WO2022036618A1 - 携带细胞因子或其多核苷酸的细菌靶向载体及其在肿瘤治疗中的应用 - Google Patents

携带细胞因子或其多核苷酸的细菌靶向载体及其在肿瘤治疗中的应用 Download PDF

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WO2022036618A1
WO2022036618A1 PCT/CN2020/110159 CN2020110159W WO2022036618A1 WO 2022036618 A1 WO2022036618 A1 WO 2022036618A1 CN 2020110159 W CN2020110159 W CN 2020110159W WO 2022036618 A1 WO2022036618 A1 WO 2022036618A1
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tumor
prokaryotic
interleukin
polynucleotide
cytokine
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刘陈立
董宇轩
郭旋
陈茜
胡勇
艾亮霞
张昊
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Shenzhen Institute of Advanced Technology of CAS
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/74—Bacteria
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/204—IL-6
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2066—IL-10
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Definitions

  • the present invention relates to the field of tumor targeted therapy, in particular to a bacterial targeting vector carrying cytokines, especially interleukin-6 and interleukin-10, or polynucleotides encoding the cytokines, such as mRNA, and their use in tumor therapy application.
  • cytokines especially interleukin-6 and interleukin-10
  • Tumor bacterial therapy relies on the characteristics of bacterial diversity, motility, tumor tropism, invasive ability and cytotoxicity to achieve tumor intervention.
  • Live bacteria have the unique ability to actively target and specifically colonize tumors, and are capable of targeting the vast majority of tumor types, including metastases. Live bacteria were also able to rely on flagella for better infiltration of intratumoral tissue.
  • traditional anticancer drugs have strong therapeutic efficacy, they spread throughout the body with the blood circulation system, and their indiscriminate attacks cause a large number of normal cells to die. The complex vasculature of tumors hinders the in-depth delivery of traditional anticancer drugs, making it difficult to exert a good tumor killing effect. Therefore, how to realize the combination of bacterial therapy and traditional therapy can make up for their respective deficiencies.
  • bacteria can express anti-tumor active molecules such as cytokines, and take advantage of the natural tumor targeting properties of bacteria to achieve targeted expression of anti-tumor active molecules in tumor areas; applying the technical concept of synthetic biology, Reconstruction and optimization of chassis cells, design and synthesis of functional modules such as targeting and regulation, can enable bacteria to have many excellent properties such as specific tumor targeting, controllable drug release, significant tumor intervention, and stronger safety.
  • anti-tumor active molecules such as cytokines
  • Attenuated ⁇ ppGpp Salmonella typhimurium was genetically engineered to secrete Vibrio vulnificus flagellin FlaB, synergistically activate TLR5 and TLR4 signaling pathways, increase the secretion of tumor suppressors, and significantly enhance the effect of Salmonella on colorectal cancer cells. destructive ability.
  • Chinese patent application CN102526760A discloses a recombinant attenuated Salmonella vaccine and pharmaceutical composition for the treatment of solid tumors, wherein the recombinant attenuated Salmonella vaccine comprises a eukaryotic expression plasmid carrying both interleukin 2 and hepatocyte growth factor antagonist NK4 double genes , so its essence is to release the plasmid itself into the human body for eukaryotic expression.
  • PCT patent application WO2015032165A1 discloses a tumor-targeted bacterial delivery and expression system for prokaryotic-eukaryotic delivery and expression of therapeutic molecules in mammalian cells.
  • the therapeutic vector in which it is delivered and expressed is pore-forming listeriolysin. And the goal of using prokaryotic carriers to deliver and release therapeutic agents is not achieved.
  • the purpose of the present invention is to provide a tumor-targeted prokaryotic delivery vector and its application in tumor therapy.
  • the present invention provides a tumor-targeting prokaryotic delivery vector, the vector is a chassis bacterium targeting the anaerobic region of a solid tumor, and comprising a cytokine-expressing polynucleotide, wherein,
  • the cytokine is selected from the group consisting of interleukin 6, interleukin 10, interleukin 8, interleukin 18, interleukin 33 and TNF- ⁇ , and
  • the polynucleotide is DNA or mRNA.
  • the chassis bacteria are selected from the group consisting of attenuated Salmonella enterica serovars Typhimurium, attenuated human Salmonella enterica serovars Typhi, E. coli and Lactic acid bacteria.
  • the chassis bacteria are attenuated Salmonella typhimurium.
  • the polynucleotide is DNA and the vector further comprises the signal peptides pelB and OmpF fused to the DNA expressing the cytokine.
  • the polynucleotide is an mRNA further comprising an m7G capping structure at the 5' end and a polyadenylation tail structure at the 3' end.
  • the cytokine is selected from the group consisting of interleukin-6 and interleukin-10.
  • the prokaryotic delivery vector comprises a tumor environment sensitive promoter.
  • the prokaryotic delivery vector may further comprise other promoters, which are constitutive promoters and inducible promoters, such as IPTG inducible promoters or aTc inducible promoters.
  • the tumor is a solid tumor found in the cervix, breast, prostate, gastrointestinal tract (eg, esophagus, oropharynx, stomach, small or large intestine, colon, rectum), bladder, Bone, skin, head or neck, liver, gallbladder, lung, pancreas, salivary glands, adrenal glands, thyroid, brain (eg glioma) ganglia, preferably colon cancer, breast cancer, melanoma, lung cancer , Glioblastoma.
  • gastrointestinal tract eg, esophagus, oropharynx, stomach, small or large intestine, colon, rectum
  • bladder e, Bone, skin, head or neck, liver, gallbladder, lung, pancreas, salivary glands, adrenal glands, thyroid, brain (eg glioma) ganglia, preferably colon cancer, breast cancer, melanoma, lung cancer , Glioblastoma.
  • the carrier is administered by intramuscular, intravenous, subcutaneous, intraperitoneal, intracerebral, buccal, nasal or oral administration.
  • the present invention provides the use of the prokaryotic delivery vehicle as described above in tumor therapy, wherein the prokaryotic delivery vehicle is used in combination with a therapeutic agent selected from the group consisting of chemical drugs, such as alkylating agents (eg, alkylating agents).
  • a therapeutic agent selected from the group consisting of chemical drugs, such as alkylating agents (eg, alkylating agents).
  • thiotepa and cyclophosphamide alkyl sulfonates, azcyclenes, ethylamines and methylamines, nitrogen mustards, antibiotics, purine analogs, pyrimidine analogs, androgens, anti-adrenaline , topoisomerase inhibitors, capecitabine; chemotherapeutic drugs: anti-hormonal drugs that modulate or inhibit the action of tumor hormones; immune-targeted drugs: gefitinib, Iressa, lorlatinib, erlotinib , Bevacizumab.
  • the combination is simultaneous administration, or sequential administration, or separate administration.
