An mRNA molecule for targeted protein degradation and its application

Abstract

The present invention discloses an mRNA molecule for protein targeted degradation and its application. The mRNA molecule for protein targeted degradation disclosed in the present invention includes a 5' cap structure, a 5' non-coding region, a Kozak element, a coding region, a 3' non-coding region and a polyadenylic acid structure in sequence, wherein the coding region includes a functional polypeptide encoding gene, and the functional polypeptide includes a VHL E3 ubiquitin ligase targeting peptide, a connecting peptide and a target protein targeting polypeptide connected in sequence. The mRNA molecule for protein targeted degradation of the present invention has higher safety, can improve the anti-tumor efficacy of peptide PROTACs, solves the key cell penetration problem, can efficiently target and degrade the target protein, and provides a new protein targeted degradation molecular pattern with broad application prospects.

Description

Technical Field

[0001] The present invention relates to an mRNA molecule for targeted protein degradation and its application in the field of biomedicine. Background Art

[0002] Cancer is the leading cause of death in countries around the world and a major obstacle to improving human life expectancy. Currently, there are many different strategies for treating cancer, mainly chemotherapy, surgery, and radiotherapy. Among them, chemotherapy has remained the main treatment for cancer since the 1940s, and anticancer drugs are currently the focus of drug research and development. However, chemotherapy is often accompanied by some toxic side effects, which are usually caused by the adverse effects of anticancer drugs on hematopoietic cells in the bone marrow, hair follicles, and cells in the digestive tract and reproductive system. Therefore, the development of anticancer drugs is committed to improving non-specificity and targeting.

[0003] During the process of tumor formation, due to significant changes in gene expression levels compared to normal cells, abnormal protein expression occurs, such as high-level expression of growth factor receptors. The overexpression of proteins has a causal relationship with tumor formation. Overexpressed proteins in tumor cells can be used as tumor markers for accurate diagnosis of early cancer, and can also be used as targets for anti-tumor drugs. Breast cancer is a common cancer. Among them, the type of breast cancer in which the estrogen receptor α (ERα) signaling pathway promotes breast cancer growth is the most common type of breast cancer, accounting for about 70% of the total number of patients. Therefore, ERα has become an important target for breast cancer treatment. For decades, estrogen inhibitors and ERα antagonists have become the main treatments for ER+ breast cancer.

[0004] Targeted protein degradation is a common strategy in cancer therapy. Intracellular protein ubiquitination and degradation is essential for maintaining cellular function. Its application to cancer therapy has led to the development of the now-representative protein degradation chimera (PROTAC) technology. PROTACs degrade target proteins by recruiting the cell's natural degradation machinery, the ubiquitin-proteasome system (UPS). PROTACs hold enormous potential, capable of degrading undruggable and non-enzymatic proteins that are beyond the reach of traditional small-molecule inhibitors, thereby overcoming the limitations of small-molecule inhibitors used in chemotherapy for decades, namely, non-specificity and drug resistance. Related reports and clinical validation data have fueled the development of this technology. With increasing attention focused on PROTACs, this promising technology is not only expanding its target range and molecular design, but also continuously innovating in environmental responsiveness and tumor targeting. This technology has been in development for two decades, primarily in the form of small chemical molecules and peptide mimetics. In recent years, biomolecules, such as nucleic acids, have emerged as ligands, emphasizing the importance of biocompatibility and safety for PROTACs.

[0005] With the success of the mRNA vaccines developed by Moderna and Pfizer / BioNTech for COVID-19, mRNA technology has received unprecedented attention. mRNA vaccines offer the potential for low-cost manufacturing and improved safety. These advantages enabled Moderna to design and produce a SARS-CoV-2 mRNA vaccine (mRNA-1273) for human use in just 42 days after obtaining the nucleotide sequence of the target antigen. More notably, the SARS-CoV-2 mRNA vaccine produced by Moderna and Pfizer / BioNTech has demonstrated very high efficacy (approximately 90% at ≤6 months of follow-up) in Phase III clinical trials and in the general population.

