WO2022166771A1 - 3'utr的构建方法和应用 - Google Patents

3'utr的构建方法和应用 Download PDF

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WO2022166771A1
WO2022166771A1 PCT/CN2022/074391 CN2022074391W WO2022166771A1 WO 2022166771 A1 WO2022166771 A1 WO 2022166771A1 CN 2022074391 W CN2022074391 W CN 2022074391W WO 2022166771 A1 WO2022166771 A1 WO 2022166771A1
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gene
sequence
utr
polynucleotide
mrna
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PCT/CN2022/074391
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French (fr)
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刘韬
王猛
高海霞
邵梅琪
蒋婷
王佩
钱其军
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上海吉量医药工程有限公司
浙江吉量科技有限公司
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Publication of WO2022166771A1 publication Critical patent/WO2022166771A1/zh

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Definitions

  • the invention belongs to the technical field of protein expression, and in particular relates to a 3'UTR region sequence for improving mRNA translation and a construction method and application thereof.
  • Messenger ribonucleic acid contains necessary untranslated sequences, such as m7G cap, 5' untranslated region (5'-untranslated region, 5'- UTR), 3'untranslated region (3'untranslated region, 3'UTR) and poly(A) tail.
  • the 3'-end untranslated region is closely connected to the coding region of messenger RNA and is closely related to post-transcriptional regulation. It affects the translation efficiency and stability of messenger RNA in the cytoplasm through the binding of other regulatory factors, such as small molecule RNA (miRNA) and The 3'-untranslated region binds, thereby inhibiting the translation of messenger RNA and even inducing its degradation; the silencer sequence in the 3'-untranslated region binds to RNA-binding proteins, which can also inhibit translation. In addition, the longer the 3' untranslated region sequence, the more likely it will negatively affect protein translation.
  • the 3'-UTR in the prior art is usually isolated from the animal itself without modification, and its effect may be affected by the host and is uncertain. There remains a need in the art for a universal 3'UTR region that enhances mRNA translation.
  • the present invention constructs 3'-end untranslated region sequences through natural genetic modification or artificial design, and these 3'-end untranslated region sequences make the messenger ribonucleic acid of the gene maintain high translation activity in different cells.
  • a first aspect of the present invention provides a polynucleotide comprising
  • 3'UTR sequence which is the sequence after knockout of one or more microRNA recognition sites in the 3'UTR region of the PCBP4 gene or PIWIL4 gene, and/or
  • the nucleotide sequence of the 3' UTR region of the PCBP4 gene is set forth in SEQ ID NO: 1 or any one of sequences having at least 80% sequence identity thereto.
  • the nucleotide sequence of the 3' UTR region of the PIWIL4 gene is as set forth in SEQ ID NO: 2 or any sequence having at least 80% sequence identity thereto.
  • the microRNA recognition site can be retrieved using http://www.targetscan.org/vert_72/.
  • the microRNA recognition site is selected from one or more of the microRNA recognition sites in Table 1 and/or Table 2.
  • the one or more microRNA recognition sites in the 3' UTR region of the PCBP4 gene are selected from Table 1.
  • the one or more microRNA recognition sites in the 3'UTR region of the PIWIL4 gene are selected from Table 2.
  • the PCBP4 gene or PIWIL4 gene is a human PCBP4 gene or a human PIWIL4 gene.
  • the sequence of the polynucleotide is selected from (1) the sequence set forth in any one of SEQ ID NOs: 3-7, (2) having at least 80% sequence identity to (1) sexual sequence.
  • the polynucleotide is DNA or RNA.
  • the first aspect of the present invention also provides a method for constructing a 3'UTR sequence, comprising the step of knocking out one or more recognition sites of microRNAs in the 3'UTR region of a gene.
  • the gene is the PCBP4 gene or the PIWIL4 gene.
  • the PCBP4 gene or PIWIL4 gene is human PCBP4 gene or human PIWIL4 gene.
  • the nucleotide sequence of the 3' UTR region of the PCBP4 gene is set forth in SEQ ID NO: 1 or any one of sequences having at least 80% sequence identity thereto.
  • the nucleotide sequence of the 3' UTR region of the PIWIL4 gene is as set forth in SEQ ID NO: 2 or any sequence having at least 80% sequence identity thereto.
  • the microRNA recognition site is retrieved from http://www.targetscan.org/vert_72/.
  • the microRNA recognition site is selected from one or more of the microRNA recognition sites in Table 1 and/or Table 2.
  • the one or more microRNA recognition sites in the 3' UTR region of the PCBP4 gene are selected from Table 1.
  • the one or more microRNA recognition sites in the 3'UTR region of the PIWIL4 gene are selected from Table 2.
  • a second aspect of the present invention provides a polynucleotide comprising
  • a 3' UTR sequence containing a sequence of at least 3 tandem repeats of an RNA methylation (m6A) site and an optional linker between any two repeats, and/or
  • the m6A site is a recognition sequence for an m6A methylase (writer) or an m6A RNA methylation recognition protein (reader).
  • the m6A methylase is selected from the group consisting of METTL3, METTL14, METTL16, WTAP, VIRMA, ZC3H13, RBM15, RBM158, HAKAI, and KIAA1492.
  • the m6A RNA methylation recognition protein is selected from the group consisting of YTHDC1-2, YTHDF1-3, IGF2BP1-3, Prrc2a, HNRNPC, HNRNPG, FMR1, SRSF2, HuR, LRPPRC, eIF3, and HNRNPA2B1.
  • the m6A site is selected from the group consisting of: GGACT, GGACC, GAACT.
  • the 3'UTR sequence contains at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 tandem of the m6A sites repeating sequence.
  • the linker is 1-5 bp in length, preferably 3 bp.
  • the linker sequence is selected from the group consisting of: TCT, TGT, GGT, CT, TT and CA.
  • the sequence of the polynucleotide is selected from (1) the sequence set forth in any of SEQ ID NOs: 8-14, (2) having at least 80% sequence identity to (1) sexual sequence.
  • the polynucleotide is DNA or RNA.
  • the second aspect of the present invention also provides a method for constructing a 3'UTR sequence, comprising the steps of connecting at least 3 RNA methylation (m6A) sites in series and optionally adding restriction enzyme cleavage sites at both ends. step.
  • m6A RNA methylation
  • connection is direct or through a linker.
  • the linker is 1-5 bp in length.
  • the m6A site is a recognition sequence of an m6A methylase or m6A RNA methylation recognition protein.
  • the m6A methylase is selected from the group consisting of METTL3, METTL14, METTL16, WTAP, VIRMA, ZC3H13, RBM15, RBM158, HAKAI, and KIAA1492.
  • the m6A RNA methylation recognition protein is selected from the group consisting of YTHDC1-2, YTHDF1-3, IGF2BP1-3, Prrc2a, HNRNPC, HNRNPG, FMR1, SRSF2, HuR, LRPPRC, eIF3, and HNRNPA2B1.
  • the m6A site is selected from the group consisting of: GGACT, GGACC, GAACT.
  • the method comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 RNA methylation (m6A) sites Steps to connect in series.
  • m6A RNA methylation
  • RNA methylation (m6A) sites in the 3'UTR sequences are identical.
  • the present invention also provides a nucleic acid construct comprising a polynucleotide as described in any of the embodiments herein and optionally a gene to be expressed.
  • the nucleic acid construct is an mRNA containing the gene to be expressed and the polynucleotide.
  • the nucleic acid construct is a vector, such as a cloning vector or an expression vector.
  • the polynucleotide is located 3' to the gene to be expressed.
  • the nucleic acid construct further comprises one or more elements selected from the group consisting of a promoter, a 5' UTR, a multiple cloning site, polyA.
  • the promoter is a T7 promoter.
  • the 5'UTR is the 5'UTR of HBB mRNA.
  • the nucleic acid construct comprises at least sequentially: a promoter, a multiple cloning site, a polynucleotide as described in any of the embodiments herein, and polyA.
  • the exogenous gene is an eGFP gene.
  • the present invention also provides a host cell comprising the polynucleotide and/or nucleic acid construct described in any of the embodiments herein.
  • the host cell is a CHO cell or a PBMC cell.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the polynucleotide, nucleic acid construct or host cell described in any of the embodiments herein, and a pharmaceutically acceptable excipient.
  • the polynucleotide is located 3' to the gene to be expressed.
  • the present invention also provides a method for increasing the intensity of gene expression, prolonging the time of gene expression, increasing the ability of mRNA to initiate translation or maintaining the duration of translation activity, comprising the step of expressing a gene carrying the polynucleotide described in any of the embodiments herein.
  • the polynucleotide is located 3' to the gene.
  • the gene is an eGFP gene.
  • the present invention also provides the polynucleotides, nucleic acid constructs and/or host cells described in any of the embodiments herein in the preparation of reagents for increasing the intensity of gene expression, prolonging the time of gene expression, increasing the ability of mRNA to initiate translation, or maintaining translation activity for a period of time applications in .
  • the polynucleotide is located 3' to the gene.
  • the gene is an eGFP gene.
  • the present invention also provides the use of the polynucleotide, nucleic acid construct and/or host cell described in any of the embodiments herein in the preparation of a medicament for treating a disease, wherein the polynucleotide is located at the 3' end of a gene, and the The expression of the gene contributes to the treatment of the disease.
  • the present invention also provides the use of the polynucleotides of any of the embodiments herein for controlling gene expression.
  • the polynucleotide is located 3' to the gene.
  • the gene is an eGFP gene.
  • Figure 1 shows an exemplary vector map containing the 3'UTR described herein.
  • Figure 2 shows the proportion of eGFP expression fluorescence in PBMC cells 24h after electroporation of mRNA under different 3'UTR conditions.
  • Figure 3 shows the mean fluorescence intensity of PBMCs electroporated with mRNAs containing different 3'UTRs for 24h.
  • Figure 4 shows the positive rate of eGFP after 72h of PBMC electrotransformed mRNA under different 3'UTR conditions.
  • Figure 5 shows the mean fluorescence intensity of eGFP mRNA with different 3'UTRs after electroporation of PBMCs for 72h.
  • Figure 6 shows the positive rate of eGFP after 24h of mRNA transfection in CHO cells under different 3'UTR conditions.
  • Figure 7 shows the mean fluorescence intensity of eGFP 24h after CHO cells were transfected with mRNA.
  • Figure 8 shows the positive rate of eGFP after 48h of mRNA transfection in CHO cells under different 3'UTR conditions.
  • Figure 9 shows the mean fluorescence intensity of eGFP 48h after CHO cells were transfected with mRNA under different 3'UTR conditions.
  • Figure 10 shows the positive rate of eGFP 96h after transfection of CHO cells under different 3'UTR conditions.
  • Figure 11 shows the mean fluorescence intensity of eGFP after 96h transfection of mRNA in CHO cells under different 3'UTR conditions.
  • the present invention constructs 3'-end untranslated region sequences through natural genetic modification or artificial design, and these 3'-end untranslated region sequences make the messenger ribonucleic acid of the gene maintain high translation activity in different cells.
  • the present invention comprises a 3'UTR polynucleotide obtained by natural genetic modification, which comprises a 3'UTR sequence or its complement, the 3'UTR sequence being a natural gene (e.g., human The sequence after knocking out one or more microRNA recognition sites in the 3'UTR region of PCBP4 gene or PIWIL4 gene.
  • the 3' UTR sequence of PCBP4 mRNA (NM_001174100) is shown in SEQ ID NO: 1
  • the 3' UTR sequence of PIWIL4 mRNA (NM_152431) is shown in SEQ ID NO: 2.
  • a microRNA recognition site is a site that is recognized by a microRNA and affects mRNA stability.
  • microRNA target sequence of the 3'UTR of PCBP4 is selected from one or more of Table 1; the microRNA target sequence of the 3'UTR of PIWIL4 is selected from one or more of Table 2.