  • the attenuated chassis bacteria with targeted solid tumors of the present invention can be used as a delivery carrier of tumor therapeutic molecules to realize the expression and controllable release of human/mouse-derived cytokines, thereby achieving anti-tumor effects.
  • Figure 1 is the IL-10 protein expression observed at the indicated time after the IL10 cmRNA prepared in Example 1 was transfected into 293T cells at different concentrations.
  • Figure 2 is a schematic diagram of the original plasmid used in Example 2 and the constructed plasmid.
  • Figure 2 (left) is the original plasmid template, pSC101-GFP-Kana;
  • Figure 2 (right) is the plasmid with the signal peptide inserted, pSC101-pelB-GFP-Kana.
  • Figure 3 shows the expression of plasmid pSC101-pelB-GFP-Kana constructed in Example 2 at the RNA level.
  • FIG. 4 is a schematic diagram of the plasmid containing IL-6 and IL-10 constructed in Example 3.
  • FIG. Figure 4 (left) is the plasmid pSC101-pelB-IL-6-Kana;
  • Figure 4 (right) is the plasmid pSC101-pelB-IL-10-Kana.
  • Figure 5 shows the construction of the plasmid of the present invention carrying its own eukaryotic signal peptide (pET30a-IL10 with eukaryotic signal peptide) and the sequencing result.
  • Figure 6 shows the construction of a plasmid carrying E.coli signal peptide (pET30a-IL10 with E.coli ompF signal peptide) and sequencing results of the interleukin 10 of the present invention.
  • Figure 7 is the construction of the interleukin 10 plasmid carrying the Salmonella signal peptide ompF of the present invention (pET30a-IL10 with the Salmonella ompF signal peptide) and the sequencing results.
  • Fig. 8 is the construction of the plasmid of the present invention carrying the pelB signal peptide (pET22b-IL10 with the pelB signal peptide) and the sequencing result.
  • FIG. 9 to FIG. 11 are respectively the results of PCR identification of bacterial liquid after transfection of cells with the four plasmids prepared in Example 4.
  • Figure 12 shows the expression levels of IL-10 carrying different signal peptides in BL21(DE3).
  • BL21(DE3)-pET22b(+)-pelB-IL-10 indicated by pelB in the figure
  • BL21(DE3)-pET30a-ompF-IL-10 indicated by ompF in the figure
  • BL21(DE3)-pET30a- IL-10 represented by eukaryon in the figure).
  • tumor-targeted prokaryotic delivery vector and its application in tumor therapy according to specific embodiments of the present invention will be explained in more detail.
  • the tumor-targeted prokaryotic delivery vector of the present invention is a chassis bacterium targeting the anaerobic region of a solid tumor, and comprises a polynucleotide expressing a cytokine, wherein the cytokine It is selected from interleukin-6, interleukin-10, interleukin-8, interleukin-18, interleukin-33 and TNF- ⁇ , preferably interleukin-6 or interleukin-10.
  • the polynucleotide is DNA or mRNA.
  • the present invention applies to the use of a prokaryotic delivery vehicle in tumor therapy, wherein the prokaryotic delivery vehicle is used in combination with a therapeutic agent selected from the group consisting of chemical drugs such as alkylation agents (such as thiotepa and cyclophosphamide), alkyl sulfonates, azcyclenes, ethylamines and methylamines, nitrogen mustards, antibiotics, purine analogs, pyrimidine analogs, androgens, Anti-epinephrine, topoisomerase inhibitors, capecitabine; chemotherapeutic drugs: anti-hormonal drugs that modulate or inhibit the action of tumor hormones; immune-targeted drugs: gefitinib, Iressa, lorlatinib, erlatinib Lotinib, bevacizumab.
  • the combination is simultaneous administration, or sequential administration, or separate administration.
  • the present invention provides a method of treating tumors using a prokaryotic delivery vector of the present invention
  • said prokaryotic delivery vector is a chassis bacterium that targets tumors, particularly anaerobic regions of solid tumors, And it comprises a polynucleotide expressing cytokines, wherein the cytokines are selected from interleukin-6, interleukin-10, interleukin-8, interleukin-18, interleukin-33 and TNF- ⁇ , preferably interleukin-6 or interleukin-10.
  • the polynucleotide is DNA or mRNA.
  • the prokaryotic delivery vehicle may be used in combination with a therapeutic agent selected from the group consisting of chemical drugs such as alkylating agents (eg thiotepa and cyclophosphamide), Alkyl sulfonates, azcyclenes, ethylamines and methylamines, nitrogen mustards, antibiotics, purine analogs, pyrimidine analogs, androgens, anti-epinephrines, topoisomerase inhibitors, cape Tabine; Chemotherapy drugs: anti-hormonal drugs that modulate or inhibit the action of tumor hormones; Immune-targeted drugs: gefitinib, Iressa, lorlatinib, erlotinib, bevacizumab.
  • the combination is simultaneous administration, or sequential administration, or separate administration.
  • the present invention also provides the use of the prokaryotic delivery vehicle of the present invention in the preparation of a medicament for treating tumors, wherein the prokaryotic delivery vehicle is combined with a therapeutic agent selected from the group consisting of: Uses: Chemical drugs, such as alkylating agents (such as thiotepa and cyclophosphamide), alkyl sulfonates, azcyclenes, ethylamines and methylamines, nitrogen mustards, antibiotics, purine analogs , pyrimidine analogs, androgens, anti-adrenergic, topoisomerase inhibitors, capecitabine; chemotherapeutic drugs: anti-hormonal drugs that regulate or inhibit the action of tumor hormones; immune-targeted drugs: gefitinib, easy Ressa, lorlatinib, erlotinib, bevacizumab.
  • the combination is simultaneous administration, or sequential administration, or separate administration.
  • the chassis bacteria are selected from attenuated Salmonella enterica serovars Typhimurium, attenuated human Salmonella enterica serovars Typhi, Escherichia coli (E. coli) and Lactic acid bacteria, preferably attenuated Salmonella typhimurium.
  • the polynucleotide is DNA and the vector further comprises the signal peptide pelB fused to the DNA expressing the cytokine.
  • the vectors of the present invention also comprise tumor environment sensitive promoters.
  • the polynucleotide is an mRNA further comprising a m7G capping structure at the 5' end and a polyadenylation tail structure at the 3' end.
  • the tumor is a solid tumor found in the cervix, breast, prostate, gastrointestinal tract (eg, esophagus, oropharynx, stomach, small or large intestine, colon, rectum) , bladder, bone, skin, head or neck, liver, gallbladder, lung, pancreas, salivary gland, adrenal gland, thyroid, brain (eg, glioma) ganglia, preferably colon cancer, breast cancer, melanoma tumor, lung cancer, glioblastoma.
  • gastrointestinal tract eg, esophagus, oropharynx, stomach, small or large intestine, colon, rectum
  • bladder bone, skin, head or neck, liver, gallbladder, lung, pancreas, salivary gland, adrenal gland, thyroid
  • brain eg, glioma
  • ganglia ganglia, preferably colon cancer, breast cancer, melanoma tumor, lung cancer, glioblastoma.
