WO2019047962A1 - VECTEUR D'EXPRESSION GÉNIQUE POUR RÉGULER L'ACTIVITÉ NF-κB DANS UNE CELLULE ET PROCÉDÉ DE RÉGULATION ASSOCIÉ ET APPLICATION CORRESPONDANTE - Google Patents

VECTEUR D'EXPRESSION GÉNIQUE POUR RÉGULER L'ACTIVITÉ NF-κB DANS UNE CELLULE ET PROCÉDÉ DE RÉGULATION ASSOCIÉ ET APPLICATION CORRESPONDANTE Download PDF

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WO2019047962A1
WO2019047962A1 PCT/CN2018/104928 CN2018104928W WO2019047962A1 WO 2019047962 A1 WO2019047962 A1 WO 2019047962A1 CN 2018104928 W CN2018104928 W CN 2018104928W WO 2019047962 A1 WO2019047962 A1 WO 2019047962A1
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gene expression
activity
mirna
cells
expression vector
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王进科
汤缓缓
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东南大学
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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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  • the invention belongs to the field of biotechnology, and particularly relates to a gene expression vector for regulating NF- ⁇ B (kappaB) activity in a cell, a method and a method for regulating the same.
  • Nuclear factor kappaB is an important class of regulatory transcription factors that play key roles in many physiological and pathological processes such as immunity, cell proliferation, apoptosis, inflammation, and especially tumorigenesis.
  • NF- ⁇ B nuclear factor kappaB
  • Several studies have shown that many diseases are associated with abnormal activation of NF- ⁇ B and its signaling pathways.
  • NF- ⁇ B is abnormally activated in many human cancers, and promotes survival and malignancy by up-regulating anti-apoptotic genes. It has been found that nuclear aggregation of NF- ⁇ B and high NF- ⁇ B target gene characteristics result in the enrichment of the NF- ⁇ B pathway in most multiple myeloma cell lines and are sensitive to apoptosis. Therefore, many pharmaceutical companies and scientists have been working to develop NF- ⁇ B inhibitors for cancer treatment.
  • Chemical drugs are an important area for the development of inhibitors of NF- ⁇ B activity.
  • NF- ⁇ B activity E.g. Kim et al. treated primary mouse microglia and BV2 microglia with a novel synthetic compound, MCAP. Then induced incubation with LPS, analysis of inducible nitric oxide synthase (iNOS), COX-2, pro-inflammatory cytokines, NF- ⁇ B and p38 MAPK signaling molecules in cells by RT-PCR, Western blot and ELISA. Expression and observation of morphological changes of microglia and nuclear translocation of NF- ⁇ B using phase contrast fluorescence microscopy.
  • iNOS inducible nitric oxide synthase
  • COX-2 pro-inflammatory cytokines
  • NF- ⁇ B and p38 MAPK signaling molecules in cells by RT-PCR, Western blot and ELISA. Expression and observation of morphological changes of microglia and nuclear translocation of NF- ⁇ B using phase contrast
  • MCAP can inhibit the expression of iNOS and COX-2 induced by LPS, indicating that MCAP exerts anti-inflammatory effects by inhibiting NF- ⁇ B signaling pathway, p38 MAPK signaling pathway and pro-inflammatory response.
  • these chemicals have low specificity.
  • the decoy strategy and the RNA interference strategy belong to the use of gene therapy to inhibit the activity of NF- ⁇ B in cells.
  • the decoy strategy refers to the synthesis of a double-stranded DNA sequence that is consistent with the sequence of the cis-regulatory element of NF- ⁇ B. After introduction into the cell, the DNA sequence can specifically bind to the activated NF- ⁇ B.
  • the activated NF- ⁇ B can be prevented from entering the nucleus and the specific binding of the NF- ⁇ B binding site to the target gene can be reduced, thereby inhibiting the expression of the target gene by inhibiting NF- ⁇ B activity.
  • the decoy sequence regulates the expression of NF- ⁇ B-related target genes by inhibiting the activity of NF- ⁇ B, leading to osteoclast apoptosis.
  • the use of synthetic decoys can inhibit fibrosarcoma cell growth by directly inhibiting NF- ⁇ B activity.
  • the synthetic scammer targeting NF- ⁇ B is introduced into the synovial cells of rheumatoid patients, the expression of cytokines and adhesion factors is reduced, and the proliferation of synovial cells is also inhibited.
  • the N- ⁇ B decoy can simultaneously regulate many genes controlled by the same cis-regulatory elements. At the same time, the decoy has a high degree of specificity and a relatively simple synthesis. However, NF- ⁇ B decoy is easily degraded by some enzymes in the body, requiring repeated and high dose administration to maintain inhibition of NF- ⁇ B and its signaling pathway activity.