  • microRNA Seed sequence Has-miR-6801-3p gcagggg Has-miR-4313 agggggc Has-miR-6745 tccaccc Has-miR-4251 ttctcag Has-miR-6777-3p agagtgg Has-miR-1976 caggaga Has-miR-92a-2-5p ccccacca Has-miR-1205 cctgcaga Has-miR-145-5p actggaa hsa-miR-548g-3p acagttt hsa-miR-548az-3p cagtttt hsa-miR-3613-3p tttttga
  • the method for constructing the above-mentioned 3'UTR sequence comprises the step of knocking out one or more recognition sites of microRNAs in the 3'UTR region of the gene and optionally adding restriction enzyme cleavage sites at both ends of the 3'UTR region. step. Knockout of the microRNA recognition site can be accomplished using any method known in the art, such as mutating the 2-8 nt target sequence in the 3'UTR complementary to the microRNA's seed.
  • primers are first designed according to the 3'UTR of the gene (such as human PCBP4 gene or PIWIL4 gene), the corresponding sequence is amplified from the RNA group by RT-PCR, and then the mutation is introduced by PCR for knockout.
  • the 3'UTR sequence of the present invention can also be obtained directly by synthesis. Exemplary sequences of such polynucleotides are set forth in any of SEQ ID NOs: 3-7.
  • the present invention comprises a 3'UTR polynucleotide obtained by artificial design, comprising a 3'UTR sequence or its complement, which contains a sequence of at least 3 tandem repeats of an RNA methylation (m6A) site and optionally a linker between any two repeats.
  • m6A site is the recognition site of m6A methylase or m6A RAN methylation recognition protein.
  • the m6A methylase can be METTL3, METTL14, METTL16, WTAP, VIRMA, ZC3H13, RBM15, RBM158, HAKAI and KIAA1492;
  • the m6A RNA methylation recognition protein can be YTHDC1-2, YTHDF1-3, IGF2BP1- 3. Prrc2a, HNRNPC, HNRNPG, FMR1, SRSF2, HuR, LRPPRC, eIF3 and HNRNPA2B1.
  • the 3'UTR sequence may contain at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 tandem repeats of the m6A site.
  • Multiple RNA methylation (m6A) sites in a 3'UTR sequence can be the same or different.
  • the method of constructing the 3'UTR sequence includes the step of linking at least 3 RNA methylation (m6A) sites in tandem and optionally adding restriction sites at both ends.
  • the ligation is direct ligation or via a linker (eg, oligonucleotides of 1-5 bp in length).
  • the artificially designed 3'UTR sequence of the present invention can also be obtained by direct synthesis. Exemplary sequences of such polynucleotides are set forth in any of SEQ ID NOs: 8-14.
  • the polynucleotide may be DNA or RNA.
  • DNA can be single-stranded or double-stranded.
  • the polynucleotides herein are generally in the form of isolated polynucleotides.
  • variant as used herein in reference to a nucleic acid can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion variants and insertion variants.
  • Nucleic acids of the invention may comprise at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 98%, At least about 99% or 100% of the nucleotide sequence.
  • the present invention also relates to nucleic acid fragments hybridizing to the above-mentioned sequences.
  • the polynucleotides described herein can generally be obtained by PCR amplification. Alternatively, the nucleic acid molecules described herein can also be synthesized directly.
  • identity refers to a comparison window or specified region, by manual alignment and visual inspection using methods known in the art, such as sequence comparison algorithms Two or more sequences or subsequences are identical or in which a certain percentage of amino acid residues or nucleotides are identical in a given region (eg, at least 60%, at least 65%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% or 100% identical).
  • BLAST and BLAST 2.0 algorithms are preferred algorithms suitable for determining percent sequence identity and percent sequence similarity.
  • the 3'UTR polynucleotides of the present invention can improve the expression of associated genes. Said improvement in expression is achieved by introducing into the cell a nucleic acid construct carrying the genes of the polynucleotides described herein. Accordingly, the present invention also provides nucleic acid constructs of the polynucleotides.
  • the nucleic acid construct can be an expression cassette containing a 3'UTR polynucleotide described herein, a multiple cloning site, and one or more regulatory sequences operably linked thereto, such as an origin of replication, a multiple cloning site, a promoter , marker genes or translational control elements, including enhancers, operators, termination sequences such as polyA, ribosome binding sites, 5'UTR.
  • the coding sequence of the gene to be expressed can be inserted into the nucleic acid construct at the 5' end of the 3' UTR polynucleotide.
  • the polynucleotides of the present invention can be manipulated in a variety of ways to ensure expression of the genes.
  • the nucleic acid construct can be manipulated according to the nature or requirements of the vector before inserting the nucleic acid construct into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.
  • the nucleic acid construct may also be an mRNA containing the gene to be expressed and the 3'UTR polynucleotide described herein.
  • the mRNA can be obtained by in vivo or in vitro transcription methods known in the art.
  • in vitro transcribed mRNA can be obtained according to the HiScribe T7 ARCA mRNA Kit operation guide.
  • operably linked means that a nucleotide sequence of interest is linked to regulatory sequences in a manner that allows expression of the nucleotide sequence.
  • Those skilled in the art are familiar with methods that can be used to construct expression vectors containing the coding sequences of the 3'UTR polynucleotides described herein and, optionally, a gene of interest and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like.
  • the promoter sequence is usually operably linked to the coding sequence for the protein to be expressed.
  • the promoter can be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, including mutated, truncated and hybrid promoters, and can be derived from extracellular coding homologous or heterologous to the host cell. Or the gene acquisition of intracellular polypeptides.
  • An example of a suitable promoter is the T7 promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Other constitutive promoter sequences can also be used, including but not limited to T3 promoter, SP6 promoter.
  • the regulatory sequence may also be a suitable transcription termination sequence, a sequence recognized by the host cell to terminate transcription.
  • a termination sequence is operably linked to the 3' end of the 3' UTR polynucleotides described herein. Any termination sequence that is functional in the host cell of choice can be used in the present invention.
  • the regulatory sequence may also be a suitable leader sequence, the 5' untranslated region of the mRNA that is important for translation by the host cell.
  • the leader sequence is operably linked to the 5' end of the gene to be expressed. Any 5' untranslated region that is functional in the host cell of choice can be used in the present invention.
  • the nucleic acid construct is a vector.
  • the vector can be a cloning vector, an expression vector, or a homologous recombination vector.
  • the polynucleotides of the present invention can be cloned into many types of vectors, eg, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Cloning vectors can be used to provide foreign genes and coding sequences for the polynucleotides described herein.
  • Expression vectors can be provided to cells in the form of viral vectors. Gene expression is typically achieved by operably linking a polynucleotide of the invention to a promoter and incorporating the construct into an expression vector.
  • the vector may be suitable for replication and integration in eukaryotic cells.
  • Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.
  • Viral vector techniques are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
  • Homologous recombination vectors are used to integrate the expression cassettes described herein into the host genome.
  • suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers.
  • the present invention uses a pT7-m5U vector, which contains an origin of replication site, a promoter, a polynucleotide described herein, and optionally a selectable marker.
  • the expression vector introduced into the cell may also contain either or both of a selectable marker gene or a reporter gene to facilitate transfection or infection from a viral vector seeking Identify and select expressing cells from a population of cells.
  • the selectable marker can be carried on a single piece of DNA and used in co-transfection procedures. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable markers include Flag, HA or V5. Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. After the DNA has been introduced into the recipient cells, the expression of the reporter gene is measured at an appropriate time.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes.
  • Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.
  • Vectors can be readily introduced into host cells, eg, mammalian, bacterial, yeast or insect cells, by any method known in the art.
  • an expression vector can be transferred into a host cell by physical, chemical or biological means.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, electroporation, etc., for example, direct in vitro transcribed mRNA is introduced into cells by electroporation.
  • Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids plastid.
  • Biological methods for introducing polynucleotides into host cells include the use of viral vectors, particularly retroviral vectors, such as those derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus and adeno-associated virus.
  • the selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to subject cells in vivo or ex vivo.
  • retroviral systems are known in the art.
  • Lentiviruses are a genus of the Retroviridae family.
  • Reagents for lentiviral packaging are well known in the art, such as conventional lentiviral vector systems including pRsv-REV, pMDlg-pRRE, pMD2G and interfering plasmids of interest.
  • host cells contain the polynucleotides or nucleic acid constructs described herein.
  • Host cells include both cells expressing the gene of interest, such as CHO or PBMC cells, as well as various cells used in the production of cells for expression, such as E. coli cells, for example, to provide the polynucleotides of the present invention or to provide herein. the carrier.
  • Cells suitable for use in the present invention may be various types of cells of various origins, including prokaryotic cells and eukaryotic cells, such as bacterial cells, yeast cells, insect cells, and mammalian cells, including, but not limited to, sf9, BHK21, COS1 , COS3, COS7, 293T, Vero.
  • the host cell is preferably a variety of cells that facilitate gene product expression or fermentative production, and such cells are well known and commonly used in the art.
  • cell preparations comprising cells described herein or extracts thereof.
  • the cell preparation can be a cell culture, pharmaceutical composition, kit, device, medium or system, such as a chip or the like, comprising the cells described herein or an extract thereof and a suitable medium.
  • Appropriate culture basics for culturing various types of cells are well known in the art.
  • nucleic acid constructs or cells described herein depends on the gene to be expressed carried therein, eg, if the gene to be expressed is a fluorescent protein, the nucleic acid constructs or cells described herein can be used for cell tracking. If the gene to be expressed is an agent that facilitates the treatment of the disease, the nucleic acid constructs or cells described herein can be used to treat the disease.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the nucleic acid construct or cell of the present invention and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant, the nucleic acid construct or A cell can produce a therapeutically effective amount of an active molecule (eg, the expression product of a gene of interest in a nucleic acid construct).
  • acceptable diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants, etc. in the pharmaceutical compositions are preferably nontoxic to recipients at the dosages and concentrations employed.
  • compositions may contain ingredients for improving, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or The rate of release, absorption or penetration of such substances.
  • ingredients for improving, maintaining or preserving for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or The rate of release, absorption or penetration of such substances.
  • These substances are known in the art, see, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition, ed. A.R. Genrmo, 1990, Mack Publishing Company.
  • the optimal pharmaceutical composition may be determined by the intended route of administration, mode of delivery, and desired dosage.
  • the route of administration of the pharmaceutical composition is according to known methods, such as injection by oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional routes; Via a sustained release system or via an implanted device.
  • the pharmaceutical compositions of the present invention may be selected for parenteral delivery. Alternatively, the composition may be selected for inhalation or delivery through the digestive tract, such as orally.
  • the preparation of such pharmaceutically acceptable compositions is within the skill of the art. Techniques for formulating a variety of other sustained or controlled delivery modes, such as liposomal vehicles, bioerodible microparticles or porous beads, and depot injections, are also known to those of skill in the art.
  • compositions for in vivo administration are usually provided in the form of sterile formulations. Sterilization is achieved by filtration through sterile filtration membranes. When the composition is lyophilized, it can be sterilized using this method before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. Parenteral compositions are usually presented in containers with sterile access ports, such as intravenous solution strips or vials with a hypodermic needle pierceable stopper.
  • kits for producing single-dose administration units may each contain a first container with dried protein and a second container with an aqueous formulation.
  • kits are provided containing single-lumen and multi-lumen pre-filled syringes (eg, liquid syringes and lyophilized syringes).
  • the present invention also provides methods of treating a patient by administering the nucleic acid constructs or cells or pharmaceutical compositions thereof of any of the embodiments of the present invention.
  • the terms "patient”, “subject”, “individual”, and “subject” are used interchangeably herein to include any organism, preferably an animal, more preferably a mammal (eg, rat, mouse, dog , cats, rabbits, etc.), and most preferably humans.
  • “Treatment” refers to the administration of a therapeutic regimen described herein to a subject to achieve at least one positive therapeutic effect. Treatment regimens that effectively treat a patient can vary depending on a variety of factors, such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anticancer response in the subject.
  • the therapeutically effective amount of a pharmaceutical composition containing a nucleic acid construct or cell of the invention to be employed will depend, for example, on the extent and purpose of treatment. Those skilled in the art will appreciate that the appropriate dosage level for treatment will depend in part on the molecule delivered (eg, the product of expression of the gene of interest in the nucleic acid construct), the indication, the route of administration, and the size of the patient (body weight, body surface or organ size) and/or condition (age and general health). In certain embodiments, the clinician can titrate the dose and alter the route of administration to obtain optimal therapeutic effect.
