each sample response. The relative expression in the samples was calculated using the 2- ⁇ ct algorithm, and then the log(2- ⁇ ct) of each sample was calculated for plotting.
  • Example 3 Analysis of the relationship between miR-31-5p gene expression and the prognosis of AML patients from the TCGA database
  • gdcSurvivalAnalysis toolkit in the R language package GDCRNATools for Kaplan Meier (KM) analysis, import miR-31-5p expression data and clinical information in the TCGA database, and group by miR-31-5p expression, in order to maximize the analysis Sensitivity and to find any potential correlation with survival independent of a preset cutoff (e.g. median), every possible cutoff between the lower and upper quartiles of expression was calculated. Each of these cutoffs was then used for a separate Kaplan Meier (KM) analysis. False discovery rate (FDR) was calculated to correct for multiple hypothesis testing, and results were considered significant only if FDR ⁇ 0.05.
  • FDR False discovery rate
  • the GDCRNAT tool was used to analyze the relationship between the expression level of miR-31-5p gene and the survival of AML patients. The results in Figure 4 show that the lower the expression level of miR-31-5p, the shorter the survival period of the patient. The above results indicated that the expression level of miR-31-5p was directly related to the prognosis of AML patients.
  • the leukemia stem cells (AML-LSC) of acute myeloid leukemia were separated according to the method in Example 1. From the 44 AML cases detected in Example 2, the LSCs of 3 different AML patients were selected to carry out the clone formation experiment, and corresponding to Patient sample numbers, the leukemia stem cell (LSC) clone formation experiments of these 3 AML patients were numbered AML-5, AML-8 and AML-9, respectively.
  • AML-LSC cell culture adopts Serum-Free Medium (STEMCELL Technologies Company) medium, in addition, three kinds of stem cell factors need to be added to the medium: rIL3 (final concentration 10 ng/mL, PeproTech Company), rFlt3 (final concentration 10 ng/mL, PeproTech Company) and rSCF (final concentration 25ng/mL, PeproTech Company), the cells were cultured at 37° C. in a 5% CO 2 incubator, and the medium was replaced or the cells were passaged every 3 days. Note that the cell culture density should be controlled at 10 3 -10 4 cells/mL to prevent stem cell differentiation.
  • control RNA in the example in Figure 5, and its DNA sequence is 5'-ttctccgaacgtgtcacgt-3'
  • miR-31-5p a DNA sequence capable of expressing miR-31-5p
  • miR-31- 5p whose DNA sequence is 5'-aggcaagatgctggcatagct-3'
  • lentiviral particles were purchased from Shanghai Gemma Company (control RNA product batch number G07AZ; miR-31-5p product batch number 170305DZ), control RNA lentivirus and expression miR-31
  • the lentivirus titer of -5p is greater than 10 9 TU (that is, the number of biologically active virus particles per milliliter) to ensure the infection efficiency.
  • B Add 5 ⁇ g/mL polybrene, and add lentivirus according to the MOI (that is, the number of virus particles infected per cell in a system) as 100. For example, 10 4 cells need to add 10 6 TU of virus.
  • MOI that is, the number of virus particles infected per cell in a system
  • step B Inoculate the cell/semi-solid medium mixture prepared in step A above in a 6-well plate, and inoculate 3 replicate wells for each AML case cell.
  • Figure 5 shows the effect of lentivirus expressing miR-31-5p on the colony formation ability after infection of AML-LSC.
  • Figure 5a three cases of AML-LSC cells infected with lentivirus expressing control RNA could form good cell clones after 14 days of culture.
  • Example 5 the effect of miR-31-5p on AML-LSC cells and bone marrow AML cells
  • the leukemia stem cells (AML-LSC) of acute myeloid leukemia are separated according to the method in Example 1, and from the 44 AML cases detected in Example 2, the LSC and bone marrow AML cells of 3 different AML patients are selected for cell death detection Experiments, and corresponding to the patient sample numbers, the cell death detection experiments of these 3 AML patients were numbered AML-5, AML-8 and AML-9, respectively.
