Breast cancer specific artificial gene line and application thereof

Abstract

The invention relates to a breast cancer specific artificial gene line and application thereof. The breast cancer specific artificial gene line comprises a fusion plasmid A, a fusion plasmid B and a reporter plasmid, the fusion plasmid A comprises a human MAFK (MAFK) promoter and a Gal4-DocS fusion protein; the fusion plasmid B comprises a fusion protein of a human RRM2 promoter and a Coh2-p65-HSF1 (Coh2-p65-HSF1); the reporter plasmid comprises a five-copy yeast upstream activation sequence, a weak promoter and a reporter gene. According to the present invention, the artificial gene line driven by the breast cancer specific promoter is designed, such that the gene treatment line is only specifically activated in the breast cancer cells and provides effects, the tumor cell targeting recognition ability is significantly improved, and the problem of poor targeting property of the existing treatment method is fundamentally solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of synthetic biology and gene therapy technology, and particularly relates to a breast cancer specific artificial gene circuit and application thereof. BACKGROUND

[0002] In today's medical field, breast cancer is one of the most malignant tumors that threatens women's life and health, and is the second leading cause of death in women. Among them, triple negative breast cancer (TNBC) has high malignancy, strong invasiveness, high metastasis risk, easy recurrence and poor prognosis, and it lacks specific target, which has always been a difficult point to be overcome in clinical. At present, the main treatment method of TNBC is still surgery and chemotherapy, and other optional drugs are targeted drugs and immune checkpoint inhibitors. However, the biggest problem of these drugs is the lack of tumor specificity, that is, while killing tumor cells, normal cells will also be damaged, resulting in serious side effects and treatment failure. Therefore, how to accurately target TNBC has become a great challenge in clinical.

[0003] So far, the development of synthetic biology has brought new ideas and solutions for the design and construction of precise controllable gene circuits. Synthetic biology is a discipline that focuses on engineering thinking, and its goal is to combine different functional elements, modules and systems to purposefully design and modify cells, so that the organism has some specific new functions. The combination of synthetic biology and biomedical engineering provides new opportunities and ideas for the treatment of diseases. At present, with the continuous improvement of synthetic biology technology and element library, people can combine different functional biomolecules and gene elements to form a variety of gene circuits. After these circuits are introduced into cells, they can sense, integrate and process molecular signals, and perform specific biological functions according to the preset logic. This method is expected to become a powerful tool to solve the bottleneck of existing disease treatment. SUMMARY

[0004] The present application provides a breast cancer specific artificial gene circuit and application thereof, which constructs a breast cancer specific artificial gene circuit through theoretical design and strict selection of promoters, significantly improving its recognition sensitivity and specificity for breast cancer, to solve the following problems existing in the prior art: 1. Insufficient specificity of gene circuit: the recognition sensitivity of traditional promoters to tumors is limited, and there is a high background leakage expression; 2. Poor clinical transformation potential: the existing gene therapy system often lacks specific expression and has large side effects.

[0005] The application provides a breast cancer specific artificial gene circuit, comprising a fusion plasmid A, a fusion plasmid B and a report plasmid; the fusion plasmid A comprises a human MAFK promoter and a Gal4-DocS fusion protein; the fusion plasmid B comprises a human RRM2 promoter and a Coh2-p65-HSF1 fusion protein; and the report plasmid comprises five copies of a yeast upstream activation sequence (UAS: Gal4 binding site), a weak promoter and a report gene.

[0006] Further, the weak promoter is P hCMVmin .

[0007] Further, the report gene is luciferase or fluorescent protein.

[0008] Further, the gene circuit is integrated in mammalian cells to drive precise expression of therapeutic proteins.

[0009] Further, the mammalian cells comprise one or more of MDA-MB-231, MDA-MB-453 and BT549 cells.

[0010] Further, the therapeutic proteins are one or more of HSV-TK and GCV.

[0011] The application also provides a use of the breast cancer specific artificial gene circuit in the preparation of a breast cancer gene therapy drug.

[0012] The application is based on the specificity of human ribonuclease reductase 2 (RRM2) and muscμLoaponeurotic fibrosarcoma oncogene homolog K (MAFK) promoters, integrates a Gal4 derived from yeast and its specific binding sequence (UAS) module, a transcription activator p65-HSF1 and a pair of proteins cohesion and dockerin (Coh2 and DocS) found in Clostridium thermocellum, which have high affinity and can spontaneously bind to each other, and constructs a gene circuit (BRAS system) capable of specifically recognizing breast cancer by reasonably assembling and splicing different module elements.

