DNA molecule for targeted degradation of target protein and use thereof

Abstract

A DNA molecule for targeted degradation of a target protein and the use thereof. The DNA molecule comprises at least one group of D-PROTAC units, wherein each group of D-PROTAC units comprises at least one first DNA segment encoding a ubiquitin ligase-targeting peptide, at least one second DNA segment encoding a target protein-targeting peptide, and a third DNA segment encoding a linker peptide which is optionally arranged between the adjacent first DNA segment and second DNA segment. A gene therapy-specific PROTAC molecular model is successfully constructed, which proves the great potential of gene therapy in the research, development and application of new anticancer PROTAC drugs, innovates the mode of action of PROTAC technique, and extends new therapeutic application scenarios for gene therapy.

Description

DNA molecule for targeted degradation of a protein of interest and use thereof TECHNICAL FIELD

[0001] The present application belongs to the field of gene therapy, and in particular relates to a DNA molecule for targeted degradation of a protein of interest, a biological material comprising the DNA molecule, and use of the DNA molecule and the biological material. BACKGROUND

[0002] In countries around the world, cancer is a major cause of death and an important obstacle to improving the average life expectancy of humans. According to the World Health Organization (WHO) estimates in 2019 for 183 countries, cancer is the first or second leading cause of death in 112 countries before the age of 70, and is in the third or fourth place in another 23 countries. There are various treatment strategies for cancer at present, mainly including chemotherapy, surgery and radiotherapy. Among them, chemotherapy has been the main treatment for cancer since the 1940s, and anticancer drugs are the focus of current drug research and development. However, chemotherapy is often accompanied by some toxic side effects, which are usually caused by adverse effects of anticancer drugs on hematopoietic cells in bone marrow, cells in hair follicles, digestive tract and reproductive system. Therefore, the research and development of anticancer drugs aims to improve non-specificity and targeting.

[0003] During the process of tumor formation, the expression level of tumor cell genes changes significantly compared with normal cells, and abnormal protein expression occurs, such as high-level expression of growth factor receptors. Excessive expression 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.

[0004] The goal of modern drug discovery is to develop highly potent, selective, and target-specific drugs. In the past few decades, the single-target theory has been successful in guiding the development of drugs for single-cause diseases, especially antibiotics and other drugs targeting specific single-pathogen protein targets. However, cancer or inflammatory diseases are usually the result of the interaction of multiple factors, involving the synergistic effect of multiple pathogenic causes or the increase of multiple receptors, and the signal network between them. In cancer, tumor cells often elevate different growth-promoting factors, which can act independently and promote each other through intracellular signal networks. By increasing alternative factors or switching the proliferation-promoting signal pathways, tumor cells are prone to develop drug resistance; therefore, treatment targeting a single target is limited. In addition to drug resistance, single-target drugs can also cause side effects, tissue toxicity, and other factors, leading to reduced efficacy and quality of life in patients. In order to overcome the shortcomings of single-target drugs, combination therapy targeting two different methods related to disease development can achieve additive or synergistic effects, reduce drug resistance, and reduce side effects due to reduced dosage of each single drug, which has become an effective method.

[0005] In current applications, one method to achieve dual-target therapy is to connect two or even multiple action sites with simple hybrid molecules, targeting multiple possible tumor-inhibiting epitope action targets. These synthetic molecules can act on multiple targets or pathways, with better efficacy and lower side effects. In the past few years, these synthetic molecules have attracted more attention due to their advantages in the field of treating complex diseases, and have gradually become an alternative to drugs, including bispecific antibodies and other dual-target small molecule drugs.

[0006] The related research of the PROTAC technology is very hot in the current field of new drug development, which induces target protein degradation through the ubiquitin-proteasome system, "event-driven", catalytic degradation mode, high activity, high selectivity, and can target more "undruggable" targets, showing great potential in new drug development. Currently, some drugs have entered the clinical stage, and in-depth research of PROTAC plays an important role in the development of new anticancer drugs.

