Linear nucleic acid structure with cGAS-STING excitation function and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a linear nucleic acid structure with a cGAS-STING excitation function and application of the linear nucleic acid structure. According to the invention, a stable DNA nanostructure is designed and synthesized, and is modified by the aptamer, so that the targeting and stability of nucleic acid are improved, and the requirement of activating a cGAS-STING pathway is met. The composite material has good biocompatibility, and can effectively activate an immune system and enhance immune recognition and killing effects on tumor cells. In vitro and mouse tumor models, the prepared nano-carrier can activate the STING pathway, can also significantly improve the tumor immunotherapy effect, and has good preclinical application prospects in the aspects of prolonging the tumor lifetime and slowing down tumor progression. The invention provides a new strategy and technical support for tumor immunotherapy, can be used as a carrier to effectively transfer drugs, and has wide application value.

Description

Technical Field

[0001] The present invention relates to a linear nucleic acid structure with cGAS-STING activation function and its application, belonging to the technical field of tumor immunotherapy. Background Art

[0002] The cGAS-STING pathway is an important part of the innate immune system in cells, responsible for recognizing abnormal DNA signals in the cytoplasm and triggering an immune response. When cGAS binds to cytoplasmic DNA, it catalyzes the generation of cGAMP, which in turn activates the STING pathway and initiates the expression of type I interferons and other pro-inflammatory factors. This pathway plays an important role in antiviral, antibacterial infections, and tumor immunity. In recent years, studies have shown that the activation of the cGAS-STING pathway can reshape the tumor microenvironment and enhance the anti-tumor immune response, thus providing a new potential strategy for cancer treatment.

[0003] In the research of cGAS-STING pathway activators, DNA nanotechnology, as an innovative method, has gradually attracted extensive attention. DNA nanostructures can specifically activate the cGAS-STING pathway by precisely designing their sequences, lengths, and spatial conformations, thereby enhancing the immune response. Research progress in this field shows that DNA nanostructures have great potential in activating the immune response, converting "cold tumors" into "hot tumors", and can effectively enhance anti-tumor immunity.

[0004] However, existing activators still face multiple challenges:

[0005] 1. Small molecule chemical drugs: Such drugs can activate the cGAS-STING pathway directly or indirectly, but often have problems such as poor targeting, large side effects, and poor drug stability, which limit their clinical applications.

[0006] 2. Cyclic dinucleotide activators (such as cGAMP): Cyclic dinucleotides can efficiently activate the STING pathway, but they are easily degraded in vivo and have low efficiency in delivering to the cytoplasm, affecting the actual application effect.

[0007] 3. DNA nanostructures: Although DNA nanostructures theoretically have good selectivity and diversity, and targeted activation effects can be achieved by finely regulating their lengths, sequences, and spatial conformations, existing DNA nanostructures still face problems such as poor stability and insufficient specificity, which affect their effects in actual treatment. In addition, DNA nanostructures of different lengths have significant differences in the activation effects on the cGAS-STING pathway. How to optimize these structures to improve the activation efficiency, especially in the application of tumor immunotherapy, remains a technical problem to be solved urgently.

[0008] Therefore, the present invention proposes a DNA nanoplatform with optimized structure. By precisely regulating the length, sequence, and spatial conformation of the DNA nanostructure, its stability, targeting ability, and activation efficacy are enhanced, overcoming the problems of poor stability, insufficient targeting ability, and ineffective activation in the prior art. In particular, the present invention has made innovations in the design of DNA structures with different lengths, significantly improving the activation effect of the cGAS-STING pathway and opening up a new direction for the immunotherapy of tumors such as triple-negative breast cancer. Summary of the Invention

[0009] In view of the deficiencies of the above prior art, the present invention provides a linear nucleic acid structure with cGAS-STING agonist function and its application, aiming to solve the technical problems of insufficient stability, targeting ability, and activation efficacy of cGAS-STING activators in the prior art.

[0010] The first technical solution provided by the present invention is a DNA nanowire, which is a double-stranded structure composed of P1 and P2, and finally the ports are closed with aptamers. P1 includes I1 and I2, P2 includes S1 and S2, and the aptamers include A1 and A2.