[0013] Advantages

[0014] (1) This invention designs an artificial gene circuit driven by a breast cancer-specific promoter, which enables the gene therapy circuit to be specifically activated and play a role only in breast cancer cells, significantly improving the ability to target and recognize tumor cells, and fundamentally solving the problem of poor targeting of existing treatment methods.

[0015] (2) The specific artificial gene circuit of the present invention can sense the molecular signals unique to tumor cells and, after integration and processing, perform preset therapeutic functions only on breast cancer cells (such as inducing tumor cell apoptosis, regulating immune response, etc.), thereby minimizing the impact on normal cells, greatly improving the precision of treatment, and reducing the incidence of side effects.

[0016] (3) This invention innovatively combines the concept of synthetic biology with gene therapy. By combining biomolecules and gene elements with different functions, it constructs gene circuits with specific logical functions, enabling them to have the systematic ability of signal perception, integration and functional execution, forming a "smart response" precision treatment mode. This breaks through the limitations of existing technologies in terms of treatment controllability and specificity, and provides a brand-new technical path for the treatment of triple-negative breast cancer. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the design principle of the BRAS system of the present invention.

[0018] Figure 2 AB is the first optimization verification of the BRAS system of this invention.

[0019] Figure 3 AB is the second optimization verification of the BRAS system of this invention.

[0020] Figure 4 AB represents the specificity of the BRAS system of this invention in TNBC cells.

[0021] Figure 5 This invention provides the specificity of the BRAS system for treating TNBC cells. Figure 6 AF represents the in vivo experiment of the BRAS system of this invention in treating TNBC model mice. Detailed Implementation

[0022] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0023] Example 1

[0024] This embodiment provides a breast cancer-specific artificial gene circuit (BRAS system), including fusion plasmid A, fusion plasmid B, and reporter plasmid; fusion plasmid A includes a human MAFK promoter and a Gal4-DocS fusion protein; fusion plasmid B includes a human RRM2 promoter and a Coh2-p65-HSF1 fusion protein; the reporter plasmid includes five copies of a yeast upstream activation sequence (UAS: Gal4 binding site) and a weak promoter (P... hCMVmin This is used to initiate the expression of Luciferase. The Luciferase reporter gene can be replaced with different output proteins depending on the application scenario.

[0025] The BRAS system works as follows: Utilizing synthetic biology principles, it introduces galactose-responsive transcription factor 4 (Gal4) and its specific binding sequence (UAS) module derived from yeast, the transcription activator p65-HSF1, and a pair of high-affinity proteins, cohesion and dockerin (referred to as Coh2 and DocS), found in Clostridium thermocellum. Through the rational assembly and splicing of different module elements, a gene circuit capable of specifically recognizing breast cancer is constructed. First, the DNA binding domains of Gal4 and DocS are fused and expressed, and Coh2 is fused and expressed with the transcription activator p65-HSF1. Additionally, a UAS-Luciferase reporter plasmid is constructed to detect transcriptional activation. This reporter plasmid contains five copies of the yeast upstream activation sequence (UAS: Gal4 binding site) and a weak promoter (P... hCMVmin This is used to initiate Luciferase expression. Because P RRM2 and P MAFK It exists in breast tumor cells, but is expressed in trace amounts in normal breast epithelial cells. Therefore, in breast cancer cells, Gal4-DocS dimers with Coh2-p65-HSF1, enters the nucleus, and recruits RNA polymerase by recognizing and specifically binding to the UAS site in the reporter plasmid, thereby initiating the expression of downstream reporter genes Luciferase or EGFP. In normal breast cells, the two fusion proteins do not dimerize, and therefore do not specifically bind to the UAS site of the reporter plasmid, terminating the transcriptional expression of downstream reporter genes. A schematic diagram of the BRAS system is provided. Figure 1 As shown.

[0026] Example 2

[0027] Characterization of the BRAS system at the cellular level

[0028] Preliminary testing of the BRAS system was performed in MDA-MB-231, MDA-MB-453, and BT549 cells. MDA-MB-231, MDA-MB-453, and BT549 cells were cultured at 3 × 10⁶ cells per well one day prior to infection. 4 Cells were seeded in 48-well plates, with 300 μl of DMEM medium containing 10% FBS added to each well. Sixteen hours after seeding, the cells were infected with three plasmids of the BRAS system. The plasmid information is shown in the table below.