  • RNA interference is another strategy for gene therapy, which refers to sequence-specific post-transcriptional gene silencing processes mediated by double-stranded RNA molecules at the mRNA level.
  • the current RNA widely used to knock out endogenous gene expression is small interfering RNA (siRNA), which has two forms of action.
  • dsRNA double-stranded RNA
  • RISC RNA-induced silencing complex
  • the other is transfection of a small hairpin RNA (shRNA) expression vector transcribed from the RNA polymerase III (Pol III) promoter and production of mature siRNA by dicer and RISC in the cells.
  • a SiRNA molecule can specifically bind to mRNA of a target gene to inhibit its expression. Studies have shown that induction of inflammatory cytokines is inhibited by inhibition of NF- ⁇ B activity by siRNA. In addition, siRNA also caused a significant increase in apoptosis of human primary synoviocytes treated with tumor necrosis factor (TNF- ⁇ ). Studies have shown that the use of RNA interference technology to treat rheumatoid arthritis has achieved good results.
  • MicroRNA is a natural small RNA ( ⁇ 22 nt) that also regulates gene expression by blocking translation of target mRNA or inducing degradation of target mRNA like siRNA.
  • the expression of MiRNA is regulated by RNA polymerase II (Pol II), and Pol II is also responsible for mRNA expression, unlike the expression of siRNA regulated by RNA Polymerase III (Pol III).
  • MiRNA is originally transcribed from Pol II as a long primary miRNA (pri-miRNA) precursor, which is then processed by Drosha and Dicer nucleases, while siRNA is transcribed into short hairpin RNA under the control of the Pol III promoter (eg U6). (shRNA).
  • miRNAs like mRNA
  • RNA polymerase II driven promoters each Pol III promoter expresses only a single shRNA, so inhibition of multiple genes requires multiple promoters or vectors. Therefore, miRNAs targeting mRNA are efficiently expressed by developing a new expression vector using a standard Pol II promoter such as the human cytomegalovirus (CMV) major immediate early promoter (MIEP).
  • CMV human cytomegalovirus
  • MIEP major immediate early promoter
  • NF- ⁇ B plays a key role in cancer cells. However, it is also essential for normal cells.
  • NF- ⁇ B inhibitors inhibits NF- ⁇ B activity in cancer cells while also blocking some essential cellular processes regulated by NF- ⁇ B in normal healthy cells, resulting in significant dose-limiting toxicity and low therapeutic index. Therefore, these conventional NF- ⁇ B inhibitors are still limited by side effects from excessive inhibition of intracellular NF- ⁇ B activity.
  • NF- ⁇ B Over-activation of the transcription factor NF- ⁇ B is a key event in diseases such as inflammation and tumor, and is closely related to physiological and pathological processes such as inflammation, immune response, and tumorigenesis. Therefore, the development of various inhibitors of NF- ⁇ B activity to inhibit NF- ⁇ B activity has become an important pathway for inflammation and tumor therapy.
  • These include small molecule nucleic acid substances that inhibit NF- ⁇ B activity, such as decoys and small interfering RNAs (siRNAs).
  • siRNAs small interfering RNAs
  • introduction of these small-molecule nucleic acid substances into cells may result in excessive inhibition of NF- ⁇ B activity due to the inability to control the number thereof, thereby causing serious side effects. Therefore, it is necessary to modify the current technical strategy of inhibiting NF- ⁇ B activity of small molecule nucleic acid substances in order to overcome the defects and develop a novel NF- ⁇ B activity inhibiting nucleic acid molecule with clinical application value.
  • the present invention relates to a gene expression vector for regulating nuclear factor NF- ⁇ B activity, which can perceive and regulate the activity of NF- ⁇ B in cells, and can gently and effectively reduce NF- ⁇ B activity is over-activated in NF- ⁇ B activity in cells, and has no significant effect on the viability of normal cells.
  • the present invention solves the problem of excessive inhibition of NF- ⁇ B activity by small molecule NF- ⁇ B inhibitors in the prior art, thereby causing serious side effects.
  • the invention also provides a method and application for regulating the gene expression vector.
  • a gene expression vector for regulating NF- ⁇ B activity in a cell comprises two sequence elements, a promoter sequence for regulating gene expression, and a miRNA downstream of a promoter (microRNA, miRNA) Also referred to as a small RNA) coding sequence; the promoter sequence consists of a NF- ⁇ B decoy sequence and a minimal promoter sequence; the miRNA coding sequence is a stretch of NF- ⁇ B messenger RNA ( Messenger RNA, mRNA) The coding sequence of the miRNA.