[0006] The expansion of mRNA applications is closely linked to the continuous improvement of in vitro transcribed mRNA (IVT-mRNA) technology. This synthetic technique improves mRNA stability and translation efficiency within cells. Compared with subunit vaccines, inactivated and attenuated viral vaccines, and DNA vaccines, the use of IVT-mRNA offers several advantages: safety. mRNA is immediately translated in the cytoplasm and directly into the target protein through cellular machinery, without entering the cell nucleus, thus eliminating the potential risk of infection or insertional mutagenesis; stability. Various modifications have made mRNA more stable and its translation efficiency higher; and low cost. mRNA is fast, inexpensive, and can be mass-produced. In addition to its use in vaccines, mRNA has also been applied in protein replacement therapies, but its potential extends far beyond this. Therefore, the rapid improvement of in vitro transcribed mRNA technology will be crucial for the future development of new mRNA-based therapies. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a PROTACs-based mRNA degradation system for target proteins.

[0008] To solve the above technical problems, the present invention first provides an mRNA molecule for targeted protein degradation, wherein the mRNA molecule includes a 5' cap structure (5'-cap), a 5' non-coding region, a Kozak element, a coding region, a 3' non-coding region and a polyadenylic acid structure (poly-A) in sequence, the coding region includes a functional polypeptide encoding gene, and the functional polypeptide includes a VHL E3 ubiquitin ligase targeting peptide, a connecting peptide and a target protein targeting polypeptide connected in sequence.

[0009] The core of the present invention is to provide a new system, namely the PROTACs-based mRNA system, specifically referring to the modular settings of the above-mentioned mRNA molecules, wherein the 5'-cap, Kozak element and poly-A are all conventional components of eukaryotic mRNA, the sequence of the Kozak element is shown in positions 51 to 56 of SEQ ID No. 7, the 5' non-coding region and the 3' non-coding region can be various conventional sequences in the art, and the present invention uses the 5' non-coding region of β-globin (the sequence is shown in positions 1 to 50 of SEQ ID No. 7) and the 3' non-coding region of α-globin (the sequence is shown in positions 897 to 1007 of SEQ ID No. 7). The coding region is an element for degrading the target protein. Based on the principle of PROTACs, it includes a VHL E3 ubiquitin ligase targeting peptide, a connecting peptide and a target protein targeting polypeptide, wherein the VHL E3 ubiquitin ligase targeting peptide sequence is a known sequence, as shown in SEQ ID No. 1, and the connecting peptide can select a conventional sequence, such as the sequence shown in SEQ ID No. 2 or SEQ ID No. 3. The target protein targeting polypeptide is a polypeptide that specifically targets the target protein and is selected or designed according to the target protein. According to the principle of PROTACs, its specific sequence does not affect the effect of the system.

[0010] According to one embodiment of the present invention, the target protein is estrogen receptor α, and the sequence of the corresponding target protein targeting polypeptide is shown in SEQ ID No. 4. Correspondingly, the sequence of the functional polypeptide is shown in SEQ ID No. 6.

[0011] According to another embodiment of the present invention, the target protein is the anti-apoptotic protein BCL-x L , the corresponding target protein targeting polypeptide sequence is shown in SEQ ID No. 5. Correspondingly, the functional polypeptide sequence is shown in SEQ ID No. 11.

[0012] In the present invention, in order to verify the feasibility of the system, a reporter protein is designed and located in the coding region, and a spacer sequence is set between the functional polypeptide to avoid mutual influence of protein expression. As long as a suitable interval is set, the coding region can contain genes encoding other proteins, for example, another functional protein encoding gene or a reporter protein encoding gene. The coding region can be composed of the functional polypeptide encoding gene, another functional protein encoding gene or a reporter protein encoding gene, and a spacer region set between them and the functional polypeptide.