[0011] In some embodiments, the nucleotide sequences of I1, I2, S1, and S2 are respectively any one of the following groups:

[0012] (1) SEQ ID NO.1 - 4;

[0013] (2) SEQ ID NO.5 - 8;

[0014] (3) SEQ ID NO.9 - 12.

[0015] In some embodiments, the nucleotide sequences of A1 and A2 are respectively as shown in SEQ ID NO.13 and 14.

[0016] The second technical solution provided by the present invention is a method for preparing a DNA nanowire. The method is to mix P1 and P2 in equal volume and incubate at room temperature to obtain a DNA double-stranded structure, and then mix the DNA double-stranded structure with aptamers and incubate to obtain a DNA nanowire.

[0017] In some embodiments, P1 is obtained by mixing I1 and I2 in equal volume and annealing at 90°C for 5 minutes, and then cooling to room temperature.

[0018] In some embodiments, P2 is obtained by mixing S1 and S2 in equal volume and annealing at 90°C for 5 minutes, and then cooling to room temperature.

[0019] In some embodiments, the DNA double-stranded structure and the aptamer are incubated at room temperature for 1 h.

[0020] The third technical solution provided by the present invention is a cGAS-STING agonist, which contains the DNA nanowire described in the first technical solution.

[0021] The fourth technical solution provided by the present invention is a method for in vitro activating cGAS-STING of dendritic cells, and the method is to co-culture the dendritic cells with the DNA nanowire described in the first technical solution.

[0022] The fifth technical solution provided by the present invention is a composition targeting breast cancer cells, and the composition includes the DNA nanowire described in the first technical solution.

[0023] The sixth technical solution provided by the present invention is the application of the DNA nanowire described in the first technical solution, or the method described in the second technical solution, or the agonist described in the third technical solution, or the composition described in the fifth technical solution in the preparation of a drug for relieving and / or treating breast cancer.

[0024] In some embodiments, the application at least includes one of the following effects:

[0025] (1) Reducing the volume and mass of solid tumors in an individual;

[0026] (2) Increasing the number of CD8+ T cells and NK cells in the spleen of an individual;

[0027] (3) Enhancing the infiltration of CD8+ T cells in the tumor tissue of an individual.

[0028] The technical effects of the present invention are as follows: [[ID=??]]

[0029] The present invention proposes an innovative DNA nanowire structure, which combines the dual advantages of nucleic acid stability and functional optimization. Compared with chemical drugs and cyclic dinucleotides, the nucleic acid structure of the present invention has the following significant advantages: high stability: by designing a stable double-stranded nucleic acid conformation and a nanoscale spatial arrangement, the anti-degradation ability is significantly improved; targeting: the surface is modified with tumor-specific aptamers, which can be accurately delivered to tumor cells and reduce the risk of non-specific immune activation; versatility: it can be used as a drug delivery carrier to co-deliver small molecule drugs and enhance the therapeutic effect; activating the STING pathway: the nucleic acid sequence length and spatial structure are optimized, which can significantly enhance the cGAS binding efficiency and the STING pathway activation efficacy. Description of the Drawings

[0030] Figure 1 It is a schematic diagram of the synthesis characterization of the DNA nanowire prepared in Example 1 of the present invention.

[0031] Figure 2 is the verification result of the stability of DNA nanowires in 10% FBS.

[0032] Figure 3 is the uptake result of DNA nanowires on 4T1 cells.

[0033] Figure 4 is the result of activating the cGAS-STING pathway by DNA nanowires of different lengths in 4T1 cells.

[0034] Figure 5 is the result of activating mouse bone marrow-derived dendritic cells (BMDC) by DNA nanowires of different lengths.

[0035] Figure 6 is the treatment result of DNA nanowires of different lengths in a mouse breast cancer model. The left figure is a photo of the mouse solid tumor, and the right figure is the tumor mass of different treatment groups in mice.