[0007] However, there are still some problems to be solved in the successful development of PROTAC, such as: large molecular weight of PROTAC, poor membrane permeability, not conducive to oral administration, off-target toxicity, non-cancer-specific toxicity, and difficulty in forming triple complexes. Among them, PROTAC has no discrimination in attacking tumor cells and normal cells when it works, which further leads to off-target effects and non-cancer-specific toxicity, which is the greatest concern about the safety of the PROTAC technology. SUMMARY

[0008] The present application provides a DNA molecule for targeted degradation of a target protein, which is a DNA form of a PROTAC molecule.

[0009] In one embodiment, the DNA PROTAC molecule of the present application has tumor specificity, which can solve the problems of poor tumor cell specificity and weak controllability. Specifically, the PROTAC molecule is designed as a DNA form including a specific expression part and a treatment part. The specific expression part uses a disease (such as a tumor) specific promoter, which can be specifically expressed in disease (such as tumor) cells, reducing non-target side effects; the treatment part uses the polypeptide translation form of gene therapy, overcoming the problems of transmembrane action concentration, etc., targeting the drug targets that small molecule PROTACs cannot target, improving drug resistance, reducing toxicity, and having good biological safety, and realizing the function of targeted protein degradation. At the same time, the present application also explores the modular replacement of different treatment parts-PI3K and ERα double-target degradation combination drug, which provides a new action model for anti-cancer new drug research and development.

[0010] To achieve the above-mentioned object, the present application provides a DNA molecule for targeted degradation of a target protein, which includes at least one group of D-PROTAC units, each group of D-PROTAC units including at least one first DNA segment encoding a ubiquitin ligase targeting peptide, at least one second DNA segment encoding a target protein targeting peptide, and optionally a third DNA segment encoding a linker peptide between adjacent first and second DNA segments.

[0011] The D-PROTAC concept and model construction are the core of the present application, which is an innovative strategy based on protein targeted degradation. D-PROTAC refers to a DNA form of PROTAC. The D-PROTAC unit can be one or more groups, and each group of D-PROTAC units can synthesize p-PROTAC (protein form of PROTAC) that plays a role by using the translation mechanism in cells, thereby inducing target protein degradation. Therefore, the D-PROTAC molecule of the present application can be single-targeted or multi-targeted.

[0012] According to one specific embodiment of the present application, the D-PROTAC can induce single-targeted degradation of B-cell lymphoma protein (Bcl-xL); according to another specific embodiment of the present application, the D-PROTAC can induce single-targeted degradation of RAD51 protein; and according to still another specific embodiment of the present application, the D-PROTAC can induce single-targeted degradation of USP30 protein.

[0013] According to another specific embodiment of the present application, the D-PROTAC can induce simultaneous degradation of the double targets of intracellular estrogen receptor ERα and phosphatidylinositol kinase PI3K.

[0014] The present application enriches the regulatory means of PROTAC by designing a PROTAC that can function in cells at the DNA level. For example, one effective design is to design a promoter upstream of all D-PROTAC units, which is preferably an organ, tissue or cell specific promoter or an environment specific promoter. The organ, tissue or cell specific promoter is preferably a disease organ, tissue or cell specific promoter, more preferably a tumor organ, tissue or cell specific promoter. The environment specific promoter is preferably selected from at least one of a hypoxia / hyperoxia response promoter, a low pH / high pH response promoter, a lactic acid promoter and a H2O2 response promoter. Due to the organ, tissue, cell selectivity or environment specificity of the promoter, the translation of D-PROTAC is also selectively completed in a specific organ, tissue, cell or under a specific environment, thereby achieving the selectivity of PROTAC treatment, such as tumor specific targeting.

[0015] The organ can be selected from muscle, central nervous system, eye, liver, heart, kidney, pancreas, lung, skin, bladder, urinary tract, spleen, myeloid cell lineage and lymphoid cell lineage or a combination thereof.

