mRNA molecules for targeted protein degradation and uses thereof

Abstract

Provided are mRNA molecules for targeted protein degradation and uses thereof. The mRNA molecule for targeted protein degradation comprises 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 that order. The coding region includes a gene encoding a functional polypeptide, and the functional polypeptide is composed of a VHL E3 ubiquitin ligase targeting peptide, a linker peptide, and a target protein targeting polypeptide, linked in sequence. The mRNA molecule for targeted protein degradation is highly safe, improves the antitumor effect of peptide-type PROTACs, solves the important problem of cell penetration, and can efficiently target and degrade target proteins. The present invention provides a novel molecular pattern for targeted protein degradation that is expected to have a wide range of applications.

Description

[Technical Field]

[0001] The present invention relates to mRNA molecules and their uses for targeted protein degradation in the biomedical field. [Background technology]

[0002] Cancer is a leading cause of death worldwide and a major obstacle to improving human life expectancy. Currently, there are various strategies for cancer treatment, primarily chemotherapy, surgery, and radiation therapy. Chemotherapy has been the mainstream of cancer treatment since the 1940s, and anticancer drugs are currently the focus of pharmaceutical research and development. However, chemotherapy often causes toxic side effects, which are usually the adverse effects of anticancer drugs on hematopoietic cells in the bone marrow, hair follicles, the gastrointestinal tract, and cells in the reproductive system. Therefore, research and development of anticancer drugs has focused on improving nonspecificity and targeting.

[0003] During tumor development, gene expression levels change significantly compared to normal cells, leading to abnormal protein expression. For example, high levels of growth factor receptor expression and protein overexpression are causally associated with tumorigenesis. Overexpressed proteins in tumor cells can be used as tumor markers for accurate early cancer diagnosis and can also be targeted by antitumor drugs. Breast cancer is a common cancer. The type in which the estrogen receptor α (ERα) signaling pathway drives breast cancer proliferation is the most common type, accounting for approximately 70% of all cases. Therefore, ERα has become an important target for breast cancer treatment. For decades, estrogen inhibitors and ERα antagonists have been the mainstay of treatment for ER+ breast cancer.

[0004] Targeted protein degradation is a commonly used strategy in tumor therapy. Ubiquitination of intracellular proteins is essential for maintaining cellular function, leading to the development of the currently representative technology, the proteolysis-targeted chimera (PROTAC) technology. PROTACs can degrade targeted proteins via the ubiquitin proteasome system (UPS), the cell's natural degradation mechanism. PROTACs have great potential for degrading undruggable and nonenzymatic proteins that are difficult to control with conventional small molecule inhibitors. This technology overcomes the shortcomings of small molecule inhibitors used in chemotherapy over the past few decades, namely nonspecificity and drug resistance. Related reports and clinical validation data have promoted the development of this technology. As attention to PROTACs has increased, this promising technology has not only expanded in target scope and molecular design, but also continuously innovated in environmental responsiveness and tumor targeting. This technology has been developed over the past 20 years, primarily in the form of small chemical molecules and peptidomimetics. In recent years, biomolecules such as nucleic acid molecules have emerged as ligands, and the need to consider biocompatibility and safety in PROTACs has been emphasized.

[0005] The success of the mRNA vaccine developed by Moderna and Pfizer / BioNTech for COVID-19 has attracted unprecedented attention to mRNA technology. mRNA vaccines can be produced at lower cost and potentially with improved safety. These advantages allowed Moderna to design and manufacture a SARS-CoV-2 mRNA vaccine (mRNA-1273) for humans in just 42 days after obtaining the nucleotide sequence of the target antigen. Even more noteworthy, the SARS-CoV-2 mRNA vaccine developed by Moderna and Pfizer / BioNTech demonstrated extremely high efficacy (approximately 90% within a six-month follow-up period) in Phase III clinical trials and in the general population.

[0006] The expansion of mRNA use is closely linked to the continuous improvement of in vitro transcribed mRNA (IVT-mRNA) technology. This synthesis technology enhances the stability and translation efficiency of mRNA within cells. Compared with subunit vaccines, inactivated and attenuated vaccines, and DNA vaccines, the use of IVT-mRNA offers the following advantages: (1) Safety: mRNA is immediately translated in the cytoplasm and directly translated into target proteins by the cellular machinery without entering the cell nucleus, eliminating the potential risks of infection and insertional mutagenesis. (2) Stability: Various modifications have improved mRNA stability and translation efficiency. (3) Low cost: mRNA has the advantages of being fast, inexpensive, and capable of mass production. In addition to vaccine applications, mRNA is also used in protein replacement therapy, and its potential is not limited to this. Therefore, with the rapid advances in in vitro transcribed mRNA technology, the development of new mRNA-based therapeutics is becoming increasingly important. Summary of the Invention

