WO2023109975A2 - 提高基因表达的rna复制子及其应用 - Google Patents

提高基因表达的rna复制子及其应用 Download PDF

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WO2023109975A2
WO2023109975A2 PCT/CN2023/073720 CN2023073720W WO2023109975A2 WO 2023109975 A2 WO2023109975 A2 WO 2023109975A2 CN 2023073720 W CN2023073720 W CN 2023073720W WO 2023109975 A2 WO2023109975 A2 WO 2023109975A2
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mutation
vee
rna
nsp1ggac
rna replicon
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张元�
林贵斌
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华南理工大学
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Definitions

  • the invention belongs to the technical field of genetic engineering, and in particular relates to an RNA replicon for improving gene expression and application thereof.
  • the strategy of introducing one or some specific genes into specific tissues and cells has been widely used in gene therapy research for a variety of diseases.
  • the in vivo delivery of genetic material usually requires the use of molecular biology methods to clone the target gene sequence into a gene carrier Above, it is usually delivered into cells in the form of DNA or RNA encoding the gene of interest.
  • the DNA molecules that enter the cytoplasm through endocytosis still need to cross the nuclear membrane to complete the delivery and expression of the target gene, and the exogenous DNA molecules delivered to the nucleus will be integrated into the genome of the cell, which is easy to induce tumorigenesis.
  • the low efficiency of delivery to the nucleus and the safety of medication have affected the clinical application of gene therapy using DNA as a carrier.
  • RNA molecules messenger RNA molecules
  • RNA molecules can be synthesized in vitro in a cell-free manner, allowing rapid, large-scale production, avoiding complex manufacturing issues associated with recombinant proteins and viral vectors, and accelerating clinical translation.
  • the mRNA expression time is relatively short, and usually the target protein is metabolized and degraded after 2 to 3 days of instantaneous expression.
  • multiple repeated administrations are usually required to effectively regulate gene expression and the efficacy of gene therapy, which limits its clinical promotion and patient compliance, and increases the cost of treatment.
  • Replicable RNA also known as RNA replicon or self-amplifying RNA
  • repRNA Replicable RNA
  • RNA replicon or self-amplifying RNA is derived from positive- or negative-strand RNA viruses. After the virus infects the host cell, it can replicate in the host cell.
  • the viral genome encodes a number of nonstructural regulatory and structural proteins.
  • RNA replicons used in gene therapy contain genes encoding nonstructural proteins of the alphavirus RNA replication machinery, but at least one gene encoding an alphavirus structural protein is deleted or does not encode a structural protein gene for virus formation, they are considered It is a "disabled" virus that cannot produce infectious offspring.
  • the alphavirus replicon includes functional elements such as the untranslated region of the alphavirus, the coding region of the non-structural protein gene, the subgenomic promoter, and the coding region of the target gene ( Figure 1)
  • the RNA-dependent RNA polymerase can use the replicable RNA released into the cytoplasm as a template to replicate and synthesize multiple transcripts, increase the transcription template, and then translate and express multiple copies of the target protein ( Figure 2). Due to the lack of structural proteins, alphavirus replicons have low intrinsic immunogenicity to the vector itself, and the same replicable RNA can be injected multiple times.
  • the object of the present invention is to provide an RNA replicon for improving gene expression and its application.
  • the first aspect of the present invention provides a RNA replicon, which includes: 5' and 3' untranslated regions; non-structural protein gene coding regions, subgenome promoters, target gene coding regions; its non-structural Any of (I) to (III) mutations in the coding region of the protein gene:
  • the 5' and 3' untranslated regions, nonstructural protein gene coding regions and subgenomic promoters are derived from alphaviruses, flaviviruses, picornaviruses, paramyxoviruses or caliciviruses .
  • the alphavirus is Venezuelan equine encephalitis virus, Sindbis virus or Semliki Forest virus; the flavivirus is dengue virus or Kunjin virus; the picornavirus It is poliovirus or human rhinovirus; the paramyxovirus is canine pox virus; and the calicivirus is feline calicivirus.
  • the alphavirus is Venezuelan equine encephalitis virus.
  • the RNA replicon from 5' to 3' end is: 5' non-translated sequence, non-structural protein White sequence, target gene coding sequence, 3' untranslated sequence.
  • the RNA replicon further comprises a subgenomic promoter, which is interposed between the nonstructural protein sequence and the coding sequence of the target gene, and regulates the translation of the target gene.
  • the RNA replicon is obtained by in vitro transcription of a phage-derived DNA-dependent RNA polymerase promoter (T7, T3, SP6), preferably, the DNA-dependent RNA polymerase promoter is the T7 promoter.
  • a phage-derived DNA-dependent RNA polymerase promoter T7, T3, SP6
  • the DNA-dependent RNA polymerase promoter is the T7 promoter.
  • the RNA replicon further comprises a 5' cap and a 3' poly-A tail, wherein the 5' cap structure is added using the vaccinia virus capping system, and a 7-formazan is added to the 5' end
  • the base guanosine cap structure, the methyltransferase uses S-adenosylmethionine (SAM) as a methyl donor, and adds it to the 2'-O of the first nucleotide of the RNA 5' end immediately adjacent to the cap structure Methyl group; E. coli poly(A) polymerase adds 20-500 A bases to the 3' end of the RNA replicon.
  • SAM S-adenosylmethionine
  • the DNA sequence of the non-structural protein region of the RNA replicon is shown in SEQ ID NO.1.
  • the target genes include tumor-specific or related antigens, pathogen-specific or related antigens, cytokines or their receptors, chemokines or their receptors, growth factors or their receptors, At least one of antibody protein, cytokine antibody fusion protein and immune checkpoint-related protein; preferably, the cytokine or chemokine is granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon- Gamma (IFN- ⁇ ), interleukin-2 (IL-2), interleukin-12 (IL-12) or interleukin-15 (IL-15).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IFN- ⁇ interferon- Gamma
  • IL-2 interleukin-2
  • IL-12 interleukin-12
  • IL-15 interleukin-15
  • the repRNA can encode any target gene sequence, such as a molecule or a vaccine antigen for disease treatment.
  • the second aspect of the present invention provides a vector comprising the RNA replicon described in the first aspect of the present invention.
  • the third aspect of the present invention provides a cell comprising the vector described in the second aspect of the present invention.
  • the recombinant cells are not plant or animal varieties.
  • the fourth aspect of the present invention provides the application of the RNA replicon described in the first aspect of the present invention in any one of (I) to (V):
  • the fifth aspect of the present invention provides a composition comprising the RNA replicon of the first aspect of the present invention or the vector of the second aspect of the present invention.
  • the composition further comprises at least one of a pharmaceutical diluent, a pharmaceutical excipient, a pharmaceutical carrier and a pharmaceutical carrier.
  • the composition can be used in combination with other drugs, including but not limited to: monoclonal antibody drugs, bispecific antibody drugs, antibody conjugated drugs, fusion protein drugs, nucleic acid drugs drugs, chemical drugs, blood product drugs, lipid drugs or Chinese medicine extracts.
  • drugs including but not limited to: monoclonal antibody drugs, bispecific antibody drugs, antibody conjugated drugs, fusion protein drugs, nucleic acid drugs drugs, chemical drugs, blood product drugs, lipid drugs or Chinese medicine extracts.
  • the pharmaceutical carrier is commercially available transfection reagents based on cationic lipids, non-viral vectors, polymer membranes, biomimetic membranes, biological membranes or viral vectors.
  • the transfection reagent includes but not limited to Lipofectamine2000, Lipofectamine3000, Lipofectamine8000, Lipofectamine LTX, Lipofectamine RNAiMAX, Lipofectamine MessengerMAX, Invivofectamine 3.0.
  • the non-viral vectors include, but are not limited to, cationic polymers, cationic liposomes, anionic liposomes, micelles, inorganic nanoparticles or microspheres.
  • the polymer membrane, biomimetic membrane or biological membrane includes but not limited to cell membrane, exosome or extracellular vesicle.
  • the viral vectors include but not limited to adenoviral vectors, retroviruses, lentiviruses, herpesviruses or virus-like particles.
  • the nanocarriers include but are not limited to polycationic peptides, cationic lipids, anionic lipids, Neutral lipids, helper lipids or amphiphilic compounds.
  • the polycationic peptide is protamine; the cationic lipid is 1,2-dioleoyl-3-trimethylammonium propane; and the auxiliary lipid is cholesterol ;
  • the amphiphilic compound is distearoylphosphatidylethanolamine-polyethylene glycol.
  • the particle size of the nano-carrier is 20-350 nm, and the charge is -40-50 mV.
  • the sixth aspect of the present invention provides a method for expressing a gene of interest in an organism, which comprises the following steps: administering the RNA replicon described in the first aspect of the present invention to the organism.
  • the organisms are prokaryotes or eukaryotes; preferably Escherichia coli, yeast, nematodes, fruit flies, mice, monkeys, pigs, cattle, dogs, rabbits, zebrafish model organisms, Human, mouse, monkey, porcine, bovine, canine, rabbit, zebrafish, mammalian cells, primary cells of Drosophila origin or related cell lines.
  • the mammalian cells include but are not limited to 293T, B16F10 or 4T1.
  • the administered RNA replicon can be transferred into cells by transfection, transformation or infection.
  • the RNA replicon can be administered by subcutaneous injection, intradermal injection, intramuscular injection, intratumoral injection, intravenous injection, intraperitoneal injection, oral administration, nasal administration, pulmonary administration Drugs or intracranial administration into the body.
  • the present invention also provides a method for performing site-directed mutation on the nonstructural protein region, specifically in the simultaneous mutation of G357, G1569, A1572, C1575 of nonstructural protein 1 and T3922 of nonstructural protein 2; G357, G1569, A1572, C1575 and A3821T, T3922 of nonstructural protein 2 were mutated at the same time; G3892 of nonstructural protein 2 and A4714 of nonstructural protein 3 were mutated at the same time.
  • the mutation method is PCR site-directed mutagenesis.
  • mutant primers are identical in some embodiments of the present invention.
  • G357C F 5'-GAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACCC-3' (SEQ ID NO.14);
  • G357C R 5'-GCTCATGACGGCGGCGAGCTCCTTCATTTTCTTGTCC-3' (SEQ ID NO.15);
  • T7VEESmaIR 5'-GCTTAAGTTAGTTGCGGCCGCCCGGGTCGACTCTAG-3' (SEQ ID NO.11);
  • T3922C F 5'-GCCCGTACGCACAATCCTTACAAGCTTTCATCAAC-3' (SEQ ID NO.4);
  • T3922C R 5'-TGAAAGCTTGTAAGGATTGTGCGTACGGGCCTTG-3' (SEQ ID NO.5);
  • G3892C F 5'-CTGTTTGTATTCATTCGGTACGATCGCAAGGCCCGTAC-3' (SEQ ID NO.6);
  • G3892C R 5'-CCTTGCGATCGTACCGAATGAATACAAACAGAACTTC-3' (SEQ ID NO.7);
  • nonstructural protein 1 initiates negative-strand RNA synthesis, participates in the capping of the 5' end of viral RNA, and is required for the binding of the RNA replicase complex to the plasma membrane; nonstructural protein 2 not only It regulates the synthesis of subgenomic RNA, and is also used as RNA helicase and protease for the processing of various proteins; nonstructural protein 3 regulates the interaction between virus and host proteins and participates in subgenomic transcription. Mutations in the nonstructural protein region may affect the function of the nonstructural protein, and then lead to changes in the expression of the target gene encoded downstream.