  • the frequency of dosing will depend on the pharmacokinetic parameters of the particular product of interest in the formulation used (eg, the product of expression of the gene of interest in the nucleic acid construct). Clinicians typically administer the compositions until a dosage is reached that achieves the desired effect.
  • the composition may thus be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion through an implanted device or catheter.
  • the 3'UTR polynucleotides described herein can increase the intensity of gene expression, prolong the time of gene expression, increase the ability of mRNA to initiate translation, or maintain translation activity for a period of time. Therefore, the present invention also provides a method for increasing the intensity of gene expression, prolonging the time of gene expression, increasing the ability of mRNA to initiate translation or maintaining the duration of translation activity, including expressing a gene carrying the 3'UTR polynucleotide described in any of the embodiments herein. A step of. Typically, the 3' UTR polynucleotide is located 3' to the gene.
  • expression is achieved by the above-described process of introducing a nucleic acid construct into a cell and incubating the cell under conditions in which the gene is expressed. GFP is used in the examples only as an example of the gene to be expressed.
  • the present invention also provides the use of the 3'UTR polynucleotide, nucleic acid construct and/or host cell described in any of the embodiments herein in the preparation of a medicament for treating a disease, wherein the 3'UTR polynucleotide is located in a gene 3' end, the expression of the gene contributes to the treatment of the disease.
  • a nucleic acid construct comprising the gene and the 3'UTR polynucleotide can be introduced into a cell by the method described above, and the gene can be expressed by incubating the cell under conditions in which the gene is expressed.
  • the expression of the gene contributes to the treatment of the disease.
  • the gene or its product can directly produce a therapeutic effect, or the expression of the gene or its product can cause the expression or activation of other genes or proteins that can produce a therapeutic effect.
  • a polynucleotide comprising:
  • 3'UTR sequence which is the sequence after knockout of one or more microRNA recognition sites in the 3'UTR region of PCBP4 gene or PIWIL4 gene
  • the PCBP4 gene or PIWIL4 gene is a human PCBP4 gene or a human PIWIL4 gene, and/or, the microRNA recognition site is as shown in the website http://www.targetscan.org/vert_72/, and/or, the The m6A site is the recognition sequence of m6A methylase or m6A RNA methylation recognition protein, and/or the 3'UTR sequence contains at least 4, at least 5, at least 6, At least 7, at least 8, at least 9, at least 10 tandemly repeated sequences, and/or, the length of the linker is 1-5 bp;
  • the nucleotide sequence of the 3'UTR region of the PCBP4 gene is as shown in SEQ ID NO:1
  • the nucleotide sequence of the 3'UTR region of the PIWIL4 gene is as shown in SEQ ID NO:2 or
  • sequence of the polynucleotide is selected from (1) a sequence shown in any one of SEQ ID NOs: 3-14, (2) a sequence having at least 80% sequence identity with (1).
  • a method for constructing a 3'UTR sequence comprising the step of knocking out the recognition site of one or more microRNAs in the 3'UTR region of a gene
  • the gene is the PCBP4 gene or the PIWIL4 gene,
  • the PCBP4 gene or the PIWIL4 gene is a human PCBP4 gene or a human PIWIL4 gene
  • nucleotide sequence of the 3'UTR region of the gene is shown in any of SEQ ID NO: 1 or 2 or a sequence having at least 80% sequence identity with it, and/or, the microRNA recognizes Sites are shown on the website http://www.targetscan.org/vert_72/.
  • a method for constructing a 3' UTR sequence comprising the steps of connecting at least 3 m6A sites in series and optionally adding restriction sites at both ends,
  • the linking is direct linking or linking through a linker
  • the m6A site is a recognition sequence of an m6A methylase or an m6A RNA methylation recognition protein
  • the method comprises adding the step of connecting at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 m6A sites in series;
  • a nucleic acid construct comprising the polynucleotide described in item 1 or 2 and an optional gene to be expressed, the polynucleotide being located at the 3' end of the gene to be expressed; preferably, the nucleic acid construct
  • the product also comprises one or more elements selected from the group consisting of a promoter, a 5'UTR, a multiple cloning site, polyA.
  • a host cell comprising the polynucleotide described in item 1 or 2 and/or the nucleic acid construct described in item 5; preferably, the host cell is a CHO cell or a PBMC cell.
  • a pharmaceutical composition comprising the polynucleotide described in item 1 or 2, the nucleic acid construct described in item 5 or the host cell described in item 6, and pharmaceutically acceptable excipients.
  • a method for improving the intensity of gene expression, prolonging the time of gene expression, improving the ability of mRNA to initiate translation of the gene or maintaining the duration of translation activity comprising the step of expressing the gene carrying the polynucleotide described in item 1 or 2, so that The polynucleotide is located at the 3' end of the gene.
  • the vector pUC57-1#3U was double digested with Sal I and Hind III, and the recovery size was 430bp fragment 1# 3U.
  • the self-constructed EGFP transcription template vector pT7-m5U-eGFP contains the T7 promoter, the 5' UTR sequence (m5U) of HBB mRNA (NM_000518), the EGFP sequence, the HBB mRNA 3' UTR sequence and the polyA sequence (SEQ ID NO: 16).
  • Figure 1 exemplarily shows the map of pT7-m5U-eGFP-1#3U, from which the map of the subsequent vector is distinguished by the use of a different 3'UTR.
  • the vector pUC57-2#3U was double digested with Sal I and Hind III, and the recovery size was 143bp fragment 2# 3U
  • the pT7-m5U-eGFP vector was double digested by Sal I and Hind III, and the above recovered fragment 2#3U was connected to the pT7-m5U-eGFP vector to obtain pT7-m5U-eGFP-2#3U.
  • the 3'-end untranslated region is an artificial random sequence expression eGFP reporter gene transcription template vector construction
  • Entrust Jinweizhi company to synthesize artificial random sequence (SEQ ID NO:8), use Sal I and Hind III double restriction enzyme to cut the vector pUC57-6#3U, the recovery size is 100bp fragment 6#3U, pT7-m5U-eGFP vector is processed by Sal I After double digestion with Hind III, the above recovered fragment 6#3U was ligated into the pT7-m5U-eGFP vector to obtain pT7-m5U-eGFP-6#3U.
  • Entrust Jinweizhi Company to synthesize artificial random sequence (SEQ ID NO:9), use Sal I and Hind III double restriction enzyme to cut the vector pUC57-7#3U, the recovery size is 116bp fragment 7#3U, the pT7-m5U-eGFP vector is processed by Sal I After double digestion with Hind III, the above recovered fragment 7#3U was ligated to the pT7-m5U-eGFP vector to obtain pT7-m5U-eGFP-7#3U.
  • Entrust Jinweizhi company to synthesize artificial random sequence (SEQ ID NO: 10), use Sal I and Hind III double restriction enzyme to cut the vector pUC57-8#3U, the recovery size is 86bp fragment 8#3U, the pT7-m5U-eGFP vector is processed by Sal I After double digestion with Hind III, the above recovered fragment 8#3U was ligated to the pT7-m5U-eGFP vector to obtain pT7-m5U-eGFP-8#3U.
  • Entrust Jinweizhi company to synthesize artificial random sequence (SEQ ID NO:11), use Sal I and Hind III double restriction enzyme to cut the vector pUC57-9#3U, the recovery size is 92bp fragment 9#3U, pT7-m5U-eGFP vector is processed by Sal I After double digestion with Hind III, the above recovered fragment 9#3U was ligated into the pT7-m5U-eGFP vector to obtain pT7-m5U-eGFP-9#3U.
  • Entrust Jinweizhi company to synthesize artificial random sequence (SEQ ID NO:12), use Sal I and Hind III double restriction enzyme to cut the vector pUC57-10#3U, the recovery size is 103bp fragment 10#3U, the pT7-m5U-eGFP vector is processed by Sal I After double digestion with Hind III, the above recovered fragment 10#3U was ligated to the pT7-m5U-eGFP vector to obtain pT7-m5U-eGFP-10#3U.
  • Entrust Jinweizhi company to synthesize artificial random sequence (SEQ ID NO: 13), use Sal I and Hind III double restriction enzyme to cut the vector pUC57-11#3U, the recovery size is 97bp fragment 11#3U, the pT7-m5U-eGFP vector is processed by Sal I After double digestion with Hind III, the above recovered fragment 11#3U was ligated into the pT7-m5U-eGFP vector to obtain pT7-m5U-eGFP-11#3U.
  • Entrusted Jinweizhi company to synthesize artificial random sequence (SEQ ID NO:14), use Sal I and Hind III double restriction enzyme to cut the vector pUC57-12#3U, the recovery size is 97bp fragment 12#3U, the pT7-m5U-eGFP vector was processed by Sal I After double digestion with Hind III, the above recovered fragment 12#3U was ligated into the pT7-m5U-eGFP vector to obtain pT7-m5U-eGFP-12#3U.
  • the PIWIL4 mRNA 3'UTR sequence (SEQ ID NO: 2) was entrusted to Jinweizhi Company to be synthesized, and the vector pUC57-PIWIL4#3U was digested with Sal I and Hind III, and the fragment PIWIL4#3U with a size of 429bp was recovered.
  • the pT7-m5U-eGFP vector was processed by After double digestion with Sal I and Hind III, the above recovered fragment PIWIL4#3U was ligated to the pT7-m5U-eGFP vector to obtain pT7-m5U-eGFP-PIWIL4#3U.
  • the IL-2 3'UTR sequence (SEQ ID NO: 15) was entrusted to Jinweizhi Company, and the vector pUC57-IL2#3U was double digested with Sal I and Hind III, and the 285bp fragment IL2#3U, pT7-m5U-eGFP was recovered. After the vector was double digested with Sal I and Hind III, the above recovered fragment IL2#3U was ligated to the pT7-m5U-eGFP vector to obtain pT7-m5U-eGFP-IL2#3U.
  • Embodiment 2 in vitro transcription contains the mRNA of artificial transformation or design 3'UTR
  • HBB mRNA 3'UTR as the control 3'UTR sequence, other 5'untranslated region (5'untranslated region, 5'UTR), eGFP coding region (coding sequence, CDS) and polyadenylation tail (poly(A) ))
  • the template sequences are all identical.
  • Embodiment 3 the constructed 3'UTR maintains gene expression detection in T cells
  • HBB mRNA 3'UTR as the control 3'UTR sequence, other 5'untranslated region (5'untranslated region, 5'UTR), coding region (coding sequence, CDS) and polyadenylation tail (poly(A) ) sequences are all the same. Resuscitate and resuspend 5 ⁇ 10 6 freshly isolated peripheral blood mononuclear cells (PBMCs), and use Lonza 4D-Nucleofector to
  • Example 1-2 20 ⁇ g mRNA (mRNA-eGFP#ct1, mRNA-eGFP1#, mRNA-eGFP2#, mRNA-eGFP3#, mRNA-eGFP4#, mRNA-eGFP5#, mRNA-eGFP6#, mRNA-eGFP4#, mRNA-eGFP5#, mRNA-eGFP6#, mRNA-eGFP7#, mRNA-eGFP8#, mRNA-eGFP9#, mRNA-eGFP10#, mRNA-eGFP11#, mRNA-eGFP12#, mRNA-eGFP PCBP4#, mRNA-eGFP PIWIL4#, mRNA-eGFPIL2#) were electroporated into PBMC The cells were cultured in a 37° C., 5% CO 2 incubator, and the medium composition was AIM-V (Gibco) and m
  • Figure 2 shows the ratio of eGFP expression fluorescence in PBMC cells 24h after electrotransformation of mRNA under different 3'UTR conditions.
  • Figure 3 is the average fluorescence intensity after 24h of PBMC electrotransformation of mRNA containing different 3'UTRs.
  • HBB mRNA 3'UTR as the control 3'UTR sequence, other 5'untranslated region (5'untranslated region, 5'UTR), coding region (coding sequence, CDS) and polyadenylation tail (poly(A) ) sequences are all the same. Resuscitate and resuspend 5 ⁇ 10 6 freshly isolated peripheral blood mononuclear cells (PBMC), and use Lonza 4D-Nucleofector to 20 ⁇ g mRNA (mRNA-eGFP#ctl) expressing EGFP protein obtained in Example 1-2.