[0016] The tissue can be selected from muscle tissue, adrenal tissue, appendix tissue, bladder tissue, bone, bowel tissue, brain tissue, breast tissue, bronchus, coronal tissue, ear tissue, esophageal tissue, eye tissue, gallbladder tissue, genital tissue, heart tissue, hypothalamic tissue, kidney tissue, large intestine tissue, intestinal tissue, laryngeal tissue, liver tissue, lung tissue, lymph node, oral tissue, nasal tissue, pancreatic tissue, parathyroid tissue, pituitary tissue, prostate tissue, rectal tissue, salivary gland tissue, skeletal muscle tissue, skin tissue, small intestine tissue, spinal cord, spleen tissue, stomach tissue, thymus tissue, tracheal tissue, thyroid tissue, ureter tissue, urethral tissue, soft and connective tissue, peritoneal tissue, vascular tissue, adipose tissue or a combination thereof.

[0017] The cell can be selected from hepatocyte, epithelial cell, endothelial cell, cardiomyocyte, skeletal muscle cell, sinusoidal cell, afferent neuron, efferent neuron, interneuron, glial cell, astrocyte, oligodendrocyte, microglial cell, ependymal cell, lung epithelial cell, Schwann cell, satellite cell, photoreceptor cell, retinal ganglion cell, T cell, B cell, NK cell, macrophage, dendritic cell or a combination thereof.

[0018] Specifically, examples of the organ-, tissue-, or cell-specific promoter include, but are not limited to, at least one of the following promoters: a liver-specific promoter selected from at least one of an albumin (ALB) promoter, a TBG promoter, a transthyretin (TTR) promoter, a human alpha-1 antitrypsin promoter, an HBV promoter, and an Afp promoter; a heart-specific promoter selected from at least one of cTNT, alpha-MHC, MLC-2v, Desmin, ANF, Tnnt2, MYH6, MYL2, and SERCA2a; a muscle-specific promoter selected from at least one of a phosphoglycolate kinase (PGK) promoter, a desmin promoter, a muscle creatine kinase promoter, a myosin light chain promoter, a myosin heavy chain promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an actin alpha promoter, an actin beta promoter, an actin gamma promoter, and a keratin promoter; a glial cell-specific promoter selected from at least one of a GFAP promoter, an ALDH1L1 promoter, an EAAT1 / GLAST promoter, a glutamine synthetase promoter, an S100β promoter, and an EAAT2 / GLT-1 promoter, an NG2 promoter, a CD68 promoter, a F4 / 80 promoter, a Slc1a3 promoter, and a Rlbp1 promoter; a cone cell, rod cell-specific promoter selected from at least one of a hGRK1p, a RedO promoter, a SynP136 promoter, a GNAT2 promoter, a SynPVI promoter, and a mCAR promoter; a pluripotent stem cell-specific promoter selected from at least one of a mEtnAhC3p, a mEtnAhS4p, a mNANOGp; a neuron-specific promoter selected from at least one of a Syn promoter, a huc promoter, a CaMKIIa promoter; an endothelial cell-specific promoter K18; a chondrocyte-specific promoter Col2a1; a macrophage-specific promoter CD11b or CD68 gene; a B cell-specific promoter ProA; a plasma cell-specific promoter ProB; an erythroid-specific promoter selected from at least one of an alpha-spectrin promoter, an ankyrin-1 promoter, a gamma-globin promoter, and a beta-globin promoter.