[0013] According to a specific embodiment of the present invention, the reporter protein is EGFP (the sequence is shown in positions 57 to 773 of SEQ ID No. 7), and the spacer region is the P2A peptide encoding gene (the sequence is shown in positions 774 to 839 of SEQ ID No. 7).

[0014] Based on the above design principles, when the target protein is estrogen receptor α, the sequence of the mRNA molecule can be shown as SEQ ID No. 7. When the target protein is BCL-x L When , the sequence of the mRNA molecule can be shown as SEQ ID No.12.

[0015] The present invention also provides biological materials related to the mRNA molecules.

[0016] The second aspect of the present invention provides a DNA molecule encoding the mRNA molecule.

[0017] When the target protein is estrogen receptor α, the sequence of the DNA molecule can be shown as SEQ ID No. 8. When the target protein is BCL-x L When , the sequence of the DNA molecule may be shown as SEQ ID No.13.

[0018] The third aspect of the present invention provides a recombinant vector comprising the aforementioned DNA molecule. The vector may be a plasmid, cosmid, phage or viral vector.

[0019] When the target protein is estrogen receptor α, the sequence of the recombinant vector can be shown as SEQ ID No. 9. When the target protein is BCL-x L When , the sequence of the recombinant vector may be shown as SEQ ID No.14.

[0020] A fourth aspect of the present invention provides a recombinant microorganism, wherein the recombinant microorganism is transfected with the above-mentioned mRNA molecule, the above-mentioned DNA molecule or the above-mentioned recombinant vector.

[0021] The fifth aspect of the present invention provides a cell transfected with the aforementioned mRNA molecule, the aforementioned DNA molecule, the aforementioned recombinant vector, or the aforementioned recombinant microorganism. The microorganism may be yeast, bacteria, algae, or fungi.

[0022] According to the present invention, the cells may be MCF-7 cells or MDA-MB-231 cells.

[0023] The present invention also provides a method for degrading a target protein for non-therapeutic purposes, the method comprising: applying the mRNA to biological cells, tissues or organs expressing the target protein to achieve degradation of the target protein in the biological cells, tissues or organs.

[0024] The biological cell can be a microbial cell, a plant cell or an isolated animal cell. The microbial cell can be a yeast, a bacterium, an algae or a fungus.

[0025] The tissue is plant tissue or isolated animal tissue.

[0026] The organ is a plant organ or an isolated animal organ.

[0027] The present invention also provides any of the following uses of the aforementioned mRNA molecule, the aforementioned DNA molecule, the aforementioned recombinant vector or the aforementioned recombinant microorganism:

[0028] M1) preparing products for treating and / or mediated diseases / conditions of the target protein;

[0029] M2) treating and / or preventing diseases / pathologies mediated by the target protein;

[0030] M3) preparing a product for inhibiting cell proliferation;

[0031] M4) inhibits cell proliferation;

[0032] M5) preparing products that promote cell apoptosis;

[0033] M6) promotes cell apoptosis.

[0034] In the above applications, the product may be a vaccine or a medicine.

[0035] According to the present invention, the disease mediated by the target protein may be cancer; the pathology mediated by the target protein may be plant pathology.

[0036] According to the present invention, the cell may be a cancer cell.

[0037] The cancer includes, but is not limited to, leukemia, lymphoma, lung cancer, breast cancer, ovarian cancer, cervical cancer, human brain glioma, melanoma, glioblastoma, nasopharyngeal cancer, liver cancer, brain cancer, pancreatic cancer, uterine cancer, testicular cancer, skin cancer, stomach cancer, colon cancer, bladder cancer or rectal cancer.

[0038] In one embodiment of the present invention, the cancer is breast cancer, the cells are breast cancer cells, and the breast cancer cells are MCF-7 cells.