  • the carrier or drug is administered by intramuscular, intravenous, subcutaneous, intraperitoneal, intracerebral, buccal, nasal, or oral administration.
  • the prokaryotic delivery vector of the present invention can be constructed and verified in the following ways: (1) using chemical modification method to modify cytokine mRNA; (2) using bacterial vector to deliver and express chemically modified cytokine mRNA; (3) heterologous expression of cytokine (4) ELISA or Western Blot to identify the expression of cytokines; (5) in vitro experiments to test the function of bacteria to secrete cytokines; (6) to test the effect of interleukin 10 on tumor therapy by tumor-bearing mouse model .
  • the cytokine mRNA of the present invention exists in the form of a plasmid in bacteria, and is released and expressed as the bacteria are delivered into the tumor, and performs the translation function in the tumor. After chemical modification, the time can be significantly prolonged to avoid degradation.
  • One aspect of the present invention relates to a method of modifying in vitro transcription of interleukin-6 or interleukin-10 cytokine mRNA, and introducing it into chassis microorganisms, so as to enter solid tumors through bacteria through blood vessels, and realize the endocrine and translation process of mRNA in solid tumors.
  • the present invention can achieve the modification and in vitro transcription of cytokine mRNA by using a bacterial vector to deliver a system for expressing chemically modified cytokine mRNA.
  • the present invention incorporates the m7G capping structure at the 5' end and the polyadenylic acid (polyA tail) structure at the 3' end when transcribing and synthesizing mRNA in vitro.
  • the capping process is achieved by co-transcription with T7 RNA polymerase; the tailing process is to add a poly(A) tail to the 3' end of the mRNA molecule with Poly(A) polymerase, and then digest the DNA with DNase enzyme template.
  • cmRNA is electro-transformed into the chassis bacteria, so as to realize the transmembrane secretion of cmRNA, and then extracellularly translate to generate IL-6/IL-10 protein.
  • Another aspect of the present invention relates to the use of signal peptides to fuse cytokines to achieve target protein expression and extracellular secretion.
  • the fusion and expression of signal peptides and cytokines were achieved by the following methods: the construction of a plasmid vector system for bacterial heterologous expression of murine cytokines IL-6 and IL-10. Heterologous expression of cytokines is easy to form inclusion bodies.
  • the present invention intends to use signal peptides to fuse and express target genes. Signal peptides can direct the extracellular transfer of synthetic proteins.
  • the present invention adopts the signal peptide pelB derived from Erwinia carotovora pectate lyase as the research of the present invention.
  • the present invention optionally adopts 6 ⁇ His tag to purify the tag as the tag detection of the subsequent target gene.
  • IL-6 and IL-10 in bacterial supernatant were detected by enzyme-linked immunosorbent assay (ELISA), thereby verifying whether IL-6 and IL-10 achieve extracellular secretion.
  • ELISA enzyme-linked immunosorbent assay
  • cytokines IL-6 and IL-10 For the chemically modified cytokine mRNA system constructed as above, and the plasmid vector system for bacterial heterologous expression of murine cytokines IL-6 and IL-10, the following solid-phase ELISA technology and/or bacterial The expression of cytokines IL-6 and IL-10 was tested in a test format for the therapeutic effect of secreted cytokines IL-6 and IL-10 on tumor-bearing mice.
  • cytokines IL-6 and IL-10 The function of cytokines IL-6 and IL-10 was identified by solid-phase enzyme-linked immunospot technique (ELISPOT) in vitro.
  • ELISPOT solid-phase enzyme-linked immunospot technique
  • CD4+T cells and CD8+T cells are cultured in vitro, and the activation of T cells is detected by solid-phase enzyme-linked immunospot technique (ELISPOT).
  • ELISPOT solid-phase enzyme-linked immunospot technique
  • a control experiment was designed to directly stimulate T cells with cytokines IL-6 and IL-10 for CD4+ T cells and CD8+ T cells cultured in vitro.
  • the present invention constructs macrophage-deficient mice, eliminates the interference of factors such as macrophage secretion of IL-10, and observes mice.
  • the tumor size of mice was collected and the secretion of cytokines such as TNF- ⁇ and IFN- ⁇ was detected by collecting mouse tumor serum.
  • the present invention can also use a delivery system constructed based on pET-22b(+) and pET30a.
  • the promoters of the present invention may be constitutive promoter J23101 and IPTG or aTc inducible expression promoters.
  • the ribosome binding site (RBS) of the present invention can be B0034, and a variety of RBSs with different expression strengths, including but not limited to more than 40 kinds such as BBa J61100, BBa J61101, and BBa J61139. These RBSs can be used to characterize the expression intensity of the protein of interest.
  • the chassis microorganisms of the present invention can be Escherichia coli BL21 (DE3), Nissle 1917 and Salmonella SL7207 (aroA-) and the like.
  • the chassis microorganisms used are mainly attenuated Salmonella strains, which have a significant killing effect on solid tumors;
  • the bacterial vector constructed in the present invention expresses a higher expression level of the cytokine interleukin-6 or interleukin-10.
  • Example 1 Preparation and intracellular expression of IL-10 mRNA
  • the DNA of IL-10 was constructed on a plasmid with a T7 promoter. After linearization, it was used as a template to synthesize RNA by in vitro transcription reaction, and the m7G cap at the 5' end was introduced in the synthesis reaction.
  • IL-10 mRNA has the function of normal translation in cells
  • the chemically modified cmRNA was transfected into 293T cells, and the transfection doses were 0.5ug and 1.5ug per well, respectively, and the cells were incubated for 24h as the time starting point.
  • Cell tissue fluid and cell supernatant were extracted at 24h, 36h, 48h, and 60h, respectively, and the expression of IL-10 protein was determined by ELISA. The results are shown in Figure 1 and Table 1.
  • Table 1 is the expression level (pg/mL) of IL-10 in transfected 293T cells.
  • the plasmid constructed above is electroporated into the attenuated Salmonella typhimurium Salmonella Typhimurium (with reference to attenuated Salmonella YB1, Chang-Xian Li etc. are disclosed in Oncology Letters, January, 2017 Volume 13 Issue 1, DOI: 10.3892/ol.2016.5453) In , the expression of GFP was tested at the RNA level.
  • RNA level analysis of GFP expression Real-time PCR test was used to design Real-time PCR forward and reverse primers, qP-pelB-F1/R1, the sequence was GGCAGCCGCTGGATTGTTAT/TGTTGCATCACCTTCACCCT, and the Tm value was 60 degrees; SL7207 ( aroA-) and SL7207 transformed into the original template plasmid were used as the control group for the experiment, and the sample before reverse transcription was selected as the control group to avoid errors caused by untreated genomic DNA.
