WO2022270946A1 - Vecteur recombinant pour améliorer l'expression d'une protéine d'intérêt par suppression de la réponse immunitaire intracellulaire et application associée - Google Patents

Vecteur recombinant pour améliorer l'expression d'une protéine d'intérêt par suppression de la réponse immunitaire intracellulaire et application associée Download PDF

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WO2022270946A1
WO2022270946A1 PCT/KR2022/008954 KR2022008954W WO2022270946A1 WO 2022270946 A1 WO2022270946 A1 WO 2022270946A1 KR 2022008954 W KR2022008954 W KR 2022008954W WO 2022270946 A1 WO2022270946 A1 WO 2022270946A1
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expression
cells
target protein
recombinant vector
vector
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김태돈
이수연
공링주
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한국생명공학연구원
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/4613—Natural-killer cells [NK or NK-T]
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    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
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Definitions

  • the present invention relates to a recombinant vector and its application for enhancing the expression of a target protein by suppressing an immune response in a host cell.
  • Non-Patent Document 1 Cyclic GMP-AMP synthase (cGAS) is activated by binding to cytosolic DNA, and the activated cGAS generates 2'3'-cGAMP (2'3'-cyclic guanosine monophosphate-adenosine monophosphate), resulting in an innate immune response (innate immune responses) are induced (Non-Patent Document 1).
  • the innate immune response occurs even when cells are damaged by various causes. In particular, when mitochondrial DNA is leaked into the cytoplasm due to mitochondrial damage, apoptosis is induced by STING pathway activation as in the case of viral invasion ( Non-Patent Document 2).
  • the cGAS pathway is presumed to be related to apoptosis of cell lines that occurs during the development and production of cell therapy products.
  • CAR chimeric antigen receptor
  • the inventors of the present invention developed a new vector system capable of studying the intact function of a target gene, and furthermore, researched intensively to increase the survival rate of cells used as cell therapy products, and to improve host cell growth by injecting ex vivo genes.
  • the present invention was completed by blocking activation of the immune response and cell death signaling.
  • Non-Patent Document 1 Ni et al. , PLoS Pathog. , 14 (8):e1007148 (2016)
  • Non-Patent Document 2 Bai et al. , Diabetes, 68 (6):1099-1108 (2019)
  • Non-Patent Document 3 Eaglesham et al. , Nature, 566 (7743):259-263 (2019)
  • Non-Patent Document 4 Xia et al. , Immunity, 48 (4): 688-701 (2018)
  • One object of the present invention is to provide a recombinant vector capable of enhancing the expression of a target protein by suppressing the innate immune response in a host cell.
  • Another object of the present invention is to provide a recombinant vector for improving the viability or production efficiency of transformants.
  • Another technical problem of the present invention is to provide cells transformed with the vector to enhance the effects of gene therapy agents and cell therapy agents.
  • one aspect of the present invention provides a first promoter and a multiple cloning site (MCS) operably linked to the first promoter and inserting a gene encoding a target protein.
  • MCS multiple cloning site
  • a first gene construct comprising; and a second gene construct comprising a gene encoding a substance that blocks or inhibits the activation of a stimulator of interferon genes (STING) pathway in a host cell.
  • STING interferon genes
  • a substance that blocks or inhibits the activation of the STING pathway may be Poxin (poxvirus immune nucleases) or cia-cGAS RNA.
  • the vector may reduce apoptosis of the host cell by suppressing the innate immune response in the host cell.
  • the vector may reduce the expression of a cytokine that appears when a gene encoding the target protein is introduced or expressed in the host cell.
  • another aspect of the present invention provides a transformant in which the recombinant vector for enhancing the expression of the target protein is introduced into a host cell.
  • another aspect of the present invention provides a cell therapy agent comprising the transformant as an active ingredient.
  • the vector of the present invention can suppress cytokine expression in the host cell to improve the expression of the target protein, and also suppress apoptosis of the host cell to increase the survival rate of the transformant. Therefore, when the vector of the present invention is used, a transformant having high expression efficiency of the target protein can be obtained with a high survival rate, and finally, the expression of the target protein can be further improved.