[0007] Research and development of anticancer drugs is a long-term endeavor, particularly in terms of improving the specificity and targeting of anticancer drugs. The present invention provides an mRNA molecule for targeted protein degradation (referred to as m-PROTAC), which comprises 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 that order, where the coding region comprises a gene encoding a functional polypeptide, and the functional polypeptide comprises a VHL E3 ubiquitin ligase targeting peptide, a linker peptide, and a target protein targeting polypeptide, linked in that order. The mRNA molecule of the present invention exhibits excellent targeting and also exhibits high therapeutic efficacy against tumors. [Problem to be solved by the invention]

[0008] The technical problem that the present invention aims to solve is to provide an mRNA system for degrading target proteins using PROTACs. [Means for solving the problem]

[0009] In order to solve the above technical problems, the mRNA molecule of the present invention (referred to as m-PROTAC) comprises 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 this order; the coding region comprises a gene encoding a functional polypeptide, and the functional polypeptide is composed of a VHL E3 ubiquitin ligase target peptide, a linker peptide, and a target protein target polypeptide linked in that order.

[0010] The core of the present invention is to provide a novel system, an mRNA system based on PROTACs. Specifically, this refers to the modular arrangement of the mRNA molecule. The 5'-cap, Kozak element, and poly-A are all basic components of eukaryotic mRNA. The Kozak element sequence is at positions 51-56 of SEQ ID NO. 7. The 5' non-coding region and 3' non-coding region may have various conventional sequences in the art, but the present invention uses the 5' non-coding region of β-globin (sequence of positions 1-50 of SEQ ID NO. 7) and the 3' non-coding region of α-globin (sequence of positions 897-1007 of SEQ ID NO. 7). The coding region is an element for target protein degradation and, based on the principle of PROTACs, includes a VHL E3 ubiquitin ligase targeting peptide, a linker peptide, and a target protein targeting polypeptide. The sequence of the VHL E3 ubiquitin ligase targeting peptide is a known sequence such as that shown in SEQ ID NO. 1, and the linker peptide may be selected from conventional sequences such as those shown in SEQ ID NO. 2 or SEQ ID NO. 3. The target protein targeting polypeptide is a polypeptide that specifically targets a target protein and is selected or designed according to the target protein. Based on the principle of PROTACs, its specific sequence does not affect the function of the system.

[0011] According to one embodiment of the present invention, the target protein is estrogen receptor alpha, 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.

[0012] According to another embodiment of the invention, the target protein is the anti-apoptotic protein BCL-x L and the sequence of the polypeptide that targets the corresponding target protein is shown in SEQ ID NO. 5. Correspondingly, the functional polypeptide sequence is shown in SEQ ID NO. 11.

[0013] In the present invention, to verify the feasibility of the system, a reporter protein is designed to be located in the coding region, and a spacer sequence is provided between the reporter protein and the functional polypeptide to prevent interactions in protein expression. As long as an appropriate spacer is provided, the coding region may also include a gene encoding another protein, for example, a gene encoding another functional protein or a gene encoding a reporter protein. The coding region may be composed of a gene encoding a functional polypeptide, a gene encoding another functional protein, or a gene encoding a reporter protein, and a spacer region provided between them and the functional polypeptide.

[0014] According to a specific embodiment of the present invention, the reporter protein is EGFP (the sequence is from positions 57 to 773 of SEQ ID NO. 7), and the spacer region is a gene encoding the P2A peptide (the sequence is from positions 774 to 839 of SEQ ID NO. 7).

[0015] Based on the above design principles, if the target protein is estrogen receptor alpha, the sequence of the mRNA molecule may be SEQ ID NO. 7. L If so, the sequence of the mRNA molecule may be SEQ ID NO. 12.

[0016] The present invention further provides biomaterials related to mRNA molecules.

[0017] A second aspect of the present invention provides a DNA molecule encoding the mRNA molecule.

[0018] If the target protein is estrogen receptor alpha, the sequence of the DNA molecule may be SEQ ID NO. 8. L If so, the sequence of the DNA molecule may be SEQ ID NO. 13.

[0019] A third aspect of the present invention provides a recombinant vector comprising the above DNA molecule. The vector may be a plasmid, cosmid, phage, or viral vector.

[0020] When the target protein is estrogen receptor alpha, the sequence of the recombinant vector may be SEQ ID NO. 9. When the target protein is BCL-x L If so, the sequence of the recombinant vector may be SEQ ID NO.14.

[0021] A fourth aspect of the present invention provides a recombinant microorganism transfected with (i.e. containing) the above mRNA molecule, the above DNA molecule, or the above recombinant vector. The microorganism may be a yeast, bacterium, algae, or fungus.

[0022] A fifth aspect of the present invention provides a cell transfected with (ie containing) the above mRNA molecule, the above DNA molecule, the above recombinant vector, or the above recombinant microorganism.

[0023] The material may (or may not) include propagation material.

[0024] According to the present invention, the cells may be MCF-7 cells or MDA-MB-231 cells.