  • the present invention uses the PCR site-directed mutagenesis technique to mutate some special sites in the non-structural protein region of the replicable RNA derived from alphavirus, specifically Enhanced replicability for simultaneous mutations of nonstructural protein 1G357C/G1569A/A1572C/C1575T with nonstructural protein 2T3922C, or nonstructural protein 1G357C/G1569A/A1572C/C1575T with nonstructural protein 2A3821T/T3922C introduced in the nonstructural protein region
  • the expression of target genes encoded downstream of subgenomic promoters of small-type RNAs may be because these mutations promote the stability of RNA structure or up-regulate the activity of RNA-dependent RNA polymerase translated from non-structural protein regions.
  • Nanoparticles have been proven to be used as nucleic acid, protein, polypeptide or drug delivery carriers for the clinical treatment of various diseases, but high concentrations of drugs are toxic and easily cause adverse reactions in the body.
  • the non-structural protein region mutant (VEE:nsP1GGAC-nsP2T or VEE:nsP1GGAC-nsP2AT) replicable RNA in the present invention can up-regulate subgenomic promoter-mediated
  • the expression of the target gene shows that the invention can reduce the dose of nanoparticle drugs while ensuring the therapeutic effect, so the invention has great clinical transformation potential and application value.
  • GM-CSF, IFN- ⁇ , IL-2, IL-12, and IL-15 are key molecules that regulate the body's immune response and play an important role in the treatment of various diseases.
  • the experimental data of the present invention shows that non-structural protein region mutants (VEE: nsP1GGAC-nsP2T or VEE: nsP1GGAC-nsP2AT) can replicate GM-CSF, IFN- ⁇ , IL-2, IL-12, IL- The expression of 15 was significantly up-regulated, suggesting the application value of this achievement in the treatment of related clinical diseases.
  • the non-structural protein region mutant replicable RNA introduced by the PCR site-directed mutagenesis technology in the present invention can be transfected into mammalian cells by Lipofectamine2000 or nanoparticles, which can significantly enhance its downstream subgenomic promoter-mediated including The expression of cytokines or chemokines including GM-CSF, IFN- ⁇ , IL-2, IL-12, IL-15 can be applied to tumors, infectious diseases, autoimmune diseases, genetic diseases, cardiovascular Treatment of diseases and other related diseases.
  • Figure 1 is a schematic diagram of the RNA replicon structure.
  • Figure 2 is a schematic diagram of RNA replicon replication and gene expression in cells.
  • Figure 3 is the T7-VEE plasmid map.
  • Figure 4 shows the mutation sites in the non-structural protein region of the T7-VEE plasmid.
  • Figure 5 shows the sequencing results of the nsP1G357C site mutation of the T7-VEE(nsP1GGAC)-GFP plasmid.
  • Fig. 6 is the sequencing result of T7-VEE(nsP1GGAC)-GFP plasmid nsP1G1569A/A1572C/C1575T site mutation.
  • Fig. 7 is the sequencing result of the nsP2T3922C site mutation of T7-VEE(nsP1GGAC-nsP2T)-GFP plasmid.
  • Fig. 8 is the sequencing result of the nsP2G3892C site mutation of the T7-VEE(nsP1GGAC-nsP2GT-nsP3A)-GFP plasmid.
  • Figure 9 shows the sequencing results of the nsP3A4714G site mutation of the T7-VEE(nsP1GGAC-nsP2GT-nsP3A)-GFP plasmid.
  • Figure 10 shows the sequencing results of the nsP2G3892C site mutation of the T7-VEE(nsP2G-nsP3A)-GFP plasmid.
  • Figure 11 shows the sequencing results of the nsP3A4714G site mutation of the T7-VEE(nsP2G-nsP3A)-GFP plasmid.
  • Fig. 12 is the sequencing result of T7-VEE(nsP1GGAC-nsP2AT)-GFP plasmid nsP2A3821T site mutation.
  • Figure 13 shows the results of ELISA detection of Lipofectamine2000 transfection of wild-type or related mutant IL-12 replicable RNA in the non-structural protein region to 293T cells.
  • Fig. 14 shows the detection results of enzyme-linked immunosorbent assay of nanoparticles transfected with wild-type or related mutant IL-12 replicable RNA into 293T cells.
  • Figure 15 shows the results of ELISA detection of Lipofectamine2000 transfection of wild-type or related mutant IL-15 replicable RNA in the non-structural protein region to 293T cells.
  • Figure 16 shows the detection results of enzyme-linked immunosorbent assay of nanoparticles transfected with wild-type or related mutant IL-15 replicable RNA into 293T cells.
  • Figure 17 shows the detection results of enzyme-linked immunosorbent assay of 293T cells transfected with Lipofectamine2000 into 293T cells with wild-type or related mutations encoding GM-CSF replicable RNA.
  • Figure 18 shows the detection results of enzyme-linked immunosorbent assay of nanoparticles transfected with wild-type or related mutant GM-CSF replicable RNA into 293T cells.
  • Figure 19 shows the detection results of enzyme-linked immunosorbent assay of Lipofectamine2000 transfection of wild-type or related mutant IFN- ⁇ replicable RNA encoding non-structural protein region to 293T cells.
  • Figure 20 shows the detection results of enzyme-linked immunosorbent assay of nanoparticles transfected with wild-type or related mutant IFN- ⁇ replicable RNA in the non-structural protein region to 293T cells.
  • Figure 21 shows the results of ELISA detection of Lipofectamine2000 transfection of wild-type or related mutant IL-2 replicable RNA in the non-structural protein region to 293T cells.
  • Figure 22 shows the detection results of enzyme-linked immunosorbent assay of nanoparticle transfection of wild-type or related mutant IL-2 replicable RNA into 293T cells.
  • the non-structural protein region is the wild-type T7-VEE-GFP (Addgene, 58977) ( Figure 3), that is, the T7-VEE(WT)-GFP plasmid is used as a template, and the DNA sequence of the non-structural protein region of the RNA replicon is as shown in SEQ ID Shown in NO.1; The sequence of the wild-type plasmid T7-VEE (WT)-GFP is shown in SEQ ID NO.25; Construction contains the T7-VEE plasmid of the nonstructural protein region point mutant, and the mutation site is shown in Figure 4 Show.
  • T7VEENdeI R 5'-ATCGATGCTGAGGGCGCCCATATGCTAGAC-3' (SEQ ID NO.3);
  • G357C F 5'-GAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACCC-3' (SEQ ID NO.14);
  • G357C R 5'-GCTCATGACGGCGGCGAGCTCCTTCATTTTCTTGTCC-3' (SEQ ID NO.15);
  • T3922C F 5'-GCCCGTACGCACAATCCTTACAAGCTTTCATCAAC-3' (SEQ ID NO.4);
  • T3922C R 5'-TGAAAGCTTGTAAGGATTGTGCGTACGGGCCTTG-3' (SEQ ID NO.5);
  • PCR amplification system 12.3 ⁇ L of ultrapure water, 4 ⁇ L of 5x HF buffer, 0.4 ⁇ L of 10mM dNTP, 1 ⁇ L of primer F, 1 ⁇ L of primer R, 0.5 ⁇ L of T7-VEE(WT)-GFP plasmid, 0.6 ⁇ L of dimethyl sulfoxide, DNA polymerase 0.2 ⁇ L;
  • Amplification program 98°C for 30s; 98°C for 10s, 55°C for 10s, 72°C for 30s/77, 30 cycles; 72°C for 8min.
  • T7-VEE(nsP1GGAC)-GFP as a template, use T7VEEBglIIF and T3922CR primers to PCR amplify the upstream fragment (1748 7p) containing the T3922C mutation, T3922CF and T7VEENdeIR to PCR amplify the downstream fragment containing the T3922C mutation (3646 7p), agar Sugar gel electrophoresis, gel recovery.
  • Transformation Add the recombinant product to the competent E. coli, and let it stand on ice for 25 minutes; 42°C, 45s; quickly put it on ice for 5 minutes; add 750 ⁇ L of LB medium without antibiotics, shake at 37°C, 200rpm, 1h; Centrifuge at 3500 rpm for 5 min, discard 600 ⁇ L of the supernatant, mix the remaining liquid, spread it on an LB plate containing ampicillin, and incubate overnight in a 37°C incubator.
  • the enzyme digestion reaction system is: 7.8 ⁇ L of ultrapure water, 1 ⁇ L of 10x buffer, 1 ⁇ L of T7-VEE(WT)-GFP plasmid, 0.1 ⁇ L of MluI, and 0.1 ⁇ L of BglII.
  • T7-VEE vector restriction site primer as in Example 1;
  • G3892C F 5'-CTGTTTGTATTCATTCGGTACGATCGCAAGGCCCGTAC-3' (SEQ ID NO.6);
  • G3892C R 5'-CCTTGCGATCGTACCGAATGAATACAAACAGAACTTC-3' (SEQ ID NO.7);
  • the PCR amplification system is the same as in Example 1, using T7-VEE(WT)-GFP as a template, using T7VEEBglIIF and G3892CR primers to PCR amplify the upstream fragment (1714 7p) containing the G3892C mutation, and G3892CF and A4714GR to PCR amplify the upstream fragment containing G3892C/
  • Transformation Add the recombinant product to the competent E. coli, and let it stand on ice for 25 minutes; 42°C, 45s; quickly put it on ice for 5 minutes; add 750 ⁇ L of LB medium without antibiotics, shake at 37°C, 200rpm, 1h; Centrifuge at 3500 rpm for 5 min, discard 600 ⁇ L of the supernatant, mix the remaining liquid, spread it on an LB plate containing ampicillin, and incubate overnight in a 37°C incubator.
  • the enzyme digestion reaction system is: 7.8 ⁇ L of ultrapure water, 1 ⁇ L of 10x buffer, 1 ⁇ L of plasmid, 0.1 ⁇ L of BglII, and 0.1 ⁇ L of XhoI.
  • T7VEESmaIR 5'-GCTTAAGTTAGTTGCGGCCGCCCGGGTCGACTCTAG-3' (SEQ ID NO. 11).
  • the PCR amplification system is the same as in Example 1, using T7-VEE(nsP1GGAC-nsP2T)-GFP as a template, using A3821TF and T7VEESmaIR primers to PCR amplify the DNA fragment (4460 7p) containing the A3821T mutation, agarose gel electrophoresis, and gel recovery .
  • Transformation Add the recombinant product to the competent E. coli, and let it stand on ice for 25 minutes; 42°C, 45s; quickly put it on ice for 5 minutes; add 750 ⁇ L of LB medium without antibiotics, shake at 37°C, 200rpm, 1h; Centrifuge at 3500 rpm for 5 min, discard 600 ⁇ L of the supernatant, mix the remaining liquid, spread it on an LB plate containing ampicillin, and incubate overnight in a 37°C incubator.
  • G357C mutation primers and G1569A/A1572C/C1575T mutation primers are the same as in Example 1.
  • the PCR amplification system is the same as in Example 1, T7-VEE(WT)-GFP is used as a template, and the upstream fragment (2227 7p) containing the G357C mutation is PCR amplified using T7VEEMluIF and G357CR primers, G357CF and G1569A/A1572C/C1575T R primers PCR amplification of the middle fragment (1258 7p) containing the G357C mutation and G1572C/C1575T mutation, G1569A/A1572C/C1575TF and T7VEEBglII R primer PCR amplification of the downstream fragment (687 7p) containing the G1569A/A1572C/C1575T mutation, agar Sugar gel electrophoresis, gel recovery.