  • the NF- ⁇ B decoy sequence also referred to as NF- ⁇ B responsive element, includes various sequences of NF- ⁇ B decoy, which is a DNA sequence that specifically binds to NF- ⁇ B protein. Its main sequence features a number of different NF- ⁇ B binding targets, such as GGGACTTTCC.
  • the minimal promoter comprises a minimal promoter sequence derived from various genes; preferably the herpes simplex virus thymidine kinase (HSV-TK) promoter minimal promoter; the main function is to basal transcription factors Binding to RNA polymerase II forms a universal transcriptional machinery that constitutes the basic conditions for gene expression.
  • HSV-TK herpes simplex virus thymidine kinase
  • the NF- ⁇ B messenger RNA is RelA mRNA, also known as p65.
  • the miRNA targeting NF- ⁇ B mRNA refers to miRNA binding to NF- ⁇ B mRNA by nucleic acid hybridization, which can cause degradation of NF- ⁇ B mRNA or prevent translation of NF- ⁇ B mRNA into NF- ⁇ B protein.
  • the miRNA targeting NF- ⁇ B mRNA comprises a native miRNA or an artificially designed miRNA, preferably an artificially designed miRNA, including amiR349, amiR531 or amiR533, the coding sequences of which are SEQ ID NO. 1: 5'-CTT CTT, respectively.
  • SEQ ID NO. 1 5'-CTT CTT
  • SEQ ID NO. 2 5'-AAG ATG GGA TGA GAA AGG ACA-3'
  • SEQ ID NO. 3 5'-CAA AGA TGG GAT GAG AAA GGA-3'.
  • the gene expression vector is a linear or circular nucleic acid molecule.
  • the nucleic acid molecule is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule, including double-stranded DNA (such as an adenoviral DNA molecule), single-stranded DNA (adeno-associated virus molecule), or a single-stranded RNA molecule (such as lentiviral RNA molecules).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • double-stranded DNA such as an adenoviral DNA molecule
  • single-stranded DNA adeno-associated virus molecule
  • a single-stranded RNA molecule such as lentiviral RNA molecules.
  • the linear nucleic acid molecule comprises a common linear DNA molecule (such as a PCR amplified fragment, a fragment), a viral DNA molecule (such as an adenovirus DNA molecule, an adeno-associated virus molecule) or a viral RNA molecule (such as a lentiviral RNA). Molecular) or the like; the circular nucleic acid molecule includes plasmid DNA and the like.
  • the method for regulating NF- ⁇ B activity in a cell according to the present invention comprises the step of introducing the gene expression vector into a cell.
  • the cell introduction method includes various types of nucleic acid cell introduction methods; preferably, introduction methods such as viral vectors, nanocarriers, liposomes, electrotransfers, gene guns, and the like.
  • the gene expression vector for regulating NF- ⁇ B activity in cells of the present invention is used for preparing NF- ⁇ B activity regulating reagent or drug molecule for gene therapy for NF- ⁇ B overactivation-related diseases (such as inflammation, tumor, autoimmune disease) Application in .
  • the method for regulating NF- ⁇ B activity in the cell of the invention is a novel NF- ⁇ B activity regulation technology, which can be used as a disease closely related to NF- ⁇ B overactivation (such as inflammation, tumor, autoimmune disease). Gene therapy technology.
  • the present invention constructs an artificial miRNA (artificial miRNA) capable of expressing NF- ⁇ B under the control of a NF- ⁇ B-specific promoter, on the basis of studying the advantages and limitations of the current NF- ⁇ B inhibition strategy of the decoy and siRNA.
  • a transgenic vector of amiRNA consisting of a NF- ⁇ B decoy (decoy) and a minimal promoter
  • the gene expression vector of the present invention is named DMP-amiRNA, ie, the "temptation" minimal promoter Decoy minimal promoter-artificial miRNA.
  • the present invention demonstrates that the vector acts as a novel NF- ⁇ B inhibitor and can sense and regulate the activity of NF- ⁇ B in cells.
  • the vector achieves self-regulation of NF- ⁇ B activity in the cell by forming a perfect feedback loop.
  • the higher the NF- ⁇ B activity the higher the DMP transcriptional activity and the more miRNA expression.
  • the vector can exert its NF- ⁇ B inhibitory function as a sensor of NF- ⁇ B activity in cells. This system combines the advantages of decoy and miRNA interference.
  • FIG. 1 The mechanism by which the novel NF- ⁇ B inhibitor of the present invention inhibits NF- ⁇ B activity is shown in the schematic diagram (Fig. 1).
  • Figure 1 reflects the novel NF- ⁇ B inhibitor (DMP-amiRNA) developed by the present invention and its principle of inhibiting NF- ⁇ B activity in cells.