[0025] The present invention further provides any of the following uses of the mRNA molecule, the DNA molecule, the recombinant vector, or the recombinant microorganism: M1) Manufacturing of products for treating and / or preventing diseases / pathologies mediated by target proteins; M2) Treatment and / or prevention of disease / pathology via target protein; M3) Manufacturing of products that inhibit cell proliferation; M4) inhibition of cell proliferation; M5) Manufacturing of products that promote apoptosis; M6) Promotion of apoptosis.

[0026] In the above uses, the product may be a vaccine or a pharmaceutical.

[0027] According to the present invention, the target protein-mediated disease may be cancer, and the target protein-mediated pathology may be a pathology in a plant or animal.

[0028] According to the present invention, the cells may be cancer cells.

[0029] Cancers include, but are 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.

[0030] In one embodiment of the invention, the cancer is breast cancer, the cells are breast cancer cells, and the breast cancer cells are MCF-7 cells.

[0031] In another embodiment of the present invention, the breast cancer cells are MDA-MB-231 cells.

[0032] In another embodiment of the present invention, the breast cancer cells are the 4T1 cell line.

[0033] The present invention further provides a method for degrading a target protein for non-therapeutic or therapeutic purposes, which method comprises administering an mRNA molecule to a biological cell, tissue or organ that expresses the target protein, thereby achieving degradation of the target protein in the biological cell, tissue or organ.

[0034] The biological cell may be a microbial cell, a plant cell, or an isolated animal cell. The microbial cell may be a yeast, bacterial, algae, or fungus. The animal cell may be a cancer cell, including, but 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 cells.

[0035] The tissue may be plant tissue or isolated animal tissue.

[0036] The organ may be a plant organ or an isolated animal organ.

[0037] The present invention further provides a method for treating and / or preventing a target protein-mediated disease / pathology, which method comprises administering an mRNA molecule to an animal suffering from the target protein-mediated disease / pathology, thereby achieving treatment and / or prevention and / or alleviation of the target protein-mediated disease / pathology in the animal.

[0038] The animal may be a human or a non-human animal.

[0039] In the above method, the target protein-mediated disease may be cancer, and the cells may be cancer cells.

[0040] The cancer may be, 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.

[0041] The present invention further provides a method for inhibiting cell proliferation for non-therapeutic or therapeutic purposes, which method comprises administering an mRNA molecule to a cell expressing a target protein to achieve inhibition of cell proliferation.

[0042] The cell may be a microbial cell, a plant cell, or an animal cell. The animal cell may be a cancer cell, including, but not limited to, a 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 cell.

[0043] The present invention further provides a method for promoting apoptosis for non-therapeutic or therapeutic purposes, which method comprises administering an mRNA molecule to cells expressing a target protein to achieve promotion of apoptosis.

[0044] The cell may be a microbial cell, a plant cell, or an animal cell. The animal cell may be a cancer cell, including, but not limited to, a 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 cell. [Effects of the Invention]

[0045] The m-PROTAC of the present invention has the following advantages: (1) Its biomolecular properties are highly safe, and its in situ expression in the cytoplasm allows for therapeutic effects through low-concentration administration and high-concentration expression. (2) It improves the antitumor effect of peptide-type PROTACs, solving the important problem of cell penetration and fully utilizing their specificity. (3) By using the PROTAC method, it can act in a catalytic-like manner and achieve efficient targeted degradation. (4) It provides a new target protein degradation molecular pattern and also provides new ideas for the therapeutic use of mRNA.

[0046] The present invention will be described in detail below with reference to specific embodiments, but the examples shown are for the purpose of illustrating the present invention and do not limit the scope of the present invention. The examples provided below can be used as a guide for those skilled in the art to make further improvements, but do not limit the present invention in any way. [Brief explanation of the drawings]

