WO2016078572A1 - 一种新的前体miRNA及其在肿瘤治疗中的应用 - Google Patents

一种新的前体miRNA及其在肿瘤治疗中的应用 Download PDF

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WO2016078572A1
WO2016078572A1 PCT/CN2015/094809 CN2015094809W WO2016078572A1 WO 2016078572 A1 WO2016078572 A1 WO 2016078572A1 CN 2015094809 W CN2015094809 W CN 2015094809W WO 2016078572 A1 WO2016078572 A1 WO 2016078572A1
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mirna
sequence
mir
precursor
complementary
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French (fr)
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张辰宇
曾科
陈熹
张峻峰
梁宏伟
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江苏命码生物科技有限公司
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Priority to CN201580062208.9A priority Critical patent/CN107109406A/zh
Priority to US15/527,258 priority patent/US20180201927A1/en
Priority to EP15861258.0A priority patent/EP3222721A4/en
Publication of WO2016078572A1 publication Critical patent/WO2016078572A1/zh
Priority to US17/817,303 priority patent/US20230056466A1/en

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Definitions

  • the present invention relates to the field of tumor therapy, and in particular to a novel precursor miRNA, and a method and application of the precursor miRNA for inhibiting tumor growth.
  • the microRNA is derived from a long-chain RNA initial transcript (Pri-miRNA) of about 1000 bp in length, and the Pri-miRNA molecule is cleaved in the nucleus by Drosha enzyme to form a miRNA precursor having a stem-loop structure of about 60-80 nt in length. After the precursor miRNA is transported to the cytoplasm, it is further cleaved to grow a double-stranded miRNA of about 18-26 nt. After the double-stranded miRNA is unfolded, the mature miRNA enters an RNA-induced silencing complex (RISC), which is completely or incompletely paired with the complementary mRNA, degrades the target mRNA or represses its expression.
  • RISC RNA-induced silencing complex
  • microRNAs account for a small proportion of total cellular RNA, they are involved in a number of important processes in life, including early embryonic development, cell proliferation and cell death, apoptosis and fat metabolism, cell differentiation, and in genes. The role of expression regulation. However, cell proliferation and apoptosis often occur abnormally in tumors, so it is speculated that abnormal deletion, mutation or overexpression of miRNA will lead to the occurrence of human diseases.
  • the present invention provides a novel precursor miRNA that expresses anti-miRNA-214 and its use in the treatment of tumors.
  • a pharmaceutical formulation comprising:
  • the expression vector of the formula I-1 expressed by the expression vector is expressed by the expression vector
  • B1 is a desired first ribonucleic acid sequence, and the first ribonucleic acid sequence comprises an anti-miRNA sequence form or an siRNA sequence form;
  • B2 is a sequence substantially complementary or completely complementary to B1, and B2 is not complementary to C;
  • C is a sequence of stem-loop structures
  • A1 and A2 are none, or optionally an RNA sequence consisting of 4-5 bases;
  • the precursor is capable of expressing (or producing or enriching) the first ribonucleic acid sequence in a host without expressing (or producing or enriching) a second ribonucleic acid complementary to the first ribonucleic acid sequence sequence.
  • the first ribonucleic acid sequence is in the 5p form and the second ribonucleic acid sequence is in the 3p form.
  • the first ribonucleic acid sequence is in the 3p form and the second ribonucleic acid sequence is in the 5p form.
  • the first ribonucleic acid sequence is an anit-miRNA and/or siRNA.
  • the B1 is an anti-tumor miRNA or siRNA.
  • the B1 is anti-miRNA-214-5p.
  • the first ribonucleic acid sequence is anti-miRNA-214-5p and the second ribonucleic acid sequence is anti-miRNA-214-3p.
  • the formulation is in a liquid dosage form.
  • the preparation is an injection.
  • the expression vector comprises a plasmid.
  • the expression vector or plasmid contains a promoter, an origin of replication, and a marker gene.
  • the expression vector contains an expression cassette that expresses an anti-miRNA and/or siRNA.
  • the expression cassette ie, a polynucleotide
  • the expression cassette is double-stranded and has the following structure:
  • Promoter-attB1-optional tagged protein e.g., GFP or emGFP
  • GFP or emGFP Promoter-attB1-optional tagged protein-5'miR flanking region sequence-sequence-5'miR flanking region sequence of formula I-attB2-optional TKPA element.
  • the formulation is a liposomal formulation.
  • a second aspect of the invention provides a method of administering a medicament comprising the steps of:
  • the pharmaceutical preparation of the first aspect of the invention is administered to a first part of a mammal such that the expression vector is processed into microvesicles in the mammal and transported to the mammal The two sites, and the anti-miRNA and/or siRNA are expressed at the second site.
  • the mammal comprises a human and a non-human mammal.
  • the first site comprises a subcutaneous, intravenous or gastrointestinal tract.
  • the second site comprises a liver, a lung, and a kidney.
  • the administration includes oral administration, subcutaneous injection, intramuscular injection, intravenous injection.
  • a third aspect of the invention provides a precursor sequence having a structure of formula I at the 5' to 3' end:
  • B1 is a desired first ribonucleic acid sequence, wherein the first ribonucleic acid sequence comprises an anti-miRNA sequence form or an siRNA sequence form;
  • B2 is a sequence substantially complementary or completely complementary to B1, and B2 is not complementary to C;
  • C is a sequence of stem-loop structures
  • A1 and A2 are none, or optionally an RNA sequence consisting of 4-5 bases;
  • the precursor is capable of expressing (or producing or enriching) the first ribonucleic acid sequence in a host without expressing (or producing or enriching) a second ribonucleic acid complementary to the first ribonucleic acid sequence sequence.
  • B1 is anti-miRNA-214-5p
  • B2 is a sequence substantially complementary or completely complementary to B1, and B2 is not complementary to C;
  • C is a stem loop structure sequence, preferably the sequence shown in SEQ ID NO.: 1 (GUUUUGGCCACUGACUGAC);
  • A1 and A2 are none, or optionally an RNA sequence consisting of 4-5 bases;
  • the precursor sequence shown can be processed into an anti-miRNA-214 in a host, and in the anti-miRNA-214, only the anti-miRNA-214-5p is expressed, and the anti-miRNA-214-3p is not expressed. .
  • the phrase "expressing only anti-miRNA-214-5p, not expressing anti-miRNA-214-3p” means that the ratio of F5/F3 is ⁇ 5, preferably ⁇ 10, more preferably ⁇ 20, optimally ⁇ 50, wherein F5 is the expression level of anti-miRNA-214-5p, and F3 is the expression level of anti-miRNA-214-3p.
  • the anti-miRNA-214-5p is represented by SEQ ID NO.: 2 (ACUGCCUGUCUGUGCCUGCCUGU).
  • the anti-miRNA-214-3p is represented by SEQ ID NO.: 3 (ACAGGCAGGC AGACAGGCAGU).
  • the B2 and B1 are not complementary to 2-8 bases.
  • the B2 and B1 are 3-5 bases which are not complementary.
  • the B2 is added or deleted 1-2 bases compared to B1.
  • the B2 is 1-2 bases deleted from B1, and more preferably, 2 bases are deleted.
  • the deleted 1-2 bases are located in the middle of B1, ie, 1-2 bases in positions 9-14, such as positions 9-10, 10-11 , 11th-12th, 12th-13th or 13th-14th.
  • the A1 is UGCUG; and/or
  • the A2 is CAGG or CAGGA.
  • A2 is preferably CAGG.
  • a polynucleotide which is transcribed by a host to form a precursor sequence as described in the fourth aspect of the invention.
  • the polynucleotide is double-stranded and has the following structure:
  • AttB1-optional tagged protein e.g., GFP or emGFP
  • attB1-optional tagged protein e.g., GFP or emGFP-5'miR flanking sequence-sequence-5'miR flanking sequence-attB2 as shown in Formula I.
  • an expression vector comprising the precursor sequence of the fourth aspect of the invention or the polynucleotide of the fifth aspect of the invention.
  • the expression vector comprises a viral vector, a non-viral vector.
  • the expression vector is a plasmid.
  • the polynucleotide of the fifth aspect of the invention is upstream of a promoter and downstream of the TKPA element.
  • the invention provides a pharmaceutical composition comprising the precursor sequence of the fourth aspect of the invention, or the expression vector of the sixth aspect of the invention, and pharmaceutically acceptable a.
  • the pharmaceutical composition comprises an anti-miR-214 plasmid.
  • the pharmaceutical composition is the expression vector of the sixth aspect of the invention, preferably a plasmid comprising the precursor sequence of the fourth aspect of the invention.
  • the pharmaceutical composition of the pharmaceutical composition comprises:
  • the pharmaceutical composition further comprises a spray, an aerosol, a powder, a volatile liquid, a topical solution, a lotion, a pour, a tincture, a paste, or Plasters, rubber ointments, ointments, plasters, pastes, eye drops, nasal drops, ophthalmic ointments, gargles, sublingual tablets or suppositories.
  • the dosage form is an injection, preferably an intravenous injection or an intraperitoneal injection.
  • the eighth aspect of the invention provides the use of the precursor sequence of the fourth aspect of the invention, or the expression vector of the sixth aspect of the invention, comprising: (i) an inhibitor for preparing miRNA-214; and/or (i i) A pharmaceutical composition for the preparation of an anti-miRNA-214 high expression malignancy.
  • the malignant tumor includes liver cancer, lung cancer, gastric cancer, esophageal cancer, ovarian cancer, colorectal cancer, cervical cancer, pancreatic cancer, prostate cancer, leukemia, or breast cancer.
  • a method for non-therapeutic inhibition of miRNA-214 high expression malignant cell growth in vitro comprising the steps of:
  • a method for treating a high expression of a malignant tumor of miRNA-214 wherein a safe and effective amount of the expression vector of the sixth aspect of the invention or the pharmaceutical composition of the seventh aspect of the invention is administered The desired subject, thereby treating miRNA-214 with high expression of malignant tumors.
  • the administered dose is from 0.05 to 10 mg/kg, preferably from 0.1 to 5 mg/kg.
  • the administration comprises: oral, respiratory, injection, transdermal, mucosal or intracavitary administration;
  • the administering comprises injecting a plasmid.