  • the top half of the figure shows the current strategy of small molecule nucleic acid substances to inhibit NF- ⁇ B activity, namely decoy (short double-stranded DNA containing NF- ⁇ B binding site) and siRNA (by shRNA in vivo) Production by chemically synthesized dsRNA that is transcribed, processed, or introduced into cells). The transcription of siRNA is done by RNA polymerase III.
  • the lower half of the figure shows a novel NF- ⁇ B inhibitory gene expression vector designed according to the present invention which combines the decoy and siRNA strategies but confers a novel function of the decoy, namely the NF- ⁇ B-specific enhancer function, which enhances Fusion with the minimal promoter to become an NF- ⁇ B specific promoter.
  • This specific promoter controls the expression of downstream genes, artificial miRNAs that target NF- ⁇ B.
  • the expressed miRNA can inhibit the production of the NF- ⁇ B protein p65 (a member of the NF- ⁇ B family having transcriptional activation function) at the translational level, thereby reducing the level of NF- ⁇ B in the cell.
  • the vector can sense the activity of NF- ⁇ B in the cell and determine the output of the miRNA.
  • the decoy in this vector still plays a decoy action, that is, binds to NF- ⁇ B in cells.
  • this scammer in the gene expression vector double-stranded circular DNA
  • the phish and enhancer dual function is more durable.
  • the present invention designed an NF- ⁇ B specific promoter (DMP), and designed and screened three artificial miRNAs (amiR349, amiR531 and amiR533) molecules.
  • DMP NF- ⁇ B specific promoter
  • miR349, amiR531 and amiR533 three artificial miRNAs
  • pDMP-mCherry-amiR349, pDMP-mCherry- a miRNA gene expression vector capable of controlling the expression level of NF- ⁇ B activity in cells was constructed.
  • amiR531, pDMP-mCherry-amiR533 pDMP-mCherry-amiR533
  • the novel NF- ⁇ B inhibitory vector of the invention can moderately inhibit the activity of NF- ⁇ B in cells with excessive activation of NF- ⁇ B activity, and has no significant effect on normal cell viability, and overcomes the shortcomings of current small molecule NF- ⁇ B inhibitors.
  • the decoy in the gene expression vector is more resistant than the traditional linear decoy.
  • the degradation of intracellular nucleases, the phish and enhancer dual functions are more durable.
  • the gene expression vector of the present invention is simple, rapid and effective, and the gene expression vector can be used as a NF- ⁇ B activity regulating reagent or drug molecule for the preparation of a gene for the treatment of NF- ⁇ B overactivation-related diseases.
  • Figure 1 is a schematic diagram showing the principle of inhibition of the vector DMP-amiRNA of the present invention; wherein the upper panel shows the NF- ⁇ B inhibition pathway (traditional inhibition) of the decoy and siRNA; the lower panel shows the NF- ⁇ B inhibition pathway of DMP-amiRNA (DMP- amiRNA inhibition);
  • Figure 2 is a plasmid vector map showing the DNA element maps of the six major plasmid vectors used in the present invention.
  • Figure 3 is the interference effect of NF- ⁇ B miRNA on reporter gene expression; from left to right: bright field, green channel, red channel and fused image, magnification: 200 ⁇ ;
  • Figure 4 is a schematic diagram of the evaluation of NF- ⁇ B-specific promoters; wherein A and C represent 293T cell images transfected with pDMP-EGFP and pCMV-EGFP, magnification: 200 ⁇ , A and C: upper field; The following is a green fluorescent channel; B and D represent the flow cytometry relationship of cell fluorescence intensity; C is a control in each figure; DE is 293T cells transfected with pDMP-EGFP; BDE is pretreated with BAY11-7082 and using pDMP-EGFP Transfected 293T cells; CE was transfected into pCMV-EGFP with 293T cells; BCE was 293T cells pretreated with BAY11-7082 and transfected with pCMV-EGFP (*, p ⁇ 0.05; **, p ⁇ 0.01);
  • Figure 5 is a schematic diagram showing the interference effect of NF- ⁇ B activity-controlled plasmid; wherein A represents a transfected 293T cell image, magnification: 200 ⁇ ; B represents a flow cytometric relationship diagram of transfected cells; A from top to bottom: Bright field, green fluorescent channel, red fluorescent channel and green and red fused images; C is the control in each figure; R is pCMV-EGFP-RelA; D is pDMP-mCherry-amiR533 and pCMV-EGFP-RelA; CR is pCMV- mCherry-amiR533 and pCMV-EGFP-RelA (*, p ⁇ 0.05; **, p ⁇ 0.01);
  • Figure 6 is a diagram showing the relationship between RelA and its target gene by pDMP-mCherry-amiR533; gene expression was detected by qPCR, wherein C is a control; T is TNF- ⁇ ; DT is pDMP-mCherry-amiR533 plus TNF- ⁇ ; CT is pCMV- mCherry-amiR533 plus TNF- ⁇ (*, p ⁇ 0.05; **, p ⁇ 0.01);
  • Figure 7 is a graph showing the effect of pDMP-mCherry-amiR533 on cell viability; flow cytometry for apoptosis; A is 93T and HepG2 cells; B is HL7702 and HepG2 cells; control: elution buffer is used as a blank control Transfected cells; BAY 11-7082: Cells were treated with 50 ⁇ M BAY 11-7082 for 1 hour; pDMP-mCherry-amiR533 and pCMV-mCherry-amiR533: cells were transfected with pDMP-mCherry-amiR533 and pCMV-mCherry-amiR533, respectively.