[0047] [Figure 1] 1 shows the structure of m-PROTAC and the principle of target protein degradation. [Figure 2] Schematic diagram of the vm-PROTAC vector. Peptide represents a functional polypeptide that targets and degrades ERα. [Figure 3] FIG. 1 shows the results of eGFP expression by flow cytometry analysis of m-PROTAC. [Figure 4] Cell imaging of MCF-7 cells 24 hours after transfection with 1 μg / mL m-PROTAC. The upper left image shows the white light channel image of m-PROTAC-treated cells, the lower left image shows the white light channel image of untreated cells, the upper right image shows the green fluorescent channel image of m-PROTAC-treated cells, and the lower right image shows the green fluorescent channel image of untreated cells. Bar=20 μm. [Figure 5] FIG. 1 shows the ERα expression levels in MCF-7 cells 24 hours after treatment with different concentrations of mRNA. [Figure 6] FIG. 1 shows the ERα expression levels in MCF-7 cells after treatment with 1 μg / mL mRNA for different times. [Figure 7] This figure shows the ERα expression levels upon co-incubation of MG-132 and m-PROTAC, where m in the right panel represents m-PROTAC. [Figure 8] Figure 1 shows the cellular activity of MCF-7 cells after treatment with different concentrations of m-PROTAC for 24 or 48 hours. [Figure 9] Figure 1 shows the results of apoptosis analysis of MCF-7 cells treated with different concentrations of m-PROTAC. [Figure 10] Figure 1 shows the results of cycle analysis of MCF-7 cells treated with different concentrations of m-PROTAC. [Figure 11] Schematic diagram of the vm-PROTAC-2 vector. Peptide-2 represents a functional polypeptide that targets and degrades BCL-xL. [Figure 12] This figure shows the concentration-dependent changes in the degradation of BCL-xL targeted by m-PROTAC-2 in MDA-MB-231 cells. [Figure 13] Figure 1 shows the results of characterization of m-PROTAC-2 / LNPs. (A) Zeta potential distribution of m-PROTAC-2 / LNPs, (B) particle size distribution (intensity) of m-PROTAC-2-LNPs, (C) Zeta potential analysis, and (D) particle size analysis. [Figure 14] Images of mice treated with luciferase mRNA-LNPs or PBS. (A) In vivo bioluminescence images of mice (n=3) 3 or 6 hours after treatment with luciferase mRNA-LNPs or PBS. (B) Ex vivo bioluminescence images of tumors and major organs 6 hours after treatment with luciferase mRNA-LNPs or PBS. [Figure 15] This figure shows the antitumor activity of m-PROTAC-2 in a 4T1 xenograft animal model. (A) shows the in vivo treatment scheme of the 4T1 mouse model, showing the changes in body weight over time when treated with m-PROTAC-2 / LNP (0.5 mg / kg), LNPs, or PBS. (B) shows the changes in tumor volume over time in the 4T1 mouse model. (C) shows photographs of tumors isolated from the mice. (E) shows the weight of tumors isolated from the mouse model. DETAILED DESCRIPTION OF THE INVENTION

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples are commercially available. The quantitative experiments in the following examples were all performed three times, and the average value was used as the result. 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, and the last position is the 3'-terminal nucleotide of the corresponding DNA.

[0049] Example 1: Preparation of m-PROTAC This example provides an mRNA PROTAC molecule (referred to as m-PROTAC) that targets ERα for degradation. This molecule functions by encoding a functional polypeptide (referred to as E3-ERα). The functional polypeptide E3-ERα is composed of a heptapeptide sequence (PIYPALA, SEQ ID NO. 1) that targets the VHL E3 ubiquitin ligase site, a linker peptide sequence (GSGS, SEQ ID NO. 2), and a polypeptide sequence (QLLRHLILH, SEQ ID NO. 4) that targets ERα protein binding, linked in sequence. The sequence is SEQ ID NO. 6 in the Sequence Listing.

[0050] The 5'-end modified cap structure (Cap) of m-PROTAC is composed of the 5' non-coding region of β-globin, a Kozak element, a coding region (enhanced green fluorescent protein (eGFP), a P2A element, and the mRNA for the functional polypeptide E3-ERα) linked in that order, the 3' non-coding region of α-globin, and a polyadenylic acid structure (Poly-A). The sequence of m-PROTAC is SEQ ID NO. 7 in the Sequence Listing, and the corresponding DNA sequence of m-PROTAC is SEQ ID NO. 8 in the Sequence Listing.

[0051] The structure of m-PROTAC and the principle of target protein degradation are shown in Figure 1. m-PROTAC mRNA was obtained by in vitro transcription of a linearized plasmid template containing m-PROTAC DNA.

[0052] 1. Preparation of recombinant vector Based on the m-PROTAC template sequence shown in SEQ ID NO. 8, an in vitro transcription vector vm-PROTAC (the sequence is SEQ ID NO. 9 in the Sequence Listing, see FIG. 2) containing the m-PROTAC template sequence shown in SEQ ID NO. 8 was synthesized. The in vitro transcription promoter is a T7 promoter, containing a downstream AscI restriction enzyme cleavage site. The vm-PROTAC was digested with AscI and purified to obtain the linearized plasmid vm-PROTAC.

[0053] 2. In vitro transcription of m-PROTAC 1) According to the in vitro transcription reagent instructions (Promega, P1300), the following was added to a 1.5 mL enzyme-free centrifuge tube in the following order: (1) 5x T7 Transfer Buffer: 20.0 μL (2)rNTP mixture: 30.0μL (3) 1-2 μg of linearized plasmid (dissolved in DEPC water): 32.5 μL (4) Cap-modified guanine (40 mM): 7.5 μL (5) Enzyme mixture: 10.0μL 2) Mix gently with a pipette tip and react in a metal bath at 37°C for 4 to 6 hours. 3) Purification: RQ1 RNase-Free DNase was added at a concentration of 1 U / μg of DNA template, and the mixture was incubated at 37°C for 30 minutes. After that, the mixture was purified using an mRNA purification kit to obtain m-PROTAC. The concentration was measured, and the mixture was aliquoted and stored in a refrigerator at -85°C.