  • PBMC peripheral blood mononuclear cells
  • Figure 4 shows the positive rate of eGFP after 72h of electrotransformed mRNA in PBMCs under different 3'UTRs. It can be seen that the expression time of artificially engineered or designed 3'UTRs in PBMCs is better than that of natural 3'UTRs such as PCBP4, PIWIL4, and IL-2.
  • Figure 5 is the average fluorescence intensity of eGFP mRNA of different 3'UTRs after electrotransformation of PBMCs for 72h. In comparison, it is found that the expression intensity of eGFP containing artificially modified or designed 3'UTR mRNAs in PBMC is higher than that of PCBP4, PIWIL4, IL-2, etc.
  • the 3'UTR of the native sequence is strong. After 72 hours, the mRNA containing the artificially engineered or designed 3'UTR still has high expression activity in PBMC, while the mRNA containing the 3'UTR of the natural sequence of IL-2 has basically no expression.
  • Embodiment 4 the constructed 3'UTR maintains gene expression detection in CHO cells
  • HBB mRNA 3 ⁇ UTR as the control 3 ⁇ UTR sequence, other 5 ⁇ untranslated region (5 ⁇ untranslated region, 5 ⁇ UTR), coding region (coding sequence, CDS) and polyadenylation tail (poly(A) ) sequences are all the same.
  • Cell plating trypsinize the well-cultured CHO cells, count them, resuspend 3 ⁇ 10 5 cells in 3 mL of culture medium, and spread them into 6-well plates for 24 hours.
  • the medium composition is 45% DMEM.
  • transfection mixture quickly add the prepared transfection reagent dropwise to the 6-well plate plated with CHO cells, shake the 6-well plate for several times, and then put it back to 37°C and incubate it in a 5% CO 2 incubator until The longest is 144h.
  • the medium was aspirated, washed once with 1 ⁇ PBS, CHO cells were digested with trypsin, counted, 3 ⁇ 10 5 cells were resuspended in 3 mL of culture medium, and plated into 6-well plates for culture.
  • the medium composition was: 45% DMEM medium + 45% RPMI-1640 medium + 10% serum + 1% L-glutamine + 1% thymidine hypoxanthine; another 3 ⁇ 10 5 cells were resuspended in 1 ⁇ PBS for Flow observation and analysis by Kaluza Analysis.
  • Figure 6 shows the positive rate of eGFP after 24h of mRNA transfection in CHO cells under different 3'UTRs. It can be seen that there is no significant difference in the transfection efficiency of mRNAs with different 3'UTRs in CHO cells.
  • Figure 7 is the average fluorescence intensity of eGFP 24h after CHO cells were transfected with mRNA. The results show that the expression intensity of eGFP in CHO cells transcribed in vitro containing mRNA containing artificially modified or designed 3'UTR is higher than that of natural sequences such as PCBP4, PIWIL4, and IL-2. The 3 ⁇ UTR is strong.
  • the medium was aspirated, washed once with 1 ⁇ PBS, CHO cells were digested with trypsin, counted, 3 ⁇ 10 5 cells were resuspended in 3 mL of culture medium, and plated into 6-well plates for culture.
  • the medium composition was: 45% DMEM medium + 45% RPMI-1640 medium + 10% serum + 1% L-glutamine + 1% thymidine hypoxanthine; another 3 ⁇ 10 5 cells were resuspended in 1 ⁇ PBS for Flow observation and analysis by FlowJo X.
  • Figure 8 shows the positive rate of eGFP after 48h of mRNA transfection in CHO cells under different 3'UTRs. It can be seen that there is no difference in the expression persistence of mRNAs of different 3'UTRs in CHO cells after 48h.
  • Figure 9 is the average fluorescence intensity of eGFP 48h after CHO cells were transfected with mRNA, the results show that after 48h, the expression intensity of eGFP in CHO cells transcribed in vitro containing mRNA containing artificially modified or designed 3'UTR is higher than that of PCBP4, PIWIL4, IL-2, etc.
  • the native sequence 3'UTR was strong, and the mRNA fluorescence intensity of the native 3'UTR decreased rapidly.
  • the medium was aspirated, washed once with 1 ⁇ PBS, CHO cells were digested with trypsin, counted, and 3 ⁇ 10 5 cells were resuspended in 1 ⁇ PBS for flow observation and analyzed by FlowJo X.
  • Figure 10 shows the positive rate of eGFP 96h after transfection into CHO cells under different 3'UTR conditions. 2, etc. mRNA of native sequence 3'UTR.
  • Figure 11 is the average fluorescence intensity of eGFP after 96h transfection of mRNA in CHO cells. The results show that the expression intensity of eGFP in CHO cells transcribed in vitro containing mRNA containing artificially engineered or designed 3'UTR is stronger than that of IL-2 3'UTR. It can be seen that after 96 hours, the mRNA containing the artificially modified or designed 3'UTR still has a high expression activity, while the expression of the mRNA containing the IL-2 3'UTR has been extremely low.

Abstract

提供了能改善基因表达的3'UTR多核苷酸、其构建方法和应用。该多核苷酸包含PCBP4基因或PIWIL4基因的3'UTR区中的一个或多个microRNA识别位点敲除后的序列，或含有至少3个串联重复的m6A位点序列和任两个重复序列之间的接头序列的3'UTR序列。

Description

3’UTR的构建方法和应用 技术领域
本发明属于蛋白表达技术领域，具体涉及提高mRNA翻译的3'UTR区序列及其构建方法和应用。
背景技术
信使核糖核酸(mRNA)除了含有翻译蛋白所必须的编码序列(coding sequences，CDS)外，还有必要的非翻译序列，如m7G帽子、5`端非翻译区(5'untranslated region，5'-UTR)、3'端非翻译区(3'untranslated region，3'UTR)与多聚腺苷酸尾(poly(A)tail)。
3'端非翻译区紧连信使核糖核酸编码区，与转录后调控密切相关，通过其他调控因子结合影响信使核糖核酸在细胞质内的翻译效率、稳定性等，例如小分子核糖核酸(miRNA)与3'端非翻译区结合，进而抑制信使核糖核酸翻译甚至诱导其降解；3'端非翻译区中的沉默子序列与RNA结合蛋白质结合，也可抑制翻译。此外，3'端非翻译区序列越长，对蛋白翻译产生负面影响的可能性越大。现有技术中的3'-UTR通常分离自动物本身而未经改造，其效果可能受宿主影响而具有不确定性。本领域仍需能提高mRNA翻译的通用3'UTR区。
发明内容
本发明通过天然基因改造或人工设计构建了3'端非翻译区序列，这些3'端非翻译区序列使基因的信使核糖核酸在不同细胞中保持较高的翻译活性。
本发明第一方面提供一种多核苷酸，所述多核苷酸包含
(1)3’UTR序列，其是PCBP4基因或PIWIL4基因的3’UTR区中的一个或多个microRNA识别位点敲除后的序列，和/或
(2)(1)的互补序列。
在一个或多个实施方案中，所述PCBP4基因的3’UTR区的核苷酸序列如 SEQ ID NO:1或与其具有至少80％序列相同性的序列中任一所示。
在一个或多个实施方案中，所述PIWIL4基因的3’UTR区的核苷酸序列如SEQ ID NO:2或与其具有至少80％序列相同性的序列中任一所示。
在一个或多个实施方案中，所述microRNA识别位点可使用http://www.targetscan.org/vert_72/网站检索获得。