  • vector pIRES2-EGFP was purchased from Clontech Laboratories, Inc., pcDNA TM 6.2-GW / EmGFP -miR-Neg purchased from Invitrogen Corporation, pEGFP-C1 available from Clontech Laboratories, Inc., pGL4.10-MP available from Promega Corporation.
  • the plasmids p-mCherry and p-RelA were constructed by Wang Jinke's laboratory of the School of Biological Science and Medical Engineering of Southeast University. The plasmids p-mCherry and p-RelA were only used to provide the coding sequences of mCherry and RelA proteins.
  • mCherry fragment was obtained by vector p-mCherry cloning, NheI was introduced upstream of this sequence, EcoRI was introduced downstream, and mCherry fragment was ligated into vector pIRES2-EGFP to construct recombinant vector pCMV-mCherry .
  • the miR-Neg fragment was ligated into the vector pCMV-mCherry constituting the control Vector pCMV-mCherry-miR-Neg.
  • RelA/p65 mRNA was uploaded to the BLOCK-iTTM RNAi online design software (https://rnaidesigner.thermofisher.com/rnaiexpress/) to design the miRNA gene sequence and homologous to the BLAST sequence alignment software.
  • Sex analysis based on the location of miRNA targeting and its inhibition efficiency, identified and synthesized three pairs of RelA gene single-stranded DNA (ssDNA) for preparation of miRNA expression vector targeting RelA, such as SEQ ID NO in Table 1. Shown in .4-9.
  • dsDNA double-stranded DNA
  • the dsDNA was then ligated to a linear pCMV-mCherry-amiR vector cleaved with BsmBI.
  • the three miRNA expression vectors targeting the RelA/p65 gene were designated as pCMV-mCherry-amiR349, pCMV-mCherry-amiR531, pCMV-mCherry-amiR533, respectively.
  • NF- ⁇ B RNAi reporter vector The full-length fragment of RelA was cloned by vector p-RelA, and EcoRI was introduced upstream of this sequence, BamHI was introduced downstream, and the RelA fragment was ligated into vector pEGFP-C1 to construct recombinant vector pCMV- EGFP-RelA ( Figure 2). The vector was verified by DNA sequencing.
  • Cell culture 293T cells were treated with DMEM containing 10% (v/v) FBS (HyClone), 100 units/mL penicillin and 100 g/mL streptomycin in a humidified incubator containing 5% (v/v) CO 2 at 37 ° C. to cultivate. The cells were seeded at a density of 1 ⁇ 10 5 cells/cm 2 in a 24-well plate and incubated for 24 hours (confluence of about 80%). All vectors for transfection were isolated using the EndoFree Plasmid Kit (CWBio). Prior to transfection, the medium was carefully removed and the cells were washed with 500 ⁇ L of PBS.
  • FBS HyClone
  • OPTI-MEM medium 500 ⁇ L was added to each well and incubated for 2 hours.
  • the expression vector was transfected into cells by using a lipid-based transfection reagent Lipofectamine 2000 (Invitrogen). After 6 hours, the mixture was removed, complete medium was added, and the cells were further cultured for 24 hours.
  • NF- ⁇ B miRNA interference vectors were successfully constructed by miRNA design, vector recombination and sequencing verification, which were pCMV-mCherry-amiR349, pCMV-mCherry-amiR531 and pCMV-mCherry-amiR533, respectively, and a negative control vector pCMV-mCherry- miR-Neg and a reporter vector pCMV-EGFP-RelA (Fig. 2).
  • three miRNA expression vectors and the reporter vector pCMV-EGFP-RelA were co-transfected into 293T cells, respectively.