  • Transformation Add the recombinant product to the competent E. coli, and let it stand on ice for 25 minutes; 42°C, 45s; quickly put it on ice for 5 minutes; add 750 ⁇ L of LB medium without antibiotics, shake at 37°C, 200rpm, 1h; Centrifuge at 3500 rpm for 5 min, discard 600 ⁇ L of the supernatant, mix the remaining liquid, spread it on an LB plate containing ampicillin, and incubate overnight in a 37°C incubator.
  • T7-VEE vector restriction site primer as in Example 1;
  • A4714G mutation primer same as Example 2.
  • the PCR amplification system is the same as in Example 1, using T7-VEE(nsP1GGAC)-GFP as a template, using T7VEEBglIIF and G3892C/T3922CR primers to PCR amplify the upstream fragment (1747 7p) containing the G3892C/T3922C mutation, G3892C/T3922CF and A4714GR
  • the middle fragment (856 7p) containing the G3892C/T3922C mutation and the A4714G mutation was amplified by PCR, and the downstream fragment (2850 7p) containing the A4714G mutation was amplified by PCR with primers A4714GF and T7VEENdeIR, followed by agarose gel electrophoresis and gel recovery.
  • Transformation Add the recombinant product to the competent E. coli, and let it stand on ice for 25 minutes; 42°C, 45s; quickly put it on ice for 5 minutes; add 750 ⁇ L of LB medium without antibiotics, shake at 37°C, 200rpm, 1h; Centrifuge at 3500 rpm for 5 min, discard 600 ⁇ L of the supernatant, mix the remaining liquid, spread it on an LB plate containing ampicillin, and incubate overnight in a 37°C incubator.
  • T7VEEGMCSFF 5'-GTCTAGTCCGCCAAGTCTAGCATATGGCCACCATGTGGCTGCAG-3' (SEQ ID NO. 20);
  • the PCR amplification system was the same as in Example 1, using T7VEEGMCSFF and 3'UTRR primers to PCR amplify GM-CSF cDNA (423 7p), agarose gel electrophoresis, and gel recovery.
  • T7VEEIFN ⁇ F 5'-GTCTAGTCCGCCAAGTCTAGCATATGGCCACCATGAACGCTACACACTGC-3' (SEQ ID NO. 22);
  • the PCR amplification system was the same as that in Example 1, using T7VEEIFN ⁇ F and 3'UTRR primers to PCR amplify IFN- ⁇ cDNA (46 57p), agarose gel electrophoresis, and gel recovery.
  • the PCR amplification system was the same as that in Example 1, using T7VEEIFN ⁇ F and 3'UTRR primers to PCR amplify IFN- ⁇ cDNA (5617p), agarose gel electrophoresis, and gel recovery.
  • T7VEEIL12F 5'-GTCTAGTCCGCCAAGTCTAGCATATGGCCACC-3' (SEQ ID NO.24);
  • the PCR amplification system was the same as that in Example 1, using T7VEEIL12F and 3'UTRR primers to PCR amplify IL-12cDNA (16457p), agarose gel electrophoresis, and gel recovery.
  • T7VEED265AF as shown in SEQ ID NO.23;
  • the PCR amplification system was the same as in Example 1, using T7VEED265AF and 3'UTRR primers to PCR amplify IL-15cDNA (753 7p), agarose gel electrophoresis, and gel recovery.
  • reaction system 1 ⁇ L of ultrapure water, 3 ⁇ L of 10x buffer, 24 ⁇ L of plasmid, 1 ⁇ L of NdeI, 1 ⁇ L of SphI;
  • GM-CSF reaction system: GM-CSF cDNA 8.46ng, NdeI and SphI digested T7-VEE-GFP plasmid 94.86ng, 2x clonExpression Mix the sum of the volume of the above DNA fragment and plasmid vector.
  • IFN- ⁇ reaction system: IFN- ⁇ cDNA 9.3ng, NdeI and SphI digested T7-VEE-GFP plasmid 94.86ng, 2x clonExpression Mix the sum of the volume of the above DNA fragment and plasmid vector.
  • IL-2 reaction system: IL-2 cDNA 11.22ng, NdeI and SphI digested T7-VEE-GFP plasmid 94.86ng, 2x clonExpression Mix the sum of the volume of the above DNA fragment and plasmid vector.
  • IL-12 reaction system: IL-12cDNA 32.9ng, NdeI and SphI digested T7-VEE-GFP plasmid 94.86ng, 2x clonExpression Mix the sum of the volume of the above DNA fragment and plasmid vector.
  • Transformation Add the recombinant product to the competent E. coli, and let it stand on ice for 25 minutes; 42°C, 45s; quickly put it on ice for 5 minutes; add 750 ⁇ L of LB medium without antibiotics, shake at 37°C, 200rpm, 1h; Centrifuge at 3500 rpm for 5 min, discard 600 ⁇ L of the supernatant, mix the remaining liquid, spread it on an LB plate containing ampicillin, and incubate overnight in a 37°C incubator.
  • reaction system 7.8 ⁇ L of ultrapure water, 1 ⁇ L of 10x buffer, 1 ⁇ L of plasmid, 0.1 ⁇ L of MluI, 0.1 ⁇ L of EcoRI.
  • T7-VEE plasmid is transcribed in vitro using the T7 promoter:
  • the reaction system is: uncapped Replicable RNA 13.5 ⁇ L (10 ⁇ g), 10x capping reaction buffer 2 ⁇ L, GTP (10 mM) 1.0 ⁇ L, S-adenosylmethionine (4 mM) 1.0 ⁇ L, vaccinia virus capping enzyme 1.0 ⁇ L, mRNA Cap2 Oxymethyltransferase 1.0 ⁇ L, RNase inhibitor 0.5 ⁇ L.
  • the replicable RNA needs to be heated at 25-70°C for 5-25min.
  • Reproducible RNA capped at the 5' end Add poly A tail (20-500 A bases) at the 3' end, RNA purification kit to purify replicable RNA, the reaction system is: capped at the 5' end Replicable RNA 15.5 ⁇ L (10 ⁇ g), 10x plus poly A tail buffer 2 ⁇ L, ATP (10 mM) 1 ⁇ L, Escherichia coli poly(A) polymerase 1 ⁇ L, RNase inhibitor 0.5 ⁇ L.
  • the RNA purification kit purifies replicable RNA with a methylated guanosine cap at the 5' end and a poly A tail at the 3' end;
  • Lipofectamine2000 or nanoparticles transfect replicable RNA into 293T cells, and enzyme-linked immunosorbent assay was used to detect the expression of the gene encoding the target gene downstream of the subgenomic promoter.
  • the cells were cultured for 36 hours, the cell culture medium was collected, and the cells were lysed.
  • Enzyme-linked immunosorbent assay was used to detect the expression of the target gene encoded downstream of the subgenomic promoter.
  • the nonstructural protein 1G357C/G1569A/A1572C/C1575T mutation first introduce the nonstructural protein 1G357C/G1569A/A1572C/C1575T mutation, the nonstructural protein 2A3821T/G3892C/T3922C mutation and the nonstructural protein 3A4714G mutation in the nonstructural protein region of the replicable RNA in vitro transcription template plasmid, and Different combinations of mutations were carried out, such as T7-VEE(nsP1GGAC); T7-VEE(nsP1GGAC-nsP2T); T7-VEE(nsP1GGAC-nsP2AT); T7-VEE(nsP1GGAC-nsP2GT-nsP3A); nsP3A).
  • the enzyme-linked immunosorbent assay detection results of Lipofectamine2000 transfecting the wild-type or related mutant IL-12 replicable RNA in the non-structural protein region to 293T cells are shown in Figure 13. Up-regulate the intracellular expression and extracellular secretion of IL-12; compared with the VEE nsP1GGAC-nsP2T mutation, the VEE nsP1GGAC-nsP2AT mutation further enhanced the intracellular expression and extracellular secretion of IL-12.
  • the enzyme-linked immunosorbent assay detection results of nanoparticles transfected with wild-type or related mutant IL-12 replicable RNA in the non-structural protein region to 293T cells are shown in Figure 14. Up-regulate the intracellular expression and extracellular secretion of IL-12; and compared with the VEE nsP1GGAC-nsP2T mutation, the VEE nsP1GGAC-nsP2AT mutation further enhanced the intracellular expression and extracellular secretion of IL-12.
  • Lipofectamine2000 transfected non-structural protein region wild-type or related mutations encoding IL-15 replicable RNA to 293T cells the results of enzyme-linked immunosorbent assay detection are shown in Figure 15.
  • the results show that VEE nsP1GGAC-nsP2T and VEE nsP1GGAC-nsP2AT mutations are both up-regulated IL-15 was expressed intracellularly and secreted extracellularly; compared with the VEE nsP1GGAC-nsP2T mutation, the VEE nsP1GGAC-nsP2AT mutation further enhanced the intracellular expression and extracellular secretion of IL-15.
  • the enzyme-linked immunosorbent assay detection results of nanoparticles transfected with wild-type or related mutant IL-15 replicable RNA in the non-structural protein region to 293T cells are shown in Figure 16.
  • the results show that VEE nsP1GGAC-nsP2T and VEE nsP1GGAC-nsP2AT mutations were all Up-regulate the intracellular expression and extracellular secretion of IL-15; compared with the VEE nsP1GGAC-nsP2T mutation, the VEE nsP1GGAC-nsP2AT mutation further enhanced the intracellular expression and extracellular secretion of IL-15.
  • Lipofectamine2000 transfected non-structural protein region wild-type or related mutant GM-CSF-encoding replicable RNA to 293T cells The results of the enzyme-linked immunosorbent assay of the cells are shown in Figure 17. The results showed that VEE nsP1GGAC-nsP2T and VEE nsP1GGAC-nsP2AT mutations both up-regulated the intracellular expression and extracellular secretion of GM-CSF; compared with the VEE nsP1GGAC-nsP2T mutation, VEE nsP1GGAC-nsP2T The nsP2AT mutation further enhanced the intracellular expression and extracellular secretion of GM-CSF.
  • the enzyme-linked immunosorbent assay detection results of nanoparticles transfected with wild-type or related mutations in the non-structural protein region encoding GM-CSF replicable RNA to 293T cells are shown in Figure 18.
  • the results show that VEE nsP1GGAC-nsP2T and VEE nsP1GGAC-nsP2AT mutations were Up-regulate the intracellular expression and extracellular secretion of GM-CSF; compared with the VEE nsP1GGAC-nsP2T mutation, the VEE nsP1GGAC-nsP2AT mutation further enhanced the intracellular expression and extracellular secretion of GM-CSF.
  • Lipofectamine2000 transfected non-structural protein region wild-type or related mutant IFN- ⁇ replicable RNA to 293T cells the enzyme-linked immunosorbent assay detection results are shown in Figure 19, the results show that VEE nsP1GGAC-nsP2AT up-regulates the extracellular secretion of IFN- ⁇ ; VEE Both nsP1GGAC-nsP2T and VEE nsP1GGAC-nsP2AT mutations up-regulated the expression of IFN- ⁇ in cells.
  • the enzyme-linked immunosorbent assay detection results of nanoparticles transfecting wild-type or related mutant IFN- ⁇ replicable RNA into 293T cells are shown in Figure 20.