  • a method for treating a high-expression malignant tumor of miRNA-214 which comprises administering an anti-miR-214 plasmid containing the precursor sequence of the fourth aspect of the present invention by intravenous injection In the desired subject, thus treating miRNA-214 with high expression of malignant tumors.
  • Figure 1 is a representative plasmid map of the invention.
  • "flanking region” means a flanking region (sequence).
  • Figure 2 shows the detection of the content of the siRNA in each tissue or organ after the injection of the anti-miR-214 overexpression plasmid from the tail vein of the mouse at 1h, 3h, 6h, 24h and other time points.
  • FIG. 3 shows the change in the number of CD4 + T cells.
  • Figure 4 shows the anti-miR-214 content of each tissue.
  • Figure 5 shows the effect of Anti-miR-214 on body weight of tumor-bearing mice.
  • Figure 6 shows a histological section of the therapeutic effect of Anti-miR-214 on mouse Lewis lung cancer, wherein A: x 40; B: x 100; C: x 400.
  • Figure 7 shows the levels of anti-miR-214 in blood cells and plasma before and after injection of the plasmid in the end-stage tumor volunteers.
  • Figure 8 shows the expression level of miR-214 in serum.
  • Figure 9 shows the map structure of the pcDNA3 plasmid.
  • the inventors have extensively and intensively studied and prepared a precursor miRNA capable of efficiently expressing anti-miRNA-214 for the first time.
  • the precursor miRNA of the present invention can efficiently express anti-miRNA-214-5p after being processed by a host cell without generating or substantially producing anti-miRNA-214-3p.
  • the interference effect of the reverse complementary sequence of the target sequence on the function of the target sequence is effectively avoided.
  • the precursor miRNA of the present invention can efficiently express the anti-miRNA-214-5p sequence in vivo and has a more effective therapeutic effect on various malignant tumors. On the basis of this, the present invention has been completed.
  • miRNA refers to a class of RNA molecules that are processed from transcripts that form miRNA precursors. Mature miRNAs typically have 18-26 nucleotides (nt) (more specifically about 19-22 nt) and do not exclude miRNA molecules with other numbers of nucleotides. miRNAs are usually detected by Northern blotting.
  • Human-derived miRNAs can be isolated from human cells.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
  • the miRNA can be processed from a precursor miRNA (Pre-miRNA), which can be folded into a stable stem-loop (hairpin) structure, which is generally 50-100 bp in length. between.
  • the precursor miRNA can be folded into a stable stem-loop structure, and the stem portion of the stem-loop structure comprises two sequences that are substantially complementary.
  • the precursor miRNA is a synthetic precursor miRNA, and the precursor miRNA has the structure shown in Formula I:
  • B1 is anti-miRNA-214-5p
  • B2 is a sequence complementary to B1 (including substantially complementary and fully complementary);
  • C is the sequence shown in SEQ ID NO.: 1 (GUUUUGGCCACUGACUGAC);
  • A1 and A2 are each no, or optionally a nucleotide sequence consisting of 4-5 bases;
  • the precursor miRNA shown can be processed into an anti-miRNA-214 in a host, and in the anti-miRNA-214, only the anti-miRNA-214-5p is expressed, and the anti-miRNA-214-3p is not expressed. .
  • a precursor miRNA that forms anti-miRNA-214-5p can be cleaved to generate a miRNA that antagonizes miRNA-214, namely anti-miRNA-214-5p (SEQ ID NO.: 2).
  • B2 and B1 are substantially complementary.
  • substantially complementary means that the sequences of the nucleotides are sufficiently complementary to interact in a predictable manner, such as to form a secondary structure (eg, a stem-loop structure).
  • a secondary structure eg, a stem-loop structure.
  • at least 70% of the nucleotides of the two "substantially complementary" nucleotide sequences are complementary to each other; preferably, at least 80% of the nucleotides are complementary; more preferably, at least 90% of the nucleotides are complementary.
  • two sufficiently complementary molecules can have up to 8 mismatched nucleotides, preferably 1, 2, 3, 4, 5 mismatched nucleotides.
  • a “stem loop” structure also referred to as a “hairpin” structure, refers to a nucleotide molecule, It can form a secondary structure comprising a double-stranded region (stem portion) formed by two regions of the nucleotide molecule (on the same molecule), the two regions being separated into double-stranded portions Both sides; it also includes at least one "loop” structure, including non-complementary nucleotide molecules, ie, single-stranded regions. Even if the two regions of the nucleotide molecule are not completely complementary, the double-stranded portion of the nucleotide can remain in a double-stranded state.
  • insertions, deletions, substitutions, etc. may result in non-complementation of a small region or the formation of a stem-loop structure or other form of secondary structure by itself, however, the two regions may still be substantially complementary and are foreseeable Interaction occurs in the manner to form a double-stranded region of the stem-loop structure.
  • Stem loop structures are well known to those skilled in the art, and typically after obtaining a nucleic acid having a nucleotide sequence of a primary structure, one skilled in the art will be able to determine whether the nucleic acid is capable of forming a stem-loop structure.
  • a “stem loop structure” may exist at the end of the precursor miRNA of Formula I, for example, since B1 and B2 form substantially complementary, C forms a fixed terminal stem-loop structure; "There may also be internal to the precursor miRNA of formula I, for example, because B1 and B2 are not fully complementary, resulting in an internal loop of B1 or B2 that does not complement the binding.
  • the miRNA-214 of the present invention refers to a microRNA-214 (miRNA-214) family comprising: miRNA-214 or a modified miRNA-214 derivative, the function of which is related to miRNA-214- Same or basically the same.
  • a polynucleotide construct which can be processed into a miRNA which can affect the expression of the corresponding mRNA can be designed, that is, the polynucleotide construct can be up-regulated in vivo.
  • the present invention provides an isolated polynucleotide (construct) which can be transcribed into a precursor miRNA by a human cell, which can be cleaved by a human cell And expressed as the miRNA.
  • the polynucleotide construct comprises a structure of formula II from the 5' to the 3' end:
  • B1 is a nucleotide sequence which can be expressed in the cell as said anti-miRNA-214-5p
  • b2 is a nucleotide sequence substantially complementary or completely complementary to b1
  • c is an interval between b1 and b2 a sequence, and the spacer sequence is not complementary to B1 and B2;
  • A1 and a2 are each no, or optionally a nucleotide sequence consisting of 4-5 bases;
  • the polynucleotide construct is located on an expression vector.
  • the invention also encompasses a vector comprising the miRNA, or the polynucleotide construct.
  • the expression vector usually further contains a promoter, an origin of replication, and/or a marker gene and the like. Methods well known to those skilled in the art can be used to construct the expression vectors required for the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as kalatinmycin, gentamicin, hygromycin, ampicillin resistance.
  • the expression vector is not particularly limited, and includes an expression vector which is commercially available or prepared by a conventional method.
  • Representative examples include, but are not limited to, pcDNATM6.2-GW/miR, pcDNA3, pMIR-REPORT miRNA, pAdTrack-CMV, pCAMBIA3101+pUC-35S, pCMVp-NEO-BAN, pBI121, pBin438, pCAMBIA1301, pSV2 , CMV4 expression vectors, pmiR-RB-Report TM, pshOK-basic, mmu-mir 300-399miRNASelect TM, pshRNA-copGFP Lentivector, GV317, GV309, GV253, GV250, GV249, GV234, GV233, GV232, GV201, GV159 , or other GV series expression vector.
  • the promoter operably linked to the expression of the precursor miRNA polynucleotide comprises a constitutive promoter or a tissue-specific promoter, preferably specific in liver tissue. Sex-initiated promoter. In other words, these promoters are used to drive expression of the precursor miRNA.
  • promoters include, but are not limited to, Pcmv promoter, U6, H1, CD43 promoter, CD45 (LCA) promoter, CD68 promoter, Endoglin (CD105) promoter, Fibronectin promoter, Flt-1 (VEGFR-1) promoter, GFAP promoter, GPIIb (Integrin ⁇ IIb) promoter, ICAM-2 (CD102) promoter, MB (Myoglobin) promoter, NphsI (Nephrin) promoter, SPB promoter, SV40/hAlb promoter Child, SYN1 promoter, WASP promoter or a combination thereof.
  • Pcmv promoter U6, H1, CD43 promoter, CD45 (LCA) promoter, CD68 promoter, Endoglin (CD105) promoter, Fibronectin promoter, Flt-1 (VEGFR-1) promoter, GFAP promoter, GPIIb (Integrin ⁇ IIb) promoter, ICAM-2 (CD102)
  • the term "effective amount” or “effective amount” refers to an amount that can produce a function or activity on a human and/or animal and that can be accepted by a human and/or animal.
  • the term "pharmaceutically acceptable” ingredient is a substance that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, a substance having a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • compositions of the present invention comprise a safe and effective amount of the active ingredient of the present invention together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched with the administration mode, and the pharmaceutical composition of the present invention is in the form of an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, and a sustained release agent.
  • it is prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. Change and wait. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, body weight of the patient, immune status of the patient, administration Ways, etc. In general, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg to 50 mg/kg of animal body weight per day (preferably 0.0001 mg to 10 mg/kg of animal body weight), a satisfactory effect can be obtained. For example, several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
  • the pharmaceutically acceptable carriers of the present invention include, but are not limited to, water, saline, liposomes, lipids, micro particles, microvesicles, exosomes, exfoliated Shedding vesicles, nanocapsules/Nanoparticles, ⁇ -cyclodextriniclusion compound proteins, protein-antibody conjugates, peptide materials, cellulose, nanogels, or combinations thereof.
  • the choice of carrier should be compatible with the mode of administration, which are well known to those of ordinary skill in the art.