在一个或多个实施方案中，所述microRNA识别位点选自表1和/或表2中的一个或多个microRNA识别位点。
在一个或多个实施方案中，所述PCBP4基因的3’UTR区中的一个或多个microRNA识别位点选自表1。
在一个或多个实施方案中，所述PIWIL4基因的3’UTR区中的一个或多个microRNA识别位点选自表2。
在一个或多个实施方案中，所述PCBP4基因或PIWIL4基因是人PCBP4基因或人PIWIL4基因。
在一个或多个实施方案中，所述多核苷酸的序列选自(1)如SEQ ID NO:3-7中任一所示的序列，(2)与(1)具有至少80％序列相同性的序列。
在一个或多个实施方案中，所述多核苷酸是DNA或RNA。
本发明第一方面还提供一种构建3’UTR序列的方法，包括将基因的3’UTR区中的一个或多个microRNA的识别位点敲除的步骤。
在一个或多个实施方案中，所述基因是PCBP4基因或PIWIL4基因。优选地，所述PCBP4基因或PIWIL4基因是人PCBP4基因或人PIWIL4基因。
在一个或多个实施方案中，所述PCBP4基因的3’UTR区的核苷酸序列如SEQ ID NO:1或与其具有至少80％序列相同性的序列中任一所示。
在一个或多个实施方案中，所述PIWIL4基因的3’UTR区的核苷酸序列如SEQ ID NO:2或与其具有至少80％序列相同性的序列中任一所示。
在一个或多个实施方案中，所述microRNA识别位点用http://www.targetscan.org/vert_72/网站检索获得。
在一个或多个实施方案中，所述microRNA识别位点选自表1和/或表2中的一个或多个microRNA识别位点。
在一个或多个实施方案中，所述PCBP4基因的3’UTR区中的一个或多个microRNA识别位点选自表1。
在一个或多个实施方案中，所述PIWIL4基因的3’UTR区中的一个或多个microRNA识别位点选自表2。
本发明第二方面提供一种多核苷酸，所述多核苷酸包含
(1)3’UTR序列，其含有RNA甲基化(m6A)位点的至少3个串联重复的序列和任选的任两个重复之间的接头，和/或
(2)(1)的互补序列。
在一个或多个实施方案中，m6A位点是m6A甲基化酶(writer)或m6A RNA甲基化识别蛋白(reader)的识别序列。在一个或多个实施方案中，m6A甲基化酶选自METTL3、METTL14、METTL16、WTAP、VIRMA、ZC3H13、RBM15、RBM158、HAKAI和KIAA1492。在一个或多个实施方案中，m6A RNA甲基化识别蛋白选自YTHDC1-2、YTHDF1-3、IGF2BP1-3、Prrc2a、HNRNPC、HNRNPG、FMR1、SRSF2、HuR、LRPPRC、eIF3和HNRNPA2B1。
在一个或多个实施方案中，m6A位点选自：AAABH、AGABH、GAABH、GGABH，其中B＝T或C或G，H＝T或A或C。
在一个或多个实施方案中，m6A位点选自：GGACT、GGACC、GAACT。
在一个或多个实施方案中，所述3’UTR序列含有所述m6A位点的至少4个、至少5个、至少6个、至少7个、至少8个、至少9个、至少10个串联重复的序列。
在一个或多个实施方案中，所述接头长度为1-5bp，优选3bp。
在一个或多个实施方案中，接头序列选自：TCT、TGT、GGT、CT、TT和CA。
在一个或多个实施方案中，所述多核苷酸的序列选自(1)如SEQ ID NO:8-14中任一所示的序列，(2)与(1)具有至少80％序列相同性的序列。
在一个或多个实施方案中，所述多核苷酸是DNA或RNA。
本发明第二方面还提供一种构建3’UTR序列的方法，包括将至少3个 RNA甲基化(m6A)位点串联连接的步骤和任选的在两端添加限制性酶切位点的步骤。
在一个或多个实施方案中，所述连接是直接连接或通过接头连接。
在一个或多个实施方案中，所述接头长度为1-5bp。
在一个或多个实施方案中，m6A位点是m6A甲基化酶或m6A RNA甲基化识别蛋白的识别序列。在一个或多个实施方案中，m6A甲基化酶选自METTL3、METTL14、METTL16、WTAP、VIRMA、ZC3H13、RBM15、RBM158、HAKAI和KIAA1492。在一个或多个实施方案中，m6A RNA甲基化识别蛋白选自YTHDC1-2、YTHDF1-3、IGF2BP1-3、Prrc2a、HNRNPC、HNRNPG、FMR1、SRSF2、HuR、LRPPRC、eIF3和HNRNPA2B1。
在一个或多个实施方案中，m6A位点选自：AAABH、AGABH、GAABH、GGABH，其中B＝T或C或G，H＝T或A或C。
在一个或多个实施方案中，m6A位点选自：GGACT、GGACC、GAACT。
在一个或多个实施方案中，所述方法包括将至少4个、至少5个、至少6个、至少7个、至少8个、至少9个、至少10个RNA甲基化(m6A)位点串联连接的步骤。
在一个或多个实施方案中，所述3’UTR序列中的RNA甲基化(m6A)位点相同。
本发明还提供一种核酸构建物，包含本文任一实施方案所述的多核苷酸和任选的待表达基因。
在一个或多个实施方案中，所述核酸构建物是含有待表达基因和所述多核苷酸的mRNA。
在一个或多个实施方案中，所述核酸构建物是载体，例如克隆载体或表达载体。
在一个或多个实施方案中，所述多核苷酸位于所述待表达基因的3’端。
在一个或多个实施方案中，所述核酸构建物还包含选自以下的一个或多个元件：启动子、5’UTR、多克隆位点、polyA。
在一个或多个实施方案中，所述启动子是T7启动子。
在一个或多个实施方案中，所述5’UTR是HBB mRNA的5’UTR。
在一个或多个实施方案中，所述核酸构建物至少依次包含：启动子、多克隆位点、本文任一实施方案所述的多核苷酸和polyA。
在一个或多个实施方案中，所述外源基因是eGFP基因。
本发明还提供一种宿主细胞，包含本文任一实施方案所述的多核苷酸和/或核酸构建物。
在一个或多个实施方案中，所述宿主细胞是CHO细胞或PBMC细胞。
本发明还提供一种药物组合物，包含本文任一实施方案所述的多核苷酸、核酸构建物或宿主细胞，和药学上可接受的辅料。
在一个或多个实施方案中，所述多核苷酸位于待表达基因的3’端。
本发明还提供一种提高基因表达强度、延长基因表达时间、提高mRNA启动翻译能力或维持翻译活性时长的方法，包括使携带有本文任一实施方案所述的多核苷酸的基因表达的步骤。
在一个或多个实施方案中，所述多核苷酸位于所述基因的3’端。
在一个或多个实施方案中，所述基因是eGFP基因。
本发明还提供本文任一实施方案所述的多核苷酸、核酸构建物和/或宿主细胞在制备用于提高基因表达强度、延长基因表达时间、提高mRNA启动翻译能力或维持翻译活性时长的试剂中的应用。
在一个或多个实施方案中，所述多核苷酸位于所述基因的3’端。
在一个或多个实施方案中，所述基因是eGFP基因。
本发明还提供本文任一实施方案所述的多核苷酸、核酸构建物和/或宿主细胞在制备用于治疗疾病的药物中的应用，所述多核苷酸位于基因的3’端，所述基因的表达有助于所述治疗疾病。
本发明还提供本文任一实施方案所述的多核苷酸在控制基因表达中的应用。
在一个或多个实施方案中，所述多核苷酸位于所述基因的3’端。
在一个或多个实施方案中，所述基因是eGFP基因。
附图说明
图1显示示例性的含有本文所述3’UTR的载体图谱。
图2显示不同3`UTR情况下，PBMC细胞电转mRNA 24h后eGFP表达荧光的比例。
图3显示PBMC电转含不同3`UTR的mRNA 24h后的平均荧光强度。
图4显示不同3`UTR情况下，PBMC电转mRNA 72h后eGFP阳性率。
图5显示不同3`UTR的eGFP mRNA电转PBMC 72h后的平均荧光强度。
图6显示不同3`UTR情况下，CHO细胞转染mRNA 24h后eGFP阳性率。
图7显示CHO细胞转染mRNA 24h后eGFP的平均荧光强度。
图8显示不同3`UTR情况下，CHO细胞转染mRNA 48h后eGFP阳性率。
图9显示不同3`UTR情况下，CHO细胞转染mRNA 48h后eGFP的平均荧光强度。
图10显示是不同3`UTR情况下，转染CHO细胞96h后的eGFP阳性率。
图11显示是不同3`UTR情况下，CHO细胞转染mRNA 96h后eGFP平均荧光强度。
具体实施方式
应理解，在本发明范围中，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，从而构成优选的技术方案。
本文和所附权利要求书所用的单数形式“一个”、“一种”和“所述”包括复数含义，除非上下文另有明确说明。同样，术语“一个”(或“一种”)、“一个或多个”和“至少一个”在本文中可互换使用。术语“包括”及其变化形式不具有限制意义，其中这些术语出现在本说明书和权利要求中。因此，术语“包括”、“包含”、和“含有”可互换使用。
本发明通过天然基因改造或人工设计构建了3'端非翻译区序列，这些3'端非翻译区序列使基因的信使核糖核酸在不同细胞中保持较高的翻译活性。
所以，在一个或多个实施方案中，本发明包含通过天然基因改造获得的3’UTR多核苷酸，其包含3’UTR序列或其互补序列，所述3’UTR序列是天然基因(例如人PCBP4基因或PIWIL4基因)的3’UTR区中的一个或多个microRNA识别位点敲除后的序列。PCBP4mRNA(NM_001174100)的3’UTR序列如SEQ ID NO:1所示，PIWIL4mRNA(NM_152431)的3’UTR序列如SEQ ID NO:2所示。本文中，microRNA识别位点是由microRNA识别并影响mRNA稳定性的位点。一些microRNA识别位点的序列可用http://www.targetscan.org/vert_72/网站进行检索获得。在具体实施方案中，PCBP4的3’UTR的microRNA靶序列选自表1中的一个或多个；PIWIL4的3’UTR的microRNA靶序列选自表2中的一个或多个。
表1，PCBP4基因3’UTR中的microRNA靶序列
microRNA 种子序列(Seed sequence)
Has-miR-6801-3p gcagggg
Has-miR-4313 agggggc
Has-miR-6745 tccaccc
Has-miR-4251 ttctcag
Has-miR-6777-3p agagtgg
Has-miR-1976 caggaga
Has-miR-92a-2-5p ccccacca
Has-miR-1205 cctgcaga
Has-miR-145-5p actggaa
hsa-miR-548g-3p acagttt
hsa-miR-548az-3p cagtttt
hsa-miR-3613-3p tttttga
表2，PIWIL4基因3’UTR中的microRNA靶序列
microRNA 种子序列(Seed sequence)
Has-miR-3912-5p atggacaa
Has-miR-4659-3p agaagaaa
Has-miR-557 gtgcaaa
Has-miR-3685 taggaaa
Has-miR-19b-2-5p gcaaaaca
Has-miR-3942-3p atctgaaa
Has-miR-548bc cagtttta
Has-miR-8485 tgtgtgt
Has-miR-3692-3p tgtggaa
Has-miR-6844 tctacaaa
Has-miR-7114-5p tccacaga
Has-miR-4698 attttga
hsa-miR-215-3p atgacaga
hsa-miR-3664-5p acagagt
Has-miR-4651 ccacccca
hsa-miR-545-3p tttgctga
hsa-miR-520b-5p tgtagag
Has-miR-1283 tttgtag
Has-miR-1305 gttgaaaa
Has-miR-12113 agttcaga
构建上述3’UTR序列的方法包括将基因的3’UTR区中的一个或多个microRNA的识别位点敲除的步骤和任选的在3’UTR区两端添加限制性酶切位点的步骤。敲除microRNA识别位点可采用本领域已知的任何方法实现，例如突变3’UTR中与microRNA的种子序列(seed)互补的2-8nt靶序列。在具体实施方案中，先根据基因(例如人PCBP4基因或PIWIL4基因)的3’UTR设计引物，从RNA组中经RT-PCR扩增出相应序列，再通过PCR引入突变进行敲除。或者，本发明的通过天然基因改造的3’UTR序列也可直接合成获得。示例性的所述多核苷酸的序列如SEQ ID NO:3-7中任一所示。