[0036] Figure 7 is the flow cytometry analysis of CD8 T cells and NK cells in the spleen of mice.

[0037] Figure 8 is the infiltration of CD3 and CD8 T cells in the tumors of mice in different treatment groups.

[0038] Figure 9 is the schematic diagram of the present invention. Detailed implementation manners

[0039] Refer to the appended Figures 1-9 , and the preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0040] Testing method:

[0041] 1. Gel electrophoresis:

[0042] Prepare a 20% non-denaturing polyacrylamide solution (acrylamide: 190 g; N,N'-methylenebisacrylamide: 10 g; 1000 mL 1×TBE). Take the above solution to prepare a 10% non-denaturing polyacrylamide gel (25 mL of 20% non-denaturing polyacrylamide solution, 25 mL of 1×TBE, 400 μL of 10% APS, 40 μL of TEMED).

[0043] Add 4 μL of 6× loading buffer to 16 μL of the sample solution to prepare a DNA staining solution. All experiments are carried out at a constant voltage of 300 V, and 1×TBE buffer is used as the working buffer. The electrophoresis runs for 90 - 120 minutes, depending on the position of the sample band.

[0044] Place the gel after electrophoresis in the staining solution prepared with GelRed and stain it in the dark for 30 minutes. Observe the band distribution in the gel using a gel imaging system and record the electrophoresis results.

[0045] 2. The protein concentration was quantitatively tested using the BCA method.

[0046] 3. Western blot analysis:

[0047] Add the corresponding volume of 5× Loading buffer to the protein sample and heat-denature it at 95 °C for 5 minutes. After the sample treatment is completed, load it onto an 8% SDS-PAGE gel for electrophoresis separation and run it at an appropriate voltage until the target protein is completely separated. After electrophoresis, transfer the protein to a PVDF membrane by wet transfer (the transfer conditions are 100 V, 1 hour). After the transfer is completed, block it with 5% skim milk powder in TBST buffer for 1 hour to reduce non-specific binding.

[0048] After blocking, incubate the PVDF membrane with the diluted primary antibodies (p-STING, p-TBK1, p-IRF3, etc., dilution ratio 1:1000) at 4 °C overnight. The next day, wash the membrane 3 times with TBST, 10 minutes each time. Then add the HRP-labeled secondary antibody (dilution ratio 1:5000) and incubate it at room temperature for 1 hour, and wash the membrane 3 times with TBST again.

[0049] Finally, cover the membrane surface with ECL developing solution, let it stand for 1 - 2 minutes, and use a gel imaging system to capture the band signal. Calculate the gray value of the target protein band through ImageJ or other image analysis software and compare it with the internal reference (such as β-actin or GAPDH). The raw materials used in the examples:

[0050] 1. The 4T1 cells were derived from the ATCC cell bank.

[0051] 2. C57BL / 6 mice were purchased from Beijing Specific Pathogen Free Biotechnology Co., Ltd.

[0052] 3. BALB / c mice were purchased from Beijing Specific Pathogen Free Biotechnology Co., Ltd.

[0053] The nucleic acid sequence information involved below is shown in Table 1.

[0054] Table 1 Sequence Information

[0055]

[0056]

[0057] Design and Preparation of the Nucleic Acid Structure in Example 1

[0058] The self-assembly of nanostructures was completed by annealing, and its stability and structural integrity were verified by acrylamide gel electrophoresis.

[0059] (1) Preparation of P1 and P2:

[0060] Equal volumes of I1 (SEQ IN NO.1) and I2 (SEQ IN NO.2) (2 μL each, concentration 10 μM) were added to 196 μL of TE buffer (100 mM NaCl, 10 mM Tris, 1 mM EDTA, pH 8.5). The mixture was annealed at 90 °C for 5 minutes and then cooled to room temperature to obtain the P1 solution. Using the same procedure as for P1, equal amounts of S1 (SEQ IN NO.3) and S2 (SEQ IN NO.4) were assembled into the P2 solution.

[0061] Figure 1 Gel electrophoresis of the DNA nanostructures (P1, P2) synthesized in the present invention. The results showed that the DNA nanostructures of the present invention were successfully synthesized.