  • the expression vector may be directly administered to a subject, and the expression vector may be administered in combination with a pharmaceutically acceptable carrier to be administered.
  • Such administration includes intravenous injection.
  • the present invention also provides a method of treating miRNA-214 high expression malignancy by administering a safe and effective amount of the expression vector or pharmaceutical composition of the present invention to a subject in need thereof, thereby treating miRNA-214 high expression malignancy.
  • miRNA-214 high expression malignant tumor means that the expression level E1 of miRNA-214 is significantly different from the amount E0 of miRNA-214 in adjacent tissues or normal tissues in the tumor, preferably.
  • the "high expression” refers to E1 ⁇ 1.5E0, and more preferably E1 ⁇ 2E0. Whether or not miRNA-214 is highly expressed in tumor tissues can be detected by a conventional method.
  • the miRNA-214 high expression malignancy includes, but is not limited to, liver cancer, lung cancer, gastric cancer, esophageal cancer, ovarian cancer, colorectal cancer, cervical cancer, pancreatic cancer, prostate cancer, leukemia, or breast cancer.
  • the precursor miRNA of the invention can effectively avoid the overexpression of the target sequence and overexpress the target sequence to obtain the reverse complement sequence, thereby effectively avoiding the interference effect of the reverse sequence complementary sequence of the target sequence on the function of the target sequence.
  • the precursor miRNA of the invention can effectively express the anti-miRNA-214-5p sequence in vivo and has an effective therapeutic effect on various malignant tumors, thereby being used for developing new tumor therapeutic drugs.
  • the microRNA vector utilizes the CMV promoter to rapidly, efficiently and continuously express pre-miRNAs in a wide range of mammalian cells, and forms small hairpin pre-miRNAs (about 70 nt) by Drosha (RNase III), and then forms a mature microRNA by Dicer. 22nt), target mRNA, Play a regulatory role.
  • the expression module of anti-miR-214 was simulated, which effectively avoided the problem of -3p, -5p when some endogenous miRNAs were expressed.
  • the synthetic oligo structure is designed as follows:
  • Competent cell DH5 ⁇ (purchased from Life)
  • Two pairs of synthesized oligo-single-stranded DNA were dissolved into 100 ⁇ M with ddH 2 O, and 5 ⁇ l of each of the complementary single strands were mixed, and the system was annealed according to the procedure given in Table 2. Two parts of the oligo mixture were heated at 95 ° C for 5 minutes and then left at room temperature for 20 minutes to form double-stranded DNA.
  • the annealed double-stranded DNA was further diluted to a concentration of 10 nM, and the system was attached at room temperature for 30 minutes as shown in Table 3.
  • 10 ⁇ l of the ligation product was transformed into 100 ⁇ l of competent cell DH5 ⁇ , and LB plate (containing 50 ⁇ g/ml spectinomycin) was applied, and then incubated at 37 °C.
  • Example 2 Cell experiments verify the efficiency of overexpression of anti-miR-214
  • the results are all the number of cycles experienced when the fluorescence signal in each reaction tube reaches the set domain value by the Ct value representing the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set domain value, wherein each template
  • the Ct value has a linear relationship with the logarithm of the initial copy number of the template. The more the initial copy number, the smaller the Ct value; the smaller the initial copy number, the larger the Ct value.
  • Table 4 shows the determination of anti-miRNA and miRNA expression levels using Real-time PCR.
  • Example 3 In vivo experiments verify the efficiency of overexpression of anti-miR-214
  • the anti-miR-214 plasmid was injected into the tail vein of C57/BL6 mice. After 24 hours, the cells were perfused, and blood, heart, liver, spleen, lung, kidney, brain and muscle tissues were taken. Total RNA was extracted, and then q-PCR was detected. Levels of miR-214-5p, anti-miR-214-3p, miR-214. The results are shown in Table 5.
  • Example 4 In vivo experiments to verify the metabolic kinetics of overexpressed siRNA
  • anti-miR-214 overexpression plasmid was injected from the tail vein of mice (anti-miR-214 refers to anti-miR-214-5p in the following experiments) at 1h, 3h, 6h, 9h, 12h, 24h, 36h, 48h
  • the content of the siRNA in plasma, heart, liver, spleen, lung, kidney, brain, skeletal muscle, and CD4+ T cells was measured, and the specific results are shown in FIG. 2 .
  • siRNA did enter the lungs and reached the highest concentration after 6 h, which was detectable within 24 h.
  • the lung cancer metastasis model was constructed by injecting LLC cell into the tail vein of the mouse. After successful model construction, the anti-miR-214 overexpression plasmid was injected into the tail vein to observe the therapeutic effect. After the mice were sacrificed, the mice were taken for Real-time PCR detection. The distribution of anti-miR-214 in each tissue of the mouse was examined by tissue sectioning to observe the therapeutic effect.
  • anti-miR-214 plasmid can effectively inhibit the number of new T cells CD25 + , Foxp3 + T cell (Treg) inhibited in tumor mice in a dose-dependent manner, with the increase of the concentration of the injected plasmid. The number of CD25 + and Foxp3 + T cell (Treg) is reduced.
  • LCC tumor cells in logarithmic growth phase were collected, and an in situ Lewis lung cancer model was established by tail vein injection at 1 ⁇ 10 6 /mouse. After the model was established, a serial concentration of Anti-miR-214 was administered intravenously, and the living conditions of the animals during the administration period were observed. After the end of the administration, the lungs were taken for pathological biopsy to observe the severity of lung tumors and the therapeutic effect of Anti-miR-214.
  • Test compound Anti-miR-214, content: 6.4 mg/ml, supplied by the School of Life Sciences, Nanjing University. The test was diluted with physiological saline for injection to the desired concentration.
  • LCC cell line provided by the School of Life Sciences of Nanjing University.
  • DMEM is a Hyclone product.
  • Fetal bovine serum is a product of Gibco.
  • the LCC cell line was cultured in a DMEM complete medium containing 10% FBS, 100 ug/ml penicillin and 100 ug/ml streptomycin at 37 ° C in a 5% CO 2 incubator.
  • the LCC cells cultured to the logarithmic growth phase were trypsinized, centrifuged at 1000 rpm, the supernatant was discarded, washed twice with sterile physiological saline, and the cells were suspended in physiological saline, and the cell viability was observed by trypan blue staining. The cell count was adjusted to a cell density of 5 ⁇ 10 6 /ml.
  • healthy C57BL/6 mice were injected slowly in 0.2 ml/tail vein. After the injection, all model mice were divided into model group (negative control group), large (5 mg/kg), medium (0.5). Mg/kg), low dose group (0.05mg/kg).
  • mice During the modeling period, the spirit, diet, defecation, weight and activities of C57BL/6 mice were observed regularly. On the 14th day, the rats were administered with a tail vein of 0.1 ml/10 g body weight, and the control group was given the corresponding physiological saline. During the administration period, it was administered once every 3 days for 7 times. On the third day after the last administration, the mice were anesthetized with ether and blood, lung and liver were taken. The lungs and liver were placed in 10% formalin for pathological sectioning to observe the formation of lung cancer and the treatment of lung cancer with Anti-miR-214.
  • C57BL/6 mice were treated with Anti-miR-214 intravenously 2 weeks after the establishment of Lewis lung cancer.
  • the animals were sacrificed on the 3rd day after the last administration, blood collection, lung and liver.
  • the other lungs and liver were fixed with formalin, and histopathological biopsy was performed to examine the tumor status of the organ.
  • the specific results are shown in Figure 6. As can be seen from the results of Fig. 6, the pathological section results showed that no tumor lesions were observed in all the liver sections of each group. In the lungs, various treatment groups showed different degrees of plaque nucleus deep-stained tumor cell aggregation.
  • Anti-miR-214 has a dose-dependent therapeutic effect on Lewis lung cancer in vivo.
  • the concentration of 5 mg/kg is the most obvious, and no abnormal drug-related reactions are observed during the administration.
  • the anti-miR-214 plasmid was injected into the end-stage of breast cancer metastasis twice a week, 1 mg each time. The volunteers were tested for one month after starting the drug.
  • Figure 8 shows the expression level of miR-214 in serum.
  • the serum of the volunteer patient miR-214 decreased significantly compared with that before the treatment, and almost decreased to a level comparable to that of the normal person, which not only suggested that the injection of anti-miR-214 plasmid could be In vivo expression, but also functional, able to adsorb miR-214 in vivo, so that its expression level is significantly reduced.
  • miR-214 inhibitor has an inhibitory effect on tumor growth. miR-214 inhibitors are useful in cancer therapy.
  • Example 8 Precursor miRNA is suitable for other overexpression vectors
  • the precursor miRNA structure was inserted into the expression vector pcDNA3, and the constructed expression module was also effective in avoiding the problem of -3p, -5p when some endogenous miRNAs were expressed.
  • the pcDNA3 map structure is shown in Figure 9.
  • Two pairs of complementary ol igo DNAs were designed and synthesized according to anti-miR-214 as described in 2.1 of Example 1, the structure of the precursor miRNA.
  • Example 1 The specific operation steps are as described in 2.2 in Example 1.
  • the efficiency verification method for overexpressing anti-miR-214 is the same as in Example 2.
  • Table 8 shows the determination of anti-miRNA and miRNA expression levels using Real-time PCR.
  • anti-miR-214 constructed according to the present invention is different from the precursor miRNA,
  • the precursor miRNA constructs of the present invention is inserted into the same vector pcDNA TM 6.2.
  • the anti-miR-214 plasmid was transfected into A549 cells, and the total RNA was used to detect the anti-miR-214-5p, anti-miR-214-3p, and miR-214 primers by ABI. -miR-214-5p, anti-miR-214-3p, miR-214 levels.
  • the synthetic oligo structure is designed as follows:
  • Examples 1 and 2 were repeated except that the structure of A2 was changed from CAGG to CAGGA.