在其他实施方案中，本发明包含通过人工设计获得的3’UTR多核苷酸，包含3’UTR序列或其互补序列，其含有RNA甲基化(m6A)位点的至少3个串联重复的序列和任选的任两个重复之间的接头。本文中，m6A位点是m6A甲基化酶或m6A RAN甲基化识别蛋白的识别位点。示例性地，m6A甲基化酶 可为METTL3、METTL14、METTL16、WTAP、VIRMA、ZC3H13、RBM15、RBM158、HAKAI和KIAA1492；m6A RNA甲基化识别蛋白可为YTHDC1-2、YTHDF1-3、IGF2BP1-3、Prrc2a、HNRNPC、HNRNPG、FMR1、SRSF2、HuR、LRPPRC、eIF3和HNRNPA2B1。可用于本发明的m6A位点选自：AAABH、AGABH、GAABH、GGABH，其中B＝T或C或G，H＝T或A或C。所述3’UTR序列可含有所述m6A位点的至少4个、至少5个、至少6个、至少7个、至少8个、至少9个、至少10个串联重复的序列。一个3’UTR序列中的多个RNA甲基化(m6A)位点可相同或不同。构建所述3’UTR序列的方法包括将至少3个RNA甲基化(m6A)位点串联连接的步骤和任选的在两端添加限制性酶切位点的步骤。所述连接是直接连接或通过接头(例如长度为1-5bp的寡核苷酸)。或者，本发明的通过人工设计的3’UTR序列也可直接合成获得。示例性的所述多核苷酸的序列如SEQ ID NO:8-14中任一所示。
本文中，多核苷酸可以是DNA或RNA。DNA可以是单链的或是双链的。因此，本文的多核苷酸一般是分离的多核苷酸形式。提到核酸时，本文所用术语“变体”可以是天然发生的等位变体或非天然发生的变体。这些核苷酸变体包括简并变体、取代变体、缺失变体和插入变体。本发明核酸可包含与所述核酸序列的序列相同性为至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约98％、至少约99％或100％的核苷酸序列。本发明还涉及与上述的序列杂交的核酸片段。本文所述的多核苷酸通常可以用PCR扩增法获得。或者，也可直接合成本文所述的核酸分子。
在两种或多种多肽或核酸分子序列中，术语“相同性”或“相同性百分数”指在比较窗口或指定区域上，采用本领域已知方法如序列比较算法，通过手工比对和目测检查来比较和比对最大对应性时，两个或多个序列或子序列相同或其中在指定区域有一定百分数的氨基酸残基或核苷酸相同(例如，至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％相同)。例如，适合测定序列相同性百分数和序列相似性百分数的优选算法是BLAST和BLAST 2.0算法，分别可参见Altschul等(1977)Nucleic Acids Res.25:3389和Altschul等(1990)J.Mol.Biol.215:403。
本领域技术人员公知，在基因克隆操作中，常常需要设计合适的酶切位点，这势必在多核苷酸末端引入了一个或多个酶切位点，而这并不影响3’UTR多核苷酸或目的蛋白的活性。本发明范围涵盖本文所述序列及其两端添加有一个或多个酶切位点的序列。
本发明的3’UTR多核苷酸可以改善关联基因的表达。所述表达的改善通过在细胞中导入载有本文所述多核苷酸的基因的核酸构建物实现。因此，本发明还提供所述多核苷酸的核酸构建物。该核酸构建物可以是表达框，含有本文所述3’UTR多核苷酸，多克隆位点，以及与之操作性连接的一个或多个调控序列，例如复制起点、多克隆位点、启动子、标记基因或翻译控制元件，包括增强子、操纵子、终止序列例如polyA、核糖体结合位点、5’UTR。待表达基因的编码序列可插入核酸构建物中所述3’UTR多核苷酸的5’端。本发明所述的多核苷酸可以多种方式被操作以保证所述基因的表达。在将核酸构建物插入载体之前可根据载体的不同或要求而对核酸构建物进行操作。利用重组DNA方法来改变多核苷酸序列的技术是本领域已知的。
所述核酸构建物还可以是含有待表达基因和本文所述3’UTR多核苷酸的mRNA。该mRNA可通过本领域已知的体内或体外转录方法获得。例如按照HiScribe T7 ARCA mRNA试剂盒操作指南获得体外转录mRNA。
在重组表达载体中，“操作性连接”是指目的的核苷酸序列与调节序列以允许核苷酸序列表达的方式连接。本领域的技术人员熟知能用于构建含本文所述3’UTR多核苷酸和任选的目的基因的编码序列和合适的转录/翻译控制信号的表达载体的方法。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。
调控序列可以是合适的启动子序列。启动子序列通常与待表达蛋白的编码序列操作性连接。启动子可以是在所选择的宿主细胞中显示转录活性的任何核苷酸序列，包括突变的、截短的和杂合启动子，并且可以从编码与该宿主细胞同源或异源的胞外或胞内多肽的基因获得。合适的启动子的例子为T7启动子序列。该启动子序列是能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。也可使用其他组成型启动子序列，包括但不限于T3启动子，SP6启动子。
调控序列也可以是合适的转录终止序列，由宿主细胞识别以终止转录的序列。终止序列与本文所述的3’UTR多核苷酸的3’末端操作性连接。在选择的宿主细胞中有功能的任何终止序列都可用于本发明。
调控序列也可以是合适的前导序列，对宿主细胞翻译重要的mRNA的5’非翻译区。前导序列与待表达基因的5’末端可操作连接。在选择的宿主细胞中有功能的任何5’非翻译区都可用于本发明。
在某些实施方案中，所述核酸构建物是载体。载体可以是克隆载体，也可以是表达载体，或者是同源重组载体。本发明的多核苷酸可被克隆入许多类型的载体，例如，质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。克隆载体可用于提供外源基因和本文所述多核苷酸的编码序列。表达载体可以以病毒载体形式提供给细胞。通常通过可操作地连接本发明的多核苷酸至启动子，并将构建体并入表达载体，实现基因表达。该载体对于复制和整合真核细胞可为合适的。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、起始序列和启动子。病毒载体技术在本领域中是公知的并在例如Sambrook等(2001，Molecular Cloning:A Laboratory Manual，Cold Spring Harbor Laboratory，New York)和其他病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。同源重组载体用于将本文所述的表达框整合到宿主基因组中。
通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记。例如，在某些实施方案中，本发明使用pT7-m5U载体，该载体含有复制起始位点，启动子，本文所述的多核苷酸，以及任选的可选择的标记。
为了评估治疗用蛋白、多肽或其部分的表达，被引入细胞的表达载体也可包含可选择的标记基因或报道基因中的任一个或两者，以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面，可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列，以便能够在宿主细胞中表达。有用的可选择标记包括Flag、HA或V5。报道基因用于鉴定潜在转染的细胞并用于评价调节序列的功能性。在DNA已经被引入受体细胞后，报道基因的表达在合适 的时间下进行测定。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白基因的基因。合适的表达系统是公知的并可利用已知技术制备或从商业上获得。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物、细菌、酵母或昆虫细胞。例如，表达载体可通过物理、化学或生物学手段转移入宿主细胞。
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔、电转等，例如直接将体外转录好的mRNA通过电转引入细胞。将多核苷酸引入宿主细胞的化学手段包括胶体分散系统，诸如大分子复合物、纳米胶囊、微球、珠；和基于脂质的系统，包括水包油乳剂、胶束、混合胶束和脂质体。
将多核苷酸引入宿主细胞的生物学方法包括使用病毒载体，特别是逆转录病毒载体，例如源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒的载体。可利用本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。许多反转录病毒系统在本领域中是已知的。慢病毒是逆转录病毒科下的属。用于慢病毒包装的试剂为本领域所周知，如常规的慢病毒载体系统包括pRsv-REV、pMDlg-pRRE、pMD2G和目的干扰质粒。
本文中，宿主细胞含有本文所述的多核苷酸或核酸构建物。宿主细胞既包括表达目标基因的细胞，例如CHO或PBMC细胞，也包括生产该表达用细胞过程中使用到的各种细胞，如大肠杆菌细胞，以用于如提供本发明多核苷酸或提供本文所述的载体。适用于本发明的细胞可以是各种来源的各种类型的细胞，包括原核细胞和真核细胞，例如细菌细胞、酵母细胞、昆虫细胞和哺乳动物细胞，包括但不限于，sf9、BHK21、COS1、COS3、COS7、293T、Vero。所述宿主细胞优选各种利于基因产物表达或发酵生产的细胞，此类细胞已为本领域熟知并常用。
本文还包括细胞制品，包含本文所述的细胞或其提取物。例如，所述细胞制品可以是包含本文所述细胞或其提取物以及合适的培养基的细胞培养物、药 物组合物、试剂盒、装置、介质或系统，例如芯片等。培养各类细胞的合适培养基本领域周知。
本文所述核酸构建物或细胞的功能取决于其中所携带的待表达基因，例如若待表达基因是荧光蛋白，则本文所述核酸构建物或细胞可用于细胞示踪。若待表达基因是有利于疾病治疗的试剂，则本文所述核酸构建物或细胞可用于治疗疾病。
本发明提供药物组合物，其包含本发明所述核酸构建物或细胞以及药学上可接受的稀释剂、载剂、增溶剂、乳化剂、防腐剂和/或佐剂，所述核酸构建物或细胞可产生治疗有效量的活性分子(例如核酸构建物中目标基因的表达产物)。在某些实施方案中，药物组合物中可接受的稀释剂、载剂、增溶剂、乳化剂、防腐剂和/或佐剂等优选地在所采用的剂量和浓度下对接受者无毒。在某些实施方案中，药物组合物可含有用于改善、维持或保留例如组合物的pH、渗透性、粘度、澄清度、颜色、等渗性、气味、无菌性、稳定性、溶解或释放速率、吸收或渗透的这类物质。这些物质为现有技术已知，例如可参见REMINGTON'S PHARMACEUTICAL SCIENCES，第18版，A.R.Genrmo编，1990，Mack Publishing Company。可视预期的施用途径、递送方式和所需的剂量来确定最佳的药物组合物。
药物组合物的施用途径是根据已知方法，例如经口、通过静脉内、腹膜内、脑内(脑实质内)、脑室内、肌肉内、眼内、动脉内、门静脉或病灶内途径注射；通过持续释放系统或通过植入装置。可选择本发明的药物组合物用于肠胃外递送。或者，可选择组合物用于吸入或通过消化道(诸如经口)递送。所述药学上可接受的组合物的制备在本领域的技术内。用于配制多种其它持续或可控传递方式的技术(诸如脂质体载剂、生物易蚀微粒或多孔珠粒和积存注射)也为本领域技术人员所知。
用于体内施用的药物组合物通常以无菌制剂的形式提供。通过经无菌过滤膜过滤来实现灭菌。在组合物冻干时，可在冻干和复水之前或之后使用此方法进行灭菌。用于肠胃外施用的组合物可以冻干形式或在溶液中储存。肠胃外组合物通常放在具有无菌进入孔的容器中，例如具有皮下注射针可刺穿的塞子的静脉内溶液带或小瓶。
药物组合物一经配制，就以溶液、悬浮液、凝胶、乳液、固体、晶体或以脱水或冻干粉末的形式储存在无菌小瓶中。所述配制物可储存成即用形式或在施用前复水的形式(例如，冻干)。本发明还提供用于产生单剂量施用单位的试剂盒。本发明的试剂盒可各自含有具有干燥蛋白的第一容器和具有含水配制物的第二容器。在本发明的某些实施方案中，提供含有单腔和多腔预填充注射器(例如，液体注射器和冻干注射器)的试剂盒。
本发明也提供通过施用本发明任一实施方案所述的核酸构建物或细胞或其药物组合物来治疗患者的方法。本文中，术语“患者”、“受试者”、“个体”、“对象”在本文中可互换使用，包括任何生物体，优选动物，更优选哺乳动物(例如大鼠、小鼠、狗、猫、兔等)，且最优选的是人。“治疗”指向受试者采用本文所述治疗方案以达到至少一种阳性治疗效果。有效治疗患者的治疗方案可根据多种因素(比如患者的疾病状态、年龄、体重及疗法激发受试者的抗癌反应的能力)而变。
将采用的含有本发明核酸构建物或细胞的药物组合物的治疗有效量将取决于例如治疗程度和目标。本领域技术人员将了解，用于治疗的适当剂量水平将部分取决于所递送的分子(例如核酸构建物中目标基因表达的产物)、适应症、施用途径和患者的大小(体重、体表或器官大小)和/或状况(年龄和一般健康状况)而变化。在某些实施方案中，临床医生可滴定剂量并改变施用途径来获得最佳的治疗效果。
给药频率将取决于所用配制物中特定目标产物(例如核酸构建物中目标基因表达的产物)的药物动力学参数。临床医生典型地施用组合物直到达到实现所需效果的剂量。组合物因此可作为单次剂量施用，或随时间以作为两次或多次剂量(可含有或不含有相同量的所需分子)施用，或通过植入装置或导管以连续输液的方式施用。
如下实施例所证实，本文所述3’UTR多核苷酸可以提高基因表达强度、延长基因表达时间、提高mRNA启动翻译能力或维持翻译活性时长。因此，本发明还提供提高基因表达强度、延长基因表达时间、提高mRNA启动翻译能力或维持翻译活性时长的方法，包括使携带有本文任一实施方案所述的3’UTR多核苷酸的基因表达的步骤。通常，所述3’UTR多核苷酸位于所述基 因的3’端。示例性地，表达通过上述将核酸构建物引入细胞并在所述基因表达的条件下孵育细胞的过程实现。实施例中使用GFP仅作为待表达基因的示例。
