Precise gene regulation and control expression system based on iPsiR

Abstract

The invention relates to the technical field of gene expression regulation, and discloses an iPsiR-based gene precise regulation expression system, which comprises: (a) a chimeric repressor protein, which is formed by fusing a PsiR protein and a repressor protein KRAB; (b) an artificial hybrid promoter, which comprises a human cytomegalovirus core promoter (PhCMV) and 3 copies of a PsiR operon sequence OPsiR connected in series at the downstream. By optimizing the artificial hybrid promoter, the expression of the PsiR gene in a eukaryotic system, especially in mammalian cells, is realized, and the successful breakthrough application of a psicose-induced gene regulation expression system in mammalian cells is promoted; therefore, the psicose-induced mammalian gene expression system has the application prospect of gene treatment of diseases.

Description

Technical Field

[0001] This invention relates to the field of gene expression regulation technology, specifically to a gene expression precision regulation system based on iPsiR. Background Technology

[0002] The statements in this section provide only background information relevant to the disclosure of this application and may not constitute prior art.

[0003] In the field of modern medicine, the rapid development of synthetic biology and cell therapy technologies has brought new ideas to the treatment of major diseases. In recent years, researchers have been committed to developing systems that can precisely regulate gene expression to achieve precise control over the expression of therapeutic proteins. Early studies discovered that small molecules (such as doxycycline) regulate transcription factor activity, achieving gene "on / off" control; however, subsequent studies found that this approach suffers from high immunogenicity and limited regulatory precision. Subsequently, gene regulation mechanisms such as light-controlled gene expression systems, protocatechuic acid (PCA)-controlled gene on / off, and thymol aroma-regulated gene loops have been discovered. However, these gene expression regulation systems have limitations, such as insufficient sensitivity to inducers, high background expression leakage rates, and difficulty in meeting the needs of long-term, precise treatment. In addition, many gene expression regulation systems rely on chemical drugs or hormones as inducers, which may trigger immune responses or side effects, limiting their clinical application.

[0004] The PsiR protein was first discovered in *Agrobacterium tumefaciens* (a bacterium that produces Agrobacterium tumefaciens In bacteria of the order Rhizobiales, such as D-allulose, PsiR is a natural transcription factor. It regulates the expression of downstream genes by specifically binding to the promoter region (PpsiA). In the absence of D-allulose, PsiR binds to PpsiA and represses the expression of downstream genes; when D-allulose is present, it binds to PsiR and dissociates it from the promoter, thereby unblocking the repression and activating gene expression.

[0005] D-Allulose is a natural, low-calorie sweetener with no toxic side effects on the human body, and can be used in the food or medical fields without additional clinical approval. Compared to tetracycline-based regulatory systems that rely on antibiotics, steroid systems (such as RU486) pose a risk of hormone interference, and while the biotin system is relatively safe, its induction efficiency is low. Furthermore, the allulose system specifically recognizes a single molecule through PsiR, is less susceptible to interference from host metabolic pathways, and has no direct impact on cellular metabolism. The natural properties of allulose give it a greater safety advantage and higher regulatory potential in biomanufacturing and gene therapy.

[0006] But as a protein expressed in prokaryotic system, it is difficult to express PsiR protein in eukaryotic cells, which makes it difficult to apply the PsiR protein D-allulose regulatory expression system in eukaryotes, especially mammals. Further hinder the application of cell therapy technology in the treatment of metabolic diseases such as diabetes, obesity and tumors, hinder the development of cell therapy technology. SUMMARY

[0007] The purpose of the present application is to solve the problem that PsiR protein is difficult to be applied in eukaryotic cells, especially mammalian cells, by providing a gene precise regulation expression system based on iPsiR, which solves the problem that PsiR protein is difficult to be applied in eukaryotic cells, especially mammalian cells. By optimizing the operon sequence, the expression of PsiR protein in mammalian cells is successfully realized, and the precise regulation of gene expression of PsiR protein D-allulose regulatory expression system in mammalian cells is successfully verified, which breaks through the technical difficulty that PsiR protein D-allulose regulatory expression system cannot be applied to mammalian gene therapy.