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Abstract

提供了一种前体miRNA及其在肿瘤治疗中的应用。该前体miRNA的5'至3'端具有式I所示的结构，B1为anti-miRNA-214-5p；B2为与B1基本互补或完全互补的序列，且B2与C不互补；C为茎环结构序列；A1和A2分别为无，任选的由4-5个碱基组成的RNA序列；其中，所示的前体miRNA能在宿主中加工形成anti-miRNA-214，且在所述anti-miRNA-214中，只表达anti-miRNA-214-5p，不表达anti-miRNA-214-3p。

Description

一种新的前体miRNA及其在肿瘤治疗中的应用 技术领域
本发明涉及肿瘤治疗领域，具体地，涉及一种新的前体miRNA，以及该前体miRNA用于抑制肿瘤生长中的方法及应用。
背景技术
microRNA来源于长度约1000bp的长链RNA初始转录产物(Pri-miRNA)，Pri-miRNA分子在细胞核中经Drosha酶剪切形成长度约60-80nt的具有茎环结构的miRNA前体。前体miRNA转运至胞质后，被进一步切割成长约18-26nt的双链miRNA。双链miRNA解开后，成熟的miRNA进入RNA诱导基因沉默复合物(RNA-induced silencing complex，RISC)，与互补mRNA完全或不完全配对，降解靶mRNA或阻遏其表达。
尽管microRNA在细胞总RNA中所占的比重很小，但由于miRNA参与生命过程中一系列的重要进程,包括早期胚胎发育、细胞增殖和细胞死亡、细胞凋亡与脂肪代谢、细胞分化以及在基因表达调控中的作用。而细胞增殖及凋亡等常在肿瘤中发生异常，因此推测miRNA的异常缺失、突变或过表达将导致人类疾病的发生。
近来，越来越多证据显示miRNA在抑制肿瘤生长、繁殖、分化中扮演者及其重要的角色。本领域迫切需要了解在肿瘤治疗领域，各种不同miRNA的作用，以开发新的肿瘤治疗药物。
发明内容
本发明提供了一种新的表达anti-miRNA-214的前体miRNA，及其在治疗肿瘤中的应用。
本发明第一方面，提供一种药物制剂，所述的制剂含有：
(a)用于表达anti-miRNA和/或siRNA的表达载体；以及
(b)药学上可接受的载体。
在另一优选例中，所述的表达载体所表达的式I－1所示的前体，
Figure PCTCN2015094809-appb-000001
    式I，
B1为所需要的第一核糖核酸序列，所述的第一核糖核酸序列包括anti-miRNA序列形式或siRNA序列形式；
B2为与B1基本互补或完全互补的序列，且B2与C不互补；
C为茎环结构序列；
A1和A2分别为无，或任选的由4-5个碱基组成的RNA序列；
其中，所述前体能在宿主中表达(或产生或富集)所述的第一核糖核酸序列，而不表达(或产生或富集)与所述第一核糖核酸序列互补的第二核糖核酸序列。
在另一优选例中，所述的第一核糖核酸序列为5p形式，而第二核糖核酸序列为3p形式。
在另一优选例中，所述的第一核糖核酸序列为3p形式，而第二核糖核酸序列为5p形式。
在另一优选例中，所述的第一核糖核酸序列为anit－miRNA和/或siRNA。
在另一优选例中，所述的B1为抗肿瘤的miRNA或siRNA。
在另一优选例中，所述的B1为anti-miRNA-214-5p。
在另一优选例中，第一核糖核酸序列为anti-miRNA-214-5p，而第二核糖核酸序列为anti-miRNA-214-3p。
在另一优选例中，所述的制剂为液体剂型。
在另一优选例中，所述的制剂为注射剂。
在另一优选例中，所述的表达载体包括质粒。
在另一优选例中，所述的表达载体或质粒含有启动子、复制起点和标记基因。
在另一优选例中，所述的表达载体中含有表达anti-miRNA和/或siRNA的表达盒。
在另一优选例中，所述的表达盒(即多核苷酸)为双链，并且具有以下结构：
启动子－attB1-任选的标签蛋白(如GFP或emGFP)－5’miR侧翼区序列－式I所示的序列－5’miR侧翼区序列－attB2－任选的TKPA元件。
在另一优选例中，所述的制剂为脂质体制剂。
本发明第二方面提供一种施用药物的方法，包括步骤：
将本发明第一方面所述的药物制剂施用于哺乳动物的第一部位，从而使得所述表达载体在所述哺乳动物体内被加工形成微粒子(microvesicle)，并被运输到所述哺乳动物的第二部位，并在所述的第二部位表达所述的anti-miRNA和/或siRNA。
在另一优选例中，所述的哺乳动物包括人和非人哺乳动物。
在另一优选例中，所述的第一部位包括皮下、静脉或肠胃道。
在另一优选例中，所述的第二部位包括肝脏、肺、肾脏。
在另一优选例中，所述的施用包括口服、皮下注射、肌内注射、静脉注射。
本发明第三方面提供一种前体序列，其5'至3'端具有式I所示的结构：
Figure PCTCN2015094809-appb-000002
     式I，
B1为所需要的第一核糖核酸序列，其中所述的第一核糖核酸序列包括anti-miRNA序列形式或siRNA序列形式；
B2为与B1基本互补或完全互补的序列，且B2与C不互补；
C为茎环结构序列；
A1和A2分别为无，或任选的由4-5个碱基组成的RNA序列；
其中，所述前体能在宿主中表达(或产生或富集)所述的第一核糖核酸序列，而不表达(或产生或富集)与所述第一核糖核酸序列互补的第二核糖核酸序列。
本发明第四方面，提供了一种前体序列，其5'至3'端具有式I所示的结构：
Figure PCTCN2015094809-appb-000003
   式I，
B1为anti-miRNA-214-5p；
B2为与B1基本互补或完全互补的序列，且B2与C不互补；
C为茎环结构序列，较佳地为SEQ ID NO.:1所示的序列(GUUUUGGCCACUGACUGAC)；
A1和A2分别为无，或任选的由4-5个碱基组成的RNA序列；
其中，所示的前体序列能在宿主中加工形成anti-miRNA-214，且在所述anti-miRNA-214中，只表达anti-miRNA-214-5p，不表达anti-miRNA-214-3p。
在另一优选例中，所述的“只表达anti-miRNA-214-5p，不表达anti-miRNA-214-3p”表示F5/F3之比≥5，较佳地≥10,更佳地≥20，最佳地≥50，其中F5为anti-miRNA-214-5p的表达量，而F3为anti-miRNA-214-3p的表达量。在另一优选例中，所述的anti-miRNA-214-5p如SEQ ID NO.:2所示(ACUGCCUGUCUGUGCCUGCCUGU)。
在另一优选例中，所述的anti-miRNA-214-3p如SEQ ID NO.:3所示(ACAGGCAGGC AGACAGGCAGU)。
在另一优选例中，所述B2与B1有2-8个碱基不互补，较佳地，所述B2与B1有3-5个碱基不互补。
在另一优选例中，所述B2较B1添加或缺失1-2个碱基。
在另一优选例中，所述的B2较B1缺失1-2个碱基，更佳地，缺失2个碱基。
在另一优选例中，所述的被缺失的1-2个碱基位于B1的中部，即9-14位中的1-2个碱基，如第9-10位，第10-11位，第11-12位，第12-13位或第13-14位。
在另一优选例中，所述的A1为UGCUG；和/或
所述的A2为CAGG或CAGGA。
在另一优选例中，A2优选为CAGG。
本发明第五方面，提供了一种多核苷酸，所述的多核苷酸能被宿主转录形成本发明第四方面中所述的前体序列。
在另一优选例中，所述的多核苷酸为双链，并且具有以下结构：
attB1-任选的标签蛋白(如GFP或emGFP)－5’miR侧翼序列－式I所示的序列－5’miR侧翼序列－attB2。
本发明第六方面，提供了一种表达载体，所述的表达载体含有本发明第四方面所述的前体序列或本发明第五方面所述的多核苷酸。
在另一优选例中，所述的表达载体包括病毒载体、非病毒载体。
在另一优选例中，所述的表达载体为质粒。
在另一优选例中，所述的本发明第五方面所述的多核苷酸的上游为启动子，并且其下游为TKPA元件。
本发明第七方面，提供了一种药物组合物，所述的药物组合物含有本发明第四方面所述的前体序列、或本发明第六方面所述的表达载体，和药学上可接受的载体。
在另一优选例中，所述的药物组合物包括anti-miR-214质粒。
在另一优选例中，所述的药物组合物为本发明第六方面所述的表达载体，较佳地，为含有本发明第四方面所述前体序列的质粒。