[0054] Example 2: Cells transfected with m-PROTACs can efficiently express target proteins MCF-7 breast cancer cell line cells were cultured in DMEM medium in 12-well plates and transfected with different concentrations of m-PROTAC (the m-PROTAC concentrations in the system were set to 0, 0.5, and 1 μg / mL). After 4 hours of incubation at 37°C, the cells were washed with PBS, replaced with complete DMEM medium, and incubated for an additional 20 hours (total incubation time: 24 hours). Cells were then harvested and the eGFP expression levels in the cells were measured using flow cytometry. The quantitative analysis results, shown in Figure 3, showed that the green fluorescent protein expression intensity at 1 μg / mL was 4.44-fold higher than the control group. MCF-7 cells transfected with 1 μg / mL of the m-PROTAC obtained above were imaged, and it was observed that the transfected cells emitted clear eGFP green fluorescence, as shown in Figure 4. This result indicates that the m-PROTAC-transfected cells can efficiently express the target protein.

[0055] Example 3: Concentration-dependent changes in m-PROTAC targeting and degradation of ERα in MCF-7 cells MCF-7 breast cancer cell line cells were cultured in DMEM medium in 12-well plates and transfected with various concentrations of m-PROTAC (m-PROTAC concentrations in the system were set to 0, 0.125, 0.25, 0.5, 1, and 2 μg / mL). After 4 hours of incubation at 37°C, the cells were washed with PBS, replaced with complete DMEM medium, and incubated for an additional 20 hours (total incubation time: 24 hours). Cells were harvested and subjected to Western blot analysis using β-actin as an internal control. Primary antibodies used were anti-ERα rabbit monoclonal antibody (Cell Signaling Technology, #8644) and anti-β-actin rabbit monoclonal antibody (ABclonal, AC026). The results are shown in Figure 5. The ERα expression level significantly decreased with increasing incubation concentration of mRNA. This indicated that ERα was degraded and that the degradation was dependent on the mRNA concentration. Because the degradation efficiency reached 60% at 1 μg / mL, 1 μg / mL of mRNA was selected for further experiments.

[0056] Example 4: Time course of m-PROTAC targeting and degrading ERα in MCF-7 cells MCF-7 breast cancer cell line cells were cultured in 12-well plates using DMEM medium and transfected with m-PROTAC (the concentration of m-PROTAC in the system was 1 μg / mL). Cells were harvested 0, 6, 12, 24, or 36 hours after transfection at 37°C and subjected to Western blot analysis. Cells treated with control mRNA at a concentration of 1 μg / mL for 24 hours served as a control. The control mRNA was an mRNA that did not contain a functional polypeptide-encoding sequence and was prepared by the following method. Following the method of Procedure 1 in Example 1, the DNA fragment shown in SEQ ID NO. 8 in the vm-PROTAC obtained in Example 1 was replaced with SEQ ID NO. 10 to obtain the recombinant vector v-control. Following the method of Procedure 1 in Example 1, vm-PROTAC was replaced with v-control, and the control mRNA was obtained without changing the other procedures. The results are shown in Figure 6. The ERα expression level significantly decreased with increasing incubation time, indicating that ERα degradation depends on the incubation time of the mRNA. Since the degradation efficiency reached 60% at 24 hours, an incubation time of 24 hours was chosen for further experiments.

[0057] Example 5: m-PROTAC utilizes the ubiquitin proteasome system to degrade ERα in MCF-7 cells MG-132 is a general proteasome inhibitor that can inhibit the function of the proteasome. MCF-7 breast cancer cell line cells were cultured in DMEM medium in 12-well plates, and MG-132 was added to the culture system at a concentration of 10 μM. m-PROTAC was transfected into MCF-7 cells (the m-PROTAC concentration in the system was set to 1 μg / mL) and incubated at 37°C for 4 hours. After 4 hours of incubation at 37°C, the cells were washed with PBS and replaced with complete DMEM medium containing 10 μM MG-132. The cells were then incubated for an additional 20 hours (total incubation time: 24 hours). Cells were then harvested and subjected to Western blot analysis. Untreated cells (blank), cells treated with m-PROTAC alone, and cells treated with MG-132 alone were used as controls. The results are shown in Figure 7. While ERα expression levels did not decrease significantly after the addition of MG-132, they did decrease significantly in cells without MG-132. This indicates that MG-132 can inhibit m-PROTAC-mediated ERα degradation. This confirms that m-PROTAC-mediated ERα degradation is mediated by the ubiquitin proteasome system.