[0062] [[ID=I3]](2) Assembly of DNA:

[0063] The prepared P1 and P2 were mixed in equal volumes and incubated at room temperature for 1 hour before use, finally obtaining a DNA double-stranded structure solution.

[0064] (3) Assembly of DNA nanowires

[0065] To 40 μL of the pre-prepared DNA double-stranded solution, 3 μL each of A1 and A2 (concentration 1 μM) were added. After thorough mixing, it was left at room temperature for 1 hour to complete the assembly.

[0066] (4) Stability investigation

[0067] 50 μL of the DNA solution (P1, P1 + P2, DNA nanowires) was taken and mixed with 10 μL of 10×TAMg buffer, 10 μL of fresh FBS, and 30 μL of DMEM to prepare a degradation solution with a total volume of 100 μL. Gently mix to ensure uniform distribution of the reaction system. Incubate at 37 °C and 400 rpm for 0, 1, 2, 4, 8, 12, 24, 36 h. At each corresponding time point, 10 μL of the sample was taken and mixed with 5 μL of glycerol, and stored in a -20 °C refrigerator to terminate the experiment. Non-denaturing acrylamide gel electrophoresis was performed after sampling.

[0068] Figure 2 It was shown that the constructed DNA nanowires had good stability in 10% FBS and could exist for more than 24 h.

[0069] Example 2: Targeted modification and verification

[0070] Equal volumes of I1 (SEQ IN NO.1) and I2 (SEQ IN NO.2) (15 μL each, concentration 10 μM), and S1 (SEQ IN NO.3) and S2 with a cy5 fluorophore added to the 5' end (cy5-S2, SEQ IN NO.4) (15 μL each, concentration 10 μM) were added to 30 μL of TE buffer (100 mM NaCl, 10 mM Tris, 1 mM EDTA, pH 8.5). The mixture was annealed at 90 °C for 5 minutes and then cooled to room temperature to obtain P1 and P2 solutions.

[0071] The incubation temperature conditions (Table 2) and nucleic acid assembly time (Table 3) were changed to find the optimal experimental conditions.

[0072] Table 2 Temperature Optimization

[0073]

[0074] Table 3 Assembly Time Optimization

[0075]

[0076]

[0077] 4T1 cells were seeded into a 6-well plate at an appropriate density and cultured at 37 °C and 5% CO2 until the cells reached 70 - 80% confluence. The assembled nucleic acid structure was co-incubated with 4T1 cells (500 nM, 4 h). The supernatant was removed, and the cells were gently washed 3 times with pre-cooled PBS to remove uningested substances and avoid interfering with the results. The cells were digested with trypsin for 5 min, and the digestion was terminated by adding 1640. The supernatant was removed by centrifugation; the cells were washed with pre-cooled PBS and the supernatant was removed again by centrifugation. The prepared flow cytometry antibody was used to stain the cells for 30 min in the dark. After staining, the staining solution was removed by centrifugation (3500 rpm, 3 min), 300 - 500 μL of PBS was added to resuspend the cells, and centrifugation was performed again to remove the supernatant. The cells were resuspended in 500 μL of PBS. The cells were filtered through a 0.4 μm filter membrane, collected in a flow cytometry tube, and subjected to flow cytometry. The results are as Figure 3 shown, indicating that the constructed DNA nanowires can enter the cells, and the amount of DNA nanowires entering the cells increases with the extension of the incubation time within 4 h.

[0078] Example 3: Verification of cGAS-STING Activation Function

[0079] (1) Western blot was used to detect p-STING, p-TBK1, and p-IRF3

[0080] First, 71-DNA nanowires (abbreviated as 71-DNA), 83-DNA nanowires (abbreviated as 83-DNA), and 96-DNA nanowires (abbreviated as 96-DNA) were prepared using I1, I2, S1, and S2 of different lengths according to the method of Example 2.