在另一优选例中，所述的药物组合物的剂型包括：
片剂、胶囊剂、粉剂、丸剂、颗粒剂、糖浆剂、溶液、混悬液、乳剂、混悬剂、注射液、或粉针剂。
在另一优选例中，所述的药物组合物的剂型还包括喷雾剂、气雾剂、粉雾剂、挥发性液体、外用溶液剂、洗剂、浇淋剂、搽剂、巴布膏剂、膏药、橡胶膏剂、软膏剂、硬膏剂、糊剂、滴眼剂、滴鼻剂、眼用软膏剂、含漱剂、舌下片剂或栓剂。
在另一优选例中，所述的剂型为注射剂，较佳地，为静脉注射剂、腹腔注射剂。
本发明第八方面，提供了本发明第四方面所述前体序列、或本发明第六方面所述表达载体的用途，包括，(i)用于制备miRNA-214的抑制剂；和/或(i i)用于制备抗miRNA-214高表达恶性肿瘤的药物组合物。
在另一优选例中，所述的恶性肿瘤包括肝癌、肺癌、胃癌、食管癌、卵巢癌、结直肠癌、宫颈癌、胰腺癌、前列腺癌、白血病、或乳腺癌。
本发明第九方面，提供了一种体外非治疗性抑制miRNA-214高表达恶性肿瘤细胞生长的方法，包括步骤：
在本发明第七方面所述药物组合物存在情况下培养miRNA-214高表达恶性肿 瘤细胞，从而抑制miRNA-214高表达恶性肿瘤细胞的生长。
本发明第十方面，提供了一种治疗miRNA-214高表达恶性肿瘤的方法，将安全有效量的本发明第六方面所述的表达载体或本发明第七方面所述的药物组合物施用于所需对象，从而治疗miRNA-214高表达恶性肿瘤。
在另一优选例中，所述施用的剂量为0.05-10mg/kg，较佳地，为0.1-5mg/kg。
在另一优选例中，所述施用包括：口服、呼吸道、注射、透皮、粘膜或腔道给药；
在另一优选例中，所述施用包括注射质粒。
本发明第十一方面，提供了一种治疗miRNA-214高表达恶性肿瘤的方法，其特征在于，将含有本发明第四方面所述的前体序列的anti-miR-214质粒通过静脉注射施用于所需对象，从而治疗miRNA-214高表达恶性肿瘤。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1本发明的代表性的质粒图谱。其中，“flanking region”表示侧翼区(序列)。
图2显示了从小鼠尾静脉注射anti-miR-214过表达质粒后，在1h,3h,6h,24h等时间节点后检测各组织或脏器中该siRNA的含量。
图3显示了CD4+T细胞的数量变化。
图4显示了各个组织anti-miR-214含量。
图5显示了Anti-miR-214对荷瘤鼠体重影响。
图6显示了Anti-miR-214对小鼠Lewis肺癌治疗作用组织切片图，其中A:×40；B:×100；C:×400。
图7显示了肿瘤末期志愿者注射质粒前后血细胞和血浆中anti-miR-214的含量。
图8显示了血清中miR-214的表达水平。
图9显示了pcDNA3质粒的图谱结构。
具体实施方式
本发明人经过广泛而深入地研究，首次设计制备了一种能够高效表达anti-miRNA-214的前体miRNA。本发明前体miRNA经过宿主细胞的加工后，能够高效地表达anti-miRNA-214-5p，而不产生或基本不产生anti-miRNA-214-3p，从 而有效地避免了目的序列的反向互补序列对目的序列发挥功能的干扰作用。实验证明，本发明前体miRNA能够在体内有效表达anti-miRNA-214-5p序列，并对多种恶性肿瘤都具有更有效的治疗作用。在此基础上，完成了本发明。
miRNA及其前体
如本文所用，所述的“miRNA”是指一类RNA分子，从可形成miRNA前体的转录物加工而来。成熟的miRNA通常具有18-26个核苷酸(nt)(更特别的约19-22nt)，也不排除具有其它数目核苷酸的miRNA分子。miRNA通常可被Northern印迹检测到。
人来源的miRNA可被从人细胞中分离。如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化的，但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
miRNA可从前体miRNA(Precursor miRNA，Pre-miRNA)加工而来，所述的前体miRNA可折叠成一种稳定的茎环(发夹)结构，所述的茎环结构长度一般在50-100bp之间。所述的前体miRNA可折叠成稳定的茎环结构，茎环结构的茎部两侧包含基本上互补的两条序列。
在本发明中，所述的前体miRNA为人工合成的前体miRNA，且所述的前体miRNA具有式I所示的结构：
Figure PCTCN2015094809-appb-000004
       式I，
作为代表性的例子，B1为anti-miRNA-214-5p；
B2为与B1互补(包括基本互补和完全互补)的序列；
C为SEQ ID NO.:1所示的序列(GUUUUGGCCACUGACUGAC)；
A1和A2分别为无，或任选的由4个-5个碱基组成的核苷酸序列；
其中，所示的前体miRNA能在宿主中加工形成anti-miRNA-214，且在所述anti-miRNA-214中，只表达anti-miRNA-214-5p，不表达anti-miRNA-214-3p。
在本发明中，形成anti-miRNA-214-5p的前体miRNA可被剪切生成与拮抗miRNA-214的miRNA，即anti-miRNA-214-5p(SEQ ID NO.:2)。
在式I中，B2和B1为基本互补。如本文所用，“基本上互补”是指核苷酸的序列是足够互补的，可以以一种可预见的方式发生相互作用，如形成二级结构(如茎环结构)。通常，两条“基本上互补”的核苷酸序列互相之间至少有70％的核苷酸是互补的；优选的，至少有80％的核苷酸是互补的；更优选的，至少有90％的核苷酸是互补的。一般地，两条足够互补的分子之间可以具有最多8个不匹配的核苷酸，优选地，具有1、2、3、4、5个不匹配的核苷酸。
如本申请所用，“茎环”结构也被称作“发夹”结构，是指一种核苷酸分子， 其可形成一种包括双链区域(茎部)的二级结构，所述的双链区域由该核苷酸分子的两个区域(位于同一分子上)形成，两个区域分列双链部分的两侧；其还包括至少一个“环”结构，包括非互补的核苷酸分子，即单链区域。即使该核苷酸分子的两个区域不是完全互补的，核苷酸的双链部分也可保持双链状态。例如，插入、缺失、取代等可导致一个小区域的不互补或该小区域自身形成茎环结构或其它形式的二级结构，然而，该两个区域仍可基本上互补，并在可预见的方式中发生相互作用，形成茎环结构的双链区域。茎环结构是本领域技术人员所熟知的，通常在获得了一条具有一级结构的核苷酸序列的核酸后，本领域技术人员能够确定该核酸是否能形成茎环结构。
本发明中，“茎环结构”可存在于式I所示前体miRNA的末端，例如由于B1和B2形成基本互补后，C会形成一固定的末端茎环结构；所述的“茎环结构”还可存在于式I所式前体miRNA内部，例如由于B1和B2之间并非完全互补，造成未互补结合的B1或B2的碱基会形成一内部的茎环(internal loop)。
本发明所述的miRNA-214是指：微小RNA-214(miRNA-214)家族，所述miRNA-214家族包括：miRNA-214或经修饰的miRNA-214衍生物，其功能与miRNA-214-相同或基本相同。
根据本发明所提供的miRNA序列，可设计出在被导入后可被加工成可影响相应的mRNA表达的miRNA的多核苷酸构建物，也即所述多核苷酸构建物能够在体内上调相应的anti-miRNA-214-5p的量，从而降低miRNA-214的量。因此，本发明提供了一种分离的多核苷酸(构建物)，所述的多核苷酸(构建物)可被人细胞转录成前体miRNA，所述的前体miRNA可被人细胞剪切且表达成所述的miRNA。
多核苷酸构建物
作为本发明的一种优选方式，所述的多核苷酸构建物自5'至3'端含有式II所示的结构：
a1-b1-c-b2-a2
式II
式II中，
b1为可在细胞中表达成所述的anti-miRNA-214-5p的核苷酸序列，b2为与b1基本上互补或完全互补的核苷酸序列；c为位于b1和b2之间的间隔序列，并且所述间隔序列与B1和B2不互补；
a1和a2分别为无，或任选的由4个-5个碱基组成的核苷酸序列；
式II所示的结构在转入细胞后，形成式I所示的二级结构：
Figure PCTCN2015094809-appb-000005
式I