  • Figure 3 shows the expression of GFP at the RNA level. The results are shown in Figure 3.
  • rho was selected as the internal reference gene, and the ratio of the sample result data to the rho internal reference gene was calculated.
  • GFP was highly expressed at the mRNA level, and neither SL7207 nor the control group of SL7207 transformed into the original template plasmid had no mRNA expression.
  • Example 3 IL-6 and IL-10 as target genes, construction of plasmid delivery system
  • the forward primer sequence is: 5'-GTCGACACTTAATTAACGGCA-3'; the reverse primer sequence: 5'-CTCGAGATTTCTCCTCTTTCG-3' Amplify the backbone vector; the mouse IL-10 gene is amplified from the Puc-IL-10-AMP plasmid, the forward primer sequence is: 5'-GAAAGAGGAGAAATCTCGAGATGA-3'; the reverse primer sequence: 5'-GCCGTTAATTAAGTGTCGACAC-3'.
  • the target fragment was inserted into the backbone by One Step-Cloning to obtain the pSC101-pelB-IL-10-kana plasmid.
  • the PCR high-fidelity amplification enzyme used PrimerSTAR MAX Premix (2 ⁇ ), a product of Takara Company, and the dNTP concentration was 0.4mM (2 ⁇ ).
  • the PCR reaction conditions were as follows:
  • pSC101-pelB-IL-6-kana was constructed in the same way, as shown in Figure 4.
  • Example 4 Construction, transformation and expression of pET30a-IL10 with ompF signal peptide and pET22b-IL10 with pelB signal peptide
  • IL-10 The protein sequence of IL-10 was analyzed, and the first 22 amino acids of IL-10 were found to be a eukaryotic signal peptide.
  • the applicant's prokaryotic signal peptides pelB and ompF The original eukaryotic sequence signal peptide was replaced, and the inducible plasmid expression vector was used to increase the soluble expression of the target protein.
  • Plasmids were successfully constructed: pET30a-IL10 with ompF signal peptide and pET22b-IL10 with pelB signal peptide.
  • pET30a and pET22b are commonly used IPTG-inducible expression vectors. The constructed plasmids are shown in Figures 5-8.
  • Plasmids pET30a-IL10 with eukaryotic signal peptide were constructed in BL21 (DE3) and ZG1 to achieve normal expression of IL-10 strains; in addition, pET30a-IL10 with E. coli ompF signal peptide and pET30a-IL10 with E.
  • E. coli BL21(DE3) carrying plasmids pET30a-ompF-IL10 and pET22b(+)-pelB-IL10 and E. coli BL21(DE3) carrying pET30a-IL10 with eukaryotic signal peptide as control were used LB medium to expand the system culture, the culture volume is 4ml, when the bacteria are cultured to the late log phase (OD value is about 0.6), IPTG with a final concentration of 1/0.5/0 mmol is added to it, and the expression is induced overnight at 16°C.

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Abstract

细菌靶向载体及其在肿瘤治疗中的应用，特别是涉及携带细胞因子，如白介素6和白介素10或携带细胞因子的多核苷酸，如mRNA的细菌靶向载体及其在肿瘤治疗中的应用。具备靶向实体瘤内的减毒底盘细菌可以作为肿瘤治疗分子的递送载体，实现人/鼠源细胞因子的表达及可控释放，从而达到抗肿瘤效果。

Description

携带细胞因子或其多核苷酸的细菌靶向载体及其在肿瘤治疗中的应用
交叉参考相关引用