[0058] Example 6: m-PROTACs exhibit inhibitory effects on the proliferation of MCF-7 cells MCF-7 breast cancer cell line cells were cultured in DMEM medium in 12-well plates and transfected with different concentrations of m-PROTAC (the m-PROTAC concentrations in the system were set to 0, 0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 μg / mL). After 4 hours of incubation at 37°C, the medium was replaced with complete DMEM and incubated for an additional 20 hours (total incubation time: 24 hours) or 44 hours (total incubation time: 48 hours), after which cell activity was measured. Changes in cell activity were measured using a CCK-8 kit (Biyuntian, C0038). The results are shown in Figure 8. The m-PROTAC exhibited a significant inhibitory effect on MCF-7 cell proliferation, with an IC50 of approximately 8.2 μg / mL at 24 hours and approximately 1.7 μg / mL at 48 hours, corresponding to molar concentrations of 24.5 nM and 5.1 nM, respectively. Previously reported ERα PROTACs were all at the micromolar level. This indicated that a given concentration of m-PROTAC could effectively kill MCF-7 cells. The m-PROTAC of the present invention was more efficient at targeting and degrading the target protein than other conventional PROTAC systems.

[0059] Example 7: m-PROTAC induces apoptosis in MCF-7 cells through S-phase arrest MCF-7 breast cancer cell line cells were cultured in DMEM medium in 12-well plates and transfected with m-PROTACs at specific concentrations (0, 0.125, 0.25, 0.5, 1, and 2 μg / mL). After 4 hours of incubation at 37°C, the cells were washed with PBS, replaced with complete DMEM medium, and incubated for an additional 20 hours (total incubation time: 24 hours). Cells were then harvested and subjected to apoptosis analysis. For apoptosis analysis, cells were stained with Annexin V-FITC and PI according to the instructions in the Annexin V-FITC Apoptosis Detection Kit (Biyuntian, C1062). For cell cycle analysis, cells were resuspended in ice-cold 70% ethanol and incubated overnight at 4°C, then centrifuged to remove the ethanol. Collected cells were treated with RNase A and propidium iodide (PI) according to the instructions in the Cell Cycle and Apoptosis Analysis Kit (Biyuntian, C1052). Finally, stained samples were measured by flow cytometry and analyzed using ModFit LT 5.0. For each experiment, more than 10,000 cells were counted for apoptosis and cell cycle analysis. The results are shown in Figures 9 and 10. As the concentration increased, the rate of late apoptosis and S-phase arrest in m-PROTAC-treated MCF-7 cells increased, indicating that m-PROTAC induced apoptosis in MCF-7 cells through S-phase arrest.

[0060] Example 8: Preparation of m-PROTAC-2 and BCL-x in MDA-MB-231 cells L Changes in concentration when targeting and degrading In this example, BCL-x L We have developed a mRNA PROTAC molecule (called m-PROTAC-2) that targets and degrades mRNA. This molecule binds to a functional polypeptide (E3-BCL-x). L It functions by encoding the functional polypeptide E3-BCL-x L contains a heptapeptide sequence (PIYPALA, SEQ ID NO. 1) that targets the VHL E3 ubiquitin ligase site, a linker peptide sequence (GGGGGG, SEQ ID NO. 3), and BCL-x L The polypeptide sequence (GQVGRQLAIIGDAINR, SEQ ID NO. 5) that targets protein binding is linked in sequence, and the sequence is SEQ ID NO. 11 in the sequence listing.

[0061] The 5'-terminal modified cap structure (Cap) of m-PROTAC-2 contains the 5' non-coding region of β-globin, a Kozak element, a coding region (enhanced green fluorescent protein (eGFP)), a P2A element, and a functional peptide E3-BCL-x. L The m-PROTAC-2 sequence is SEQ ID NO. 12 in the Sequence Listing, and the corresponding m-PROTAC-2 DNA sequence is SEQ ID NO. 13 in the Sequence Listing.

[0062] m-PROTAC-2 mRNA was obtained by in vitro transcription of a linearized plasmid template containing m-PROTAC-2 DNA.

[0063] Based on the m-PROTAC-2 template sequence shown in SEQ ID NO. 13, an in vitro transcription vector vm-PROTAC-2 (sequence is SEQ ID NO. 14 in the Sequence Listing, see FIG. 11) containing the m-PROTAC-2 template sequence shown in SEQ ID NO. 13 was synthesized. The in vitro transcription promoter is a T7 promoter, containing a downstream AscI restriction enzyme cleavage site. vm-PROTAC-2 was digested with AscI and purified to obtain the linearized plasmid vm-PROTAC-2.

[0064] Following the method of Procedure 2 in Example 1, vm-PROTAC was replaced with vm-PROTAC-2 to obtain m-PROTAC-2.

[0065] Breast cancer cell line MDA-MB-231 cells were cultured in L-15 medium in 12-well plates and transfected with m-PROTAC-2 at different concentrations (the m-PROTAC-2 concentrations in the system were set to 0, 0.125, 0.25, 0.5, 1, and 2 μg / mL). After 4 hours of incubation at 37°C, the cells were washed with PBS, replaced with complete L-15 medium, and incubated for an additional 20 hours (total incubation time: 24 hours). Cells were harvested and subjected to Western blot analysis using the β-actin gene as an internal control. The primary antibody used was anti-BCL-x. L Recombinant rabbit monoclonal antibody (Nishisei Bio, 31011ES) and anti-β-actin rabbit monoclonal antibody (ABclonal, AC026) were used.