[0039] In another embodiment of the present invention, the breast cancer cells are MDA-MB-231 cells.

[0040] The m-PROTAC of the present invention has the following advantages: (1) The biomolecular properties of the drug itself have higher safety, and in situ expression in the cytoplasm can achieve the therapeutic effect of low-concentration administration and high-concentration expression; (2) The drug can improve the anti-tumor efficacy of peptide PROTACs, solve the key cell penetration problem, and fully utilize its specific advantages; (3) The drug uses the PROTACs method to act in a mode similar to catalysis to achieve efficient targeted degradation function; (4) The drug provides a new protein targeted degradation molecular pattern and also provides new ideas for the therapeutic use of mRNA.

[0041] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of the composition of m-PROTAC and the degradation of target proteins.

[0043] Figure 2 Schematic diagram of the vm-PROTAC vector, where Peptide represents a functional peptide that targets and degrades ERα.

[0044] Figure 3 Shown are the results of eGFP expression detection by m-PROTAC flow cytometry analysis.

[0045] Figure 4 Cell imaging images of MCF-7 cells 24 hours after transfection with 1 μg / mL m-PROTAC. The upper left corner shows the white light channel image of m-PROTAC-treated cells, while the lower left corner shows the white light channel image of untreated cells. The upper right corner shows the green fluorescence channel image of m-PROTAC-treated cells, while the lower right corner shows the green fluorescence channel image of untreated cells. Bar = 20 μm.

[0046] Figure 5 Shown are the ERα expression levels in MCF-7 cells after treatment with different concentrations of mRNA for 24 h.

[0047] Figure 6 Shown are the ERα expression levels in MCF-7 cells after treatment with 1 μg / mL mRNA for different time periods.

[0048] Figure 7 The expression levels of ERα in MG-132 cells co-incubated with m-PROTAC are shown. In the right panel, m represents m-PROTAC.

[0049] Figure 8 Shown are the cell viabilities of MCF-7 cells treated with different concentrations of m-PROTAC for 24 h or 48 h.

[0050] Figure 9 Shown are the results of apoptosis analysis of MCF-7 cells treated with different concentrations of m-PROTAC.

[0051] Figure 10 Shown are the cell cycle analysis results of MCF-7 cells treated with different concentrations of m-PROTAC.

[0052] Figure 11 Schematic diagram of the vm-PROTAC-2 vector, where Peptide-2 represents a functional peptide that targets and degrades BCL-xL.

[0053] Figure 12 Shown is the targeted degradation of BCL-x by m-PROTAC-2 in MDA-MB-231 cells L Variation with concentration. DETAILED DESCRIPTION

[0054] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples were all repeated three times, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0055] Example 1. Preparation of m-PROTAC

[0056] This example provides an mRNA PROTAC molecule targeting the degradation of ERα, referred to as m-PROTAC, which works by encoding a functional polypeptide (the functional polypeptide is denoted as E3-ERα). The functional polypeptide E3-ERα is obtained by sequentially connecting a heptapeptide sequence (PIYPALA, SEQ ID No. 1) targeting the VHL E3 ubiquitin ligase portion, a connecting peptide sequence (GSGS, SEQ ID No. 2), and a polypeptide sequence targeting ERα protein binding (QLLRHLILH, SEQ ID No. 4). Its sequence is SEQ ID No. 6 in the sequence list.

[0057] The 5'-end modified cap structure (Cap) of m-PROTAC is obtained by sequentially connecting the 5' non-coding region of β-globin, the Kozak element, the coding region (obtained by sequentially connecting the enhanced green fluorescent protein (eGFP), the P2A element, and the mRNA of the functional polypeptide E3-ERα), the 3' non-coding region of α-globin, and the polyadenylation structure (Poly-A). The sequence of m-PROTAC is SEQ ID No. 7 in the sequence listing, and the corresponding m-PROTAC DNA sequence is SEQ ID No. 8 in the sequence listing.