  • the interference effects were judged by the green and red fluorescence intensities produced by EGFP and red fluorescent protein (RFP).
  • the results showed that the red fluorescence intensity produced by the three miRNAs was basically the same, indicating that the three miRNAs in the transfected cells were similarly expressed (Fig. 3).
  • the green fluorescence intensity produced by the three miRNAs was significantly different.
  • the green control fluorescence of the negative control cells was the strongest, indicating that the negative control miRNA did not affect the reporter gene expression.
  • three miRNAs targeting NF- ⁇ B interfered with reporter gene expression.
  • amiR533 caused the most significant interference effect (Figure 3).
  • the results indicate that the expression of the RelA gene is interfered by these artificial miRNAs, especially amiR533. Therefore, these miRNAs can be used in subsequent experiments.
  • Vector construction A 52-bp fragment containing the NF- ⁇ B classical binding site on the cloning vector pGL4.32 and designated as a scammer, sequence SEQ ID NO. 10: 5'-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC C-3', which is used as an NF- ⁇ B specific regulatory element.
  • Two single-stranded oligonucleotides were designed and synthesized according to this sequence, and the sequences thereof are shown in SEQ ID NO. 11-12 of Table 2.
  • the two single-stranded oligonucleotides are denatured and annealed to form double-stranded DNA; sequences upstream of and downstream of the double-stranded DNA are annealed to the KpnI and HindIII digested sticky end sequences, respectively.
  • the double-stranded DNA was then ligated with KpnI and HindIII double-enzyme-cleaved linear pGL4.10-MP (MP, minimal promoter) and the recombinant vector was designated pGL4.10-DMP.
  • pGL4.10-DMP contains the smallest promoter (sequence SEQ ID NO.
  • Cell culture 293T cells were treated with DMEM containing 10% (v/v) FBS (HyClone), 100 units/mL penicillin and 100 g/mL streptomycin in a humidified incubator containing 5% (v/v) CO 2 at 37 ° C. to cultivate. The cells were seeded at a density of 1 ⁇ 10 5 cells/cm 2 in a 24-well plate and incubated for 24 hours (confluence of about 80%). All vectors for transfection were isolated using the EndoFree Plasmid Kit (CWBio). Prior to transfection, the medium was carefully removed and the cells were washed with 500 ⁇ L of PBS.
  • FBS HyClone
  • OPTI-MEM medium 500 ⁇ L was added to each well and incubated for 2 hours.
  • the expression vector was transfected into cells by using a lipid-based transfection reagent Lipofectamine 2000 (Invitrogen). After 6 hours, the mixture was removed, complete medium was added and the cells were cultured for an additional 18 h or 42 h.
  • Transfected cells were imaged by fluorescence microscopy and then digested with 10 mg/mL trypsin/EDTA solution for flow cytometry analysis.
  • the NF- ⁇ B decoy sequence was ligated to the minimal promoter to construct an NF- ⁇ B specific promoter, designated DMP promoter. Then, a DMP-regulated EGFP-expressing reporter vector pDMP-EGFP was constructed to evaluate the transcriptional activity of DMP. This example also constructed a comparison of the reporter vector pCMV-EGFP expressing EGFP under the regulation of human CMV MIEP. The two vectors were transfected into 293T cells, respectively. The results indicated that DMP successfully induced the expression of the reporter gene; however, its transcriptional activity was much lower than that of the strong promoter MIEP (Fig. 4).
  • the DMP fragment was cloned by the vector pDMP-EGFP, and the AseI and NheI cleavage sites were inserted upstream and downstream, respectively.
  • the DMP fragment was ligated into the recombinant vector pCMV-mCherry-amiR533 from which the CMV promoter was removed, and an NF- ⁇ B self-controlled miRNA expression vector was constructed and designated as pDMP-mCherry-amiR533 (Fig. 2).
  • Vector validation was performed by PCR amplification and gene sequencing.
  • pCMV-mCherry-amiR533 was also constructed as a control (Fig. 2), and the vector was verified by DNA sequencing.
  • 293T cells were co-transfected with pCMV-EGFP-RelA and pDMP-mCherry-amiR533 or pCMV-mCherry-amiR533 and incubated for 24 hours.
  • pCMV-EGFP-RelA and elution buffer were used as positive and blank controls, respectively.
  • NF- ⁇ B-specific promoter DMP
  • the targeted NF- ⁇ B miRNA miR533
  • pDMP-mCherry-amiR533 a new plasmid vector pDMP-mCherry-amiR533.
  • amiR533 expression levels are expected to be regulated by intracellular NF- ⁇ B itself.
  • the level of expression of amiR533 will depend on the NF- ⁇ B activity in the cell.