[0066] The results are shown in Figure 12. L The expression level significantly decreased with increasing mRNA incubation concentration. Lwas degraded and showed that the degradation depended on the mRNA concentration.

[0067] Example 9: m-PROTAC-2 inhibits tumor growth in vivo To confirm the antitumor potential of m-PROTACs, BCL-x L Xenograft experiments were performed using the 4T1 cell line (a mouse breast cancer cell line) overexpressing α-glucan.

[0068] The BALB / c mice used in this example were obtained from the Guangdong Provincial Medical Experimental Animal Center. Four-week-old female BALB / c mice were inoculated with 1 × 10 mice per mouse. 5 4T1 cells were injected subcutaneously, and the tumor size was 100 mm 3 A 4T1 mouse tumor model was obtained at this time. All animal experiments were conducted in accordance with the guidelines and approval of the Animal Care and Use Committee of the Shenzhen International Research Institute of Tsinghua University and the Guangdong Provincial Medical Laboratory Animal Center (Permit Number: 2023).

[0069] In this study, cationic lipid nanoparticles (LNPs) SM-102 (Shenzhen Xinhe Biomedical Technology Co., Ltd., CAS: 2089251-47-6) were used as an in vivo delivery carrier for m-PROTAC. Cationic lipid nanoparticles SM-102, distearoylphosphatidylcholine (DSPC), cholesterol, and DMG-PEG2000 were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 and shaken thoroughly to prepare a lipid mixture. Firefly luciferase mRNA (SEQ ID No. 15 in the Sequence Listing) or m-PROTAC-2 was diluted with 50 mM citrate buffer (pH 4) to obtain an mRNA aqueous solution. The lipid mixture was mixed with the mRNA aqueous solution at a volume ratio of 1:3 using a microfluidic device (nanoE, Micro&Nano) and dialyzed against PBS (pH 7.4) for 18 hours to remove ethanol and complete the exchange of citrate buffer to form mRNA / LNPs (m-PROTAC-2 / LNPs or Luciferase mRNA / LNPs).

[0070] LNPs carriers were obtained in the same manner as above, except that no mRNA was added.

[0071] Litesizer TM The size distribution, polydispersity index (PDI), and zeta potential (Zeta) of the mRNA / LNPs were measured using an Anton Paar 500. The encapsulation efficiency of the lipid nanoparticles was evaluated using the RiboGreen assay (characterization results are shown in Figure 13).

[0072] The results showed that the particle size distribution was unimodal and uniform.

[0073] (1) Luciferase mRNA verification experiment After establishing tumor-bearing mouse models, luciferase mRNA encapsulated in SM-102 (LNPs) was injected into the tumors of 4T1 mice. Bioluminescence signals were recorded 3 or 6 hours after injection using a small animal in vivo imaging system (IVIS-Spectrum, PerkinElmer). Six hours after injection, one mouse from each group was dissected, and the tumor and major organs (heart, liver, spleen, lungs, and kidneys) were removed and subjected to ex vivo imaging (In-Vivo F Pro, Bruker). Mice injected with PBS alone served as controls. The results are shown in Figure 14. Clear luciferase signals were observed in the mRNA-LNP group, confirming efficient transfection of mRNA by SM-102. Analysis of signal distribution in the tumor and major organs (heart, liver, spleen, lungs, and kidneys) after 6 hours revealed efficient delivery and expression of mRNA within the tumor.

[0074] (2) m-PROTAC inhibits tumor growth in vivo To verify the therapeutic effect of m-PROTAC in vivo, tumors with a volume of approximately 100 mm 3The 4T1 mouse tumor model was randomly divided into three groups of four mice each. Every four days, an equal volume of PBS, LNP vector, or m-PROTAC-2 / LNP (0.5 mg / kg) was injected into the tumor of the 4T1 mouse tumor model. The in vivo treatment scheme of the 4T1 mouse tumor model is shown in Figure 15(A). Before each injection, the tumor volume and mouse body weight were measured, and the tumor volume was calculated using the formula V = 1 / 2ab 2 (a is the maximum diameter, b is the minimum diameter) was used for calculations. After the final injection, the mice were monitored for 4 days, after which they were euthanized with CO2. The tumors and major organs of the 4T1 mouse tumor model were excised, and their weights and tumor volumes were measured. The results are shown in Figure 15. The weight of the m-PROTAC-2 / LNPs-treated 4T1 mouse tumor model remained stable throughout the experimental period, consistent with that of the LNP carrier and PBS-treated control groups. Furthermore, the m-PROTAC-2 / LNPs group demonstrated significant tumor growth inhibition (Figure 15B and C). This indicates that m-PROTAC-2 has low toxicity and high antitumor activity. After dissection, the tumors in the m-PROTAC-2 / LNPs-treated 4T1 mouse tumor model were significantly smaller than those in the control group (Figure 15D and E). Furthermore, the effective in vivo dose of m-PROTAC-2 was significantly lower than the effective dose (15 mg / kg) of DT2216 (a novel BCL-xL-specific PROTAC) used to treat the 4T1 mouse tumor model, demonstrating its superior tumor treatment efficacy. These results demonstrate the in vivo efficacy of m-PROTACs and their significant potential in the treatment of related diseases. [Industrial Applicability]