[0058] The composition of m-PROTAC and the principle of degradation of target protein are as follows Figure 1 Figure 2: m-PROTAC mRNA was obtained by in vitro transcription from a linearized plasmid template containing m-PROTAC DNA.

[0059] 1. Preparation of recombinant vector

[0060] Based on the m-PROTAC template sequence shown in SEQ ID No. 8, an in vitro transcription vector vector vm-PROTAC (whose sequence is SEQ ID No. 9 in the sequence list, as shown in FIG. 8 ) containing the m-PROTAC template sequence shown in SEQ ID No. 8 was synthesized. Figure 2 The in vitro transcription promoter is T7 promoter, and the downstream contains AscI restriction site.

[0061] vm-PROTAC was digested with AscI and purified to obtain the linear plasmid vm-PROTAC.

[0062] 2. In vitro transcription of m-PROTAC

[0063] 1) Based on the instructions for the in vitro transcription reagent (Promega, P1300), add the following to a 1.5 mL enzyme-free centrifuge tube in the following order:

[0064] ①5×T7 transcription buffer: 20.0 μL

[0065] ② rNTP mixture: 30.0 μL

[0066] ③1-2μg linear plasmid (DEPC water solution): 32.5μL

[0067] ④Cap-modified guanine (40mM): 7.5μL

[0068] ⑤ Enzyme mixture: 10.0 μL;

[0069] 2) Mix gently with a pipette tip and place in a 37°C metal bath for 4-6 hours;

[0070] 3) Purification: Add RQ1 RNase-Free DNase at a concentration of 1 U / μg DNA template, incubate at 37°C for 30 min, and then purify using an mRNA purification kit to obtain m-PROTAC. Measure the concentration and store in aliquots at -85°C.

[0071] Example 2. Cells transfected with m-PROTAC can efficiently express the target protein

[0072] The breast cancer cell line MCF-7 cells were cultured in DMEM medium in a 12-well plate and transfected with different concentrations of m-PROTAC (the concentration of m-PROTAC in the system was set to 0, 0.5, and 1 μg / mL). After incubation at 37°C for 4 hours, the cells were washed with PBS and replaced with DMEM complete medium for a further 20 hours (total incubation time was 24 hours). The cells were then collected and flow cytometry was performed to detect the expression level of eGFP in the cells. The quantitative analysis results are shown in Figure 2. Figure 3 As shown, the intensity of green fluorescent protein expressed at 1 μg / mL was 4.44 times that of the control group.

[0073] The MCF-7 cells transfected with 1 μg / mL m-PROTAC obtained above were imaged. The results are as follows. Figure 4 As shown in the figure, the transfected cells produce obvious eGFP green fluorescence. The results show that cells transfected with m-PROTAC can efficiently express the target protein.

[0074] Example 3. Changes in m-PROTAC concentration in MCF-7 cells targeting ERα degradation

[0075] MCF-7 cells, a breast cancer cell line, were cultured in DMEM medium in 12-well plates and transfected with m-PROTAC at different concentrations (the concentration of m-PROTAC in the system was set to 0, 0.125, 0.25, 0.5, 1, and 2 μg / mL). After incubation at 37°C for 4 hours, the cells were washed with PBS and replaced with complete DMEM medium. The cells were then collected and incubated for a further 20 hours (total incubation time of 24 hours). Western Blot analysis was performed using the β-actin gene as an internal control. The primary antibodies used were ERα rabbit monoclonal antibody (CellSignaling Technology, #8644) and β-actin rabbit monoclonal antibody (ABclonal, AC026).

[0076] The results are as follows Figure 5As shown, ERα expression levels decreased significantly with increasing mRNA incubation concentrations, indicating that ERα degradation occurred in an mRNA concentration-dependent manner. Degradation efficiency reached 60% at 1 μg / mL, and 1 μg / mL mRNA was subsequently used for further analysis.