[0019] The tumor organ, tissue or cell-specific promoter includes, but is not limited to, at least one of a human telomerase reverse transcriptase (hTERT) promoter, a Survivin promoter, an alpha-fetoprotein (AFP) promoter, an osteocalcin (OC) promoter, a neuron-specific enolase (NSE) promoter, a carcinoembryonic antigen (CEA) promoter, a cyclooxygenase-2 (COX-2) promoter, a prostate-specific antigen (PSA) promoter, an E2F-1 promoter, a cholecystokinin promoter, a C-erbB2 / neu oncogene promoter, a CXCR4 promoter, a HE4 promoter, a hexokinase type II promoter, a L-plastin promoter, a MUC1 promoter, a TRP1 promoter, a tyrosinase promoter, a hypoxia-inducible factor-1 promoter, a tumor antigen mucin promoter, a secretory leukocyte protease inhibitor promoter, a survivin promoter, and a MetR-specific promoter. The promoter element of the corresponding specificity can be selected as required.

[0020] The ubiquitin ligase targeting peptide is an essential component of the p-PROTAC, but the specific type thereof can be flexible, and the present application does not have a specific limitation thereon, for example, can be at least one of a VHL E3 ubiquitin ligase targeting peptide, a CRBN E3 ubiquitin ligase targeting peptide, an MDM2 E3 ubiquitin ligase targeting peptide, an IAPs E3 ubiquitin ligase targeting peptide, a DCAF E3 ubiquitin ligase targeting peptide, and an RNF E3 ubiquitin ligase targeting peptide.

[0021] The VHL E3 ubiquitin ligase targeting peptide which is widely used is selected in the embodiment of the present application, and the amino acid sequence thereof is PIYPALA (SEQ ID NO: 1), and accordingly, the sequence of the first DNA segment encoding the ubiquitin ligase targeting peptide is preferably shown in SEQ ID NO: 2: 5'-CCC ATC TAT CCT GCT CTG GCT-3'.

[0022] Based on the principle of PROTAC, one skilled in the art can determine that the protein of interest is arbitrarily selected depending on the degradation target. Specifically, the protein of interest includes, but is not limited to, at least one of B-cell lymphoma protein (Bcl-xL, Human B-cell lymphoma XL), estrogen receptor alpha (Erα, Estrogen Receptor Alpha), intracellular phosphatidylinositol kinase (PI3K, Phosphatidylinositide 3-kinases), estrogen receptor 1 (ESR1, Estrogen Receptor 1), RAD51 protein, USP30 protein (Ubiquitin specific peptidase 30), androgen receptor (AR, Androgen receptor), BTK tyrosine-protein kinase (BTK, Tyrosine-protein kinase BTK), interleukin-1 receptor-associated kinase 4 (IRAK4, Interleukin-1 receptor-associated kinase 4), RAC alpha-serine / threonine-protein kinase (AKT1, RAC-alpha serine / threonine-protein kinase), RAC beta-serine / threonine-protein kinase (AKT2, RAC-beta serine / threonine-protein kinase), bromodomain-containing protein 4 (BRD4, Bromodomain-containing protein 4), cyclin-dependent kinase 9 (CDK9, Cyclin-dependent kinase 9), receptor-interacting serine / threonine-protein kinase 1 (RIPK1, Receptor-interacting serine / threonine-protein kinase 1), transcription intermediary factor 1-alpha (TRIM24, Transcription intermediary factor 1-alpha), epidemal growth factor receptor (EGFR, Epidemal growth factor receptor), hepatocyte growth factor receptor (MET, Hepatocyte growth factor receptor), and mast / stem cell growth factor receptor (KIT, Mast / stem cell growth factor receptor).

[0023] Some embodiments of the present application take several highly expressed proteins commonly found in tumor cells as examples, such as B-cell lymphoma protein (Bcl-xL), estrogen receptor alpha (ERa), and intracellular phosphatidylinositol kinase (PI3K). Other embodiments target other important target proteins in cells, such as RAD51 protein and USP30 protein. Among them, the sequence of the Bcl-xL targeting peptide is GQVGRQLAIIGDAINR (SEQ ID NO: 3), the sequence of the ERa targeting peptide is HKILHRLLQ (SEQ ID NO: 4), the sequence of the PI3K targeting peptide is PGGDYAAMGACPASEQGYEEMRA (SEQ ID NO: 5), the sequence of the RAD51 protein targeting peptide is LLGFHTASGKKVKIAK (SEQ ID NO: 6), and the sequence of the USP30 protein targeting peptide is GIYVIWGPITERKKRRKG (SEQ ID NO: 7). The DNA coding sequence of the five target peptides of the proteins of interest can be determined according to the above-mentioned amino acid sequences, which is a very conventional technical means for those skilled in the art.