[0081] 4T1 cells were cultured until they reached approximately 70%-80% confluence. The cells were divided into different treatment groups, including a negative control group (untreated), 71-DNA, 83-DNA, 96-DNA, Mn2+, and 96+Mn2+ (DNA: 500 Nm, Mn2+: 50 μM). The drugs of each group were added to the cell culture medium, and incubation was continued for 24 hours.

[0082] After treatment, the culture medium was discarded, and the cells were washed 2-3 times with ice-cold PBS buffer to remove the residual culture medium. Pre-cooled RIPA lysis buffer (added with protease and phosphatase inhibitors) was added, and the cells were lysed on ice for 5-10 minutes. After lysis was completed, a cell scraper was used to collect the samples. After ultrasonic fragmentation in an ice bath for 30 s, the samples were centrifuged at 15,000 rpm at 4°C for 20 minutes, and the supernatant was collected for subsequent protein analysis. The changes in the expression levels of the target proteins in different treatment groups were analyzed to verify whether the nucleic acid nanostructure activated the cGAS-STING pathway. The results are as Figure 4 shown. In the untreated control group, the expression levels of p-STING, p-TBK1, and p-IRF3 were extremely low, indicating that the cGAS-STING signaling pathway was not significantly activated. In the treatment groups, the expression levels of these three phosphorylated proteins were significantly up-regulated, and with the increase in DNA length, the activation effect of the signaling pathway gradually enhanced. Specifically, the 71bp DNA nanostructure caused a slight up-regulation of protein expression, the 83bp DNA nanostructure showed a medium-strength activation effect, and the phosphorylation levels of the three proteins in the 96bp DNA nanostructure treatment group were significantly higher than those in other groups, indicating that its activation effect was the most significant. (2) The promoting effect of the nucleic acid structure on the maturation of mouse bone marrow-derived dendritic cells.

[0083] C57BL / 6 mice were sacrificed and femurs and tibias were aseptically obtained. The bone marrow cavities were flushed with PBS to collect bone marrow cells. Subsequently, the collected bone marrow cells were passed through a 70-μm cell strainer to remove impurities. After adjusting the cell concentration, the cells were seeded into RPMI-1640 medium containing 10% FBS, and 20 ng / mL of GM-CSF and 10 ng / mL of IL-4 were added to induce the differentiation of BMDCs. During the culture period, half of the medium was changed every two days and fresh GM-CSF and IL-4 were supplemented to maintain cell viability. On the 6th day, the differentiated and mature BMDCs were seeded into 6-well plates. Subsequently, nucleic acid structures of different lengths (71-DNA, 83-DNA, 96-DNA) were added according to the experimental design, and a blank control group was set up. After culturing the cells at 37 °C and 5% CO2 for 24 hours, the cell culture supernatant was collected for detecting the level of cytokine (IFN-β), and the expression levels of surface maturation markers (CD80, CD86) of BMDCs were detected by flow cytometry to evaluate the promoting effect of nucleic acid structures on the maturation of BMDCs. After the experiment was completed, the differences in the effects of different nucleic acid structures on stimulating the maturation of BMDCs were analyzed according to the detection results. The results are as Figure 5 shown, indicating that the expression levels of the maturation markers CD80 and CD-86 were significantly upregulated in BMDCs treated with DNA nanostructures. It promoted the maturation of dendritic cells in immunity in vitro. The proportion of mature DC cells composed of 96-DNA was 50.9%. Compared with the untreated control group, the 96-DNA group could significantly enhance the immune activity of BMDCs, further indicating that it promoted the maturation of dendritic cells through the cGAS-STING pathway and strengthened the antigen presentation ability, which played a key role in initiating the anti-tumor immune response.