[0077] Example 4. Changes in ERα targeted degradation by m-PROTAC in MCF-7 cells over time

[0078] MCF-7 cells, a breast cancer cell line, were cultured in DMEM medium in 12-well plates and transfected with m-PROTAC (at a concentration of 1 μg / mL). Cells were harvested at 37°C after 0, 6, 12, 24, or 36 hours of transfection and analyzed by Western Blot. Cells treated with a control mRNA at a concentration of 1 μg / mL for 24 hours served as a control.

[0079] The control mRNA is an mRNA that does not contain a functional polypeptide coding sequence and is prepared as follows: according to the method of step 1 in Example 1, the DNA fragment represented by SEQ ID No. 8 in the vm-PROTAC obtained in Example 1 is replaced with SEQ ID No. 10 to obtain a recombinant vector v-control; according to the method of step 1 in Example 1, vm-PROTAC is replaced with v-control, and the other steps remain unchanged to obtain a control mRNA.

[0080] The results are as follows Figure 6 As shown, ERα expression levels decreased significantly with increasing incubation time, indicating that ERα degradation is dependent on mRNA incubation time. The degradation efficiency reached 60% after 24 hours, so a 24-hour incubation time was selected for further experiments.

[0081] Example 5: m-PROTAC uses the ubiquitin-protease system to degrade ERα in MCF-7 cells

[0082] MG-132 can inhibit the function of proteasome and is a common proteasome inhibitor.

[0083] MCF-7 cells, a breast cancer cell line, were cultured in 12-well plates using DMEM medium. MG-132 was added to the culture system at a concentration of 10 μM. MCF-7 cells were transfected with m-PROTAC (the concentration of m-PROTAC in the system was set to 1 μg / mL) and incubated at 37°C for 4 h. After washing with PBS and replacing with DMEM complete medium containing 10 μM MG132, the cells were collected and incubated for a further 20 h (total incubation time of 24 h). Cells were then collected and analyzed by Western Blot. Untreated cells (blank), cells treated with m-PROTAC alone, and cells treated with MG-132 alone were set as controls.

[0084] The results are as follows Figure 7 The expression level of ERα did not decrease significantly after the addition of MG-132, while the expression level of ERα in cells not treated with MG-132 decreased significantly, indicating that MG-132 could block the degradation of ERα by m-PROTAC, further verifying that m-PROTAC-induced ERα degradation is carried out by the ubiquitin-proteasome system.

[0085] Example 6: m-PROTAC inhibits the proliferation of MCF-7 cells

[0086] MCF-7 cells, a breast cancer cell line, were cultured in DMEM medium in 12-well plates and transfected with m-PROTAC at varying concentrations (0, 0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 μg / mL). After incubation at 37°C for 4 h, the cells were replaced with complete DMEM medium and incubated for a further 20 h (total incubation time of 24 h) or 44 h (total incubation time of 48 h), and cell viability was measured. Cell viability was assessed using a CCK-8 kit (Beyotime, C0038).

[0087] The results are as follows Figure 8 As shown, m-PROTAC has a significant inhibitory effect on the proliferation of MCF-7 cells, with an IC50 of approximately 8.2 μg / mL at 24 hours and an IC50 of approximately 1.7 μg / mL at 48 hours of treatment, corresponding to molar concentrations of 24.5 nM and 5.1 nM, while the currently reported ERα PROTACs are all at the micromolar level. This shows that a certain concentration of m-PROTAC can effectively kill MCF-7 cells. The m-PROTAC of the present invention is more efficient in targeting and degrading target proteins than other existing PROTACs systems.

[0088] Example 7: m-PROTAC induces apoptosis in MCF-7 cells through S phase arrest

[0089] The breast cancer cell line MCF-7 cells were cultured in DMEM medium in a 12-well plate and transfected with m-PROTAC at specific concentrations (the concentrations were set to 0, 0.125, 0.25, 0.5, 1, and 2 μg / mL). After incubation at 37°C for 4 h, the cells were washed with PBS and replaced with DMEM complete medium. The cells were then collected and subjected to cell apoptosis analysis.