  • the transformant prepared using the vector of the present invention not only has a high expression rate of the target protein, but also has a high survival rate, so it can show long-term effects in vivo.
  • it since it has the advantage of being easy to store and distribute, it can be usefully used as an active ingredient in cell therapy products.
  • FIG. 1 is a schematic diagram showing the structure of a recombinant vector for enhancing the expression of a target protein according to an embodiment of the present invention.
  • FIG. 2 is a schematic diagram showing the structure of a recombinant vector for enhancing the expression of a target protein according to another embodiment of the present invention.
  • Figure 3 shows the result of confirming the effect of the introduction of a foreign gene on the activation of the STING pathway and the expression of the target protein.
  • Figure 4 shows the results of confirming the effect of activation of the STING pathway on cell apoptosis of host cells.
  • Figure 5 shows the result of confirming the expression change of the target protein in the host cell according to the treatment of H-151, a STING pathway inhibitor.
  • Figure 6 shows the result of confirming the effect of Poxin expression on the expression of the target protein in the host cell.
  • Figure 7 shows the results confirming the effect of Poxin expression on the activation of the STING pathway in the host cell.
  • Figure 8 shows the result of confirming the successful production of Poxin-NK92 cells, which are NK cells expressing Poxin, and changes in the STING pathway in the Poxin-NK92 cells.
  • Figure 9 shows the results of measuring apoptosis and cytotoxicity after doxorubicin or radiation treatment to the Poxin-NK92 cells.
  • Figure 12 shows the results of confirming changes in the STING pathway by human cia-cGAS.
  • the 'target protein' referred to in the present invention may be any protein to be expressed in a host cell through the vector of the present invention.
  • the protein of interest of the present invention may be an antigen, a fragment of an antigen, a hormone, a hormone analogue, an enzyme, an enzyme inhibitor, a receptor, a fragment of a receptor, an antibody, a monoclonal antibody, a fragment of an antibody, a structural protein, a toxin protein, or any of these It may be a variant, or a protein in which some or all of them are fused.
  • the 'host cell' referred to in the present invention refers to a cell capable of utilizing its own transcription system by introducing the vector of the present invention into the inside in order to express the target protein, and any known host cell having a STING pathway. It can be any kind of cell.
  • 'promoter' in the present invention refers to a DNA sequence that regulates the expression of an operably linked gene in a specific host cell.
  • a possible enhancer may be further included.
  • a 'vector' refers to a DNA product in which essential elements capable of expressing a gene in a host cell are operably linked.
  • Suitable vectors include expression control sequences such as promoters, operators, initiation codons, stop codons, polyadenylation signals and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and may be prepared in various ways depending on the purpose.
  • One aspect of the present invention provides a recombinant vector for enhancing the expression of a target protein.
  • the recombinant vector of the present invention includes a first gene construct including a gene encoding a substance that blocks or inhibits the activation of a stimulator of interferon genes (STING) pathway in a host cell.
  • STING interferon genes
  • the STING pathway is a signaling pathway for cell innate immunity.
  • an external genome such as a viral nucleic acid
  • cGAS recognizes the foreign nucleic acid.
  • cGAS combined with foreign nucleic acid generates cGAMP, which activates STING and induces apoptosis and cytokine expression.
  • Substances that block or inhibit the activation of the STING pathway in the host cells are specifically substances that inhibit the expression of cGAS, antagonists that bind to cGAS to inhibit activity, and substances that degrade 2'3'-cGAMP produced by cGAS. It may be the like, but is not limited thereto, and any substance that inhibits the activation of the STING pathway is included.
  • Substances that block or inhibit activation of the intracellular STING pathway may be, for example, Poxin (poxvirus immune nucleases) or cia-cGAS RNA.