本申请要求2020年08月18日递交的申请号为202010830264.5、发明名称为“携带细胞因子或其多核苷酸的细菌靶向载体及其在肿瘤治疗中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及肿瘤靶向治疗领域，具体而言涉及携带细胞因子，特别是白介素6和白介素10，或编码所述细胞因子的多核苷酸，如mRNA的细菌靶向载体及其在肿瘤治疗中的应用。
背景技术
目前常规的肿瘤治疗方法，如手术疗法、放疗和化疗等，都暴露出对正常组织的损害和严重的毒副作用等缺陷，已无法满足患者的临床需求。新兴的诸如CAR-T技术，检查点抑制剂等疗法为肿瘤治疗带来了全新的希望，但是肿瘤免疫疗法也存在着一定局限性，并且其对实体瘤治疗效果较差，这限制了免疫疗法进一步的开发应用。所以，如何扩大免疫疗法的选择范围，是实现免疫疗法突破的关键。
肿瘤细菌疗法的研究历史可追溯到150年前。肿瘤细菌疗法是依赖于细菌繁多的种类、运动性、肿瘤趋向性、侵袭能力和细胞毒性等特征来实现肿瘤干预。活细菌具有主动靶向肿瘤并特异性定植瘤内的独特能力，而且能够靶向包括转移瘤在内的绝大多数肿瘤类型。活细菌还能够依赖鞭毛更好地渗入瘤内组织。而传统抗癌药物虽然治疗毒性效力强，但其随血液循环系统遍及周身，其无差别的攻击使正常细胞大量死亡。肿瘤复杂的脉管系统又阻碍着传统抗癌药物的深入递送，使其难以发挥良好的肿瘤杀伤效果。所以，如何实现将细菌疗法与传统疗法联合便能很好地弥补两者各自的不足。
基因工程技术和合成生物学理念的发展成熟，为肿瘤细菌疗法与免疫疗法的联合提供了更有效、更安全的治疗策略。通过基因工程技术改造细菌能够表达细胞因子等抗肿瘤活性分子，利用细菌天然的肿瘤靶向性等诸多优点，实现抗肿瘤活性分子在肿瘤区的 靶向性表达；应用合成生物学的技术理念，对底盘细胞重构优化，设计合成靶向和调控等功能模块，能够使细菌具备特异靶向肿瘤、可控释放药物、显著肿瘤干预和更强安全性等诸多优良性质。
目前已有多篇文章报道了细菌与免疫联合疗法的实例。通过改造减毒细菌靶向递送细胞因子等抗肿瘤药剂，从而进一步增强抗肿瘤效果。Xiao-qin Ha等(“Inhibitory Effects of the Attenuated Salmonella typhimurium Containing the IL-2 Gene on Hepatic Tumors inMice”,Journal of Biomedicine and Biotechnology,Volume 2012,Article ID 946139)证明了表达IL-2的减毒沙门氏菌相比沙门氏菌对照组具有更好的抑制肝癌细胞增殖的潜力。与IL-2的工作相似，M Loeffler等于2008年报道了表达IL-18的减毒鼠伤寒沙门氏菌(“IL-18-producing Salmonella inhibit tumor growth”,Cancer Gene Therapy(2008)15,787–794)。LIGHT、IL-18和CCL21等细胞因子在实体瘤的原位表达，通过诱导肿瘤区T细胞、树突状细胞和自然杀伤细胞等增殖，激活肿瘤内的免疫抑制环境，对于肝癌等实体瘤的干预及转移瘤的抑制都具有一定作用。另外，韩国全南国立大学的研究人员利用改造的沙门氏菌，开发了一种组成型治疗系统中。对减毒的ΔppGpp鼠伤寒沙门氏菌进行基因工程化改造，使其分泌创伤弧菌鞭毛蛋白FlaB，对TLR5和TLR4信号通路协同活化，提高了肿瘤抑制因子的分泌，显著增强了沙门氏菌对结肠直肠癌细胞的杀伤能力。
中国专利申请CN102526760A公开了一种治疗实体瘤的重组减毒沙门氏菌疫苗和药物组合物，其中重组减毒沙门氏菌疫苗包含同时携带白细胞介素2和肝细胞生长因子拮抗剂NK4双基因的真核表达质粒，因此其实质是将质粒本身释放到人体中进行真核表达。
PCT专利申请WO2015032165A1公开了一种靶向肿瘤的细菌递送和表达系统，用于进行原核-真核递送，并在哺乳动物细胞中表达治疗分子。其所递送和表达的治疗载体是成孔李斯特菌溶血素(pore-forming listeriolysin)。并且没有实现采用原核载体完成递送和释放治疗剂的目的。
Christoph
Figure PCTCN2020110159-appb-000001
等(“Periplasmic Delivery of Biologically Active Human Interleukin-10 in Escherichia coli via a Sec-Dependent Signal Peptide”,J Mol Microbiol Biotechnol 2012；22:1–9)用大肠杆菌分泌表达具备生物活性的人源细胞因子白介素10，并用于炎性肠病的治疗。其将克隆自E.coli K-12 MG1655的信号肽ompF基因替换人源白介素10基因的真核生物信号序列，由此实现原核信号肽ompF基因与人源白介素10基因的融合，并克隆到BAC pSG1107质粒载体中，由T7启动子启动表达， 实现异源蛋白的跨膜递送，引导细胞外分泌。其在大肠杆菌BL21(DE3)中表达后，在培养上清液和胞内中的表达量分别为5.8±1.7和355.8±86.3ng/ml。
目前还没有采用针对靶向肿瘤优化的用于精准递送和释放细胞因子的原核载体，和采用该载体的治疗应用。
发明内容
为了解决现有技术的问题，本发明的目的是提供一种靶向肿瘤的原核递送载体及其在肿瘤治疗中的应用。
在第一方面，本发明提供了一种靶向肿瘤的原核递送载体，所述载体是靶向实体瘤厌氧区的底盘细菌，并且包含表达细胞因子的多核苷酸，其中，
所述细胞因子选自白介素6，白介素10，白介素8，白介素18，白介素33和TNF-α，并且
所述多核苷酸是DNA或mRNA。
在本发明的第一方面中，所述底盘细菌选自减毒的鼠伤寒沙门氏菌(Salmonella enterica serovars Typhimurium)、减毒的人源伤寒沙门氏菌(Salmonella enterica serovars Typhi)、大肠杆菌(E.coli)和乳酸菌(Lactic acid bacteria)。
在本发明的第一方面中，所述底盘细菌是减毒的鼠伤寒沙门氏菌。
在本发明的第一方面中，所述多核苷酸是DNA，所述载体还包含与表达所述细胞因子的DNA融合的信号肽pelB和OmpF。
在本发明的第一方面中，所述多核苷酸是mRNA，所述mRNA还包含5’端的m7G加帽结构和3’端的多聚腺苷酸尾结构。
在本发明的第一方面中，所述细胞因子选自白介素6和白介素10。
在本发明的第一方面中，所述原核递送载体包含肿瘤环境敏感的启动子。所述原核递送载体还可以包含其他启动子，所述其他启动子为组成型启动子和诱导型启动子，所述诱导型启动子例如为IPTG诱导型启动子或aTc诱导型启动子。
在本发明的第一方面中，所述肿瘤为发现在以下部位的实体瘤：宫颈、乳腺、前列腺,胃肠道(例如食道、口咽、胃、小肠或大肠,结肠、直肠)、膀胱、骨、皮肤、头部或颈部、肝脏、胆囊、肺、胰腺、唾液腺、肾上腺、甲状腺、大脑(如神经胶质瘤)神经节，所述肿瘤优选为结肠癌、乳腺癌、黑色素瘤、肺癌、胶质母细胞瘤。