  • AML-LSC culture method is the same as embodiment 4.
  • Bone marrow AML cells were cultured in 1640 medium containing 20% fetal bovine blood at 37°C and 5% CO 2 .
  • the method of lentivirus infection of AML-LSC is the same as that in Example 4.
  • the method for infecting bone marrow AML cells with lentivirus was the same as that for infecting AML-LSC in Experimental Example 4.
  • the cell death rate was detected (using the LIVE/DEAD cell viability detection kit from THERMO FISHER company):
  • FIG. 6 shows the results of cell death induced by lentivirus expressing miR-31-5p after infection of AML-LSC and bone marrow AML cells. As shown in Figure 6a, the expression of miR-31-5p caused the cells to have a strong fluorescence peak, indicating that the cells had died.
  • Example 6 Effects of miR-31-5p combined with chemotherapy drugs on AML-LSC cells and bone marrow AML cells
  • the leukemia stem cells (AML-LSC) of acute myeloid leukemia are separated according to the method in Example 1, and from the 44 AML cases detected in Example 2, the LSC and bone marrow AML cells of 3 different AML patients are selected for cell death detection Experiments, and corresponding to the patient sample numbers, the cell death detection experiments of these 3 AML patients were numbered AML-5, AML-8 and AML-9, respectively.
  • AML-LSC culture method is the same as embodiment 4.
  • Bone marrow AML cells were cultured in 1640 medium containing 20% fetal bovine blood at 37°C and 5% CO 2 .
  • the method of lentivirus infection of AML-LSC is the same as that in Example 4.
  • the method for infecting bone marrow AML cells with lentivirus was the same as that for infecting AML-LSC in Experimental Example 4.
  • the lentivirus-infected AML-LSC and bone marrow AML cells were cultured for 72 hours, they were cultured for 24 hours in the medium containing or not containing 5 ⁇ M chemotherapeutic drug cytarabine (Ara-C), and then the cell death rate was detected.
  • the specific detection method is the same as in Example 5.
  • Figure 7 shows the results of cell death after the lentivirus expressing miR-31-5p infected AML-LSC and bone marrow AML cells treated with cytarabine. As shown in Figures 7a and 7b, whether it was AML-LSC cells or bone marrow AML cells, the cell death rate in the miR-31-5p expression group increased. However, the death rate of AML-LSC cells and bone marrow AML cells was further increased after the chemotherapy drug cytarabine was used in combination.
  • AML-LSC (36 ⁇ 2.9 increased to 72 ⁇ 6.6, 33.6 ⁇ 4.5 increased to 64.6 ⁇ 6.9, and 29.3 ⁇ 3.6 increased to 47.3 ⁇ 4.6)
  • bone marrow AML cells (41.6 ⁇ 3.3 increased to 65.3 ⁇ 6.6, 39.3 ⁇ 6.5 increased to 73.6 ⁇ 4.1, and 46.3 ⁇ 7.1 increased to 69.6 ⁇ 5.7). Therefore, miR-31-5p expression can effectively enhance chemotherapy drug-induced AML-LSC and bone marrow AML cell death.
  • Example 7 In vivo experiments in animals verify the effect of miR-31-5p expression on AML and AML-LSC
  • AML-LSC culture and lentivirus infection methods are the same as in Example 4.
  • mice aged 4-6 weeks were purchased from Beijing Biocytogen Biotechnology Co., Ltd. After the mice were purchased, they were kept in an SPF-grade animal room, and the experiment was carried out after one week of adaptation to the environment.
  • B-NDG immunodeficient mice were irradiated by Rad Source RS2000 series X-ray bioirradiation apparatus, and received a radiation dose of 2Gy to destroy the residual immune system.
  • AML-LSC cells infected with lentiviruses expressing control RNA (Control RNA) and miR-31-5p (miR-31-5p) were injected into mice through the tail vein, and the number of injected cells was 1 ⁇ 10 6 cells/mouse to establish B-NDG mouse leukemia model.