  • the substance that blocks or inhibits the activation of the STING pathway in the host cell is a foreign gene expressed in the host cell, but this is for increasing the survival rate of the host cell and enhancing the expression of the target protein, which is the target protein to be improved in the present invention. is distinguished from
  • the poxin protein is derived from smallpox virus, and signaling through the STING pathway can be restricted by cutting 2'3'-cGAMP.
  • the circular RNA (circRNA) known as Cia-cGAS binds to cGAS and suppresses the activation of cGAS by foreign nucleic acids exposed to the cytoplasm, thereby blocking the STING pathway similarly to poxin.
  • the first gene construct may further include a first promoter capable of controlling the expression of a gene encoding a substance that blocks or inhibits the activation of the STING pathway in the host cell.
  • a first promoter any known promoter may be used as long as it can control the expression of a gene encoding a substance that blocks or inhibits the activation of the STING pathway in the host cell.
  • a gene encoding a substance that blocks or inhibits activation of the STING pathway in the host cell may be operably linked to the first promoter.
  • the operably linked means that the expression of a gene encoding a substance that blocks or inhibits the activation of the STING pathway in the host cell is regulated by the first promoter.
  • the first gene construct may further include an internal ribosome entry site (IRES) region upstream of a gene encoding a substance that blocks or inhibits activation of the STING pathway in the host cell.
  • IRES region is an internal ribosome entry site or a ribosome binding site, and forms a loop structure on mRNA to initiate translation of mRNA.
  • a gene encoding a substance that blocks or inhibits activation of the STING pathway in the host cell is transcribed into mRNA and then the efficiency of the translation process can be further improved. Therefore, the IRES region can be used to further enhance the expression of a protein such as Poxin, which blocks or inhibits the activation of the STING pathway in the host cell.
  • the recombinant vector of the present invention can be used to enhance the expression of a target protein or to improve the survival rate or production efficiency of a transformant.
  • a gene encoding the target protein or a vector containing the gene is introduced into a host cell to express the target protein, the gene or vector itself is recognized as an exogenous nucleic acid exposed to the cytoplasm of the host cell and activates cGAS. and, eventually, by activating the STING pathway, the innate immune response of the host cell can be induced.
  • a substance that blocks or inhibits the activation of the STING pathway in the host cell is expressed from the second gene construct, thereby blocking or inhibiting the activation of the STING pathway by introduction of foreign nucleic acid in the host cell. Inhibition not only reduces the expression of cytokines in the host cell, but also can reduce apoptosis of the host cell.
  • the signal transduction by cGAMP induced by the foreign nucleic acid as described above has a very strong crosstalk with other signal transduction pathways, it is very likely to interfere with the expression of the target protein.
  • the recombinant vector of the present invention blocks or inhibits the activation of the STING pathway and thus reduces the cross-talk caused by cGAMP signaling, the target protein can be expressed without interference by other factors. Therefore, when using the recombinant vector of the present invention, the role of the target protein in cells and organisms can be accurately analyzed.
  • the target protein may be expressed in the recombinant vector of the present invention by including the gene encoding the target protein in the recombinant vector of the present invention, but is not limited thereto, and is separate from the recombinant vector of the present invention. It can be introduced into the host cell by a vector of or by another method that is distinguished.
  • the recombinant vector of the present invention includes a multiple cloning site (MCS) It may further include a second gene construct.
  • MCS multiple cloning site
  • the multi-cloning site is a site into which a gene encoding a target protein to be expressed through the vector of the present invention is inserted, contains a number of restriction enzyme recognition sites, and is generally used during a process including cloning or subcloning.
  • Restriction enzyme recognition sites in the multi-cloning site can be configured in various ways according to the purpose, and due to the flexibility of such restriction enzyme recognition sites, cloning of target genes for various uses may be possible.
  • the second gene construct may further include a second promoter for regulating expression of a gene encoding a target protein inserted into the multiple cloning site.
  • a second promoter for regulating expression of a gene encoding a target protein inserted into the multiple cloning site.
  • the second promoter any known promoter may be used as long as it can control the expression of the gene encoding the target protein inserted into the multiple cloning site.