通常，所述的多核苷酸构建物位于表达载体上。因此，本发明还包括一种载体，它含有所述的miRNA，或所述的多核苷酸构建物。所述的表达载体通常还含有启动子、复制起点和/或标记基因等。本领域的技术人员熟知的方法能用于构建本发明所需的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如卡拉霉素、庆大霉素、潮霉素、氨苄青霉素抗性。
在本发明中，所述表达载体没有特别限制，包括市售的或用常规制备的表达载体。代表性的例子包括(但并不限于)：pcDNATM6.2-GW/miR、pcDNA3、pMIR-REPORT miRNA、pAdTrack-CMV、pCAMBIA3101+pUC-35S、pCMVp-NEO-BAN、pBI121、pBin438、pCAMBIA1301、pSV2、CMV4表达载体、pmiR-RB-ReportTM、pshOK-basic、mmu-mir 300-399miRNASelectTM、pshRNA-copGFP Lentivector、GV317、GV309、GV253、GV250、GV249、GV234、GV233、GV232、GV201、GV159或其他GV系列表达载体。
在另一优选例中，在所述表达载体中，与所述表达所述前体miRNA多核苷酸操作性相连的启动子包括组成型启动子或组织特异性启动子，优选在肝脏组织中特异性启动的启动子。换言之，这些启动子用于驱动前体miRNA的表达。
代表性的启动子包括(但并不限于)：Pcmv启动子、U6、H1、CD43启动子、CD45(LCA)启动子、CD68启动子、Endoglin(CD105)启动子、Fibronectin启动子、Flt-1(VEGFR-1)启动子、GFAP启动子、GPIIb(IntegrinαIIb)启动子、ICAM-2(CD102)启动子、MB(Myoglobin)启动子、NphsI(Nephrin)启动子、SPB启动子、SV40/hAlb启动子、SYN1启动子、WASP启动子或其组合。
药物组合物及施用方法
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，术语“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
本发明的药物组合物含有安全有效量的本发明的活性成分以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物的剂型为注射剂、口服制剂(片剂、胶囊、口服液)、透皮剂、缓释剂。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。
本发明所述的活性成分的有效量可随给药的模式和待治疗的疾病的严重程度 等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，当本发明的活性成分每天以约0.00001mg-50mg/kg动物体重(较佳的0.0001mg-10mg/kg动物体重)的剂量给予，能得到令人满意的效果。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
本发明所述的药学上可接受的载体包括(但不限于)：水、盐水、脂质体、脂质、微粒子(micro particle)、微泡(micro vesicle)、外泌体(exosomes)、脱落囊泡(shedding vesicle)、纳米胶囊(Nanocapsules/Nanoparticles)、β环糊精胶囊(β-cyclodextriniclusion compound)蛋白、蛋白-抗体缀合物、肽类物质、纤维素、纳米凝胶、或其组合。载体的选择应与给药方式相匹配，这些都是本领域的普通技术人员所熟知的。
在本发明中，可将所述的表达载体直接施用于对象，也可将所述的表达载体与药学上可接受的载体制备成药物组合后进行施用。所述的施用包括静脉注射。
治疗方法
本发明还提供了一种治疗miRNA-214高表达恶性肿瘤的方法，即，将安全有效量的本发明表达载体或药物组合物施用于所需对象，从而治疗miRNA-214高表达恶性肿瘤。通常，“miRNA-214高表达恶性肿瘤”指的是所述的肿瘤中，miRNA-214的表达量E1与癌旁组织或正常组织中miRNA-214的量E0相比具有显著性差异，较佳地，所述“高表达”指的是E1≥1.5E0，更佳地E1≥2E0。肿瘤组织中，miRNA-214是否高表达可以通过常规方法检测。通常，所述的miRNA-214高表达恶性肿瘤包括(但不限于)肝癌、肺癌、胃癌、食管癌、卵巢癌、结直肠癌、宫颈癌、胰腺癌、前列腺癌、白血病、或乳腺癌。
本发明有益效果
本发明前体miRNA能够有效的避免在过表达目的序列得同时也过表达目的序列得反向互补序列，从而有效避免了目的序列得反向互补序列对目的序列发挥功能的干扰作用。
本发明前体miRNA能够在体内有效表达anti-miRNA-214-5p序列，并对多种恶性肿瘤都具有有效的治疗作用，从而用于开发新的肿瘤治疗药物。
实施例1：Anti-miR-214过表达载体构建
MicroRNA载体利用CMV启动子可以在众多哺乳动物细胞中快速、高效、持续表达pre-miRNA，经过Drosha(RNaseⅢ)作用形成small hairpin pre-miRNAs(约70nt)，再过Dicer作用形成成熟的microRNA(约22nt)，作用靶mRNA， 起到表达调控作用。利用优化过的载体，模拟anti-miR-214的表达模块，有效的避免了有些内源miRNA表达时产生-3p，-5p的问题。
1.anti-miR-214-5p序列
>anti-miR-214-5p
5’-ACUGCCUGUCUGUGCCUGCCUGU-3’(SEQ ID NO.:2)
2.anti-miR-214载体
2.1.Oligo DNA的设计与合成
根据基因序列设计并合成2对互补oligo DNA(oligo设计方法及架构请参见产品使用说明第6条)，序列见表1。
设计合成的oligo结构如下：
Figure PCTCN2015094809-appb-000006
表1、oligo DNA序列及其对应的前体miRNA元件
Figure PCTCN2015094809-appb-000007
2.2miRNA载体的构建与验证
2.2.1材料
含有EmGFP的BLOCK-iTTM Pol II miR RNAi表达载体盒(购自Life)
oligo DNA(购自Life)
感受态细胞DH5α(购自Life)
2.2.2方法
(1)退火
将2对合成好的寡聚单链DNA用ddH2O溶解成100μM，互补单链各取5μl两两混合，按表2给出体系进行退火。将2份oligo混合物在95℃加热5分钟，然后放置室温20分钟，形成双链DNA。
表2、oligo DNA退火体系
Figure PCTCN2015094809-appb-000008
(2)连接
将退火的双链DNA继续稀释成10nM浓度，按表3给出体系在室温连接30分钟。
表3、酶连接体系
Figure PCTCN2015094809-appb-000009
注：pcDNA6.2-GW/EmGFP-miR和pcDNA6.2-GW/miR的结构如图1所示。
(3)转化
取10μl连接产物转化100μl感受态细胞DH5α，涂LB平板(含50μg/ml壮观霉素)后，37℃孵育。
(4)测序验证
每个转化平板分别挑取3个克隆，摇菌抽提质粒后进行测序，以验证重组克隆中插入片段序列是否与设计的寡聚单链DNA序列一致。
实施例2：细胞实验验证过表达anti-miR-214的效率
在A549细胞(购自中国科学院上海生命科学研究院细胞资源中心)中转染 anti-miR-214质粒，统一总RNA后分别利用从ABI公司定制的anti-miR-214-5p，anti-miR-214-3p，miR-214的引物Real-time-PCR检测anti-miR-214-5p、anti-miR-214-3p、miR-214的水平。结果均以Ct值代表每个反应管内的荧光信号到达设定的域值时所经历的循环数每个反应管内的荧光信号到达设定的域值时所经历的循环数，其中,每个模板的Ct值与该模板的起始拷贝数的对数存在线性关系，起始拷贝数越多，Ct值越小；起始拷贝数越少，Ct值越大。表4显示了利用Real-time PCR对anti-miRNA和miRNA表达水平进行测定。
表4、anti-miR-214-5p、anti-miR-214-3p、miR-214的表达水平
(Ct) anti-miR-214-5p
空白质粒 39.63±0.2928
anti-miR-214质粒 24.55±0.5052
   