在本发明的第一方面中，所述载体通过肌肉注射、静脉注射、皮下注射、腹腔注射、 经脑内施用、经口腔施用、经鼻腔施用或口服施用进行施用。
在第二方面，本发明提供了如上所述的原核递送载体在肿瘤治疗中的应用，其中，所述原核递送载体与选自以下的治疗剂联用：化学药物，如烷基化剂(如硫代特帕和环磷酰胺)、烷基磺酸盐、阿兹环烯类药物、乙胺和甲胺、氮芥类、抗生素、嘌呤类似物、嘧啶类似物、雄酮类、抗肾上腺素、拓扑异构酶抑制剂、卡培他滨；化疗药物：调节或抑制肿瘤激素作用的抗激素药物；免疫靶向药物：吉非替尼、易瑞沙、劳拉替尼、厄洛替尼、贝伐珠单抗。
在本发明的第二方面中，所述联用为同时施用，或依次施用，或分开施用。
本发明的具备靶向实体瘤内的减毒底盘细菌可以作为肿瘤治疗分子的递送载体，实现人/鼠源细胞因子的表达及可控释放，从而达到抗肿瘤效果。
附图说明
图1是实施例1制备的IL10 cmRNA以不同浓度转染293T细胞后在所示时间观察到的IL-10蛋白的表达情况。
图2是实施例2采用的原始质粒和所构建的质粒的示意图。图2(左)为原始质粒模板，pSC101-GFP-Kana；图2(右)为插入信号肽的质粒，pSC101-pelB-GFP-Kana。
图3是实施例2所构建的质粒pSC101-pelB-GFP-Kana在RNA水平上的表达情况。
图4是实施例3所构建的包含IL-6和IL-10的质粒示意图。图4(左)为质粒pSC101-pelB-IL-6-Kana；图4(右)为质粒pSC101-pelB-IL-10-Kana。
图5是本发明的白介素10携带自身真核信号肽的质粒构建(带有真核信号肽的pET30a-IL10)及测序结果。
图6是本发明的白介素10携带E.coli信号肽的质粒构建(带有E.coli ompF信号肽的pET30a-IL10)及测序结果。
图7是本发明的白介素10携带沙门氏菌信号肽ompF质粒构建(带有沙门氏菌ompF信号肽的pET30a-IL10)及测序结果。
图8是本发明的白介素10携带pelB信号肽质粒构建(带有pelB信号肽的pET22b-IL10)及测序结果。
图9至图11分别是实施例4制备的4种质粒的转染细胞后的菌液PCR鉴定结果。
图12显示携带不同信号肽的IL-10在BL21(DE3)中的表达水平。BL21(DE3)携带不同信号肽的裂解上清液中的IL-10的表达水平(左)和LB培养基上清液中的表达水平 (右)。BL21(DE3)-pET22b(+)-pelB-IL-10(图中用pelB表示)，BL21(DE3)-pET30a-ompF-IL-10(图中用ompF表示)，BL21(DE3)-pET30a-IL-10(图中用eukaryon表示)。
具体实施方式
尽管可以对本发明进行各种修改并且本发明可以具有各种形式，但是下面将详细说明和解释具体实例。然而，应当理解的是，这些并不旨在将本发明限制于特定的公开内容，并且本发明包括其所有修改、等同物或替代物而不脱离本发明的精神和技术范围。
在下文中，将更详细地解释根据本发明具体实施方式的靶向肿瘤的原核递送载体及其在肿瘤治疗中的应用。
在本发明的一个或多个实施方式中，本发明的靶向肿瘤的原核递送载体是靶向实体瘤厌氧区的底盘细菌，并且包含表达细胞因子的多核苷酸，其中，所述细胞因子选自白介素6，白介素10，白介素8，白介素18，白介素33和TNF-α，优选白介素6或白介素10。并且所述多核苷酸是DNA或mRNA。
在本发明的一个或多个实施方式中，本发明应用于原核递送载体在肿瘤治疗中的应用，其中，所述原核递送载体与选自以下的治疗剂联用：化学药物，如烷基化剂(如硫代特帕和环磷酰胺)、烷基磺酸盐、阿兹环烯类药物、乙胺和甲胺、氮芥类、抗生素、嘌呤类似物、嘧啶类似物、雄酮类、抗肾上腺素、拓扑异构酶抑制剂、卡培他滨；化疗药物：调节或抑制肿瘤激素作用的抗激素药物；免疫靶向药物：吉非替尼、易瑞沙、劳拉替尼、厄洛替尼、贝伐珠单抗。所述联用为同时施用，或依次施用，或分开施用。
在本发明的一个或多个实施方式中，本发明提供了使用本发明原核递送载体治疗肿瘤的方法，所述原核递送载体是靶向肿瘤，特别是靶向实体瘤厌氧区的底盘细菌，并且包含表达细胞因子的多核苷酸，其中，所述细胞因子选自白介素6，白介素10，白介素8，白介素18，白介素33和TNF-α，优选白介素6或白介素10。并且所述多核苷酸是DNA或mRNA。
在本发明治疗方法的一个或多个实施方式中，所述原核递送载体可以与选自以下的治疗剂联用：化学药物，如烷基化剂(如硫代特帕和环磷酰胺)、烷基磺酸盐、阿兹环烯类药物、乙胺和甲胺、氮芥类、抗生素、嘌呤类似物、嘧啶类似物、雄酮类、抗肾上腺素、拓扑异构酶抑制剂、卡培他滨；化疗药物：调节或抑制肿瘤激素作用的抗激素药物；免疫靶向药物：吉非替尼、易瑞沙、劳拉替尼、厄洛替尼、贝伐珠单抗。所述联用为同 时施用，或依次施用，或分开施用。
在本发明的一个或多个实施方式中，本发明还提供了本发明的原核递送载体在制备用于治疗肿瘤的药物中的应用，其中，所述原核递送载体与选自以下的治疗剂联用：化学药物，如烷基化剂(如硫代特帕和环磷酰胺)、烷基磺酸盐、阿兹环烯类药物、乙胺和甲胺、氮芥类、抗生素、嘌呤类似物、嘧啶类似物、雄酮类、抗肾上腺素、拓扑异构酶抑制剂、卡培他滨；化疗药物：调节或抑制肿瘤激素作用的抗激素药物；免疫靶向药物：吉非替尼、易瑞沙、劳拉替尼、厄洛替尼、贝伐珠单抗。所述联用为同时施用，或依次施用，或分开施用。
在本发明的一个或多个实施方式中，所述底盘细菌选自减毒的鼠伤寒沙门氏菌(Salmonella enterica serovars Typhimurium)、减毒的人源伤寒沙门氏菌(Salmonella enterica serovars Typhi)、大肠杆菌(E.coli)和乳酸菌(Lactic acid bacteria)，优选减毒的鼠伤寒沙门氏菌。
在本发明的一个或多个实施方式中，所述多核苷酸是DNA，所述载体还包含与表达所述细胞因子的DNA融合的信号肽pelB。本发明的载体还包含肿瘤环境敏感的启动子。
在本发明的一个或多个实施方式中，所述多核苷酸是mRNA，所述mRNA还包含5’端的m7G加帽结构和3’端的多聚腺苷酸尾结构。
在本发明的一个或多个实施方式中，所述肿瘤为发现在以下部位的实体瘤：宫颈、乳腺、前列腺,胃肠道(例如食道、口咽、胃、小肠或大肠,结肠、直肠)、膀胱、骨、皮肤、头部或颈部、肝脏、胆囊、肺、胰腺、唾液腺、肾上腺、甲状腺、大脑(如神经胶质瘤)神经节，所述肿瘤优选为结肠癌、乳腺癌、黑色素瘤、肺癌、胶质母细胞瘤。
在本发明的一个或多个实施方式中，所述载体或药物通过肌肉注射、静脉注射、皮下注射、腹腔注射、经脑内施用、经口腔施用、经鼻腔施用或口服施用进行施用。