  • the multiple cloning site may be operably linked to the first promoter.
  • the multi-cloning site is operably linked to the first promoter, it means that the expression of the gene encoding the target protein inserted into the multi-cloning site is regulated by the first promoter. Therefore, in the case of the first gene construct, even if a gene encoding a target protein is inserted anywhere in the multiple cloning site, its expression can be controlled by the first promoter.
  • the vector may be introduced into cells by a method such as transfection or transduction.
  • Transfection is performed by calcium phosphate-DNA co-precipitation method, DEAE-dextran-mediated transfection method, polybrene-mediated transfection method, electroporation method, microinjection method, liposome fusion method, lipofectamine and protoplast fusion method, etc. This can be done by several methods known in the art.
  • transfection means the transfer of a gene into a cell using a virus or viral vector particles by infection (infection).
  • transfection and transduction may be used interchangeably, but both are preferably interpreted as transformation of transfer of a foreign gene to a host cell in a broad sense, and cells into which a foreign gene has been introduced by transformation or transduction is called a transformant.
  • the present invention provides a composition comprising the recombinant vector for confirming the function of a target protein or for improving the production efficiency of an immune cell therapeutic agent.
  • the recombinant vector included in the composition of the present invention is as described above. Since the vector can suppress the response by cGAMP signaling generated by the innate immune response of the host cell, the composition of the present invention can be used to investigate the function of a target protein or to enhance the viability of the host cell.
  • the present invention provides a transformant or cell transformed with the recombinant vector.
  • the transformant is characterized in that the STING pathway activation is blocked, cell death and cytokine expression are suppressed, and the innate immune response of the host cell itself is suppressed, and the survival rate is improved.
  • the host cell may be an immune cell, and the transformant may be provided as an immune cell therapy agent.
  • the host cells may be immune cells (eg, T cells, NK cells, macrophages, dendritic cells, etc.).
  • cell therapy product refers to cells and tissues manufactured through isolation, culture, and special manipulation from a subject, and is a drug (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention, and is used to restore the function of cells or tissues. It refers to drugs used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferation and selection of living autologous, allogeneic, or heterogeneous cells in vitro or changing the biological characteristics of cells in other ways.
  • the cell therapy agent of the present invention may be used for anticancer or infectious disease treatment or prevention, but is not limited thereto, and all possible uses of general cell therapy agents may be applied.
  • Anticancer agents used in chemotherapy induce apoptosis of proliferating cells, and radiation to enhance the therapeutic effect of anticancer agents increases such apoptosis.
  • cells whose apoptosis is induced by anticancer agents and radiation are not limited to cancer cells, and may also affect immune cell therapy agents administered to individuals for immunotherapy.
  • cell therapy products are injected into a subject to be treated after radiation treatment of cells for reasons such as suppression of cancer-inducing factors, and there is a high possibility that apoptosis of cell lines, which are active ingredients of cell therapy products, is induced by such radiation treatment
  • NK cells were transformed with the vector of the present invention and treated with doxorubicin or radiation in order to confirm whether the immunocytotherapeutic agent injected with the poxin or Cia-cGAS RNA expression vector can be combined with chemotherapy and radiation therapy. After irradiation, the degree of apoptosis, viability, and cytotoxicity of NK cells were confirmed. As a result, NK cells whose cGAS-STING pathway was blocked by poxin or Cia-cGAS RNA showed high survival rates despite doxorubicin treatment and irradiation. It was confirmed that the cytotoxicity to cancer cells was maintained.
  • the immunotherapeutic agent has resistance to anticancer agents (eg, doxorubicin, etc.) and radiation that induce apoptosis, and is administered in combination with known anticancer agents or combined with radiation therapy. It can be provided as a treatment method.
  • anticancer agents eg, doxorubicin, etc.
  • the host cell may express a chimeric antigen receptor (CAR). That is, the target protein additionally provided to the vector of the present invention may be a CAR.