  • a similar pCMV-mCherry-amiR533 was constructed as a control vector.
  • 293T cells were co-transfected with pDMP-mCherry-amiR533, pCMV-mCherry-amiR533 and pCMV-EGFP-RelA, respectively.
  • the results showed that pCMV-mCherry-amiR533 resulted in higher expression of amiR533 than pDMP-mCherry-amiR533. Accordingly, the inhibitory effect of pCMV-mCherry-amiR533 on reporter gene expression was more pronounced than that of pDMP-mCherry-amiR533 (Fig. 5A).
  • pDMP-mCherry-amiR533 transfection only resulted in a significant decrease in the number of fluorescent cells (p ⁇ 0.01), but the relative fluorescence intensity of the cells was not significantly attenuated.
  • pCMV-mCherry-amiR533 transfection resulted in a significant decrease in the number of fluorescent cells and relative fluorescence intensity (Fig. 5B).
  • the data indicate that the regulatory activity of pDMP-mCherry-amiR533 is much milder than that of pCMV-mCherry-amiR533, indicating that pDMP-mCherry-amiR533 can be used to modulate the intracellular activity of NF- ⁇ B.
  • a strong promoter such as human CMV MIEP cannot be used to regulate the expression of NF- ⁇ B-targeted miRNAs, as its inherently high transcriptional activity inevitably over-inhibits NF- ⁇ B activity, thereby impairing the normality of important transcription factors.
  • Physiological function the results of pDMP-mCherry-amiR349 and pDMP-mCherry-amiR531 are similar to those of pCMV-mCherry-amiR533, and can also be used to gently regulate the intracellular activity of NF- ⁇ B.
  • HepG2 cells were treated with DMEM containing 10% (v/v) FBS (HyClone), 100 units/mL penicillin and 100 g/mL streptomycin in a humidified incubator containing 5% (v/v) CO 2 at 37 ° C. to cultivate.
  • the cells were seeded in a 6-well plate at a density of 1 ⁇ 10 5 cells/cm 2 and incubated for 24 hours (confluence of about 80%). All vectors for transfection were isolated using the EndoFree Plasmid Kit (CWBio). Prior to transfection, the medium was carefully removed and the cells were washed with 500 ⁇ L of PBS.
  • OPTI-MEM medium 500 ⁇ L was added to each well and incubated for 2 hours.
  • the expression vector was transfected into cells by using a lipid-based transfection reagent Lipofectamine 2000 (Invitrogen). After 6 hours, the mixture was removed, complete medium was added, and the cells were further cultured for 48 h.
  • HepG2 cells in 6-well plates were transfected with 4000 ng of pDMP-mCherry-amiR533 or pCMV-mCherry-amiR533, respectively, and the elution buffer was a blank control. After 48 hours of culture, the cells were stimulated with TNF- ⁇ (10 ng/mL) for 1 hour.
  • Gene expression assay Cells were harvested, total RNA was extracted with Trizol, and complementary DNA (cDNA) was synthesized by reverse transcription. The expression of RelA and its target genes, including BCL3, NFKB1, CD54, NFKBIA, CXCL1, PTGS2, CCL2, NFKB2 and MMP9, was quantitatively analyzed by qPCR. The upper and lower primers for qPCR are shown in SEQ ID NO. 14-35, as shown in Table 3.
  • pDMP-mCherry-amiR533 regulates the expression of endogenous NF- ⁇ B and its target genes
  • pDMP-mCherry-amiR533, pCMV-mCherry-amiR533 were transfected into HepG2 cells, respectively.
  • TNF- ⁇ a known NF- ⁇ B stimulator.
  • the expression of RelA and some of its typical target genes was quantitatively analyzed by qPCR. The results showed that TNF- ⁇ successfully induced the expression of RelA and its target genes (Fig. 6).
  • Both amiRNA expression vectors inhibited the induced expression of RelA and its target genes to varying degrees (Fig. 6).
  • Cell culture 293T, HepG2 and HL7702 cells were humidified with 5% (v/v) CO 2 in DMEM containing 10% (v/v) FBS (HyClone), 100 units/mL penicillin and 100 g/mL streptomycin. Incubate at 37 ° C in an incubator. The cells were seeded at a density of 1 ⁇ 10 5 cells/cm 2 in a 24-well plate or a 6-well plate and incubated for 24 hours (confluence of about 80%). All vectors for transfection were isolated using the EndoFree Plasmid Kit (CWBio). Prior to transfection, the medium was carefully removed and the cells were washed with 500 ⁇ L of PBS.
  • CWBio EndoFree Plasmid Kit
  • OPTI-MEM medium 500 ⁇ L was added to each well and incubated for 2 hours.