[0090] For apoptosis analysis, cells were stained with Annexin V-FITC and PI according to the instructions of the Annexin V-FITC Apoptosis Detection Kit (Biyuntian, C1062). For cell cycle analysis, the cells were resuspended in 70% pre-cooled ethanol aqueous solution, placed at 4°C overnight, and then centrifuged to remove ethanol. The collected cells were treated with RNase A and propidium iodide (PI) according to the instructions of the Cell Cycle and Apoptosis Analysis Kit (Biyuntian, C1052). Finally, the stained samples were tested using a flow cytometer and analyzed using ModFit LT 5.0. For each individual experiment, more than 10,000 cells were counted for apoptosis and cycle detection.

[0091] The results are as follows Figure 9 、 10 As shown in Figure 3, with increasing concentrations, the proportion of late apoptosis in MCF-7 cells treated with m-PROTAC increased, and the number of cells trapped in the S phase increased, indicating that m-PROTAC caused MCF-7 cell apoptosis by trapping them in the S phase.

[0092] Example 8. Preparation of m-PROTAC-2 and its targeted degradation of BCL-x in MDA-MB-231 cells L Changes with concentration

[0093] This embodiment provides a method for the targeted degradation of BCL-x L The mRNA PROTAC molecule, referred to as m-PROTAC-2, encodes a functional polypeptide (the functional polypeptide is denoted as E3-BCL-x L ) plays a role, the functional polypeptide E3-BCL-x L The peptide sequence targeting VHL E3 ubiquitin ligase (PIYPALA, SEQ ID No. 1), the connecting peptide sequence (GGGGGG, SEQ ID No. 3), and the peptide sequence targeting BCL-x L The protein-binding polypeptide sequence (GQVGRQLAIIGDAINR, SEQ ID No. 5) was sequentially connected to obtain a sequence of SEQ ID No. 11 in the sequence listing.

[0094] The 5' end of m-PROTAC-2 is modified with a cap structure (Cap), which is composed of the 5' non-coding region of β-globin, a Kozak element, a coding region (composed of enhanced green fluorescent protein (eGFP), a P2A element, and a functional peptide E3-BCL-x L The mRNA of m-PROTAC-2 is connected in sequence, the 3' non-coding region of α-globin, and the polyadenylation structure (Poly-A) are connected in sequence. The sequence of m-PROTAC-2 is SEQ ID No. 12 in the sequence listing, and the corresponding DNA sequence of m-PROTAC-2 is SEQ ID No. 13 in the sequence listing.

[0095] m-PROTAC-2 mRNA was obtained by in vitro transcription from a linearized plasmid template containing m-PROTAC-2 DNA.

[0096] Based on the m-PROTAC-2 template sequence shown in SEQ ID No. 13, an in vitro transcription vector vector vm-PROTAC-2 (whose sequence is SEQ ID No. 14 in the sequence list) containing the m-PROTAC-2 template sequence shown in SEQ ID No. 13 was synthesized. Figure 11 The in vitro transcription promoter is T7 promoter, and the downstream contains AscI restriction site.

[0097] vm-PROTAC-2 was digested with AscI and purified to obtain the linear plasmid vm-PROTAC-2.

[0098] According to the method of step 2 in Example 1, vm-PROTAC was replaced with vm-PROTAC-2 to obtain m-PROTAC-2.