  • the CAR may be contained within the vector of the present invention, or may be expressed in immune cells by a vector separate from the vector of the present invention or by other distinct methods. Since a gene is introduced from outside the immune cell for the expression of the CAR, the introduction of such a foreign gene can induce the apoptosis pathway of the immune cell, but the present invention blocks it to inhibit apoptosis, thereby increasing the survival rate of CAR-immune cells can make it
  • the transformants may be specifically CAR-T cells or CAR-NK cells with increased viability.
  • the present invention provides a method for preparing a transformant with increased viability and a method for increasing the viability of a transformant, comprising the step of injecting the vector into a host cell.
  • the present invention provides a method for preparing an immune cell therapy product with increased survival rate, comprising the step of injecting the vector into immune cells.
  • the present invention provides a pharmaceutical composition for treating cancer comprising the CAR-T cells or CAR-NK cells.
  • a cancer treatment method comprising administering the CAR-T cells or CAR-NK cells to a subject.
  • the method may further include administering a known anticancer agent to the subject and/or irradiating the subject with radiation.
  • each of the above steps may be performed sequentially or simultaneously, and there is no limitation on the precedence or precedence of each step.
  • the present invention provides a use of the vector for producing a cell therapy product.
  • Anticancer agents used in chemotherapy induce apoptosis of proliferating cells, and radiation to enhance the therapeutic effect of anticancer agents increases such apoptosis.
  • cells whose apoptosis is induced by anticancer agents and radiation are not limited to cancer cells, and may also affect immune cell therapy agents administered to individuals for immunotherapy.
  • the present inventors transformed NK cells with the vectors to confirm that immunocytotherapeutic agents injected with poxin or Cia-cGAS RNA expression vectors can be combined with chemotherapy and radiation therapy, and apoptosis after doxorubicin treatment or irradiation. The degree, viability, and cytotoxicity of NK cells were confirmed. As a result, it was confirmed that NK cells in which the cGAS-STING pathway was blocked by poxin or Cia-cGAS RNA exhibited a high survival rate despite doxorubicin treatment and irradiation and maintained cytotoxicity to cancer cells.
  • the present invention provides a method for preparing a transformant with increased viability using the vector, and provides an immune cell therapy agent that can be combined with chemotherapy and/or radiation therapy by injecting the vector into immune cells.
  • a pEGFP-N1 vector (Addene, USA) was purchased and prepared.
  • an IRES-Kozak-FLAG-Poxin gene construct having the nucleotide sequence of SEQ ID NO: 1 between the EcoR I restriction enzyme site and the Xba I restriction enzyme site of the pcDNA3.1 TM /HisA vector (Invitrogen, USA) multi-cloning site
  • a pcN-IP vector expressing Poxin was constructed by inserting each of the vectors.
  • pSK expressing cia-cGAS by inserting the human cia-cGAS gene having the nucleotide sequence of SEQ ID NO: 2 into the Xba I restriction enzyme site and the Not I restriction enzyme site of the multiple cloning site of the pBluescript SK vector (Addene, USA) -hcia-cGAS vectors were respectively constructed.
  • FIGS. 1 and 2 Detailed vector drawings, related sequences, and protein expression confirmation of the recombinant vectors are shown in FIGS. 1 and 2 .
  • THP1 cells Korea Cell Line Bank
  • HEK293T cells ATCC
  • -1 was introduced in an amount of 0, 1, 2, 4, 6, and 8UG and cultured for 24 hours, and then changes in the STING pathway were confirmed through the expression of proteins on the STING pathway.
  • Type 1 IFN a sub-signal system of the STING pathway
  • the pEGFP-N1 vector was prepared in 0, 1, and 2UG amounts. In each of the cells introduced into and cultured for 24 hours, activation of the STING pathway was confirmed once again, and changes in apoptosis-related factors were also confirmed.