(Ct) anti-miR-214-3p
空白质粒 30.45±0.7865
anti-miR-214质粒 33.87±0.5683
   
(Ct) miR-214
空白质粒 20.55±0.6822
anti-miR-214质粒 23.63±0.5119
结论：在A549细胞中转染anti-miR-214质粒后，能检测到anti-miR-214-5p的急剧上升(p<0.01)，基本检测不到anti-miR-214-3p(p＞0.05)，且不会引起miR-214的上升，相反会导致miR-214下降(p<0.05)。基于Ct值的差异，可以判断出F5/F3之比远大于50/1。
实施例3：体内实验验证过表达anti-miR-214的效率
给C57/BL6小鼠尾静脉注射anti-miR-214质粒，24h后灌流，取血、心、肝、脾、肺、肾、脑、肌肉组织，统一总RNA，然后进行q-PCR检测anti-miR-214-5p、anti-miR-214-3p、miR-214的水平。结果见表5。
表5、anti-miR-214-5p、anti-miR-214-3p的表达水平
Figure PCTCN2015094809-appb-000010
Figure PCTCN2015094809-appb-000011
实验结果说明：
从实验结果来看，注射0.1mg质粒后，小鼠全血，肝，肺中anti-miR-214-5p水平已经显著升高(p＜0.05),而检测不到anti-miR-214-3p的变化(p＜0.05)。
实施例4：体内实验验证过表达siRNA的代谢动力学
从小鼠尾静脉注射anti-miR-214过表达质粒(以下实验中，anti-miR-214均指anti-miR-214-5p)，在1h,3h,6h,9h,12h,24h,36h,48h后检测血浆、心脏、肝、脾、肺、肾、脑、骨骼肌、CD4+T细胞中该siRNA的含量，具体结果见图2。从图2可以看出，siRNA确实可以进入肺部并且在6h后达到最高浓度，在24h内均能够检测到。
实施例5：植瘤小鼠实验
从小鼠尾静脉注射LLC cell,构建肺癌转移模型，模型构建成功后，尾静脉注射anti-miR-214过表达质粒，观察治疗效果，处死小鼠后取小鼠各个组织进行Real-time PCR检测小鼠各个组织内anti-miR-214的分布，并进行组织切片检测以观察治疗效果。
(1)CD25+,Foxp3+T cell数量变化
具体结果见图3。从图3可以看出，anti-miR-214质粒可以有效抑制肿瘤小鼠体内抑制新T细胞CD25+,Foxp3+T cell(Treg)的数量，并且呈剂量依赖性，随着注射质粒浓度的提高，CD25+,Foxp3+T cell(Treg)的数量随之减少。
(2)各个组织anti-miR-214含量
具体结果见图4。从图4可以看出，注射anti-miR-214质粒后，anti-miR-214可以被肝脏运送到多个组织，并且使多个组织anti-miR-214水平急剧升高。
实施例6：Anti-miR-214对小鼠Lewis肺癌治疗作用研究
通过C57BL/6小鼠在体肿瘤造模，Anti-miR-214静脉给药对小鼠Lewis肺癌的治疗作用，为后续研究提供参考。
收集对数生长期的LCC肿瘤细胞，按1×106/鼠通过尾静脉注射建立原位Lewis肺癌模型。成模后，静脉注射给予系列浓度的Anti-miR-214，并观察给药期动物生活状况。给药结束后，取肺脏进行病理切片检查，观察肺肿瘤严重程度及Anti-miR-214治疗效果。
1.试验材料与方法
1.1试验材料
受试化合物：Anti-miR-214，含量：6.4mg/ml，由南京大学生命科学学院提供。试验时用注射用生理盐水稀释成所需浓度。
LCC细胞系：由南京大学生命科学学院提供。DMEM为Hyclone公司产品。胎牛血清为Gibco公司产品。试验时，LCC细胞系用含10％FBS、100ug/ml青霉素和100ug/ml链霉素的DMEM完全培养基于37℃、5％CO2培养箱中培养。
动物：C57BL/6小鼠40只，雌雄各半，6周龄，由扬州大学实验动物中心提供，合格证号：SCXK(苏)2007-0001。
1.2试验方法
将培养至对数生长期的LCC细胞用胰酶消化后，1000rpm离心，弃上清，用无菌生理盐水洗涤两次，将细胞悬浮于生理盐水中，台盼蓝染色观察细胞活力，并进行细胞计数，调整细胞密度为5×106/ml。试验时，取健康C57BL/6鼠，按0.2ml/只尾静脉缓慢注射，注射结束后，将所有造模小鼠分为模型组(阴性对照组)、大(5mg/kg)、中(0.5mg/kg)、小剂量组(0.05mg/kg)。造模期间，定期观察C57BL/6小鼠的精神、饮食、排便、体重和活动等情况。第14天开始，按0.1ml/10g体重尾静脉注射给药，对照组给予相应的生理盐水。给药期间，每3天给药一次，共7次。末次给药后第3天，小鼠乙醚麻醉，取血、肺及肝脏。将肺、肝脏放10％福尔马林中，做病理切片，观察肺癌成模情况及Anti-miR-214对肺癌治疗情况。
肺部肿瘤病理切片及评分
对所有肺组织进行病理切片观察。根据肉眼大体观察状况，取3个不同病变部位进行切片，根据肺部成瘤情况及严重程度，分为“-”表示镜下未见肿瘤灶；“±”表示轻微肿瘤灶；“+”表示轻度肿瘤灶；“++”表示中度肿瘤灶；“+++”表示重度肿瘤灶，并相应评分为0-4分。
1.3统计学处理
所有计量资料采用
Figure PCTCN2015094809-appb-000012
表示。采用SPSS 16.0软件包进行统计学分析处理，多组间比较采用方差分析F检验，组间比较采用成组t检验，P<0.05为具有统计学意义。
2.结果
2.1造模及给药期间动物一般状况观察
造模期间，所有动物生活状态良好，未见竖毛、目光呆滞、呼吸异常、活动迟缓、粪便异常等不良反应。造模后两周期间，动物体重均有增加，但与造模前相比无统计学差异(P>0.05)。给药期间，各给药组所有动物均未见药物引起的异常反应。给药期间，所有动物体重均出现不同程度的增加，且给药组体重增加较为明显，与造模前相比，从第3w开始具有显著的统计学差异(P<0.05)；而阴性对照组(即模型组)增加较为缓慢，在第5w时才与给药前动物体重出现差异。 大、小剂量组动物体重与给药前(即第2w)相比，具有显著的统计学差异(P<0.05)。具体结果见图5。
2.2Anti-miR-214对小鼠Lewis肺癌治疗作用
C57BL/6小鼠Lewis肺癌造模2w后静脉注射给予Anti-miR-214治疗，给药期间，每3天一次，于末次给药后的第3天处死动物，采血、肺脏及肝脏。除部分肺脏用于Anti-miR-214含量测定外，其余肺脏与肝脏用福尔马林固定，进行组织病理切片检查该脏器肿瘤状况。具体结果见图6。从图6的结果可以看出，病理切片结果表明：所有各组肝脏切片未见有肿瘤灶。在肺部，各治疗组可见不同程度的片状核大深染的肿瘤细胞聚集灶。各给药组中，大剂量即5mg/kg micro-214对在体Lewis肺癌有很好的治疗作用，其分值与严重程度均较对照组轻，光镜下未见肿瘤灶个体鼠数也多于对照组；小剂量组即0.05mg/kg对原位Lewis肺癌亦有一定的治疗作用，可能与该品体内复制有关，结果见表6、7。
表6、Anti-miR-214对小鼠Lewis肺癌的治疗作用
Figure PCTCN2015094809-appb-000013
表7、Anti-miR-214对Lewis肺癌的治疗作用
Figure PCTCN2015094809-appb-000014
3.结论
给药期间，所有动物生活状况良好，未见与用药相关的异常反应，且给药组动物体重明显增加，与造模或给药前相比，有显著差异(P<0.05)。病理切片表明，各给药组肿瘤严重程度均较对照组有所减轻，Anti-miR-214在5mg/kg时抑瘤作用明显。
Anti-miR-214对在体小鼠Lewis肺癌有一定剂量依赖性的治疗作用，其中浓度到达5mg/kg治疗作用最明显，给药期间未见与用药相关的异常反应。
实施例7、临床实验
给乳腺癌转移终末期的志愿者注射anti-miR-214质粒，每周两次点滴，每次1mg。在开始用药一个月后检测志愿者的各项指标。
(1)anti-miR-214检测
具体结果见图7。从图7可以看出，肿瘤末期志愿者注射质粒后(治疗后)，肿瘤末期志愿者血细胞和血浆中都能够检测到高含量的anti-miR-214，这充分说明通过静脉注射anti-miR-214质粒，确实可以在体内高表达anti-miR-214。
(2)血清中miR-214结果
图8显示了血清中miR-214的表达水平。从图8可以看出，注射质粒后，志愿者患者血清中miR-214相比于治疗前显著下降，几乎降至和正常人相当的水平，这不仅提示了注射anti-miR-214质粒可以在体内表达，而且还具有功能，能够吸附体内miR-214,从而使它的表达水平显著下降。
(3)肿瘤的变化情况
注射anti-miR-214质粒后，乳腺癌转移终末期志愿者的情况得到一定改善，肿瘤大小、体积没有变化，肿瘤没有发生转移，由此可见，miR-214抑制剂对肿瘤生长有抑制作用，miR-214抑制剂可用于肿瘤治疗。
实施例8前体miRNA适用于其他过表达载体
前体miRNA结构插入表达载体pcDNA3，构建的表达模块，同样有效的避免了有些内源miRNA表达时产生-3p，-5p的问题。pcDNA3图谱结构如图9所示。
1.anti-miR-214-5p序列
>anti-miR-214-5p
5’-ACUGCCUGUCUGUGCCUGCCUGU-3’(SEQ ID NO.:2)
2.anti-miR-214载体
2.1.Oligo DNA的设计与合成
根据anti-miR-214设计并合成2对互补ol igo DNA如实施例1中的2.1所述，即前体miRNA的结构。
2.2miRNA载体的构建与验证
具体操作步骤如实施例1中的2.2所述。过表达anti-miR-214的效率验证方法同实施例2。表8显示了利用Real-time PCR对anti-miRNA和miRNA表达水平进行测定。
表8、anti-miR-214-5p、anti-miR-214-3p、miR-214的表达水平
(Ct) anti-miR-214-5p
空白质粒 38.25±0.2564
anti-miR-214质粒 25.43±0.5123
   