本发明的原核递送载体可采取以下方式构建并验证：(1)利用化学修饰方法修饰细胞因子mRNA；(2)利用细菌载体递送表达经化学修饰的细胞因子mRNA；(3)异源表达细胞因子的质粒载体系统的构建；(4)ELISA或Western Blot鉴定细胞因子的表达情况；(5)体外实验测试细菌分泌细胞因子的功能；(6)通过荷瘤小鼠模型测试白介素10治疗肿瘤的作用。
本发明的细胞因子mRNA在细菌中以质粒的方式存在，随着细菌递送进瘤内后，释放表达，在瘤内行使翻译功能，经化学修饰之后会显著延长时间，避免降解。
本发明的一个方面涉及到采用一种修饰体外转录白介素6或白介素10细胞因子 mRNA的方法，将其导入底盘微生物，从而通过细菌经血管进入实体瘤，实现mRNA在实体瘤内分泌及翻译过程。
具体而言，例如本发明可采用以下方式实现细胞因子mRNA的修饰和体外转录：利用细菌载体递送表达经化学修饰的细胞因子mRNA的系统构建。为了实现mRNA在体外有效翻译，本发明在体外转录合成mRNA时，掺入了5’端的m7G加帽结构和3’端的多聚腺苷酸(polyA尾)结构。加帽的过程是由T7 RNA聚合酶共转录来实现的；加尾的过程，是用Poly(A)聚合酶在mRNA分子3’端，加上poly(A)尾，再用DNase酶消化DNA模板。cmRNA经过质粒表达，电转进底盘细菌，从而实现cmRNA的跨膜分泌，随即在胞外翻译生成IL-6/IL-10蛋白质。
本发明的另一方面涉及利用信号肽融合细胞因子的方式，实现目的蛋白表达及胞外分泌。
具体而言，信号肽与细胞因子的融合和表达通过以下方式实现：细菌异源表达鼠源细胞因子IL-6和IL-10的质粒载体系统的构建。细胞因子的异源表达极易形成包涵体，为避免此问题的出现，本发明拟采用信号肽融合表达的目的基因。信号肽能够引导合成蛋白质的胞外转移。本发明采用来源于胡萝卜软腐欧文氏菌果胶裂解酶的信号肽pelB作为本发明研究。另外，本发明可选地采用6×His tag纯化标签，作为后续目的基因的标记检测。通过酶联免疫吸附实验(ELISA)检测细菌上清液中的IL-6和IL-10，由此验证IL-6和IL-10是否实现胞外分泌。
对于如上构建的经化学修饰的细胞因子mRNA的系统，和细菌异源表达鼠源细胞因子IL-6和IL-10的质粒载体系统，均可以按照以下固相酶联免疫斑点技术和/或细菌分泌细胞因子IL-6和IL-10对荷瘤小鼠治疗作用的测试方式来确定所表达细胞因子IL-6和IL-10的活性。
固相酶联免疫斑点技术(ELISPOT)体外鉴定细胞因子IL-6和IL-10的功能作用为明确细菌分泌的细胞因子IL-6和IL-10是否具备T细胞激活功能。本发明采用CD4+T细胞和CD8+T细胞体外培养，并通过固相酶联免疫斑点技术(ELISPOT)检测T细胞激活情况。同时，设计对照实验，对体外培养的CD4+T细胞和CD8+T细胞，直接使用细胞因子IL-6和IL-10刺激T细胞。
细菌分泌细胞因子IL-6和IL-10对荷瘤小鼠治疗作用的测试。为证明表达细胞因子IL-6和IL-10的减毒沙门氏菌对实体瘤的抑制作用，本发明通过构建巨噬细胞缺陷小鼠，排除巨噬细胞分泌IL-10等的因素干扰，观察小鼠的肿瘤大小并通过收集小鼠肿瘤血清， 检测TNF-α、IFN-γ等细胞因子的分泌情况。
本发明还可以采用基于pET-22b(+)和pET30a改造构建的递送系统。
本发明启动子可以是组成型启动子J23101和IPTG或aTc诱导表达启动子。
本发明的核糖体结合位点(RBS)可以是B0034，以及多种不同表达强度的RBS，包括但不限于BBa J61100、BBa J61101、BBa J61139等40余种。这些RBS可用于表征目的蛋白的表达强度。
本发明的底盘微生物可以是大肠杆菌BL21(DE3)，Nissle 1917和沙门氏菌SL7207(aroA-)等。
本发明的以上各种实施方式具有以下有益效果：
(1)应用信号肽融合表达白介素10细胞因子，应用于肿瘤治疗，开拓了一个新的应用方向；
(2)所使用的底盘微生物主要是减毒的沙门氏菌菌株，对实体瘤的杀伤效果显著；
(3)应用化学修饰白介素6或白介素10细胞因子mRNA的方法，能够有效增强细胞因子的半衰期，延长作用时间，提高治疗效果；
(4)本发明中构建的细菌载体表达细胞因子白介素6或白介素10的表达量更高。
实施例
提供以下非限制性实施例。
实施例1：IL-10 mRNA的制备和细胞内表达
采用IL-10的DNA为模板，将IL-10的DNA构建在具有T7启动子的质粒上，线性化后以其为模板，通过体外转录反应合成RNA，在合成反应中引入5’端的m7G帽子结构和3’端的多聚腺苷酸(polyA)结构，同时加入m5C，Pseudo-UTP等化学修饰碱基提高其稳定性和翻译效率。加帽的过程是在20μl体外转录体系中加入1μg的模板DNA，并加入150nmol ATP，m5C，Pseudo-UTP的同时以4:1的比例添加ARCA(ARCA:GTP＝4:1，总量仍为150nmol)，经由T7 RNA聚合酶在37摄氏度条件下体外转录6小时实现。接着用TURBO DNase消化DNA模版在37摄氏度下反应30分钟后进行加尾反应，加尾反应1小时。扩大反应体系5倍，在100μl反应体系中外源添加100nmol ATP和250nmol MnCl 2，37摄氏度反应条件下利用Poly(A)聚合酶在RNA的3’端合成多聚腺苷酸。最后通过LiCl沉淀法纯化合成的mRNA。
为了验证IL-10 mRNA在细胞中具备正常翻译的功能，将经过化学修饰的cmRNA 转染至293T细胞，转染剂量分别为每孔0.5ug和1.5ug，并以细胞孵育24h后为时间起点，在24h，36h，48h，60h分别提取细胞组织液及细胞上清液，ELISA测定IL-10蛋白的表达量，结果如图1和表1所示。表1是转染293T细胞IL-10的表达量(pg/mL)。
从图1和表1可以看出，与未转染RNA的细胞对照组相比，转染RNA的细胞组织液中的IL-10蛋白表达量显著提高，说明IL-10 cmRNA在细胞中正常翻译；上清液中的IL-10蛋白表达量处在相对较高水平，说明蛋白正常翻译并大量分泌到胞外。
表1
Figure PCTCN2020110159-appb-000002
实施例2：GFP作为目的基因对pelB信号肽的功能鉴定
(1)pSC101-pelB-GFP-kana质粒的构建
选择国际基因工程机器大赛iGEM基因元件库中的pSC101-GFP-Kana质粒(UT Austin iGEM 2019:Characterization of metabolic burden of the Anderson Series，见网址 http://parts.igem.org/wiki/index.php？title＝Part:BBa_J23101)为模板，该质粒由J23101组成型启动子启动表达目的基因GFP。通过一步克隆One Step-Cloning的方式，设计正反向引物扩增出信号肽pelB以及骨架片段，并根据同源重组的方式，将pelB片段插入质粒，得到pSC101-pelB-GFP-kana质粒，如下图2所示。图2(左)为原始质粒模板，pSC101-GFP-Kana；图(右)为插入信号肽的质粒，pSC101-pelB-GFP-Kana。