  • the introduction of the pEGFP-N1 vector not only activated the STING pathway only in THP1 cells in which the STING pathway existed, as was confirmed in Example [2-1], but also activated the STING pathway in the cells. It was confirmed that the expression of Bim, P21, Pim1, Noxa, and PARP, which are apoptotic factors, increased, and the expression of Bcl-xl, an anti-apoptotic factor, decreased.
  • the expression level of GFP according to the presence or absence of the treatment was measured using the STING inhibitor H-151.
  • the pEGFP-N1 vector was introduced into THP1 cells in an amount of 1 UG, treated with 1 uM H-151, cultured for 24 hours or 48 hours, and then the STING pathway was changed.
  • the expression level of the target protein, GFP was confirmed.
  • the STING pathway which was activated by the introduction of foreign genes, was inhibited by the treatment of H-151, and accordingly, the expression level of the target protein, GFP, was also increased (No. 3 in FIG. 5). , lane 4 and lanes 7 and 8), it was confirmed that the expression level of the target protein, GFP, gradually increased over time as the STING pathway was inhibited as described above (lanes 4 and 8 in FIG. 5). .
  • the pEGFP-N1 vector prepared in Example [1-1] was introduced into THP1 cells in an amount of 2 UG, while the pcN-IP vector prepared in Example [1-2] was used as an empty vector, pcDNA3.1 TM /HisA vector was combined and introduced together in a total amount of 4 UG, cultured for 48 hours, and then the expression level of the target protein, GFP, was confirmed.
  • the pcN-IP vector prepared in Example [1-2] and the pcN-IP vector prepared by changing the Poxin gene in the pcN-IP vector to the gene of a Poxin (H17A) variant in which Poxin activity was removed were used in THP1 cells.
  • -ImutP vectors were introduced in an amount of 2 UG, respectively, and cultured for 12, 24, 48, and 72 hours, and changes in the STING pathway were respectively confirmed.
  • the poxin gene having the nucleotide sequence of SEQ ID NO: 3 was inserted into the pLVX-EF1 ⁇ -IRES-ZsGreen1 vector (Clonetech, USA) and introduced into NK92 cells (ATCC) to construct transformed NK92 cells expressing Poxin.
  • Example [1-1] the pEGFP-N1 vector used in Example [1-1] was introduced into the transformed Poxin-NK92 cells in an amount of 0, 5, and 10 UG, cultured for 48 hours, and changes in the STING pathway were observed. Confirmed.
  • NK92 cells expressing Poxin and NK92 cells not expressing Poxin were treated with doxorubicin at different concentrations, cultured for 24 hours and 48 hours, and each cell was then treated with AnnexinV and 7AAD, and using flow cytometry, the degree of apoptosis of each cell was confirmed.
  • NK92 cells that do not express Poxin show an increased degree of apoptosis in a doxorubicin concentration-dependent manner after 24 hours of doxorubicin treatment, whereas NK92 cells that express Poxin (poxin-NK92) doxorubicin After 48 hours of treatment, apoptosis was increased, and the degree was also confirmed to be less than that of NK92 cells that do not express Poxin.
  • NK92 cells that do not express Poxin and NK92 cells that express Poxin were irradiated with 10 Gy of ⁇ -rays, and the degree of apoptosis of cells over time was confirmed.
  • NK92 cells that do not express Poxin show increased apoptosis over time from 1 day after ⁇ -irradiation, whereas NK92 cells that express Poxin (poxin-NK92) There was no difference in apoptosis after 1 day and 2 days, and it was confirmed that the degree was less than that of NK92 cells that do not express Poxin.
  • cytotoxicity of NK92 cells not expressing Poxin and NK92 cells expressing Poxin (poxin-NK92) to K562 cells was compared.
  • calcein-stained K562 cells were used as target cells, and the NK cells and the K562 cells were used at ratios of 10: 1, 5: 1, 2.5: 1, 1.25: 1 and 0.625: 1 ( After reacting for 4 hours with E: T), 100ul of the supernatant was taken to confirm the amount of calcein present in the supernatant.