  • the results show that VEE nsP1GGAC-nsP2T and VEE nsP1GGAC-nsP2AT mutations are Up-regulate the intracellular expression and extracellular secretion of IFN- ⁇ ; compared with the VEE nsP1GGAC-nsP2T mutation, the VEE nsP1GGAC-nsP2AT mutation further enhanced the intracellular expression and extracellular secretion of IFN- ⁇ .
  • Lipofectamine2000 transfected non-structural protein region wild-type or related mutant IL-2 replicable RNA to 293T cells for enzyme-linked immunosorbent assay detection results are shown in Figure 21, the results show that VEE nsP1GGAC-nsP2T and VEE nsP1GGAC-nsP2AT mutations are both up-regulated IL-2 is expressed in cells and secreted extracellularly; compared with VEE nsP1GGAC-nsP2T mutations, VEE nsP1GGAC-nsP2AT mutations further enhance the expression of IL-2 in cells (VEE nsP1GGAC-nsP2AT mutations have a higher IL-2 intracellular expression than VEE nsP1GGAC-nsP2T mutant IL-2 intracellular expression ⁇ 20 times) and extracellular secretion (VEE nsP1GGAC-nsP2AT mutant IL-2 extracellular secretion is ⁇ 12 times the VEE nsP1GG
  • VEE nsP2G-nsP3A mutation up-regulated the intracellular expression and extracellular secretion of IL-2, and its expression level was between that of VEE nsP1GGAC-nsP2T mutation and VEE nsP1GGAC-nsP2AT.
  • the results of the enzyme-linked immunosorbent assay detection results of the nanoparticle transfection of wild-type or related mutations in the non-structural protein region encoding IL-2 replicable RNA to 293T cells are shown in Figure 22.
  • VEE nsP2G-nsP3A mutation up-regulated the intracellular expression and extracellular secretion of IL-2, and its expression level was between that of VEE nsP1GGAC-nsP2T mutation and VEE nsP1GGAC-nsP2AT.

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Abstract

本发明公开了一种提高基因表达的RNA复制子及其应用，所述RNA复制子包括：5'和3'非翻译区；非结构蛋白基因编码区、亚基因组启动子、目的基因编码区。本发明通过PCR定点突变技术引入的非结构蛋白区域突变体可复制型RNA，通过Lipofectamine2000或者纳米粒转染至哺乳动物真核生物细胞，可显著增强其下游亚基因组启动子介导的包括GM-CSF、IFN-γ、IL-2、IL-12、IL-15在内的细胞因子、趋化因子表达，可以应用于肿瘤、传染性疾病、自身免疫性疾病、遗传性疾病或心血管疾病的治疗。

Description

提高基因表达的RNA复制子及其应用 技术领域
本发明属于基因工程技术领域，具体涉及一种提高基因表达的RNA复制子及其应用。
背景技术
将某一个或某些特定基因导入特定的组织和细胞的策略已经被广泛用于针对多种疾病的基因治疗研究，基因物质的体内传送通常需要借助分子生物学手段将目的基因序列克隆到基因载体上，通常以编码目的基因的DNA或RNA的形式向细胞内递送。但经内吞作用进入细胞质内的DNA分子还需要跨越细胞核膜才能完成目的基因的递送和表达，且递送到细胞核内的外源DNA分子会整合到细胞的基因组中，易诱导肿瘤发生，故DNA向细胞核内的递送效率低以及用药安全性等问题影响了以DNA为载体的基因治疗在临床中的应用。信使RNA分子(mRNA)的转录和翻译只发生在靶细胞的细胞质中，不需要跨越细胞核膜，消除了基因组整合的风险，提高了基因治疗的安全性。RNA分子可以在体外以无细胞的方式合成，允许快速、大规模的生产，避免了与重组蛋白和病毒载体相关的复杂的生产制造等问题，可加速临床转化。但mRNA表达时间较短，通常目标蛋白质瞬间表达2～3天后，即被代谢降解。为维持治疗效果，通常需要多次重复给药才能有效调控基因表达和基因治疗的疗效，限制了其在临床上的推广和患者顺应性，且提高了治疗成本。
可复制型RNA(repRNA)，又称为RNA复制子或自我扩增RNA，来源于正链或负链RNA病毒。该病毒感染宿主细胞后，可在宿主细胞内进行复制。病毒基因组编码了一些非结构调节蛋白和结构蛋白。在基因治疗中使用的RNA复制子包含有编码甲病毒RNA复制机制的非结构蛋白基因，但至少一个编码甲病毒结构蛋白的基因被删除或不编码用于病毒形成的结构蛋白基因，它们被认为是“失能”病毒，无法产生具有传染性的后代，甲病毒复制子包括甲病毒的非翻译区、非结构蛋白基因编码区、亚基因组启动子和目的基因编码区等功能元件(图1)，repRNA导入细胞后，RNA依赖的RNA聚合酶可以将释放到细胞质中的可复制型RNA作为模板复制合成多份转录本，增加了转录模板，进而翻译表达出多份目标蛋白(图2)。由于缺乏结构蛋白，甲病毒复制子对载体本身具有较低的内在免疫原性，同一可复制型RNA可以被多次重复注射。
发明内容
本发明的目的在于提供一种提高基因表达的RNA复制子及其应用。
本发明所采取的技术方案是：
本发明的第一方面，提供一种RNA复制子，所述RNA复制子包括：5’和3’非翻译区；非结构蛋白基因编码区、亚基因组启动子、目的基因编码区；其非结构蛋白基因编码区发生(I)～(III)任意一种突变：
(I)选自非结构蛋白1的G357、G1569、A1572、C1575和非结构蛋白2的T3922位点中的至少一个位点的突变；优选为同时突变；
(II)选自非结构蛋白1的G357、G1569、A1572、C1575和非结构蛋白2的A3821、T3922位点中的至少一个位点的突变；优选为同时突变；
(III)包括但不限于非结构蛋白2的G3892和非结构蛋白3的A4714位点中的至少一个位点的突变；优选为同时突变。
在本发明的一些实施方式中，所述5’和3’非翻译区、非结构蛋白基因编码区和亚基因组启动子来源于甲病毒、黄病毒、小RNA病毒、副粘病毒或杯状病毒。在本发明的一些优选实施方式中，所述甲病毒为委内瑞拉马脑炎病毒、辛德比斯病毒或塞姆利基森林病毒；所述黄病毒为登革热病毒或昆津病毒；所述小RNA病毒为脊髓灰质炎病毒或人鼻病毒；所述副粘病毒为犬痘热病毒；所述杯状病毒为猫杯状病毒。
在本发明的一些更优选实施方式在，所述甲病毒为委内瑞拉马脑炎病毒。
在本发明的一些优选实施方式中，所述RNA复制子从5’至3’末端为：5’非翻译序列、非结构蛋 白序列、目的基因编码序列、3’非翻译序列。
在本发明的一些实施方式中，所述RNA复制子还包含亚基因组启动子，所述亚基因组启动子介于非结构蛋白序列、目的基因编码序列之间，调节目的基因翻译。
在本发明的一些实施方式中，所述RNA复制子通过噬菌体来源的DNA依赖的RNA聚合酶启动子(T7，T3，SP6)体外转录获得，优选地，所述DNA依赖的RNA聚合酶启动子为T7启动子。
在本发明的一些实施方式中，所述RNA复制子还包含5’帽和3’poly-A尾，其中5’帽结构的添加采用牛痘病毒加帽体系，在5’末端加上7-甲基鸟苷帽结构，甲基转移酶以S-腺苷甲硫氨酸(SAM)作为甲基供体，在RNA 5’末端紧邻帽结构的第一个核苷酸的2'-O上添加甲基基团；大肠杆菌聚(A)聚合酶在RNA复制子3’末端加上20-500个A碱基。
在本发明的一些实施方式中，所述RNA复制子的非结构蛋白区域DNA序列如SEQ ID NO.1所示。
在本发明的一些实施方式中，所述目的基因包括肿瘤特异性或相关抗原、病原体特异性或相关抗原、细胞因子或其受体、趋化因子或其受体、生长因子或其受体、抗体蛋白、细胞因子抗体融合蛋白和免疫检查点相关蛋白中的至少一种；优选地，所述细胞因子或者趋化因子为粒细胞-巨噬细胞集落刺激因子(GM-CSF)、干扰素-γ(IFN-γ)、白细胞介素-2(IL-2)、白细胞介素-12(IL-12)或白细胞介素-15(IL-15)。细胞因子可以增强免疫反应，趋化因子可以诱导附近的应答细胞(例如白细胞)向感染部位迁移，它们在感染、免疫反应、炎症、创伤、败血症或癌症的发生发展和治疗方面起重要有用。
其中repRNA可编码任意的目的基因序列，例如用于疾病治疗的分子或疫苗抗原。
本发明的第二方面，提供一种载体，包含本发明第一方面所述RNA复制子。
本发明的第三方面，提供一种细胞，包含本发明第二方面所述载体。
在本发明的一些实施方式中，所述重组细胞非植物或动物新品种。
本发明的第四方面，提供本发明第一方面所述RNA复制子在(I)～(V)任一项中的应用：
(I)递送目的基因；
(II)实现目的基因的长效表达；
(III)提高目的基因的表达量；
(IV)基因治疗；
(V)疫苗研发。
本发明的第五方面，提供一种组合物，包含本发明第一方面所述RNA复制子或本发明第二方面所述载体。
在本发明的一些实施方式中，所述组合物还包含药用稀释剂、药用赋形剂、药用载体和药用载剂中的至少一种。
在本发明的一些优选实施方式中，所述组合物可搭配其他药物联合使用，所述药物包括但不限于：单克隆抗体药物、双特异性抗体药物、抗体偶联药物、融合蛋白药物、核酸类药物、化学药物、血液制品药物、脂类药物或中药提取物。
在本发明的一些实施方式中，所述药用载体为基于阳离子脂质的市售转染试剂、非病毒载体、高分子膜、仿生膜、生物膜或病毒载体。
在本发明的一些实施方式中，所述转染试剂，包括但不限于Lipofectamine2000、Lipofectamine3000、Lipofectamine8000、Lipofectamine LTX、Lipofectamine RNAiMAX、Lipofectamine MessengerMAX、Invivofectamine 3.0。
在本发明的一些实施方式中，所述非病毒载体包括但不限于阳离子聚合物、阳离子脂质体、阴离子脂质体、胶束、无机纳米颗粒或微球。