[0024] According to a specific embodiment of the present application, the DNA sequence of the Bcl-xL targeting peptide is located at positions 600-647 in the TERT-Bcl-xL-PROTAC plasmid shown in SEQ ID NO: 16. The DNA sequence of the ERa targeting peptide is located at positions 1311-1337 in the TERT-Dual-PROTAC plasmid shown in SEQ ID NO: 17. The DNA sequence of the PI3K targeting peptide is located at positions 1353-1421 in the TERT-Dual-PROTAC plasmid shown in SEQ ID NO: 17. The DNA sequence of the RAD51 protein targeting peptide is located at positions 641-688 in the RAD51-PROTAC plasmid shown in SEQ ID NO: 18. The DNA sequence of the USP30 protein targeting peptide is located at positions 641-694 in the USP30-PROTAC plasmid shown in SEQ ID NO: 19.

[0025] In the present application, the linker peptide is used to separate the two polypeptides to avoid their mutual influence, so any sequence that meets this function can be used. There are many known linker peptide sequences in the art, which can be used in the present application, such as (G) m , (GS) n . In which m and n can be integers from 2 to 20, which can be specifically listed as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0026] According to the design of the present application, the D-PROTAC unit can be one or more groups, when it is one group, the formed D-PROTAC is a single target, and when it is more groups, the formed D-PROTAC is a multi-target. Generally, the number of D-PROTAC units is 1-6 groups, for example, 1 group, 2 groups, 3 groups, 4 groups, 5 groups, 6 groups, and each group of D-PROTAC can be the same or different, that is, the whole DNA molecule can express multiple identical p-PROTAC or multiple different p-PROTAC.

[0027] When it is more groups, a spacer region needs to be provided between adjacent D-PROTAC units, which is the coding sequence of a controllable cleavage peptide chain, which can realize peptide chain cleavage under controllable conditions (such as a specific cell environment) to make the translation products of each D-PROTAC unit exist independently. In this way, the D-PROTAC can form multiple independent p-PROTAC after translation in the cell, avoiding the influence of protein steric hindrance on the degradation effect. Preferably, the controllable cleavage peptide chain is a cell protease recognition site or a self-cleavage peptide, and more preferably, a disease cell-specific highly expressed protease recognition site is used, so that it can be selectively cleaved in disease cells, combined with the selection of the aforementioned promoter, to realize double or even multiple selection. According to one specific embodiment of the present application, the spacer region is designed as a cathepsin B recognition site that is highly expressed in tumor cells.

[0028] According to one specific embodiment of the present application, the DNA molecule includes a group of D-PROTAC units, including a Bcl-xL targeting peptide coding sequence, a connecting peptide coding sequence, and a VHL targeting peptide coding sequence, the sequence of which is shown in SEQ ID NO: 8:

[0029] 5'-CCCATCTATCCTGCTCTGGCTGGAGGAGGGGGCGGCGGCGGACAGGTGGGAAGACAGCTGGCTATCATTGGCGATGCTATTAATAGA-3' (SEQ ID NO: 8).

[0030] According to another specific embodiment of the present application, the DNA molecule includes a group of D-PROTAC units, including a RAD51 protein targeting peptide coding sequence, a connecting peptide coding sequence, and a VHL targeting peptide coding sequence, the sequence of which is shown in SEQ ID NO: 9:

[0031] 5'-CTGCTGGGCTTTCACACCGCCTCTGGCAAAAAAGTGAAGATTGCCAAAAGCGGCTCCGGCGCCCTGGCCCCATACATCCCC-3' (SEQ ID NO: 9).