[0075] The m-PROTAC of the present invention can efficiently target and degrade target proteins and can be used to treat and / or prevent diseases / pathologies mediated by target proteins. It can also treat tumors by inhibiting tumor cell proliferation or promoting apoptosis in tumor cells.

Claims

1. 1. An mRNA molecule for targeted protein degradation, comprising, in this order, 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, wherein the coding region comprises a gene encoding a functional polypeptide, and the functional polypeptide is formed by sequentially linking a VHL E3 ubiquitin ligase target peptide, a linker peptide, and a target protein target polypeptide.

2. The sequence of the VHL E3 ubiquitin ligase target peptide is shown in SEQ ID NO. 1, The sequence of the linker peptide is shown in SEQ ID NO. 2 or SEQ ID NO. 3, 2. The mRNA molecule for targeted protein degradation according to claim 1, wherein the target protein-targeting polypeptide is a polypeptide that specifically targets the target protein, and the sequence thereof is set forth in SEQ ID NO. 4 or SEQ ID NO.

5.

3. 3. The mRNA molecule for targeted protein degradation according to claim 2, wherein the sequence of the functional polypeptide is set forth in SEQ ID NO. 6 or SEQ ID NO.

11.

4. The mRNA molecule for targeted protein degradation according to any one of claims 1 to 3, characterized in that the 5' non-coding region is the 5' non-coding region of beta globin and the 3' non-coding region is the 3' non-coding region of alpha globin.

5. The mRNA molecule for targeted protein degradation according to any one of claims 1 to 3, characterized in that the coding region further comprises a gene encoding another functional protein or a gene encoding a reporter protein, and a spacer region provided between the gene encoding another functional protein or a reporter protein and the functional polypeptide.

6. The mRNA molecule for targeted protein degradation according to any one of claims 1 to 3, characterized in that the sequence of the mRNA molecule is set forth in SEQ ID NO. 7 or SEQ ID NO.

12.

7. A DNA molecule encoding the mRNA molecule of any one of claims 1 to 5, wherein the sequence of the DNA molecule is set forth in SEQ ID NO. 8 or SEQ ID NO.

13.

8. A recombinant vector comprising the DNA molecule of claim 7.

9. A recombinant microorganism comprising the mRNA molecule of any one of claims 1 to 6, the DNA molecule of claim 7, or the recombinant vector of claim 8.

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

11. Use of the mRNA molecule according to any one of claims 1 to 6, the DNA molecule according to claim 7, the recombinant vector according to claim 8, or the recombinant microorganism according to claim 9 in any of the following: M1) Manufacturing a product for treating and / or preventing a disease / pathology mediated by a target protein according to any one of claims 1 to 6; M2) treatment and / or prevention of a disease / pathology mediated by the target protein; M3) Manufacturing products that inhibit cell proliferation; M4) inhibition of cell proliferation; M5) Manufacturing products that promote apoptosis; M6) Promotion of apoptosis.

12. The use according to claim 11, wherein the target protein-mediated disease is cancer and the cells are cancer cells.

13. 13. The use according to claim 12, characterized in that the cancer is 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.

14. A method for degrading a target protein, comprising administering an mRNA molecule according to any one of claims 1 to 6 to a biological cell, tissue or organ that expresses the target protein, thereby achieving degradation of the target protein in the biological cell, tissue or organ.

15. The method for degrading a target protein according to claim 14, wherein the biological cells are microbial cells, plant cells, or animal cells.

16. A method for treating and / or preventing a target protein-mediated disease / pathology, comprising administering an mRNA molecule according to any one of claims 1 to 6 to an animal suffering from the target protein-mediated disease / pathology, thereby achieving treatment and / or prevention and / or alleviation of the target protein-mediated disease / pathology in the animal.

17. 17. The method of claim 16, wherein the target protein-mediated disease is cancer and the cell is a cancer cell.

18. 18. The method of claim 17, wherein the cancer is 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.

19. 10. A method for inhibiting cell proliferation, comprising administering an mRNA molecule according to any one of claims 1 to 6 to a cell expressing a target protein, thereby achieving said inhibition of cell proliferation.

20. A method for promoting apoptosis, comprising administering an mRNA molecule according to any one of claims 1 to 6 to a cell expressing a target protein, thereby achieving said promotion of apoptosis.

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