  • the expression vector was transfected into cells by using a lipid-based transfection reagent Lipofectamine 2000 (Invitrogen). After 6 hours, the mixture was removed, complete medium was added and the cells were cultured for an additional 18 h or 42 h.
  • NF- ⁇ B often exerts an anti-apoptotic effect through a target gene that inhibits apoptosis. Therefore, the effect of pDMP-mCherry-amiR533 on apoptosis was evaluated by flow cytokine V-FITC/PI dual channel. Cells treated with BAY 11-7082 were used as positive controls. The results showed that pDMP-mCherry-amiR533 caused the early (lower right quadrant) and late (upper right quadrant) apoptotic cells of the two cells to represent the lowest total apoptosis rate (Fig. 7A).
  • the vector pCMV-mCherry-amiR533 resulted in a higher apoptotic rate relative to pDMP-mCherry-amiR533.
  • BAY 11-7082 a common inhibitor of NF- ⁇ B activity, resulted in the highest rate of apoptosis, much higher than the two miR533 expression vectors (Fig. 7A). It is clear that small molecule chemical inhibitors cause high apoptosis by strongly over-inhibiting NF- ⁇ B activity. NF- ⁇ B miRNA interference produces much less apoptosis than small molecule chemical inhibitors.
  • NF- ⁇ B miRNA interference results in higher apoptosis than that regulated by the NF- ⁇ B-specific promoter (DMP).
  • DMP NF- ⁇ B-specific promoter
  • the NF- ⁇ B miRNA interference of pDMP-amiR533 has the least effect on cell viability.
  • Apoptosis analysis also showed that miRNA interference resulted in apoptosis of 293T cells much lower than that of HepG2 cells. The reason is that 293T is a genetically modified human embryonic kidney cell, and HepG2 is a human hepatoma cell, the former having a lower NF- ⁇ B activity than the latter.
  • HL7702 normal human hepatocyte cell line HL7702 was further evaluated.
  • HL7702 was used as a negative control for NF- ⁇ B overactivation (38-40) and compared to human hepatoma cell HepG2.
  • two cell lines were treated with pCMV-mCherry-amiR533, pDMP-mCherry-amiR533 and BAY 11-7082, and apoptosis was analyzed by Annexin V-FITC/PI two-channel flow cytometry. The results showed that pDMP-mCherry-amiR533 did not induce apoptosis of HL7702 cells (Fig.
  • pDMP-mCherry-amiR533 The effect of pDMP-mCherry-amiR533 on normal human hepatocyte HL7702 activity means that this new NF- ⁇ B inhibitor can abolish the excessive activation of NF- ⁇ B in NF- ⁇ B over-activated cells such as cancer or inflammatory cells. But it does not affect normal cells.
  • the results of pDMP-mCherry-amiR349/amiR531 are similar to those of pCMV-mCherry-amiR533, which can eliminate the excessive activation of NF- ⁇ B in NF- ⁇ B over-activated cells, but does not affect normal cells.

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Abstract

L'invention concerne un vecteur d'expression génique pour réguler l'activité NF-κB dans une cellule et un procédé de régulation associé et une application correspondante, le vecteur comprenant deux éléments de séquence, une séquence de promoteur qui régule l'expression génique et une séquence de codage de miARN en aval de promoteur; la séquence de promoteur est constituée d'une séquence de leurre NF-κB et d'une séquence de promoteur minimale; la séquence de codage de miARN est une séquence de codage capable de coder le miARN de l'ARNm cible de NF-κB. La transfection du vecteur d'expression génique peut réduire modérément et efficacement l'activité NF-κB dans une cellule présentant une activité NF-κB suractive et n'a pas d'effets évidents sur l'activité de cellules normales, ce qui permet d'éviter les effets secondaires correspondants. Le procédé de régulation pour un vecteur d'expression génique est simple, rapide et efficace. Le vecteur d'expression génique est utilisé pour préparer un réactif de régulation d'activité NF-κB ou une molécule de médicament pour la thérapie génique de maladies étroitement liées à la suractivité de NF-κB, en étant un nouveau réactif de thérapie génique.
PCT/CN2018/104928 2017-09-11 2018-09-11 VECTEUR D'EXPRESSION GÉNIQUE POUR RÉGULER L'ACTIVITÉ NF-κB DANS UNE CELLULE ET PROCÉDÉ DE RÉGULATION ASSOCIÉ ET APPLICATION CORRESPONDANTE WO2019047962A1 (fr)

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CN108220336A (zh) * 2017-12-14 2018-06-29 东南大学 基于细胞内NF-κB活性激活效应基因在NF-κB过度活化细胞内的基因表达及应用
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