(2)pelB信号肽在RNA水平上的功能鉴定
将上述构建的质粒电转到减毒的鼠伤寒沙门氏菌Salmonella Typhimurium(参考减毒沙门氏菌YB1，有Chang-Xian Li等公开于Oncology Letters，2017年1月Volume 13 Issue 1，DOI:10.3892/ol.2016.5453)中，在RNA水平测试GFP的表达情况。其中，对于GFP表达的RNA水平分析，借助Real-time PCR测试，设计Real-time PCR正反向引物，qP-pelB-F1/R1，序列为GGCAGCCGCTGGATTGTTAT/TGTTGCATCACCTTCACCCT，Tm值为60度；选择SL7207(aroA-)和转入原始模板质粒的SL7207为对照组进行试验，并同时选择反转录前的样品作为对照组，避免由于基因组DNA未处理干净而造成的误差。图3显示 了GFP在RNA水平上的表达情况。结果如图3所示，Real-time PCR测试时，选择了rho作为内参基因，计算得出样本结果数据与rho内参基因的比值，结果如下：电转质粒PSC101-pelB-GFP-Kana的细菌中，GFP在mRNA水平高表达，SL7207和转入原始模板质粒的SL7207的对照组均无mRNA表达。
实施例3：IL-6和IL-10作为目的基因，质粒递送系统的构建
选择上述说明的的pSC101-pelB-GFP-Kana质粒为模板，反向设计正反向引物，正向引物序列是：5’-GTCGACACTTAATTAACGGCA-3’；反向引物序列：5’-CTCGAGATTTCTCCTCTTTCG-3’扩增骨架载体；鼠源IL-10基因扩增自Puc-IL-10-AMP质粒，正向引物序列是：5’-GAAAGAGGAGAAATCTCGAGATGA-3’；反向引物序列：5’-GCCGTTAATTAAGTGTCGACAC-3’。通过一步克隆One Step-Cloning的方式，将目的片段插入进骨架中，得到pSC101-pelB-IL-10-kana质粒。
PCR高保真扩增酶使用Takara公司产品PrimerSTAR MAX Premix(2×)，dNTP浓度0.4mM(2×)，PCR反应条件如下：
Figure PCTCN2020110159-appb-000003
pSC101-pelB-IL-6-kana构建方式同理所述，如图4所示。
实施例4：带有ompF信号肽的pET30a-IL10和带有pelB信号肽的pET22b-IL10的构建、转化和表达
构建：分析IL-10的蛋白序列，发现IL-10前22个氨基酸是一个真核信号肽，为促进IL-10在原核载体的成功表达及跨膜递送，申请人原核信号肽pelB和ompF将原真核序列信号肽替换掉，并采用诱导型质粒表达载体提高目的蛋白的可溶表达量，成功构建质粒：带有ompF信号肽的pET30a-IL10和带有pelB信号肽的pET22b-IL10。pET30a和 pET22b是常用的IPTG诱导表达载体。构建的质粒见图5-图8。
转化：分别将上述质粒电转入BL21(DE3)和减毒沙门氏菌ZG1。分别将质粒带有真核信号肽的pET30a-IL10构建在BL21(DE3)和ZG1中能够实现正常表达IL-10的菌株；另外，将带有E.coli ompF信号肽的pET30a-IL10和带有沙门氏菌ompF信号肽的pET30a-IL10构建在BL21(DE3)和ZG1中实现IL-10的表达及胞质分泌；以及将带有pelB信号肽的pET22b-IL10质粒构建在BL21(DE3)和ZG1中实现IL-10的表达及胞质分泌。菌液PCR鉴定结果见图9-图11。
分别将携带pET30a-ompF-IL10和pET22b(+)-pelB-IL10质粒的大肠杆菌BL21(DE3)以及用作对照的携带带有真核信号肽的pET30a-IL10的的大肠杆菌BL21(DE3)用LB培养基扩大体系培养，培养体积4ml，当细菌培养到对数期后期的时候(OD值0.6左右)，向其中分别加入终浓度为1/0.5/0毫摩的IPTG，16℃诱导表达过夜(24h)，收集菌体和LB培养液，菌体用蛋白裂解液裂解完全，14000g离心30分钟，分别收集裂解液上清液和LB培养基上清液。通过免疫荧光吸附实验ELISA定量分析白介素10的表达情况。结果见图12。结论：说明IL-10在pelB和OmpF信号肽的作用下，实现可溶蛋白的表达水平在70-80ng/(10 8CFU)。

Claims (10)

  1. 一种靶向肿瘤的原核递送载体，所述载体是靶向实体瘤厌氧区的底盘细菌，并且包含表达细胞因子的多核苷酸，其中，
    所述细胞因子选自白介素6，白介素10，白介素8，白介素18，白介素33和TNF-α，并且
    所述多核苷酸是DNA或mRNA。
  2. 如权利要求1所述的原核递送载体，所述底盘细菌选自减毒的鼠伤寒沙门氏菌、减毒的人源伤寒沙门氏菌、大肠杆菌和乳酸菌。
  3. 如权利要求1或2所述的原核递送载体，所述多核苷酸是DNA，所述载体还包含与表达所述细胞因子的DNA融合的信号肽pelB和OmpF。
  4. 如权利要求1或2所述的原核递送载体，所述多核苷酸是mRNA，所述mRNA还包含5’端的m7G加帽结构和3’端的多聚腺苷酸尾结构。
  5. 如权利要求1至3中任一项所述的原核递送载体，当所述多核苷酸是DNA时，所述原核递送载体包含肿瘤环境敏感的启动子，以及其他启动子，所述其他启动子为组成型启动子和诱导型启动子，所述诱导型启动子例如为IPTG诱导型启动子或aTc诱导型启动子。
  6. 如权利要求1至5中任一项所述的原核递送载体，所述肿瘤为发现在以下部位的实体瘤：宫颈、乳腺、前列腺、肠道、膀胱、骨、皮肤、头部或颈部、肝脏、胆囊、肺、胰腺、唾液腺、肾上腺、甲状腺、大脑神经节，所述肿瘤优选为结肠癌、乳腺癌、黑色素瘤、肺癌、胶质母细胞瘤。
  7. 如权利要求1至6中任一项所述的原核递送载体，所述载体通过肌肉注射、静脉注射、皮下注射、腹腔注射、经脑内施用、经口腔施用、经鼻腔施用或口服施用进行施用。
  8. 权利要求1至7中任一项所述的原核递送载体在肿瘤治疗中的应用，其中，所述原核递送载体与选自以下的治疗剂联用：化学药物、化疗药物、免疫靶向药物。
  9. 如权利要求8所述的应用，其中，所述化学药物为烷基化剂、烷基磺酸盐、阿兹环烯类药物、乙胺和甲胺、氮芥类、抗生素、嘌呤类似物、嘧啶类似物、雄 酮类、抗肾上腺素、拓扑异构酶抑制剂、卡培他滨；所述化疗药物为调节或抑制肿瘤激素作用的抗激素药物；所述免疫靶向药物为吉非替尼、易瑞沙、劳拉替尼、厄洛替尼、贝伐珠单抗。
  10. 如权利要求9所述的应用，其中，所述烷基化剂为硫代特帕和环磷酰胺。
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