[0099] The breast cancer cell line MDA-MB-231 cells were cultured in L-15 medium in 12-well plates and transfected with different concentrations of m-PROTAC-2 (the concentration of m-PROTAC-2 in the system was set to 0, 0.125, 0.25, 0.5, 1, and 2 μg / mL). After incubation at 37°C for 4 hours, PBS was washed and replaced with L-15 complete medium for a further 20 hours (total incubation time was 24 hours). After that, the cells were collected and analyzed by Western Blot. The internal reference was the β-actin gene. The primary antibody used was BCL-x L Recombinant rabbit monoclonal antibody (Yisheng Bio, 31011ES), β-actin rabbit monoclonal antibody (ABclonal, AC026).

[0100] The results are as follows Figure 12 As shown. BCL-x LAs the mRNA concentration increased, the expression level decreased significantly, indicating that BCL-x L The degradation was observed to be mRNA concentration-dependent.

[0101] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. An mRNA molecule for targeted protein degradation, the mRNA molecule comprising, in sequence, a 5' cap structure, a 5' non-coding region, a Kozak element, a coding region, a 3' non-coding region, and a polyadenylation structure; the coding region comprising a gene encoding a functional polypeptide, wherein the functional polypeptide comprises, in sequence, a VHL E3 ubiquitin ligase targeting peptide, a linker peptide, and a target protein targeting polypeptide; the coding region further comprising a gene encoding a reporter protein, and a spacer disposed between the reporter protein and the functional polypeptide; the reporter protein is EGFP, and the spacer is a gene encoding a P2A peptide; The target protein targeting polypeptide is a polypeptide that specifically targets the target protein, and its sequence is shown in SEQ ID No. 4 or SEQ ID No.

5.

2. The mRNA molecule for protein targeted degradation according to claim 1, wherein The sequence of the VHL E3 ubiquitin ligase targeting peptide is shown in SEQ ID No. 1; The sequence of the connecting peptide is shown as SEQ ID No. 2 or SEQ ID No.

3.

3. The mRNA molecule for protein targeted degradation according to claim 1, wherein The sequence of the functional polypeptide is shown as SEQ ID No.6 or SEQ ID No.

11.

4. The mRNA molecule for protein targeted degradation according to claim 1, wherein The 5' non-coding region is the 5' non-coding region of β-globin, and the 3' non-coding region is the 3' non-coding region of α-globin.

5. The mRNA molecule for protein targeted degradation according to claim 1, wherein The sequence of the mRNA molecule is shown as SEQ ID No.7 or SEQ ID No.

12.

6. A DNA molecule encoding the mRNA molecule according to any one of claims 1 to 5.

7. The DNA molecule according to claim 6, wherein The sequence of the DNA molecule is shown as SEQ ID No.8 or SEQ ID No.

13. A recombinant vector comprising the DNA molecule according to claim 6 or 7.

9. The recombinant vector according to claim 8, wherein The sequence of the recombinant vector is shown as SEQ ID No.9 or SEQ ID No.

14.

10. A recombinant microorganism, wherein the recombinant microorganism is transfected with the mRNA molecule according to any one of claims 1 to 5, the DNA molecule according to claim 6 or 7, or the recombinant vector according to claim 8 or 9.

11. A cell transfected with the mRNA molecule according to any one of claims 1 to 5, the DNA molecule according to claim 6 or 7, the recombinant vector according to claim 8 or 9, or the recombinant microorganism according to claim 10.

12. The cell according to claim 11, wherein The cells are MCF-7 cells or MDA-MB-231 cells.

13. Any of the following uses of the mRNA molecule according to any one of claims 1 to 5, the DNA molecule according to claim 6 or 7, the recombinant vector according to claim 8 or 9, or the recombinant microorganism according to claim 10: M1) preparing products for treating and / or preventing diseases / pathologies mediated by the target protein; M2) Preparation of products that inhibit cell proliferation; M3) Preparation of products that promote cell apoptosis; The sequence of the target protein targeting polypeptide is shown as SEQ ID No. 4, the disease / lesion mediated by the target protein is breast cancer, and the cell is a breast cancer cell.

14. The use according to claim 13, wherein: The product is a vaccine or a medicine.

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