(Ct) anti-miR-214-3p
空白质粒 31.25±0.4585
anti-miR-214质粒 32.89±0.6245
   
(Ct) miR-214
空白质粒 21.03±0.5874
anti-miR-214质粒 24.08±0.1254
结论：在A549细胞中转染前体miRNA构建的其他过表达载体质粒后，能检测到anti-miR-214-5p的急剧上升(p<0.01)，检测不到anti-miR-214-3p，且不会引起miR-214的上升，相反会导致miR-214下降(p<0.05)。前体miRNA结构插入其他表达载体，同样有效的避免了有些内源miRNA表达时产生-3p，-5p的问题。
对比例1
利用anti-miR-214构建与本发明不同的前体miRNA,将所构建的前体miRNA插入与本发明相同的载体pcDNATM 6.2。在A549细胞中转染anti-miR-214质粒，统一总RNA后分别利用从ABI公司定制的anti-miR-214-5p，anti-miR-214-3p，miR-214的引物q-PCR检测anti-miR-214-5p、anti-miR-214-3p、miR-214的水平。
1.anti-miR-214-5p序列
>anti-miR-214-5p
5’-ACUGCCUGUCUGUGCCUGCCUGU-3’(SEQ ID NO.2:)
2.anti-miR-214载体
2.1.Oligo DNA的设计与合成
根据基因序列设计并合成2对互补oligo DNA，序列见表9。
设计合成的oligo结构如下：
Figure PCTCN2015094809-appb-000015
表9、oligo DNA序列及其对应的前体miRNA元件
Figure PCTCN2015094809-appb-000016
Figure PCTCN2015094809-appb-000017
2.2miRNA载体的构建与验证
具体操作步骤如实施例1中的2.2所述。结果见表10。
表10、anti-miR-214-5p、anti-miR-214-3p、miR-214的表达水平
(Ct) anti-miR-214-5p
空白质粒 35.92±0.1545
anti-miR-214质粒 26.45±0.1535
   
(Ct) anti-miR-214-3p
空白质粒 32.12±0.1657
anti-miR-214质粒 25.48±0.1456
   
(Ct) miR-214
空白质粒 21.26±0.5652
anti-miR-214质粒 21.43±0.4586
从表10可以看出，在A549细胞中转染与本发明不同的前体miRNA质粒后，虽然能检测到anti-miR-214-5p的急剧上升(p<0.05)，但是anti-miR-214-3p表达水平也非常高(p<0.05)，且miR-214水平没有发生显著改变。这是因为与本发明不同的前体miRNA质粒不仅能够表达anti-miR-214-5p，也能够表达anti-miR-214-3p，由于竞争性结合，大量的anti-miR-214-5p被anti-miR-214-3p吸附，从而不能降低miR-214的表达水平。
实施例9
重复实施例1和2,不同在于:将A2的结构从CAGG改为CAGGA。
结果表明，将A2的结构从CAGG改为CAGGA，F5/F3之比进一步上升了约50％。这表明，采用CAGGA更有助于提高F5/F3之比。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (12)

  1. 一种药物制剂，其特征在于，所述的制剂含有：
    (a)用于表达anti-miRNA和/或siRNA的表达载体；以及
    (b)药学上可接受的载体。
  2. 一种施用药物的方法，其特征在于，包括步骤：
    将权利要求1所述的药物制剂施用于哺乳动物的第一部位，从而使得所述表达载体在所述哺乳动物体内被加工形成微粒子(microvesicle)，并被运输到所述哺乳动物的第二部位，并在所述的第二部位表达所述的anti-miRNA和/或siRNA。
  3. 一种前体序列，其特征在于，其5'至3'端具有式I所示的结构：
    Figure PCTCN2015094809-appb-100001
    B1为所需要的第一核糖核酸序列，其中所述的第一核糖核酸序列包括anti-miRNA序列形式或siRNA序列形式；
    B2为与B1基本互补或完全互补的序列，且B2与C不互补；
    C为茎环结构序列；
    A1和A2分别为无，或任选的由4-5个碱基组成的RNA序列；
    其中，所述前体能在宿主中表达(或产生或富集)所述的第一核糖核酸序列，而不表达(或产生或富集)与所述第一核糖核酸序列互补的第二核糖核酸序列。
  4. 如权利要求3所述的前体序列，其特征在于，其5'至3'端具有式I所示的结构：
    Figure PCTCN2015094809-appb-100002
    B1为anti-miRNA-214-5p；
    B2为与B1基本互补或完全互补的序列，且B2与C不互补；
    C为茎环结构序列，优选为SEQ ID NO.:1所示的序列；
    A1和A2分别为无，或任选的由4-5个碱基组成的RNA序列；
    其中，所示的前体序列能在宿主中加工形成anti-miRNA-214，且在所述anti-miRNA-214中，只表达anti-miRNA-214-5p，不表达anti-miRNA-214-3p。
  5. 如权利要求4所述的前体序列，其特征在于，所述的基本互补指所述B2 与B1有2-8个碱基不互补，较佳地，所述B2与B1有3-5个碱基不互补；更佳地，所述的B2较B1缺失1-2个碱基。
  6. 如权利要求4所述的前体序列，其特征在于，所述的A1为UGCUG；和/或
    所述的A2为CAGG或CAGGA。
  7. 一种多核苷酸，其特征在于，所述的多核苷酸能被宿主转录形成权利要求4中所述的前体序列。
  8. 一种表达载体，其特征在于，所述的表达载体含有权利要求4所述的前体序列或权利要求7所述的多核苷酸。
  9. 一种药物组合物，其特征在于，所述的药物组合物含有权利要求4所述的前体序列、或权利要求8所述的表达载体，和药学上可接受的载体。
  10. 如权利要求9所述的药物组合物，其特征在于，所述的药物组合物为权利要求8所述的表达载体，较佳地，为含有权利要求4所述前体序列的质粒；和/或
    所述药物组合物的剂型包括片剂、胶囊剂、粉剂、丸剂、颗粒剂、糖浆剂、溶液、混悬液、乳剂、混悬剂、注射液、或粉针剂；较佳地，所述的剂型为注射剂，如静脉注射剂、腹腔注射剂。
  11. 如权利要求9所述的药物组合物，其特征在于，所述的药物组合物的施用方法包括：口服、呼吸道、注射、透皮、粘膜或腔道给药；较佳地，所述施用方法包括直接注射质粒。
  12. 权利要求4所述前体序列、或权利要求8所述表达载体的用途，其特征在于，(i)用于制备miRNA-214的抑制剂；和/或(ii)用于制备抗miRNA-214高表达恶性肿瘤的药物组合物；
    较佳地，所述的恶性肿瘤包括肝癌、肺癌、胃癌、食管癌、卵巢癌、结直肠癌、宫颈癌、胰腺癌、前列腺癌、白血病、或乳腺癌。
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