[0008] The technical scheme of the present application is as follows: In one aspect, the present application provides a gene precise regulation expression system based on iPsiR, comprising: (a) a chimeric repressor protein, which is formed by fusion of the PsiR protein (amino acid sequence as shown in SEQ ID NO: 1) and a repressor protein; (b) an artificial hybrid promoter, which comprises a human cytomegalovirus core promoter (P hCMV ) and a downstream tandem 3 copies of PsiR operon sequence O PsiR (base sequence as shown in SEQ ID NO: 5).

[0009] Preferably, the repressor protein is Polycomb Group protein complex, DNA methyltransferase, chromatin protein 1 family related protein, SMRT / NCoR complex, Sin3A / HDAC complex or KRAB. The amino acid sequence of the KRAB is shown in SEQ ID NO: 3.

[0010] The present application optimizes and modifies the expression operon of the PsiR gene. Without the guidance of the existing technology, it is confirmed that the expression sequence O PsiR of the PsiR gene in the mammalian system is O PsiR The O PsiR sequence needs to be repeated three times to finally realize accurate regulation of gene expression.

[0011] Another aspect of the present application provides an application of the iPsiR-based gene precise regulation expression system in driving the expression of a reporter gene SEAP, thymic stromal lymphopoietin, uric acid oxidase or parathyroid hormone gene.

[0012] Another aspect of the present application provides a genetically engineered cell, wherein the cell is integrated with the iPsiR-based gene precise regulation expression system as described above.

[0013] Preferably, the cell is a HEK-293T, HeLa, BHK-21, hMSC-TERT or B16F10 cell.

[0014] Another aspect of the present application provides an application of the genetically engineered cell as described above in preparing a drug for treating a genetic disease.

[0015] Preferably, the genetic disease is a neurodegenerative disease, a monogenic genetic disease, cancer, diabetes, obesity, organic acidemia, hereditary tyrosinemia type I, phenylketonuria, glycogen storage disease or lipoprotein lipase deficiency, arthritis, anemia, cardiovascular and cerebrovascular diseases, and tumors, etc.

[0016] Preferably, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease or Huntington's disease; the monogenic genetic disease is cystic fibrosis or hemophilia; and the cancer is liver cancer or brain cancer.

[0017] Another aspect of the present application provides a PsiR protein artificial promoter, comprising P hCMV -(O PsiR )3, wherein the O PsiR has a base sequence as shown in SEQ ID NO: 5.

[0018] Compared with the prior art, the present application has the following beneficial effects: 1. An iPsiR-based gene precise regulation expression system, which provides an optimized artificial promoter sequence, the sequence being capable of realizing the expression of PsiR protein in mammalian cells, thereby laying a foundation for the successful operation of the allulose-induced mammalian gene expression system; and breaking through the prior art problem that PsiR cannot be expressed in mammalian cells, thereby opening up a new idea for gene therapy. 2. The iPsiR-based gene precise regulation expression system improves the convenience and patient compliance of treatment by using the low-calorie sweetener D-psicose as an inducer, and achieves precise and long-term control of the expression of therapeutic proteins. This innovative treatment mode is expected to provide a new solution for the treatment of metabolic diseases such as diabetes and obesity, and tumors, and to promote the development of cell therapy technology towards more precise, safe and efficient directions. 3. The iPsiR-based gene precise regulation expression system constructed in the present application not only realizes the successful operation of the mammalian gene expression system induced by D-psicose, but also can precisely regulate the expression of genes under different concentrations of D-psicose. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Figure 1 is a schematic diagram of the design principle of the iPsiR-based gene precise regulation expression system (PURE system); Figure 2 Figure 2 is the expression amount of the SEAP reporter gene induced by different concentrations of D-psicose; Figure 3 Figure 3 is the SEAP reporter gene produced by the mammalian gene expression system induced by D-psicose induced by different sugar substances; Figure 4 Figure 4 is the working condition of the iPsiR-based gene precise regulation expression system (PURE system) in different cell lines. DETAILED DESCRIPTION

[0020] The specific examples listed in the present application are only as examples of the present application, and the present application is not limited to the specific examples described below. Any equivalent modifications and alternatives to the examples described below are also within the scope of the present application for those skilled in the art. Therefore, any equivalent transformation and modification without departing from the spirit and scope of the present application should be covered within the scope of the present application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments not mentioned by the manufacturer are conventional products that can be purchased on the market. In order to better illustrate the present application, numerous specific details are given in the following specific embodiments. Those skilled in the art should understand that the present application can also be implemented without some specific details. In some other embodiments, methods, means, apparatus and steps well known to those skilled in the art are not described in detail in order to highlight the main idea of the present application.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0022] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0023] Example 1: Construction of an allulose-induced mammalian gene expression system The system consists of two plasmids. First, the human EF1α promoter initiates the expression of the KRAB-PsiR fusion protein; second, an inducible promoter drives the expression of the SEAP reporter gene, which is composed of the human CMV promoter and three copies of the PsiR operon sequence O. PsiR (hCMV-(O) PsiR 3) The SEAP reporter gene can be replaced with different output proteins depending on the application scenario.