[0084] Example 4: Application in in vivo anti-tumor effect

[0085] A breast cancer mouse model was established using 4T1 cells. 4T1 cells in the logarithmic growth phase were prepared into a single-cell suspension and adjusted to an appropriate concentration with PBS (about 1×106 cells / 100 μL). Subsequently, the cells were inoculated into female BALB / c mice. When the tumor volume reached about 50-100 mm 3 , the mice were randomly grouped and the designed nucleic acid nanostructures and the corresponding control group (PBS) were delivered by tail vein injection respectively. Injection was performed once every 2 days for 3 times. During the injection period, the long and short diameters of the tumor were regularly measured using calipers, the volume was calculated (volume formula: V = long diameter × short diameter2 / 2), and the tumor growth curve was recorded. At the end of the experiment, the mice were sacrificed, the tumor tissues and spleens were removed, and the infiltration of splenic immune cells, including CD8+ T cells, NK cells, etc., was analyzed by flow cytometry.

[0086] The results of the tumor mass of the treated mice are asFigure 6 As shown, it indicates that the anti-tumor effect of 71-DNA in vivo is weak, which may be related to its efficiency in activating the cGAS-STING pathway, or its stability and targeting in vivo are not as good as those of 83-DNA and 96-DNA structures. After injection, the tumor growth rate of mice in the 96-DNA treatment group slowed down, showing a certain tumor inhibitory effect. The tumor mass at the end of treatment decreased by 39.8% compared with the PBS group, while the change in tumor mass in the 71-DNA group was small and almost equivalent to that of the control group. The therapeutic effect of the 96-DNA group far exceeded that of the 71-DNA group, demonstrating the advantage of 96-DNA in anti-tumor immunotherapy. The in vivo immune activation effect is as Figures 7-8 , and the results show that the proportion of CD8+ T cells in the spleens of mice in the 96-DNA group increased (p < 0.05), and the number of NK cells increased more significantly (p < 0.001), providing stronger support for anti-tumor immunity. In the immunofluorescence results of mouse tumor tissues, the infiltration of CD8+ T cells in the tumor tissues of mice in the 96-DNA group was higher than that of the control group. These results are consistent with the anti-tumor effect in mice in vivo, supporting the role of 96-DNA in activating T cells and improving immune surveillance ability.

[0087] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A DNA nanowire, characterized in that, The DNA nanowire is a double-stranded structure composed of P1 and P2, and the ports are blocked with aptamers. P1 includes I1 and I2, P2 includes S1 and S2, and the aptamer includes A1 and A2; The nucleotide sequences of I1, I2, S1, and S2 are each shown as any one of the following groups: (1) SEQ ID NO.1-4; (2) SEQ ID NO.5-8; (3) SEQ ID NO.9-12; The nucleotide sequences of A1 and A2 are shown as SEQ ID NO.13 and SEQ ID NO.14, respectively.

2. The preparation method of the DNA nanowire according to claim 1, characterized in that, The method is to mix P1 and P2 in equal volumes, incubate at room temperature to obtain a DNA double-stranded structure, mix the DNA double-stranded structure with the aptamer and incubate to obtain a DNA nanowire.

3. The preparation method according to claim 2, characterized in that, P1 is obtained by mixing equal volumes of I1 and I2, annealing at 90 °C for 5 minutes, and cooling to room temperature.

4. The preparation method according to claim 2, characterized in that, P2 is obtained by mixing equal volumes of S1 and S2, annealing at 90 °C for 5 minutes, and cooling to room temperature.

5. The preparation method according to claim 2, wherein The DNA double-stranded structure and the aptamer are incubated at room temperature for 1 h.

6. A cGAS-STING agonist, characterized in that, Containing the DNA nanowire described in claim 1.

7. A method for in vitro activation of dendritic cell cGAS-STING, characterized in that, The method is to co-culture with dendritic cells using the DNA nanowire described in claim 1.

8. A composition targeting breast cancer cells, characterized in that, The composition includes the DNA nanowire described in claim 1.

9. Use of the DNA nanowire described in claim 1, or the method described in any one of claims 2-5, or the agonist described in claim 6, or the composition described in claim 8 in the preparation of a drug for relieving and / or treating breast cancer.

10. The application according to claim 9, characterized in that, The application at least includes the following functions: (1) Reducing the volume and mass of the individual's solid tumor; (2) Increasing the number of CD8+ T cells and NK cells in the individual's spleen; (3) Enhancing the infiltration of CD8+ T cells in the individual's tumor tissue.

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