在本发明的一些实施方式中，所述高分子膜、仿生膜或生物膜，包括但不限于细胞膜、外泌体或细胞外囊泡。
在本发明的一些实施方式中，所述病毒载体，包括但不限于腺病毒载体、逆转录病毒、慢病毒、疱疹病毒或病毒样颗粒。
在本发明的一些实施方式中，所述纳米载体包括但不限于多聚阳离子肽、阳离子脂质、阴离子脂质、 中性脂质、辅助脂质或两亲性化合物。
在本发明的一些实施方式中，所述多聚阳离子肽为鱼精蛋白；所述阳离子脂质为1,2-二油酰-3-三甲基铵盐丙烷；所述辅助脂质为胆固醇；所述两亲性化合物为二硬脂酰基磷脂酰乙醇胺-聚乙二醇。
在本发明的一些实施方式中，所述纳米载体的粒径为20～350nm，电荷为-40～50mV。
本发明的第六方面，提供一种用于在生物体表达目的基因的方法，其包含以下步骤：向所述生物体中施用本发明第一方面所述的RNA复制子。
在本发明的一些实施方式中，所述生物体为原核生物或真核生物；优选为大肠杆菌、酵母、线虫、果蝇、鼠、猴、猪、牛、犬、兔、斑马鱼模式生物、人、鼠、猴、猪、牛、犬、兔、斑马鱼、哺乳动物细胞、果蝇来源的原代细胞或相关细胞系。
在本发明的一些实施方式中，所述哺乳动物细胞，包括但不限于293T、B16F10或4T1。
在本发明的一些实施方式中，所述对施用RNA复制子可以通过转染、转化或感染方式转入细胞。
在本发明的一些实施方式中，所述对施用RNA复制子可以通过皮下注射、皮内注射、肌内注射、瘤内注射、静脉注射、腹腔注射、口服给药、鼻腔给药、肺部给药或颅内给药导入机体。
本发明还提供一种对非结构蛋白区域进行定点突变的方法，具体在非结构蛋白1的G357、G1569、A1572、C1575和非结构蛋白2的T3922同时突变；非结构蛋白1的G357、G1569、A1572、C1575和非结构蛋白2的A3821T、T3922同时突变；非结构蛋白2的G3892和非结构蛋白3的A4714同时突变。在本发明的一些实施方式中，所述突变的方法为PCR定点突变。
在本发明的一些实施方式中，所述突变引物：
G357C突变引物：
G357C F：5’-GAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACCC-3’(SEQ ID NO.14)；
G357C R：5’-GCTCATGACGGCGGCGAGCTCCTTCATTTTCTTGTCC-3’(SEQ ID NO.15)；
G1569A/A1572C/C1575T突变引物：
G1569A/A1572C/C1575T F：
5’-GGAGCCCACTCTGGAAGCCGATGTCGACTTGATGTTACAAGAGG-3’(SEQ ID NO.16)；
G1569A/A1572C/C1575T R：
5’-TAACATCAAGTCGACATCGGCTTCCAGAGTGGGCTCCTCAACATC-3’(SEQ ID NO.17)；
A3821T突变引物：
A3821T F:
5’-CATTGGTGCTATAGCGCGGCTGTTCAAGTTTTCCCGGGTATGCAAAC-3’(SEQ ID NO.10)；
T7VEESmaI R:5’-GCTTAAGTTAGTTGCGGCCGCCCGGGTCGACTCTAG-3’(SEQ ID NO.11)；
T3922C突变引物：
T3922C F：5’-GCCCGTACGCACAATCCTTACAAGCTTTCATCAAC-3’(SEQ ID NO.4)；
T3922C R：5’-TGAAAGCTTGTAAGGATTGTGCGTACGGGCCTTG-3’(SEQ ID NO.5)；
G3892C突变引物：
G3892C F 5’-CTGTTTGTATTCATTCGGTACGATCGCAAGGCCCGTAC-3’(SEQ ID NO.6)；
G3892C R 5’-CCTTGCGATCGTACCGAATGAATACAAACAGAACTTC-3’(SEQ ID NO.7)；
A4714G突变引物：
A4714G F 5’-TATATCCTCGGAGAAGGCATGAGCAGTATTAGGTCG-3’(SEQ ID NO.8)；
A4714G R 5’-TAATACTGCTCATGCCTTCTCCGAGGATATACATGC-3’(SEQ ID NO.9)。
本发明的有益效果是：
在可复制型RNA非结构蛋白区域中，非结构蛋白1启动负链RNA合成，参与病毒RNA 5’末端加帽，并且是RNA复制酶复合体与细胞质膜结合所必需的；非结构蛋白2不仅调节亚基因组RNA的合成，还作为RNA解旋酶和蛋白酶用于多种蛋白的加工；非结构蛋白3调节病毒与宿主蛋白的相互作用，参与亚基因组转录。非结构蛋白区域位点的突变，可能影响非结构蛋白的功能，进而导致其下游编码的目的基因表达的改变。
为了进一步增强可复制型RNA编码的抗原表达，促进RNA疫苗引起的免疫反应，本发明利用PCR定点突变技术在甲病毒来源的可复制型RNA非结构蛋白区域进行了一些特殊位点的突变，具体为在非结构蛋白区域中引入的非结构蛋白1G357C/G1569A/A1572C/C1575T与非结构蛋白2T3922C同时突变，或非结构蛋白1G357C/G1569A/A1572C/C1575T与非结构蛋白2A3821T/T3922C，增强了可复制型RNA亚基因组启动子下游编码的目的基因表达，可能是因为这些突变促进了RNA结构的稳定性或上调了非结构蛋白区域翻译出的RNA依赖的RNA聚合酶的活性。酶联免疫吸附测定结果显示可复制型RNA非结构蛋白区域中非结构蛋白1G357C/G1569A/A1572C/C1575T与非结构蛋白2T3922C，或非结构蛋白1G357C/G1569A/A1572C/C1575T与非结构蛋白2A3821T/T3922C同时突变，可显著上调亚基因组启动子下游GM-CSF、IFN-γ、IL-2、IL-12、IL-15表达，其中非结构蛋白1G357C/G1569A/A1572C/C1575T与非结构蛋白2A3821T/T3922C同时突变效果更加明显。
纳米粒已被证实可作为核酸、蛋白、多肽或药物递送载体，应用于多种疾病的临床治疗，但高浓度的药物具有毒性，容易引起机体不良反应。本发明中的非结构蛋白区域突变体(VEE:nsP1GGAC-nsP2T或VEE:nsP1GGAC-nsP2AT)可复制型RNA，使用Lipofectamine2000或者纳米粒转染哺乳动物细胞293T，均可上调亚基因组启动子介导的目的基因表达，表明该发明可在保证治疗效果的同时，降低纳米粒药物剂量，因此该发明具备极大地临床转化潜力与应用价值。GM-CSF、IFN-γ、IL-2、IL-12、IL-15是调控机体免疫反应的关键分子，在多种疾病的治疗中发挥重要作用。本发明的实验数据显示，非结构蛋白区域突变体(VEE:nsP1GGAC-nsP2T或VEE:nsP1GGAC-nsP2AT)可复制型RNA编码的GM-CSF、IFN-γ、IL-2、IL-12、IL-15表达显著上调，提示了该成果在相关临床疾病治疗中的应用价值。
综上所述，本发明通过PCR定点突变技术引入的非结构蛋白区域突变体可复制型RNA，通过Lipofectamine2000或者纳米粒转染至哺乳动物细胞，可显著增强其下游亚基因组启动子介导的包括GM-CSF、IFN-γ、IL-2、IL-12、IL-15在内的细胞因子或趋化因子表达，可以应用于肿瘤、传染性疾病、自身免疫性疾病、遗传性疾病、心血管疾病等相关疾病的治疗。
附图说明
图1为RNA复制子结构示意图。
图2为RNA复制子在细胞内复制及基因表达示意图。
图3为T7-VEE质粒图谱。
图4为T7-VEE质粒非结构蛋白区突变位点。
图5为T7-VEE(nsP1GGAC)-GFP质粒nsP1G357C位点突变测序结果。
图6为T7-VEE(nsP1GGAC)-GFP质粒nsP1G1569A/A1572C/C1575T位点突变测序结果。
图7为T7-VEE(nsP1GGAC-nsP2T)-GFP质粒nsP2T3922C位点突变测序结果。
图8为T7-VEE(nsP1GGAC-nsP2GT-nsP3A)-GFP质粒nsP2G3892C位点突变测序结果。
图9为T7-VEE(nsP1GGAC-nsP2GT-nsP3A)-GFP质粒nsP3A4714G位点突变测序结果。
图10为T7-VEE(nsP2G-nsP3A)-GFP质粒nsP2G3892C位点突变测序结果。
图11为T7-VEE(nsP2G-nsP3A)-GFP质粒nsP3A4714G位点突变测序结果。
图12为T7-VEE(nsP1GGAC-nsP2AT)-GFP质粒nsP2A3821T位点突变测序结果。
图13为Lipofectamine2000转染非结构蛋白区域野生型或相关突变的编码IL-12可复制型RNA至293T细胞的酶联免疫吸附测定检测结果。
图14为纳米粒转染非结构蛋白区域野生型或相关突变的编码IL-12可复制型RNA至293T细胞的酶联免疫吸附测定检测结果。
图15为Lipofectamine2000转染非结构蛋白区域野生型或相关突变的编码IL-15可复制型RNA至293T细胞的酶联免疫吸附测定检测结果。
图16为纳米粒转染非结构蛋白区域野生型或相关突变的编码IL-15可复制型RNA至293T细胞的酶联免疫吸附测定检测结果。
图17为Lipofectamine2000转染非结构蛋白区域野生型或相关突变的编码GM-CSF可复制型RNA至293T细胞的酶联免疫吸附测定检测结果。
图18为纳米粒转染非结构蛋白区域野生型或相关突变的编码GM-CSF可复制型RNA至293T细胞的酶联免疫吸附测定检测结果。
图19为Lipofectamine2000转染非结构蛋白区域野生型或相关突变的编码IFN-γ可复制型RNA至293T细胞的酶联免疫吸附测定检测结果。
图20为纳米粒转染非结构蛋白区域野生型或相关突变的编码IFN-γ可复制型RNA至293T细胞的酶联免疫吸附测定检测结果。
图21为Lipofectamine2000转染非结构蛋白区域野生型或相关突变的编码IL-2可复制型RNA至293T细胞的酶联免疫吸附测定检测结果。
图22为纳米粒转染非结构蛋白区域野生型或相关突变的编码IL-2可复制型RNA至293T细胞的酶联免疫吸附测定检测结果。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本发明的目的、特征和效果。显然，所描述的实施例只是本发明的一部分实施例，而不是全部实施例，基于本发明的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本发明保护的范围。
以非结构蛋白区域为野生型的T7-VEE-GFP(Addgene,58977)(图3)即T7-VEE(WT)-GFP质粒为模板，其中RNA复制子的非结构蛋白区域DNA序列如SEQ ID NO.1所示；野生型质粒T7-VEE(WT)-GFP的序列如SEQ ID NO.25所示；构建含有非结构蛋白区域点突变体的T7-VEE质粒，突变位点如图4所示。
实施例1构建nsP1G357C/G1569A/A1572C/C1575T-nsP2T3922C突变体
即T7-VEE(nsP1GGAC-nsP2T)-GFP：
1)T7-VEE载体酶切位点引物：
T7VEEBglII F 5’-AAAAGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCC-3’(SEQ ID NO.2)；
T7VEENdeI R 5’-ATCGATGCTGAGGGCGCGCCCATATGCTAGAC-3’(SEQ ID NO.3)；
G357C突变引物：
G357C F 5’-GAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACCC-3’(SEQ ID NO.14)；