[0024] As attached Figure 1 As shown. The system works as follows: In the absence of D-allulose, the KRAB-PsiR fusion protein binds to (O PsiR 3. Operator, KRAB protein represses P hCMV Initially, the SEAP reporter gene is not expressed during gene transcription and expression. However, when D-allulose is present, it binds to the PsiR protein, causing the PsiR protein to dissociate from the operon. The repressor protein KRAB then leaves the DNA, the CMV promoter repression is released, and downstream reporter gene expression is initiated.

[0025] Example 2: Validation of the allulose-induced mammalian gene expression system The system was tested in HEK-293T cells (human embryonic kidney cells).

[0026] One day before transfection, HEK293T cells were stored at 5 × 10⁶ cells per well. 4 Cells were seeded into 24-well plates, with 500 μl of DMEM medium containing 10% FBS added to each well. Sixteen hours after seeding, two plasmids from the transfection system were transfected. Plasmid information is shown in the table below.

[0027]

[0028] The plasmid was premixed with PEI transfection reagent (plasmid to PEI mass ratio 1:3) at a total volume of 200 ng per well in 50 μL of serum-free and antibiotic-free DMEM at a ratio of 3:1. After standing for 15 minutes, the DNA-PEI premix was added dropwise to each well of cells. Six hours after transfection, the culture medium for each well was replaced with 500 μL of DMEM medium containing 10% FBS and different concentrations of D-allulose (0-20 μM).

[0029] Forty-eight hours post-transfection, cell supernatant was collected and heated in a 65°C oven for 30 minutes to remove endogenous alkaline phosphatase. Next, 100 μL of 2×buffer buffer and 20 μL of PNPP substrate were added to each well of the pre-detection working solution, and the plate was incubated at 37°C for preheating. After heating the cell supernatant for 30 minutes, 80 μL of the cell supernatant was transferred to a clear 96-well plate, and 120 μL of the pre-detection working solution was added. The 96-well plate was then quickly placed in a microplate reader for detection. The specific detection method was as follows: absorbance of the sample was measured at 405 nm (10 consecutive measurements, 1 minute apart). Finally, the detection time was plotted on the x-axis, and the corresponding absorbance value on the y-axis, and the slope of the curve was calculated. The formula for calculating enzyme activity is: Enzyme activity (U / L) = Curve slope * 256.8.

[0030] Experimental results showed that as the concentration of D-allulose in the culture medium increased, the expression level of the reporter gene SEAP also gradually increased. Figure 2 ).

[0031] Example 3: Characterization of the system at the cellular level To verify the specificity of the system's response to D-allulose, after transfecting HEK-293T cells with the PURE system plasmid, several different sugars were added to the culture medium 6 hours later to observe whether they could activate the PURE system to produce a reporter gene. The results showed that only D-allulose could effectively activate reporter gene expression; other sugars failed to initiate reporter gene expression. Figure 3 This result clearly demonstrates that the system is highly specific, responding only to D-allulose.

[0032] To verify the system's versatility across various cell lines, the system plasmid was transfected into different cell lines, including HeLa, BHK-21, hMSC-TERT, and B16F10. Six hours after transfection, the medium was replaced with fresh medium containing D-allulose. The system's efficacy was assessed by detecting the expression level of the SEAP reporter gene. The results showed that, regardless of the cell line, the expression level of the SEAP reporter gene was significantly increased in the presence of D-allulose. Figure 4This indicates that the system can operate effectively in a variety of cell lines, demonstrating good versatility and broad application prospects.