[0029]

[0030] The virus was premixed with the infection reagent PB at a total volume of 15 μL per well, and the premix was added dropwise to each well of cells. Sixteen hours after transfection, the culture medium for each well was replaced with DMEM medium containing 300 μL of 10% FBS at different concentrations. Experimental results showed that the BRAS system was specifically highly expressed in TNBC cells (…). Figure 2 ).

[0031] To improve the specificity of the BRAS system for TNBC, miR205, which is lowly expressed in tumor cells but highly expressed in normal cells, was screened. Subsequently, it was tested in MDA-MB-231 and BT549 cells. The results showed that miR205 significantly improved the specificity of the BRAS system in TNBC. Figure 3 ).

[0032] To verify the specificity of the BRAS system in TNBC, the BRAS system plasmid was transfected into different cell lines, including HeLa, HepG2, RPE, MCF7, and SKBR3, and cultured in fresh DMEM medium for 24 hours post-infection. System efficacy was evaluated by detecting the expression level of the Luc reporter gene. The results showed that the BRAS system specifically expressed (…) in TNBC cells. Figure 4 It has good specificity and broad application prospects.

[0033] Example 3

[0034] The BRAS system's specificity in treating TNBC

[0035] First, the output protein of the BRAS system was replaced from Luc to HSV-TK / GCV, and HSV-TK was constructed as the output reporter plasmid for the gene circuit: pST108C [LTR-5×UAS-PhCMVmin-HSV-TK-LTR]. This plasmid, along with the pST110 module, was transduced into MCF10A, MDA-MB-231, and MDA-MB-453 cells. After 48 h, 100 μg / mL ganciclovir (GCV) was added. CCK-8 cell proliferation assays showed that this artificial AND-gate logic gene circuit significantly and specifically killed triple-negative breast cancer cells (MDA-MB-453, MDA-MB-231, BT549) without damaging normal breast epithelial cells (MCF10A). Simultaneously, this gene circuit also achieved specific killing of primary breast cancer cells without damaging primary normal breast epithelial cells. Figure 5 ).

[0036] Example 4

[0037] Application of BRAS system in the treatment of TNBC model mice

[0038] After confirming the in vitro efficacy of the BRAS system, its in vivo therapeutic potential was further evaluated using an in situ triple-negative breast cancer (TNBC) model. First, an in situ breast cancer mouse model was established by injecting tumor cells into the mammary fat pads of immunodeficient mice. Ten days post-injection, when the tumor volume reached 50-100 mm... 3 Mice were randomly divided into 4 groups and received the following treatments via intratumoral injection: (1) phosphate-buffered saline (PBS, G1 group), (2) BRAS system vector containing only HSV-TK output (G2 group), (3) ganciclovir only (GCV, G3 group), and (4) BRAS system vector containing HSV-TK output combined with GCV (G4 group). Figure 6 A shows the experimental timeline of the BRAS system in treating TNBC model mice. (For example...) Figure 6 As shown in the BF diagram, compared with all control groups, only the G4 group showed significant tumor growth inhibition, reduced tumor weight, and significant tumor cell apoptosis.

Claims

1. A breast cancer-specific artificial gene circuit, characterized by: The gene circuit comprises a fusion plasmid A, a fusion plasmid B and a reporter plasmid; the fusion plasmid A comprises a human MAFK promoter and a Gal4-DocS fusion protein; the fusion plasmid B comprises a human RRM2 promoter and a Coh2-p65-HSF1 fusion protein; and the reporter plasmid comprises five copies of a yeast upstream activating sequence, a weak promoter and a reporter gene.

2. The breast cancer specific artificial gene circuit of claim 1, wherein: The weak promoter is P hCMVmin .

3. The breast cancer-specific artificial gene circuit according to claim 1, characterized in that: The reporter gene is luciferase or fluorescent protein.

4. The breast cancer specific artificial gene circuit of claim 1, wherein: The gene circuit is integrated in mammalian cells to drive precise expression of therapeutic proteins.

5. The breast cancer specific artificial gene circuit of claim 4, wherein: The mammalian cells comprise one or more of MDA-MB-231, MDA-MB-453 and BT549 cells.

6. The breast cancer specific artificial gene circuit of claim 4, wherein: The therapeutic proteins are one or more of HSV-TK and GCV.

7. Use of the breast cancer-specific artificial gene circuit of claim 1 in the preparation of a breast cancer gene therapy drug.

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