G357C R 5’-GCTCATGACGGCGGCGAGCTCCTTCATTTTCTTGTCC-3’(SEQ ID NO.15)；
G1569A/A1572C/C1575T突变引物：
G1569A/A1572C/C1575T F
5’-GGAGCCCACTCTGGAAGCCGATGTCGACTTGATGTTACAAGAGG-3’(SEQ ID NO.16)；
G1569A/A1572C/C1575T R
5’-TAACATCAAGTCGACATCGGCTTCCAGAGTGGGCTCCTCAACATC-3’(SEQ ID NO.17)；
T3922C突变引物：
T3922C F 5’-GCCCGTACGCACAATCCTTACAAGCTTTCATCAAC-3’(SEQ ID NO.4)；
T3922C R 5’-TGAAAGCTTGTAAGGATTGTGCGTACGGGCCTTG-3’(SEQ ID NO.5)；
PCR扩增体系：超纯水12.3μL、5x HF缓冲液4μL、10mM dNTP 0.4μL、引物F 1μL、引物R 1μL、T7-VEE(WT)-GFP质粒0.5μL、二甲基亚砜0.6μL、DNA聚合酶0.2μL；
扩增程序：98℃30s；98℃10s、55℃10s、72℃30s/77、30个循环；72℃8min。
以T7-VEE(nsP1GGAC)-GFP为模板，使用T7VEEBglIIF与T3922CR引物PCR扩增含有T3922C突变的上游片段(1748 7p)，T3922CF与T7VEENdeIR物PCR扩增含有T3922C突变的下游片段(3646 7p)，琼脂糖凝胶电泳，胶回收。
2)BglII，NdeI与XhoI酶切T7-VEE(nsP1GGAC)-GFP质粒载体，酶切体系：10x缓冲液3μL、T7-VEE(nsP1GGAC)-GFP质粒24μL、BglII 1μL、NdeI 1μL、XhoI 1μL；
37℃，2h后琼脂糖凝胶电泳，胶回收6212 7p片段。
3)同源重组过程，具体反应体系：含有T3922C突变的上游片段0.01x 1748 7p＝17.48ng、含有T3922C突变的下游片段0.01x 3646 7p＝36.46ng、BglII，NdeI与XhoI酶切的T7-VEE(nsP1GGAC)-GFP质粒载 体0.01x 6212 7p＝62.12ng、2x clonExpression Mix上述DNA片段和质粒载体的体积之和。
50℃，15min后，立即置于冰上，静置5min。
4)转化：将重组产物加入大肠杆菌感受态中，冰上静置25min；42℃，45s；迅速置于冰上5min；加入750μL无抗生素的LB培养基，37℃摇床，200rpm，1h；3500rpm，离心5min，弃上清600μL，剩余液体混匀，涂于含氨苄青霉素的LB平板中，37℃培养箱，倒置培养过夜。
5)挑选单克隆，MluI与EcoRI酶切鉴定，酶切反应体系为：超纯水7.8μL、10x缓冲液1μL、T7-VEE(WT)-GFP质粒1μL、MluI 0.1μL、BglII 0.1μL。
37℃，1h后琼脂糖凝胶电泳，鉴定正确的质粒测序，T3922C位点突变测序结果如图7所示。
实施例2构建nsP2G3892C-nsP3A4714G突变体
即T7-VEE(nsP2G-nsP3A)-GFP：
1)T7-VEE载体酶切位点引物：如实施例1；
G3892C突变引物：
G3892C F 5’-CTGTTTGTATTCATTCGGTACGATCGCAAGGCCCGTAC-3’(SEQ ID NO.6)；
G3892C R 5’-CCTTGCGATCGTACCGAATGAATACAAACAGAACTTC-3’(SEQ ID NO.7)；
A4714G突变引物：
A4714G F 5’-TATATCCTCGGAGAAGGCATGAGCAGTATTAGGTCG-3’(SEQ ID NO.8)；
A4714G R 5’-TAATACTGCTCATGCCTTCTCCGAGGATATACATGC-3’(SEQ ID NO.9)。
PCR扩增体系同实施例1，以T7-VEE(WT)-GFP为模板，使用T7VEEBglIIF与G3892CR引物PCR扩增含有G3892C突变的上游片段(1714 7p)，G3892CF与A4714GR物PCR扩增含有G3892C/T3922C突变与A4714G突变的中间片段(853 7p)，A4714GF与T7VEENdeIR引物PCR扩增含有A4714G突变的下游片段(2850 7p)，琼脂糖凝胶电泳，胶回收。
2)BglII，NdeI与XhoI酶切T7-VEE(WT)-GFP质粒载体，酶切体系同实施例1。
37℃，2h后琼脂糖凝胶电泳，胶回收62127p片段。
3)同源重组，反应体系：含有G3892C突变的上游片段17.14ng、含有G3892C突变与A4714G突变的中间片段8.53ng、含有A4714G突变的下游片段28.5ng、BglII，NdeI与XhoI酶切的T7-VEE(WT)-GFP质粒载体62.12ng、2x clonExpression Mix上述DNA片段和质粒载体的体积之和。
50℃，15min后，立即置于冰上，静置5min。
4)转化：将重组产物加入大肠杆菌感受态中，冰上静置25min；42℃，45s；迅速置于冰上5min；加入750μL无抗生素的LB培养基，37℃摇床，200rpm，1h；3500rpm，离心5min，弃上清600μL，剩余液体混匀，涂于含氨苄青霉素的LB平板中，37℃培养箱，倒置培养过夜。
5)挑选单克隆，BglII与XhoI酶切鉴定，酶切反应体系为：超纯水7.8μL、10x缓冲液1μL、质粒1μL、BglII 0.1μL、XhoI 0.1μL。
37℃，1h后琼脂糖凝胶电泳，鉴定正确的质粒测序，G3892C位点突变测序结果如图10所示，A4714G位点突变测序结果如图11所示。
实施例3构建nsP1G357C/G1569A/A1572C/C1575T-nsP2A3821T/T3922C突变体
即T7-VEE(nsP1GGAC-nsP2AT)-GFP：
A3821T突变引物：
A3821T F 5’-CATTGGTGCTATAGCGCGGCTGTTCAAGTTTTCCCGGGTATGCAAAC-3’(SEQ ID NO.10)；
T7VEESmaI R 5’-GCTTAAGTTAGTTGCGGCCGCCCGGGTCGACTCTAG-3’(SEQ ID NO.11)。
PCR扩增体系同实施例1，以T7-VEE(nsP1GGAC-nsP2T)-GFP为模板，使用A3821TF与T7VEESmaIR引物PCR扩增含有A3821T突变的DNA片段(4460 7p)，琼脂糖凝胶电泳，胶回收。
2)SmaI酶切T7-VEE(nsP1GGAC-nsP2T)-GFP质粒载体，酶切体系：10x缓冲液3μL、T7-VEE(nsP1GGAC-nsP2T)-GFP 26μL、SmaI 1μL；
37℃，2h后琼脂糖凝胶电泳，胶回收7062 7p片段。
3)同源重组，反应体系：含有A3821T突变的PCR扩增片段44.6ng、SmaI酶切的T7-VEE(nsP1GGAC-nsP2T)-GFP质粒载体70.62ng、2x clonExpression Mix上述DNA片段和质粒载体的体积之和。
50℃，15min后，立即置于冰上，静置5min。
4)转化：将重组产物加入大肠杆菌感受态中，冰上静置25min；42℃，45s；迅速置于冰上5min；加入750μL无抗生素的LB培养基，37℃摇床，200rpm，1h；3500rpm，离心5min，弃上清600μL，剩余液体混匀，涂于含氨苄青霉素的LB平板中，37℃培养箱，倒置培养过夜。
5)挑选单克隆，SmaI酶切鉴定，酶切反应体系：超纯水7.9μL、10x缓冲液1μL、质粒1μL、SmaI0.1μL。
37℃，1h后琼脂糖凝胶电泳，鉴定正确的质粒测序，A3821T位点突变测序结果如图12所示。
对比例1构建nsP1G357C/G1569A/A1572C/C1575T突变体
即T7-VEE(nsP1GGAC)-GFP：
1)T7-VEE载体酶切位点引物：
T7-VEE载体酶切位点引物：
T7VEEMluI F
5’-AAAAAAAAAAAAAAAAAAAACGCGTCGAGGGGAATTAATTCTTGAAGACG-3’(SEQ ID NO.12)；
T7VEEBglII R
5’-CTTTCTTGGCGCTCACCACTAGATCTTTTTTGGTGACTGCGCTTTTAATG-3’(SEQ ID NO.13)；
G357C突变引物、G1569A/A1572C/C1575T突变引物同实施例1。
PCR扩增体系同实施例1，T7-VEE(WT)-GFP为模板，使用T7VEEMluI F与G357CR引物PCR扩增含有G357C突变的上游片段(2227 7p)，G357C F与G1569A/A1572C/C1575T R引物PCR扩增含有G357C突变与G1569A/A1572C/C1575T突变的中间片段(1258 7p)，G1569A/A1572C/C1575T F与T7VEEBglII R引物PCR扩增含有G1569A/A1572C/C1575T突变的下游片段(687 7p)，琼脂糖凝胶电泳，胶回收。
2)MluI与BglII酶切T7-VEE(WT)-GFP质粒载体，酶切体系：10x缓冲液3μL、T7-VEE(WT)-GFP质粒25μL、MluI 1μL、BglII 1μL。
37℃，2h后琼脂糖凝胶电泳，胶回收74387p片段。
3)同源重组，反应体系：含有G357C突变的上游片段22.27ng、含有G357C突变与G1569A/A1572C/C1575T突变的中间片段12.58ng、含有G1569A/A1572C/C1575T突变的下游片段6.87ng、MluI与BglII酶切的T7-VEE(WT)-GFP质粒载体74.38ng、2x clonExpression Mix上述DNA片段和质粒载体的体积之和。
50℃，15min后，立即置于冰上，静置5min。
4)转化：将重组产物加入大肠杆菌感受态中，冰上静置25min；42℃，45s；迅速置于冰上5min；加入750μL无抗生素的LB培养基，37℃摇床，200rpm，1h；3500rpm，离心5min，弃上清600μL，剩余液体混匀，涂于含氨苄青霉素的LB平板中，37℃培养箱，倒置培养过夜。
5)挑选单克隆，MluI与EcoRI酶切鉴定，酶切反应体系：超纯水7.8μL、10x缓冲液1μL、质粒1μL、MluI 0.1μL、EcoRI 0.1μL。
37℃1h后琼脂糖凝胶电泳，鉴定正确的质粒测序，G357C位点突变测序结果如图5所示，G1569A/A1572C/C1575T位点突变测序结果如图6所示。
对比例2构建nsP1G357C/G1569A/A1572C/C1575T-nsP2G3892C/T3922C-nsP3A4714G突变体
即T7-VEE(nsP1GGAC-nsP2GT-nsP3A)-GFP：
1)T7-VEE载体酶切位点引物：如实施例1；
G3892C/T3922C突变引物：
G3892C/T3922C F
5’-GTTCTGTTTGTATTCATTCGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAAC-3’(SEQ ID NO.18)；
G3892C/T3922C R
5’-TTGATGAAAGCTTGTAAGGATTGTGCGTACGGGCCTTGCGATCGTACCGAATGAATACAAACAGAAC-3’(SEQ ID NO.19)；