本发明还提供本文任一实施方案所述的3’UTR多核苷酸、核酸构建物和/或宿主细胞在制备用于治疗疾病的药物中的应用，所述3’UTR多核苷酸位于基因的3’端，所述基因的表达有助于所述治疗疾病。可通过如上所述的方法，将包含所述基因和所述3’UTR多核苷酸的核酸构建物导入细胞，通过在所述基因表达的条件下孵育细胞来使基因表达。所述基因的表达有助于所述治疗疾病可以是基因或其产物直接产生治疗效果，或者也可以是基因或其产物的表达引起其他能产生治疗效果的基因或蛋白的表达或活化。
本发明包括如下具体实施方案：
1、一种多核苷酸，所述多核苷酸包含：
(1)3’UTR序列，其是PCBP4基因或PIWIL4基因的3’UTR区中的一个或多个microRNA识别位点敲除后的序列，
(2)3’UTR序列，其含有m6A位点的至少3个串联重复的序列和任选的任两个重复之间的接头，和/或
(3)(1)或(2)的互补序列。
2、如项目1所述的多核苷酸，其特征在于，
所述PCBP4基因或PIWIL4基因是人PCBP4基因或人PIWIL4基因，和/或，所述microRNA识别位点如网站http://www.targetscan.org/vert_72/中所示，和/或，所述m6A位点是m6A甲基化酶或m6A RNA甲基化识别蛋白的识别序列，和/或，所述3’UTR序列含有所述m6A位点的至少4个、至少5个、至少6个、至少7个、至少8个、至少9个、至少10个串联重复的序列，和/或，所述接头长度为1-5bp；
优选地，所述PCBP4基因的3’UTR区的核苷酸序列如SEQ ID NO:1所示，和/或所述PIWIL4基因的3’UTR区的核苷酸序列如SEQ ID NO:2所示；或，所述m6A位点选自：AAABH、AGABH、GAABH、GGABH，其中B＝T或C或G，H＝T或A或C；
更优选地，所述多核苷酸的序列选自(1)如SEQ ID NO:3-14中任一所示的序列，(2)与(1)具有至少80％序列相同性的序列。
3、一种构建3’UTR序列的方法，包括将基因的3’UTR区中的一个或多个microRNA的识别位点敲除的步骤，
优选地，所述基因是PCBP4基因或PIWIL4基因，
更优选地，所述PCBP4基因或PIWIL4基因是人PCBP4基因或人PIWIL4基因，
进一步优选地，所述基因的3’UTR区的核苷酸序列如SEQ ID NO:1或2或与其具有至少80％序列相同性的序列中任一所示，和/或，所述microRNA识别位点如网站http://www.targetscan.org/vert_72/中所示。
4、一种构建3’UTR序列的方法，包括将至少3个m6A位点串联连接的步骤和任选的在两端添加限制性酶切位点的步骤，
优选地，所述连接是直接连接或通过接头连接，和/或，所述m6A位点是m6A甲基化酶或m6A RNA甲基化识别蛋白的识别序列，和/或，所述方法包括将至少4个、至少5个、至少6个、至少7个、至少8个、至少9个、至少10个m6A位点串联连接的步骤；
更优选地，所述接头长度为1-5bp，和/或，m6A位点选自：AAABH、AGABH、GAABH、GGABH，其中B＝T或C或G，H＝T或A或C，和/或，所述3’UTR序列中的m6A位点相同。
5、一种核酸构建物，包含项目1或2所述的多核苷酸和任选的待表达基因，所述多核苷酸位于所述待表达基因的3’端；优选地，所述核酸构建物还包含选自以下的一个或多个元件：启动子、5’UTR、多克隆位点、polyA。
6、一种宿主细胞，包含项目1或2所述的多核苷酸和/或项目5所述的核酸构建物；优选地，所述宿主细胞是CHO细胞或PBMC细胞。
7、一种药物组合物，包含项目1或2所述的多核苷酸、项目5所述的核酸构建物或项目6所述的宿主细胞，和药学上可接受的辅料。
8、一种提高基因表达强度、延长基因表达时间、提高基因的mRNA启动翻译能力或维持翻译活性时长的方法，包括使携带有项目1或2所述的多核苷酸的基因表达的步骤，所述多核苷酸位于所述基因的3’端。
9、项目1或2所述的多核苷酸、项目5所述的核酸构建物或项目6所述的宿主细胞在制备用于治疗疾病的药物中的应用，所述多核苷酸位于基因的3’ 端，所述基因的表达有助于所述疾病的治疗。
10、项目1或2所述的多核苷酸在控制基因表达中的应用，所述多核苷酸位于所述基因的3’端。
实施例
实施例1，3'端非翻译区转录模板载体构建
1)基于人源天然基因改造的3'端非翻译区表达eGFP报告基因转录模板载体构建
构建载体pT7-m5U-eGFP-1#3U
委托金唯智公司合成基于人PCBP4基因3'端非翻译区改造后的序列(SEQ ID NO:3)，用Sal I和Hind Ⅲ双酶切载体pUC57-1#3U，回收大小为430bp片段1#3U。自构建EGFP转录模板载体pT7-m5U-eGFP含T7启动子、HBBmRNA(NM_000518)的5’UTR序列(m5U)、EGFP序列、HBB mRNA3’UTR序列和polyA序列(SEQ ID NO:16)。经Sal I和Hind Ⅲ双酶切后，将上述回收片段1#3U连接到pT7-m5U-eGFP载体上，替换原HBB mRNA 3’UTR，即得到pT7-m5U-eGFP-1#3U。
图1示例性显示pT7-m5U-eGFP-1#3U的图谱，后续载体的图谱与之区别在于使用不同的3’UTR。
构建载体pT7-m5U-eGFP-2#3U
委托金唯智公司合成基于人PCBP4基因3'端非翻译区改造后的序列(SEQ ID NO:4)，用Sal I和Hind Ⅲ双酶切载体pUC57-2#3U，回收大小为143bp片段2#3U，pT7-m5U-eGFP载体经Sal I和Hind Ⅲ双酶切后，将上述回收片段2#3U连接到pT7-m5U-eGFP载体上，即得到pT7-m5U-eGFP-2#3U。
构建载体pT7-m5U-eGFP-3#3U
委托金唯智公司合成基于人PCBP4基因3'端非翻译区改造后的序列(SEQ ID NO:5)，用Sal I和Hind Ⅲ双酶切载体pUC57-3#3U，回收大小为443bp片段3#3U，pT7-m5U-eGFP载体经Sal I和Hind Ⅲ双酶切后，将上述回收片段3#3U连接到pT7-m5U-eGFP载体上，即得到pT7-m5U-eGFP-3#3U。
构建载体pT7-m5U-eGFP-4#3U
委托金唯智公司合成基于人PCBP4基因3'端非翻译区改造后的序列(SEQ ID NO:6),用Sal I和Hind Ⅲ双酶切载体pUC57-4#3U,回收大小为430bp片段4#3U，pT7-m5U-eGFP载体经Sal I和Hind Ⅲ双酶切后，将上述回收片段4#3U连接到pT7-m5U-eGFP载体上，即得到pT7-m5U-eGFP-4#3U。
构建载体pT7-m5U-eGFP-5#3U
委托金唯智公司合基于成人PIWIL4基因3'端非翻译区改造后的序列(SEQ ID NO:7)，用Sal I和Hind Ⅲ双酶切载体pUC57-5#3U，回收大小为124bp片段5#3U，pT7-m5U-eGFP载体经Sal I和Hind Ⅲ双酶切后，将上述回收片段5#3U连接到pT7-m5U-eGFP载体上，即得到pT7-m5U-eGFP-5#3U。
2)3'端非翻译区为人工随机序列的表达eGFP报告基因转录模板载体构建
构建载体pT7-m5U-eGFP-6#3U
委托金唯智公司合成人工随机序列(SEQ ID NO:8)，用Sal I和Hind Ⅲ双酶切载体pUC57-6#3U，回收大小为100bp片段6#3U，pT7-m5U-eGFP载体经Sal I和Hind Ⅲ双酶切后，将上述回收片段6#3U连接到pT7-m5U-eGFP载体上，即得到pT7-m5U-eGFP-6#3U。
构建载体pT7-m5U-eGFP-7#3U
委托金唯智公司合成人工随机序列(SEQ ID NO:9)，用Sal I和Hind Ⅲ双酶切载体pUC57-7#3U，回收大小为116bp片段7#3U，pT7-m5U-eGFP载体经Sal I和Hind Ⅲ双酶切后，将上述回收片段7#3U连接到pT7-m5U-eGFP载体上，即得到pT7-m5U-eGFP-7#3U。
构建载体pT7-m5U-eGFP-8#3U
委托金唯智公司合成人工随机序列(SEQ ID NO:10)，用Sal I和Hind Ⅲ双酶切载体pUC57-8#3U，回收大小为86bp片段8#3U，pT7-m5U-eGFP载体经Sal I和Hind Ⅲ双酶切后，将上述回收片段8#3U连接到pT7-m5U-eGFP载体上，即得到pT7-m5U-eGFP-8#3U。
构建载体pT7-m5U-eGFP-9#3U
委托金唯智公司合成人工随机序列(SEQ ID NO:11)，用Sal I和Hind Ⅲ双酶切载体pUC57-9#3U，回收大小为92bp片段9#3U，pT7-m5U-eGFP载体 经Sal I和Hind Ⅲ双酶切后，将上述回收片段9#3U连接到pT7-m5U-eGFP载体上，即得到pT7-m5U-eGFP-9#3U。
构建载体pT7-m5U-eGFP-10#3U
委托金唯智公司合成人工随机序列(SEQ ID NO:12)，用Sal I和Hind Ⅲ双酶切载体pUC57-10#3U，回收大小为103bp片段10#3U，pT7-m5U-eGFP载体经Sal I和Hind Ⅲ双酶切后，将上述回收片段10#3U连接到pT7-m5U-eGFP载体上，即得到pT7-m5U-eGFP-10#3U。
构建载体pT7-m5U-eGFP-11#3U
委托金唯智公司合成人工随机序列(SEQ ID NO:13)，用Sal I和Hind Ⅲ双酶切载体pUC57-11#3U，回收大小为97bp片段11#3U，pT7-m5U-eGFP载体经Sal I和Hind Ⅲ双酶切后，将上述回收片段11#3U连接到pT7-m5U-eGFP载体上，即得到pT7-m5U-eGFP-11#3U。
构建载体pT7-m5U-eGFP-12#3U
委托金唯智公司合成人工随机序列(SEQ ID NO:14)，用Sal I和Hind Ⅲ双酶切载体pUC57-12#3U，回收大小为97bp片段12#3U，pT7-m5U-eGFP载体经Sal I和Hind Ⅲ双酶切后，将上述回收片段12#3U连接到pT7-m5U-eGFP载体上，即得到pT7-m5U-eGFP-12#3U。
3)3'端非翻译区为天然基因序列的表达eGFP报告基因转录模板载体构建
构建载体pT7-m5U-eGFP-PCBP4-3U
委托金唯智公司合成PCBP4mRNA 3`UTR序列(SEQ ID NO:1)，用Sal I和Hind Ⅲ双酶切载体pUC57-PCBP4#3U，回收大小为609bp片段PCBP4#3U，pT7-m5U-eGFP载体经Sal I和Hind Ⅲ双酶切后，将上述回收片段PCBP4#3U连接到pT7-m5U-eGFP载体上，即得到pT7-m5U-eGFP-PCBP4#3U。
构建载体pT7-m5U-eGFP-PIWIL4-3U
委托金唯智公司合成PIWIL4mRNA 3`UTR序列(SEQ ID NO:2)，用Sal I和Hind Ⅲ双酶切载体pUC57-PIWIL4#3U，回收大小为429bp片段PIWIL4#3U，pT7-m5U-eGFP载体经Sal I和Hind Ⅲ双酶切后，将上述回收片段PIWIL4#3U连接到pT7-m5U-eGFP载体上，即得到 pT7-m5U-eGFP-PIWIL4#3U。
构建载体pT7-m5U-eGFP-IL2#3U
委托金唯智公司合成IL-2 3`UTR序列(SEQ ID NO:15)，用Sal I和Hind Ⅲ双酶切载体pUC57-IL2#3U，回收大小为285bp片段IL2#3U，pT7-m5U-eGFP载体经Sal I和Hind Ⅲ双酶切后，将上述回收片段IL2#3U连接到pT7-m5U-eGFP载体上，即得到pT7-m5U-eGFP-IL2#3U。
实施例2，体外转录含有人工改造或设计3`UTR的mRNA
以HBB mRNA 3’UTR为对照3`UTR序列，其他5`非翻译区(5`untranslated region，5`UTR)、eGFP编码区(coding sequence，CDS)和多聚腺苷酸尾巴(poly(A))模板序列全部相同。使用BsaI限制性内切酶分别消化pT7-m5U-eGFP、pT7-m5U-eGFP-1#3U、pT7-m5U-eGFP-2#3U、pT7-m5U-eGFP-3#3U、pT7-m5U-eGFP-4#3U、pT7-m5U-eGFP-5#3U、pT7-m5U-eGFP-6#3U、pT7-m5U-eGFP-7#3U、pT7-m5U-eGFP-8#3U、pT7-m5U-eGFP-9#3U、pT7-m5U-eGFP-10#3U、pT7-m5U-eGFP-11#3U、pT7-m5U-eGFP-12#3U、pT7-m5U-eGFP-PCBP4#3U、pT7-m5U-eGFP-PIWIL4#3U、pT7-m5U-eGFP-IL2#3U，按照HiScribe T7 ARCA mRNA试剂盒(NEB,E2065S)操作指南获得体外转录mRNA：在无核酸酶的试管中加入以下试剂：2×ARCA/NTP Mix 10μL，BsaI线性化的质粒模板1μg，T7 RNA Polymerase Mix 2μl，H 2O至20μL；37℃孵育30分钟；加入2μL DNA酶，消化15min；使用随试剂盒提供的LiCl溶液沉淀2h；12000rpm离心2min；75％乙醇清洗2次，吸走全部上清，室温晾干；H 2O溶解沉淀mRNA，最终得到mRNA-eGFP#ctl、mRNA-eGFP1#、mRNA-eGFP2#、mRNA-eGFP3#、mRNA-eGFP4#、mRNA-eGFP5#、mRNA-eGFP6#、mRNA-eGFP7#、mRNA-eGFP8#、mRNA-eGFP9#、mRNA-eGFP10#、mRNA-eGFP11#、mRNA-eGFP12#、mRNA-eGFP PCBP4#、mRNA-eGFP PIWIL4#、mRNA-eGFP IL2#。