  • calcein-stained K562 cells were treated with only RPMI1640 (10% FBS) (spontaneous value) and 2% Triton X-100 (maximum value), and the degree of cytotoxicity was as follows. calculated in the same way.
  • Poxin-expressing NK92 cells maintained higher cytotoxicity than Poxin-non-expressing NK92 cells even after ⁇ -ray irradiation.
  • a single cell of NK cells (Cot-CAR-NK cells) expressing an anti-cotinine chimeric antigen receptor prepared in Korean Patent No. 2,122,546 was isolated by automated high-speed flow cytometry (FacsAria Fusion), and the isolated cells (clone No.: M2) was transformed to express Poxin in the same manner as in Example [3-2] (Poxin-M2 cells). Transformed Poxin-M2 cells and control M2 cells that do not express Poxin were respectively irradiated with ⁇ -rays, and NK cell cytotoxicity was confirmed over time.
  • Example [3-3] In order to confirm cytotoxicity, the same calcein method as in Example [3-3] was used, and the NK cells and the K562 cells were 5: 1, 1: 1 and 0.5: After reacting for 4 hours at a ratio of 1 (E:T), the degree of cytotoxicity was evaluated.
  • Poxin-expressing M2 cells (Poxin-M2 cells) have high cytotoxicity for a longer period of time despite ⁇ -ray irradiation compared to M2 cells that do not express Poxin. was confirmed to be maintained.
  • Human cia-cGAS mRNA was prepared by in vitro transcription using the pSK-hcia-cGAS vector prepared in Example [1-2]. 5ug of the mRNA obtained as above was introduced into NK92 cells together with 8ug of the pEGFP-N1 vector prepared in Example [1-1], and the mRNA levels of elements related to the STING pathway were measured in the transformed NK92 cells. did At this time, as a control for cia-cGAS, antisense cia-cGAS (AS cia-cGAS) having the nucleotide sequence of SEQ ID NO: 4 prepared in the same way as the human cia-cGAS mRNA was used.
  • AS cia-cGAS antisense cia-cGAS having the nucleotide sequence of SEQ ID NO: 4 prepared in the same way as the human cia-cGAS mRNA was used.

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Abstract

La présente invention concerne un vecteur recombinant pour améliorer l'expression d'une protéine d'intérêt, le vecteur recombinant comprenant une première construction de gène contenant un gène codant pour un matériau pour bloquer ou supprimer l'activation du stimulateur de la voie de gènes d'interféron (STING) dans des cellules hôtes, ce qui permet d'améliorer l'expression d'une protéine d'intérêt par la suppression de l'activation de la voie de STING dans les cellules hôtes et d'améliorer le taux de survie des cellules hôtes.
PCT/KR2022/008954 2021-06-23 2022-06-23 Vecteur recombinant pour améliorer l'expression d'une protéine d'intérêt par suppression de la réponse immunitaire intracellulaire et application associée WO2022270946A1 (fr)

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KR20170053500A (ko) * 2015-11-06 2017-05-16 한국생명공학연구원 목적 단백질 생산을 위한 발현 벡터 및 이를 이용한 목적 단백질의 과발현 방법
WO2019035901A1 (fr) * 2017-08-15 2019-02-21 University Of Miami Compositions et méthodes pour la modulation de la protéine sting
KR20190123257A (ko) * 2016-11-28 2019-10-31 아라팅가.바이오 에이아이오 Sting 경로를 활성화하는 유전자 보조제의 발현을 위한 바이러스 벡터 작제물

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KR20170053500A (ko) * 2015-11-06 2017-05-16 한국생명공학연구원 목적 단백질 생산을 위한 발현 벡터 및 이를 이용한 목적 단백질의 과발현 방법
KR20190123257A (ko) * 2016-11-28 2019-10-31 아라팅가.바이오 에이아이오 Sting 경로를 활성화하는 유전자 보조제의 발현을 위한 바이러스 벡터 작제물
WO2019035901A1 (fr) * 2017-08-15 2019-02-21 University Of Miami Compositions et méthodes pour la modulation de la protéine sting

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