A4714G突变引物：同实施例2。
PCR扩增体系同实施例1，以T7-VEE(nsP1GGAC)-GFP为模板，使用T7VEEBglIIF与G3892C/T3922CR引物PCR扩增含有G3892C/T3922C突变的上游片段(1747 7p)，G3892C/T3922CF与A4714GR物PCR扩增含有G3892C/T3922C突变与A4714G突变的中间片段(856 7p)，A4714GF与T7VEENdeIR引物PCR扩增含有A4714G突变的下游片段(2850 7p)，琼脂糖凝胶电泳，胶回收。
2)BglII，NdeI与XhoI酶切T7-VEE(nsP1GGAC)-GFP质粒载体，酶切体系为：10x缓冲液3μL、T7-VEE(nsP1GGAC)-GFP质粒24μL、BglII 1μL、NdeI 1μL、XhoI 1μL。
37℃，2h后琼脂糖凝胶电泳，胶回收6212 7p片段。
3)同源重组，反应体系：含有G3892C/T3922C突变的上游片段17.47ng、含有G3892C/T3922C突变与A4714G突变的中间片段8.56ng、含有A4714G突变的下游片段28.5ng、BglII，NdeI与XhoI酶切的T7-VEE(nsP1GGAC)-GFP质粒载体62.12ng、2x clonExpression Mix上述DNA片段和质粒载体的体积之和。
50℃，15min后，立即置于冰上，静置5min。
4)转化：将重组产物加入大肠杆菌感受态中，冰上静置25min；42℃，45s；迅速置于冰上5min；加入750μL无抗生素的LB培养基，37℃摇床，200rpm，1h；3500rpm，离心5min，弃上清600μL，剩余液体混匀，涂于含氨苄青霉素的LB平板中，37℃培养箱，倒置培养过夜。
5)挑选单克隆，BglII与XhoI酶切鉴定，酶切反应体系：超纯水7.8μL、10x缓冲液1μL、质粒1μL、BglII 0.1μL、XhoI 0.1μL。
37℃，1h后琼脂糖凝胶电泳，鉴定正确的质粒测序，G3892C位点突变测序结果如图8所示，A4714G位点突变测序结果如图9所示。
效果例
检测方法：
将不同的目的基因(包括细胞因子和趋化因子)克隆至结构蛋白区。
1)PCR扩增目的基因
(i)GM-CSF
T7-VEE载体酶切位点引物：
T7VEEGMCSFF 5’-GTCTAGTCCGCCAAGTCTAGCATATGGCCACCATGTGGCTGCAG-3’(SEQ ID NO.20)；
3'UTRR 5’-AAAATAAAAATTTTAAGGCGGCATGCCAATCGCCGCGAGTTCTATGTAAGCAG-3’(SEQ ID NO.21)；
PCR扩增体系同实施例1，使用T7VEEGMCSFF与3'UTRR引物PCR扩增GM-CSF cDNA(423 7p)，琼脂糖凝胶电泳，胶回收。
(ii)IFN-γ
T7-VEE载体酶切位点引物：
T7VEEIFNγF 5’-GTCTAGTCCGCCAAGTCTAGCATATGGCCACCATGAACGCTACACACTGC-3’(SEQ ID NO.22)；
3'UTRR：如SEQ ID NO.21所示；
PCR扩增体系同实施例1，使用T7VEEIFNγF与3'UTRR引物PCR扩增IFN-γcDNA(46 57p)，琼脂糖凝胶电泳，胶回收。
(iii)IL-2
T7-VEE载体酶切位点引物：
T7VEED265AF：5’-GTCTAGTCCGCCAAGTCTAGCATATGGCCACCATGGAGACAGACACAC-3’ (SEQ ID NO.23)；
3'UTRR：如SEQ ID NO.21所示。
PCR扩增体系同实施例1，使用T7VEEIFNγF与3'UTRR引物PCR扩增IFN-γcDNA(5617p)，琼脂糖凝胶电泳，胶回收。
(iv)IL-12
T7-VEE载体酶切位点引物：
T7VEEIL12F：5’-GTCTAGTCCGCCAAGTCTAGCATATGGCCACC-3’(SEQ ID NO.24)；
3'UTRR：如SEQ ID NO.21所示。
PCR扩增体系同实施例1，使用T7VEEIL12F与3'UTRR引物PCR扩增IL-12cDNA(16457p)，琼脂糖凝胶电泳，胶回收。
(v)IL-15
T7-VEE载体酶切位点引物：
T7VEED265AF：如SEQ ID NO.23所示；
3'UTRR：如SEQ ID NO.21所示。
PCR扩增体系同实施例1，使用T7VEED265AF与3'UTRR引物PCR扩增IL-15cDNA(753 7p)，琼脂糖凝胶电泳，胶回收。
2)NdeI与SphI酶切上述T7-VEE-GFP质粒，反应体系：超纯水1μL、10x缓冲液3μL、质粒24μL、NdeI 1μL、SphI 1μL；
37℃，2h后琼脂糖凝胶电泳，胶回收94867p片段。
3)同源重组
(i)GM-CSF，反应体系：GM-CSF cDNA 8.46ng、NdeI与SphI酶切的上述T7-VEE-GFP质粒94.86ng、2x clonExpression Mix上述DNA片段和质粒载体的体积之和。
(ii)IFN-γ，反应体系：IFN-γcDNA 9.3ng、NdeI与SphI酶切的上述T7-VEE-GFP质粒94.86ng、2x clonExpression Mix上述DNA片段和质粒载体的体积之和。
(iii)IL-2，反应体系：IL-2cDNA 11.22ng、NdeI与SphI酶切的上述T7-VEE-GFP质粒94.86ng、2x clonExpression Mix上述DNA片段和质粒载体的体积之和。
(iv)IL-12，反应体系：IL-12cDNA 32.9ng、NdeI与SphI酶切的上述T7-VEE-GFP质粒94.86ng、2x clonExpression Mix上述DNA片段和质粒载体的体积之和。
(v)IL-15，反应体系：IL-15cDNA 15.06ng、NdeI与SphI酶切的上述T7-VEE-GFP质粒94.86ng、2x clonExpression Mix上述DNA片段和质粒载体的体积之和。
50℃，15min后，立即置于冰上，静置5min。
4)转化：将重组产物加入大肠杆菌感受态中，冰上静置25min；42℃，45s；迅速置于冰上5min；加入750μL无抗生素的LB培养基，37℃摇床，200rpm，1h；3500rpm，离心5min，弃上清600μL，剩余液体混匀，涂于含氨苄青霉素的LB平板中，37℃培养箱，倒置培养过夜。
5)挑选单克隆，MluI与EcoRI酶切鉴定，反应体系：超纯水7.8μL、10x缓冲液1μL、质粒1μL、MluI 0.1μL、EcoRI 0.1μL。
37℃，1h后琼脂糖凝胶电泳，鉴定正确的质粒测序。
6)MluI单酶切线性化T7-VEE质粒，并去除DNA模板RNase，反应体系：10x缓冲液8μL、质粒70μL、MluI 2μL。
7)纯化后的T7-VEE质粒，使用T7启动子体外转录：
室温条件下，向1.5mL无RNA酶离心管中，按照顺序加入：5x T7转录缓冲液2μL、rNTPs(25mM ATP,CTP,GTP,UTP)3μL、线性化DNA模板3.8μL(1μg)、体外转录酶(T7)1μL、RNA酶抑制剂0.2μL。37℃反应3-6h。
8)无RNA酶的DNA酶消化T7启动子体外转录体系的T7-VEE质粒模板，氯化锂纯化可复制型RNA。
9)可复制型RNA 5’端加甲基化鸟嘌呤核苷帽子，氯化锂纯化可复制型RNA，反应体系为：未加帽 的可复制型RNA 13.5μL(10μg)、10x加帽反应缓冲液2μL、GTP(10mM)1.0μL、S-腺苷甲硫氨酸(4mM)1.0μL、牛痘病毒加帽酶1.0μL、mRNA Cap2氧甲基转移酶1.0μL、RNA酶抑制剂0.5μL。加帽反应前，可复制型RNA需在25-70℃加热5-25min。
10)5’端加帽的可复制型RNA 3’端加多聚A尾巴(20-500个A碱基)，RNA纯化试剂盒纯化可复制型RNA，反应体系为：5’端加帽的可复制型RNA 15.5μL(10μg)、10x加多聚A尾巴缓冲液2μL、ATP(10mM)1μL、大肠杆菌聚(A)聚合酶1μL、RNA酶抑制剂0.5μL。
37℃，1h。RNA纯化试剂盒纯化5’端加甲基化鸟嘌呤核苷帽子，3’端加多聚A尾巴的可复制型RNA；
11)Lipofectamine2000或纳米粒转染可复制型RNA至293T细胞，酶联免疫吸附测定检测亚基因组启动子下游编码目的基因表达。
11.1Lipofectamine2000转染可复制型RNA至293T细胞。
293T细胞中48孔板，约60％满。
1.5mL离心管A：12.5μL opti-MEM培养基，加入500ng可复制型RNA。
1.5mL离心管B：12.5μL opti-MEM培养基，加入1μL Lipofectamine2000。
将管A加入管B混匀，室温，5min，加入293T细胞培养基中。
细胞培养36h，收集细胞培养基，并裂解细胞。
11.2纳米粒包载可复制型RNA处理293T细胞。
无核酸酶水10μL、可复制型RNA 500ng、鱼精蛋白375ng室温10-15min后添加48.475nmol 1,2-二油酰-3-三甲基铵盐丙烷/胆固醇，室温10-15min后添加2.776μg二硬脂酰基磷脂酰乙醇胺-聚乙二醇，50℃12-15min。
11.3酶联免疫吸附测定检测亚基因组启动子下游编码目的基因表达。
实验结果
使用PCR定点突变技术首先在可复制型RNA体外转录模板质粒非结构蛋白区域中引入非结构蛋白1G357C/G1569A/A1572C/C1575T突变，非结构蛋白2A3821T/G3892C/T3922C突变和非结构蛋白3A4714G突变，并进行了不同的突变组合，如T7-VEE(nsP1GGAC)；T7-VEE(nsP1GGAC-nsP2T)；T7-VEE(nsP1GGAC-nsP2AT)；T7-VEE(nsP1GGAC-nsP2GT-nsP3A)；T7-VEE(nsP2G-nsP3A)。
并在将不同的目的基因克隆至结构蛋白区，其中主要包括IL-12、IL-15、GM-CSF、IFN-γ、IL-2看其表达情况，主要通过Lipofectamine2000和纳米粒转染的方式。
其中Lipofectamine2000转染非结构蛋白区域野生型或相关突变的编码IL-12可复制型RNA至293T细胞的酶联免疫吸附测定检测结果见图13，结果显示VEE nsP1GGAC-nsP2T与VEE nsP1GGAC-nsP2AT突变均上调IL-12在细胞内表达与细胞外分泌；与VEE nsP1GGAC-nsP2T突变相比较，VEE nsP1GGAC-nsP2AT突变进一步增强IL-12在细胞内表达与细胞外分泌。
纳米粒转染非结构蛋白区域野生型或相关突变的编码IL-12可复制型RNA至293T细胞的酶联免疫吸附测定检测结果见图14，结果显示VEE nsP1GGAC-nsP2T与VEE nsP1GGAC-nsP2AT突变均上调IL-12在细胞内表达与细胞外分泌；而且与VEE nsP1GGAC-nsP2T突变相比较，VEE nsP1GGAC-nsP2AT突变进一步增强IL-12在细胞内表达与细胞外分泌。
Lipofectamine2000转染非结构蛋白区域野生型或相关突变的编码IL-15可复制型RNA至293T细胞的酶联免疫吸附测定检测结果见图15、结果显示VEE nsP1GGAC-nsP2T与VEE nsP1GGAC-nsP2AT突变均上调IL-15在细胞内表达与细胞外分泌；与VEE nsP1GGAC-nsP2T突变相比较，VEE nsP1GGAC-nsP2AT突变进一步增强IL-15在细胞内表达与细胞外分泌。