[0033] SEQ ID NO:1 PsiR protein sequence MTGISSKKATIYDLSILSGASASTVSAVLNGSWRKRRISEETADKILSLAKAQRYTTNLQARGLRSSKSGLVGLLVPVYDNRFFSSMAQTFEGQARKRGLSPMVVSGRRDPEEERRTVETLIAYSIDALFIAGSNDPDGVHQVCARAALPHVNIDLPGKFASSVISNNRHGAEILTAAIL AHAAKGGSLGPDDVILFGGHDDHASRERIDGFHAAKADYFGVEGGDDIEITGYSPHMTEMAFERFFGRRGRLPRCFFVNSSINFEGLLRFMGRHDGEAFGDIVVGCFDYDPFASFLPFPVYMIKPDIAQMLEKGFELLEENRTEPEVTIIEPQLIPPRTALEGPLDDIWDPVALRRMAK* SEQ ID NO:2: PsiR gene sequence SEQ ID NO:3: Amino acid sequence of the repressor KRAB MAPDPKKKRKVDPKKKRKVDPKKKRKVMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSVSSRSIFKDKQSCDIKMEGMARNDLW SEQ ID NO:4: Gene sequence of the repressor KRAB ATGGCGCCAGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAATGGATGCTAAGTCACTAACTGCCTGGTCCCGGACACTGGTGACCTTCAAGGATGTATTTGTGGACTTCACCAGGGAGGAGTGGAAGCTGCTGGACACTGCTCAGCAGATCGTGTACAGAAATGTGATGCTGGAGAACTATAAGAACCTGGTTTCCTTGGGTTATCAGCTTACTAAGCCAGATGTGATCCTCCGGTTGGAGAAGGGAGAAGAGCCCTGGCTGGTGGAGAGAGAAATTCACCAAGAGACCCATCCTGATTCAGAGACTGCATTTGAAATCAAATCATCAGTTTCCAGCAGGAGCATTTTTAAAGATAAGCAATCCTGTGACATTAAAATGGAAGGAATGGCAAGGAATGATCTCTGGGCCAGCGGAAGTGGCGGAGGAGGCGACGTC SEQ ID NO:5: Base sequence of the PsiR operon O PsiR ATTGCACAATCGATGGTGCAA SEQ ID NO:6: The gene sequence of PhCMV is: GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACC SEQ ID NO:7: The SEAP reporter gene sequence is as follows: The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A gene expression precision regulation system based on iPsiR, characterized in that, include: (a) A chimeric repressor protein, formed by fusing the PsiR protein with a repressor protein; (b) Artificial hybrid promoters, including the human cytomegalovirus core promoter (P... hCMV ) and downstream tandem 3-copy PsiR operon sequence O PsiR ; The amino acid sequence of the PsiR protein is shown in SEQ ID NO:1; The O PsiR The base sequence is shown in SEQ ID NO:

5.

2. A gene expression precision regulation system based on iPsiR, characterized in that, The repressor protein is a Polycomb Group protein complex, DNA methyltransferase, chromatin protein 1 family-associated protein, SMRT / NCoR complex, Sin3A / HDAC complex, or KRAB. The amino acid sequence of the KRAB is shown in SEQ ID NO:

3.

3. The application of the iPsiR-based gene precise regulation and expression system according to claim 1 or 2 in driving the expression of reporter genes SEAP, thymic stromal lymphopoietin, uricase, or parathyroid hormone.

4. A genetically engineered cell, characterized in that, The cell integrates the iPsiR-based gene precision regulation expression system as described in claim 1.

5. A genetically engineered cell according to claim 4, characterized in that, The cells are HEK-293T, HeLa, BHK-21, hMSC-TERT, or B16F10 cells.

6. The use of a genetically engineered cell as described in claim 4 or 5 in the preparation of a medicament for treating genetic diseases.

7. The application according to claim 6, characterized in that, The genetic diseases mentioned include neurodegenerative diseases, single-gene hereditary diseases, cancer, diabetes, obesity, organic acidemia, hereditary tyrosinemia type I, phenylketonuria, glycogen storage disease or lipoprotein lipase deficiency, arthritis, anemia, cardiovascular and cerebrovascular diseases, and tumors.

8. An artificial promoter for the PsiR protein, comprising P hCMV -(O PsiR )3, the O PsiR The base sequence is shown in SEQ ID NO:

5.

9. The application of the artificial promoter for PsiR protein according to claim 8 in the activation of PsiR protein in a eukaryotic expression system.

10. The application according to claim 9, characterized in that, The eukaryotic expression system includes a mammalian expression system.

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