实施例3，构建的3`UTR在T细胞中维持基因表达检测
1)通过eGFP 24h表达强度测定3`非翻译区序列对mRNA翻译影响：
以HBB mRNA 3’UTR为对照3`UTR序列，其他5`非翻译区(5`untranslated region，5`UTR)、编码区(coding sequence，CDS)和多聚腺苷酸尾巴(poly(A))序列全部相同。复苏并重悬5×10 6新鲜分离获得的外周血单个核细胞(peripheral blood mononuclear cell，PBMC)，使用Lonza 4D-Nucleofector将
实施例1-2中得到表达EGFP蛋白的20μg mRNA(mRNA-eGFP#ctl、mRNA-eGFP1#、mRNA-eGFP2#、mRNA-eGFP3#、mRNA-eGFP4#、mRNA-eGFP5#、mRNA-eGFP6#、mRNA-eGFP7#、mRNA-eGFP8#、mRNA-eGFP9#、mRNA-eGFP10#、mRNA-eGFP11#、mRNA-eGFP12#、mRNA-eGFP PCBP4#、mRNA-eGFP PIWIL4#、mRNA-eGFPIL2#)电转到PBMC中，置37℃、5％CO 2培养箱中培养，培养基成分为AIM-V(Gibco)、2％胎牛血清(Gibco)。24小时后细胞计数并取3×10 5细胞用流式细胞术分析荧光表达情况。
图2是不同3`UTR情况下，PBMC细胞电转mRNA 24h后eGFP表达荧光的比例，可见各组mRNA在PBMC中的电转效率没有显著差异。图3是PBMC电转含不同3`UTR的mRNA 24h后的平均荧光强度，比较之下发现体外转录含有人工改造或设计3`UTR的mRNA的eGFP表达强度在PBMC中较含PCBP4、PIWIL4、IL-2等天然3`UTR的mRNA强，其中mRNA-eGFP8#、mRNA-eGFP9#和mRNA-eGFP10#显著优于含HBB 3`UTR的mRNA。
2)通过eGFP 72h表达强度测定3`非翻译区序列对T细胞中mRNA翻译时间影响
以HBB mRNA 3’UTR为对照3`UTR序列，其他5`非翻译区(5`untranslated region，5`UTR)、编码区(coding sequence，CDS)和多聚腺苷酸尾巴(poly(A))序列全部相同。复苏并重悬5×10 6新鲜分离获得的外周血单个核细胞(peripheral blood mononuclear cell，PBMC)，使用Lonza 4D-Nucleofector将实施例1-2中得到表达EGFP蛋白的20μg mRNA(mRNA-eGFP#ctl、mRNA-eGFP1#、mRNA-eGFP2#、mRNA-eGFP3#、mRNA-eGFP4#、mRNA-eGFP5#、mRNA-eGFP6#、mRNA-eGFP7#、mRNA-eGFP8#、mRNA-eGFP9#、mRNA-eGFP10#、mRNA-eGFP11#、mRNA-eGFP12#、 mRNA-eGFP PCBP4#、mRNA-eGFP PIWIL4#、mRNA-eGFPIL2#)电转到PBMC中，置37℃、5％CO2培养箱中培养，培养基成分为AIM-V(Gibco)、2％胎牛血清(Gibco)。72h后细胞计数，并取3×10 5细胞重悬于PBS中，用流式细胞术分析细胞阳性率和平均荧光强度。
图4是不同3`UTR情况下，PBMC电转mRNA 72h后eGFP阳性率，可见人工改造或设计3`UTR的mRNA在PBMC中的表达时间优于PCBP4、PIWIL4、IL-2等天然3`UTR。图5是不同3’UTR的eGFP mRNA电转PBMC72h后的平均荧光强度，比较之下发现体外转录含有人工改造或设计3`UTR的mRNA的eGFP在PBMC中表达强度较PCBP4、PIWIL4、IL-2等天然序列的3`UTR强。72小时后含有人工改造或设计3`UTR的mRNA在PBMC中仍具有较高的表达活性，而含有IL-2天然序列的3`UTR的mRNA已基本无表达。
实施例4，构建的3`UTR在CHO细胞中维持基因表达检测
1)CHO细胞转染(Lipo2000转染试剂盒)：
以HBB mRNA 3`UTR为对照3`UTR序列，其他5`非翻译区(5`untranslated region，5`UTR)、编码区(coding sequence，CDS)和多聚腺苷酸尾巴(poly(A))序列全部相同。
a、细胞铺板：用胰酶消化培养状态良好的CHO细胞，计数，取3×10 5细胞重悬到3mL培液中，铺到6孔板中培养24小时，培养基成分为45％DMEM培养基+45％RPMI-1640培养基+10％血清+1％L-谷氨酰胺+1％胸苷次黄嘌呤(Hypoxanthine-Thymidine)；
b、换培液：CHO细胞于6孔板培养24h后，弃尽原细胞培养液，加1mL PBS缓冲液清洗细胞，弃尽洗液，向其中加入2mL新培液，并将6孔板重新放回37℃培养箱中培养；
c、配转染试剂(每孔)：3μg(1μg/μL)实例1-2中得到的eGFP mRNA(mRNA-eGFP#ctl、mRNA-eGFP1#、mRNA-eGFP2#、mRNA-eGFP3#、mRNA-eGFP4#、mRNA-eGFP5#、mRNA-eGFP6#、mRNA-eGFP7#、mRNA-eGFP8#、mRNA-eGFP9#、mRNA-eGFP10#、mRNA-eGFP11#、mRNA-eGFP12#、mRNA-eGFP PCBP4#、mRNA-eGFP PIWIL4#、 mRNA-eGFPIL2#)+6μL Lipo2000(mRNA：Lipo2000＝1:2)，溶于200μL opti-MEM培养基中，涡旋振荡10s，室温孵育5-20min，形成复合物；
d、加转染混合物：迅速将配置好的转染试剂滴加到铺有CHO细胞的6孔板中，将6孔板摇晃数次后放回37℃，5％CO 2培养箱中培养至最长144h。
2)通过eGFP表达强度测定3`非翻译区序列对CHO细胞中mRNA翻译时间与强度影响
24h后吸走培养基，用1×PBS清洗1次，用胰酶消化CHO细胞，计数，取3×10 5细胞重悬到3mL培液中，铺到6孔板中培养，培养基成分为45％DMEM培养基+45％RPMI-1640培养基+10％血清+1％L-谷氨酰胺+1％胸苷次黄嘌呤；另取3×10 5细胞重悬到1×PBS中，进行流式观察，并通过Kaluza Analysis分析。
图6是不同3`UTR情况下，CHO细胞转染mRNA 24h后eGFP阳性率，可见不同3’UTR的mRNA在CHO细胞中转染效率没有显著差异。图7是CHO细胞转染mRNA 24h后eGFP的平均荧光强度，结果显示，体外转录含有人工改造或设计3`UTR的mRNA在CHO细胞中的eGFP表达强度较PCBP4、PIWIL4、IL-2等天然序列的3`UTR强。
48h后吸走培养基，用1×PBS清洗1次，用胰酶消化CHO细胞，计数，取3×10 5细胞重悬到3mL培液中，铺到6孔板中培养，培养基成分为45％DMEM培养基+45％RPMI-1640培养基+10％血清+1％L-谷氨酰胺+1％胸苷次黄嘌呤；另取3×10 5细胞重悬到1×PBS中，进行流式观察，并通过FlowJo X分析。
图8是不同3`UTR情况下，CHO细胞转染mRNA 48h后eGFP阳性率，可见48h后不同3’UTR的mRNA在CHO细胞中的表达持续性未有差异。图9是CHO细胞转染mRNA 48h后eGFP的平均荧光强度，结果显示，48h后体外转录含有人工改造或设计3`UTR的mRNA在CHO细胞中的eGFP表达强度较PCBP4、PIWIL4、IL-2等天然序列3’UTR强，且天然3’UTR的mRNA荧光强度下降迅速。
96h后吸走培养基，用1×PBS清洗1次，用胰酶消化CHO细胞，计数，取3×10 5细胞重悬到1×PBS中，进行流式观察，并通过FlowJo X分析。
图10是不同3`UTR情况下，转染CHO细胞96h后的eGFP阳性率，可见含有人工改造或设计3’UTR的eGFP mRNA在CHO细胞中的表达持续性优于含PCBP4、PIWIL4、IL-2等天然序列3’UTR的mRNA。图11是CHO细胞转染mRNA 96h后eGFP平均荧光强度，结果显示，体外转录含有人工改造或设计3`UTR的mRNA在CHO细胞中的eGFP表达强度较IL-2 3`UTR强。可以看出，96个小时后含有人工改造或设计3`UTR的mRNA仍具有较高的表达活性，而含有IL-2 3`UTR的mRNA表达已极低。

Claims (10)

  1. 一种多核苷酸，所述多核苷酸包含：
    (1)3’UTR序列，其是PCBP4基因或PIWIL4基因的3’UTR区中的一个或多个microRNA识别位点敲除后的序列，
    (2)3’UTR序列，其含有m6A位点的至少3个串联重复的序列和任选的任两个重复之间的接头，和/或
    (3)(1)或(2)的互补序列。
  2. 如权利要求1所述的多核苷酸，其特征在于，
    所述PCBP4基因或PIWIL4基因是人PCBP4基因或人PIWIL4基因，和/或
    所述microRNA识别位点如网站http://www.targetscan.org/vert_72/中所示，和/或
    所述m6A位点是m6A甲基化酶或m6A RNA甲基化识别蛋白的识别序列，和/或
    所述3’UTR序列含有所述m6A位点的至少4个、至少5个、至少6个、至少7个、至少8个、至少9个、至少10个串联重复的序列，和/或
    所述接头长度为1-5bp；
    优选地，
    所述PCBP4基因的3’UTR区的核苷酸序列如SEQ ID NO:1所示，和/或所述PIWIL4基因的3’UTR区的核苷酸序列如SEQ ID NO:2所示，或
    所述m6A位点选自：AAABH、AGABH、GAABH、GGABH，其中B＝T或C或G，H＝T或A或C；
    更优选地，所述多核苷酸的序列选自(1)如SEQ ID NO:3-14中任一所示的序列，(2)与(1)具有至少80％序列相同性的序列。
  3. 一种构建3’UTR序列的方法，包括将基因的3’UTR区中的一个或多个microRNA的识别位点敲除的步骤，
    优选地，所述基因是PCBP4基因或PIWIL4基因，
    更优选地，所述PCBP4基因或PIWIL4基因是人PCBP4基因或人PIWIL4基因，
    进一步优选地，所述基因的3’UTR区的核苷酸序列如SEQ ID NO:1或2或与其具有至少80％序列相同性的序列中任一所示，和/或，所述microRNA识别位点如网站http://www.targetscan.org/vert_72/中所示。
  4. 一种构建3’UTR序列的方法，包括将至少3个m6A位点串联连接的步骤和任选的在两端添加限制性酶切位点的步骤，
    优选地，
    所述连接是直接连接或通过接头连接，和/或
    所述m6A位点是m6A甲基化酶或m6A RNA甲基化识别蛋白的识别序列，和/或
    所述方法包括将至少4个、至少5个、至少6个、至少7个、至少8个、至少9个、至少10个m6A位点串联连接的步骤；
    更优选地，
    所述接头长度为1-5bp，和/或
    m6A位点选自：AAABH、AGABH、GAABH、GGABH，其中B＝T或C或G，H＝T或A或C，和/或
    所述3’UTR序列中的m6A位点相同。
  5. 一种核酸构建物，包含权利要求1或2所述的多核苷酸和任选的待表达基因，所述多核苷酸位于所述待表达基因的3’端，
    优选地，所述核酸构建物还包含选自以下的一个或多个元件：启动子、5’UTR、多克隆位点、polyA。
  6. 一种宿主细胞，包含权利要求1或2所述的多核苷酸和/或权利要求5所述的核酸构建物，
    优选地，所述宿主细胞是CHO细胞或PBMC细胞。
  7. 一种药物组合物，包含权利要求1或2所述的多核苷酸、权利要求5所述的核酸构建物或权利要求6所述的宿主细胞，和药学上可接受的辅料。
  8. 一种提高基因表达强度、延长基因表达时间、提高基因的mRNA启动翻译能力或维持翻译活性时长的方法，包括使携带有权利要求1或2所述的多核苷酸的基因表达的步骤，所述多核苷酸位于所述基因的3’端。
  9. 权利要求1或2所述的多核苷酸、权利要求5所述的核酸构建物或权利要求6所述的宿主细胞在制备用于治疗疾病的药物中的应用，所述多核苷酸位于基因的3’端，所述基因的表达有助于所述疾病的治疗。
  10. 权利要求1或2所述的多核苷酸在控制基因表达中的应用，所述多核苷酸位于所述基因的3’端。
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