纳米粒转染非结构蛋白区域野生型或相关突变的编码IL-15可复制型RNA至293T细胞的酶联免疫吸附测定检测结果见图16，结果显示VEE nsP1GGAC-nsP2T与VEE nsP1GGAC-nsP2AT突变均上调IL-15在细胞内表达与细胞外分泌；与VEE nsP1GGAC-nsP2T突变相比较，VEE nsP1GGAC-nsP2AT突变进一步增强IL-15在细胞内表达与细胞外分泌。
Lipofectamine2000转染非结构蛋白区域野生型或相关突变的编码GM-CSF可复制型RNA至293T细 胞的酶联免疫吸附测定检测结果见图17，结果显示VEE nsP1GGAC-nsP2T与VEE nsP1GGAC-nsP2AT突变均上调GM-CSF在细胞内表达与细胞外分泌；与VEE nsP1GGAC-nsP2T突变相比较，VEE nsP1GGAC-nsP2AT突变进一步增强GM-CSF在细胞内表达与细胞外分泌。
纳米粒转染非结构蛋白区域野生型或相关突变的编码GM-CSF可复制型RNA至293T细胞的酶联免疫吸附测定检测结果见图18，结果显示VEE nsP1GGAC-nsP2T与VEE nsP1GGAC-nsP2AT突变均上调GM-CSF在细胞内表达与细胞外分泌；与VEE nsP1GGAC-nsP2T突变相比较，VEE nsP1GGAC-nsP2AT突变进一步增强GM-CSF在细胞内表达与细胞外分泌。
Lipofectamine2000转染非结构蛋白区域野生型或相关突变的编码IFN-γ可复制型RNA至293T细胞的酶联免疫吸附测定检测结果见图19，结果显示VEE nsP1GGAC-nsP2AT上调IFN-γ细胞外分泌；VEE nsP1GGAC-nsP2T与VEE nsP1GGAC-nsP2AT突变均上调IFN-γ在细胞内表达。
纳米粒转染非结构蛋白区域野生型或相关突变的编码IFN-γ可复制型RNA至293T细胞的酶联免疫吸附测定检测结果见图20，结果显示VEE nsP1GGAC-nsP2T与VEE nsP1GGAC-nsP2AT突变均上调IFN-γ在细胞内表达与细胞外分泌；与VEE nsP1GGAC-nsP2T突变相比较，VEE nsP1GGAC-nsP2AT突变进一步增强IFN-γ在细胞内表达与细胞外分泌。
Lipofectamine2000转染非结构蛋白区域野生型或相关突变的编码IL-2可复制型RNA至293T细胞的酶联免疫吸附测定检测结果见图21，结果显示VEE nsP1GGAC-nsP2T与VEE nsP1GGAC-nsP2AT突变均上调IL-2在细胞内表达与细胞外分泌；与VEE nsP1GGAC-nsP2T突变相比较，VEE nsP1GGAC-nsP2AT突变进一步增强IL-2在细胞内表达(VEE nsP1GGAC-nsP2AT突变的IL-2细胞内表达量是VEE nsP1GGAC-nsP2T突变的IL-2细胞内表达量的～20倍)与细胞外分泌(VEE nsP1GGAC-nsP2AT突变的IL-2细胞外分泌量是VEE nsP1GGAC-nsP2T突变的IL-2细胞外分泌量的～12倍)。其中VEE nsP2G-nsP3A突变上调IL-2在细胞内表达与细胞外分泌，其表达水平介于VEE nsP1GGAC-nsP2T突变与VEE nsP1GGAC-nsP2AT之间。
纳米粒转染非结构蛋白区域野生型或相关突变的编码IL-2可复制型RNA至293T细胞的酶联免疫吸附测定检测结果见图22，结果显示VEE nsP1GGAC-nsP2T与VEE nsP1GGAC-nsP2AT突变均上调IL-2在细胞内表达与细胞外分泌；与VEE nsP1GGAC-nsP2T突变相比较，VEE nsP1GGAC-nsP2AT突变进一步增强IL-2在细胞内表达(VEE nsP1GGAC-nsP2AT突变的IL-2细胞内表达量是VEE nsP1GGAC-nsP2T突变的IL-2细胞内表达量的～30倍)与细胞外分泌(VEE nsP1GGAC-nsP2AT突变的IL-2细胞外分泌量是VEE nsP1GGAC-nsP2T突变的IL-2细胞外分泌量的～30倍)。其中VEE nsP2G-nsP3A突变上调IL-2在细胞内表达与细胞外分泌，其表达水平介于VEE nsP1GGAC-nsP2T突变与VEE nsP1GGAC-nsP2AT之间。
综上所述，可以看出使用Lipofectamine2000或纳米粒载体将体外转录的非结构蛋白区域野生型或突变体的可复制型RNA转染至哺乳动物细胞293T，酶联免疫吸附测定结果显示可复制型RNA非结构蛋白区域nsP1G357C/G1569A/A1572C/C1575T-nsP2T3922C同时突变，即VEE(nsP1GGAC-nsP2T)，与nsP1G357C/G1569A/A1572C/C1575T-nsP2A3821T/T3922C突变体，即VEE(nsP1GGAC-nsP2AT)均可显著增强其下游亚基因组启动子介导的趋化因子或细胞因子，如GM-CSF、IFN-γ、IL-2、IL-12或IL-15的细胞内表达与细胞外分泌；与VEE nsP1GGAC-nsP2T突变相比较，VEE nsP1GGAC-nsP2AT突变进一步上调上述趋化因子或细胞因子在细胞内表达与细胞外分泌；此外，可复制型RNA非结构蛋白区域nsP2G3892C-nsP3A4714G同时突变，即VEE nsP2G-nsP3A突变上调IL-2在细胞内表达与细胞外分泌，其上调IL-2表达能力介于VEE nsP1GGAC-nsP2T突变与VEE nsP1GGAC-nsP2AT之间。
上述具体实施方式对本发明作了详细说明，但是本发明不限于上述实施例，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下作出各种变化。此外，在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合。

Claims (10)

  1. 一种RNA复制子，按5’-3’方向，所述RNA复制子包括：5’和3’非翻译区；非结构蛋白基因编码区、亚基因组启动子、目的基因编码区；其非结构蛋白基因编码区发生(I)～(III)任意一种突变：
    (I)选自非结构蛋白1的G357、G1569、A1572、C1575和非结构蛋白2的T3922位点中的至少一个位点的突变；优选为同时突变；
    (II)选自非结构蛋白1的G357、G1569、A1572、C1575和非结构蛋白2的A3821、T3922位点中的至少一个位点的突变；优选为同时突变；
    (III)包括但不限于非结构蛋白2的G3892和非结构蛋白3的A4714位点中的至少一个位点的突变；优选为同时突变。
  2. 根据权利要求1所述的RNA复制子，其特征在于，所述5’和3’非翻译区、非结构蛋白基因编码区和亚基因组启动子来源于甲病毒、黄病毒、小RNA病毒、副粘病毒或杯状病毒；
    优选地，所述甲病毒为委内瑞拉马脑炎病毒、辛德比斯病毒或塞姆利基森林病毒；所述黄病毒为登革热病毒或昆津病毒；所述小RNA病毒为脊髓灰质炎病毒或人鼻病毒；所述副粘病毒为犬痘热病毒；所述杯状病毒为猫杯状病毒；
    更优选地，所述甲病毒为委内瑞拉马脑炎病毒。
  3. 根据权利要求1所述的RNA复制子，其特征在于，所述目的基因包括肿瘤特异性或相关抗原、病原体特异性或相关抗原、细胞因子或其受体、趋化因子或其受体、生长因子或其受体、抗体蛋白、双特异性抗体蛋白、细胞因子抗体融合蛋白和免疫检查点相关蛋白中的至少一种；优选地，所述细胞因子为GM-CSF、IFN-γ、IL-2、IL-12或IL-15。
  4. 根据权利要求1所述的RNA复制子，其特征在于，所述突变的方法为PCR定点突变；
    优选地，所述PCR定点突变的引物包括：
    G357C F：5’-GAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACCC-3’；
    G357C R：5’-GCTCATGACGGCGGCGAGCTCCTTCATTTTCTTGTCC-3’；
    G1569A/A1572C/C1575T F：
    5’-GGAGCCCACTCTGGAAGCCGATGTCGACTTGATGTTACAAGAGG-3’；
    G1569A/A1572C/C1575T R：
    5’-TAACATCAAGTCGACATCGGCTTCCAGAGTGGGCTCCTCAACATC-3’；
    A3821T F:
    5’-CATTGGTGCTATAGCGCGGCTGTTCAAGTTTTCCCGGGTATGCAAAC-3’；
    T7VEESmaI R:5’-GCTTAAGTTAGTTGCGGCCGCCCGGGTCGACTCTAG-3’；
    T3922C F：5’-GCCCGTACGCACAATCCTTACAAGCTTTCATCAAC-3’；
    T3922C R：5’-TGAAAGCTTGTAAGGATTGTGCGTACGGGCCTTG-3’；
    G3892C F 5’-CTGTTTGTATTCATTCGGTACGATCGCAAGGCCCGTAC-3’；
    G3892C R 5’-CCTTGCGATCGTACCGAATGAATACAAACAGAACTTC-3’；
    A4714G F 5’-TATATCCTCGGAGAAGGCATGAGCAGTATTAGGTCG-3’；
    A4714G R 5’-TAATACTGCTCATGCCTTCTCCGAGGATATACATGC-3’。
  5. 一种载体，包含权利要求1～4任一项所述RNA复制子。
  6. 一种细胞，包含权利要求5所述载体。
  7. 权利要求1～4任一项所述RNA复制子在(I)～(V)任一项中的应用：
    (I)递送目的基因；
    (II)实现目的基因的长效表达；
    (III)提高目的基因的表达量；
    (IV)基因治疗；
    (V)疫苗研发。
  8. 一种组合物，包含权利要求1～4任一项所述RNA复制子或权利要求5所述载体。
  9. 根据权利要求8所述的组合物，其特征在于，所述组合物还包含药用稀释剂、药用赋形剂、药用载体和药用载剂中的至少一种；优选地，所述组合物中还可包含其他药物联合使用，所述药物包括但不限于：单克隆抗体药物、双特异性抗体药物、抗体偶联药物、融合蛋白药物、核酸类药物、化学药物、血液制品药物、脂类药物或中药提取物；
    优选地，所述药用载体为基于阳离子脂质的市售转染试剂、非病毒载体、高分子膜、仿生膜、生物膜或病毒载体；
    所述转染试剂包括但不限于Lipofectamine2000、Lipofectamine3000、Lipofectamine8000、Lipofectamine LTX、Lipofectamine RNAiMAX、Lipofectamine MessengerMAX或Invivofectamine 3.0；
    所述非病毒载体包括但不限于阳离子聚合物、阳离子脂质体、阴离子脂质体、胶束、无机纳米颗粒或微球；
    所述高分子膜、仿生膜或生物膜，包括但不限于细胞膜、外泌体或细胞外囊泡；所述病毒载体，包括但不限于腺病毒载体、逆转录病毒、慢病毒、疱疹病毒或病毒样颗粒；
    优选地，所述纳米载体包括但不限于多聚阳离子肽、阳离子脂质、阴离子脂质、中性脂 质、辅助脂质或两亲性化合物；
    优选地，所述纳米载体的粒径为20～350nm，电荷为-40～50mV。
  10. 一种用于在生物体表达目的基因的方法，其包含以下步骤：向所述生物体中施用权利要求1～4任一项所述的RNA复制子或权利要求5所述的载体；优选地，所述生物体为原核生物或真核生物。
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