  • AML-LSC cells of each case were injected into 12 mice.
  • mice in each group were randomly selected for dissection, bone marrow coelomocytes were isolated, stained with anti-human CD45 and CD34 antibodies, and the proportion of positive cells was detected by flow cytometry (CD45 molecules were present on all leukocytes).
  • CD45 molecules were present on all leukocytes.
  • the anti-human CD45 antibody can specifically recognize the human leukocytes transplanted in mice, so the ratio of CD45 + cells reflects the ratio of human AML cells transplanted into mice, and the CD45 + CD34 + cells reflect the transplanted human leukocytes AML-LSC).
  • the specific method is as follows:
  • Cell staining buffer Biolegend Company
  • Cell density was adjusted to 10 6 cells/mL.
  • mice The remaining 6 mice continued to be fed, the time of death was recorded, and the survival curve was drawn.
  • Figure 8 shows the verification of the therapeutic effect of miR-31-5p expression on AML disease in B-NDG mice. in:
  • FIG. 8a show that, after 2 weeks of transplantation, AML cells (hCD45 + cells) were successfully implanted in the bone marrow cavity of the mice treated by the control group, and the three cases of AML-5, AML-8 and AML-9
  • the implantation rates of AML cells were: 40.5 ⁇ 6.9, 32.5 ⁇ 6.2 and 32.3 ⁇ 7.3; on the contrary, the cell implantation rates in the miR-31-5p expression group were significantly decreased, respectively: 4 ⁇ 3, 6.3 ⁇ 4.5 and 7.7 ⁇ 4.4.
  • FIG. 8b show that, 2 weeks after transplantation, AML-LSC cells (hCD45 + CD34 + cells) were successfully implanted in the bone marrow cavity of mice treated with the control group, and AML-5, AML-8 and AML-9
  • the engraftment rates of AML-LSC cells in the three cases were: 9.6 ⁇ 3.9, 11.2 ⁇ 4.6, and 8.1 ⁇ 3.8; on the contrary, the cell engraftment rates in the miR-31-5p expression group were significantly decreased, respectively: 1.5 ⁇ 1.1 , 1.1 ⁇ 0.7 and 2.4 ⁇ 1.5.
  • mice treated with the control group died from the 15th day after transplantation, and all mice died within 30 days.
  • the median survival periods were 19 days, 20 days and 19 days, respectively; in contrast, only a few mice in the miR-31-5p expression group died, and most of the mice survived for more than 40 days.

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Abstract

La présente divulgation concerne une application du miR-31-5p dans le traitement de la leucémie myéloïde aiguë (LMA), en particulier, une utilisation d'un produit pour détecter le miR-31-5p dans la préparation d'un réactif, d'une puce, ou d'un kit de réactifs pour le diagnostic auxiliaire d'un sujet souffrant de la LMA et/ou pour le pronostic du temps de survie du sujet, et une utilisation d'un miARN primaire/précurseur du miR-31 (pri/pre-miR-31) et/ou d'un miARN mature du miR-31 (miR-31-5p) dans la préparation d'un médicament pour le traitement de la LMA. L'introduction du miR-31-5p dans les cellules par transduction génique peut induire la mort cellulaire des cellules de LAM de la moelle osseuse et des cellules souches leucémiques de la LAM (LSC de la LAM) et améliorer la cytotoxicité de la cytosine arabinoside en tant que médicament chimiothérapeutique, et l'expression du miR-31-5p peut inhiber la croissance des cellules de LAM et des LSC de la LAM, et prolonger la durée de vie des animaux. La présente invention apporte un nouveau procédé pour le diagnostic et l'évaluation du pronostic de la LAM, et apporte en outre un médicament de thérapie génique pour la LAM.
PCT/CN2022/101571 2021-07-05 2022-06-27 Application de mir-31-5p dans le traitement de la leucémie myéloïde aiguë Ceased WO2023280001A1 (fr)

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