[0032] According to another specific embodiment of the present application, the DNA molecule comprises two groups of D-PROTAC units, the first group of D-PROTAC units comprising an ERa targeting peptide coding sequence, a linker peptide coding sequence, a VHL targeting peptide coding sequence, and the second group of D-PROTAC units comprising a PI3K targeting peptide coding sequence, a linker peptide coding sequence, a VHL targeting peptide coding sequence, and the two groups of D-PROTAC units are connected by a Cathepsin B coding gene, and the sequence is shown in SEQ ID NO: 11:

[0033] 5'-GGCATCTACGTGATTTGGGGCCCCATCACCGAGAGGAAAAAGAGGAGAAAGGGAAGCGGCAGCGGCGCCCTGGCTCCTTACATCCCT-3' (SEQ ID NO: 10).

[0034] According to another specific embodiment of the present application, the DNA molecule comprises two groups of D-PROTAC units, the first group of D-PROTAC units comprising an ERa targeting peptide coding sequence, a linker peptide coding sequence, a VHL targeting peptide coding sequence, and the second group of D-PROTAC units comprising a PI3K targeting peptide coding sequence, a linker peptide coding sequence, a VHL targeting peptide coding sequence, and the two groups of D-PROTAC units are connected by a Cathepsin B coding gene, and the sequence is shown in SEQ ID NO: 11:

[0035] 5'-ATCTACCCAGCCCTGGCTTCTGGATCTGGCCATAAAATTCTGCATAGACTGCTGCAGGGCCTGTTTGGCGGACCCGGCGGCGATTACGCTGCCATGGGAGCCTGTCCTGCCTCTGAGCAGGGCTATGAAGAGATGAGAGCTTCCGGCTCCGGTGCCCTGGCCCCTTACATCCCATAG-3' (SEQ ID NO: 11).

[0036] In the case of verifying the system of the present application or other needs, the DNA molecule further comprises a reporter protein coding gene, and a spacer is arranged between the reporter protein coding gene and the D-PROTAC unit, the spacer is a sequence encoding a self-cleavage peptide or an intracellular protease recognition site, which is used to separate the functional proteins connected thereto and self-cleavage or cleavage under suitable conditions, so that the functional proteins (i.e. the reporter protein and the p-PROTAC) connected thereto exist independently. In the case of verifying the system of the present application or other needs, the DNA molecule further comprises a reporter protein coding gene, and a spacer is arranged between the reporter protein coding gene and the D-PROTAC unit, the spacer is a sequence encoding a self-cleavage peptide or an intracellular protease recognition site, which is used to separate the functional proteins connected thereto and self-cleavage or cleavage under suitable conditions, so that the functional proteins (i.e. the reporter protein and the p-PROTAC) connected thereto exist independently.

[0037] According to a specific embodiment of the present application, the reporter protein coding gene is an m-Cherry coding gene or an EGFP coding gene, and the spacer is a coding sequence of a P2A peptide. Other reporter proteins and self-cleaving peptides can be selected by those skilled in the art as needed, and the present application does not particularly limit the same.

[0038] The present application also provides a biological material, which is any one of A1) to A5) described below:

[0039] A1) an expression cassette containing the DNA molecule described above;

[0040] A2) a recombinant vector containing the DNA molecule described above;

[0041] A3) a recombinant microorganism containing the DNA molecule described above, or a recombinant microorganism containing the expression cassette of A1), or a recombinant microorganism containing the recombinant vector of A2);

[0042] A4) a cell transfected with the DNA molecule described above, or the expression cassette of A1), or the recombinant vector of A2), or the recombinant microorganism of A3);

[0043] A5) a virus transfected with the DNA molecule described above, or the expression cassette of A1), or the recombinant vector of A2).

[0044] According to the present application, the recombinant vector further comprises a functional element, which can be selected from one or more of a replication origin, an enhancer, a Kozak sequence, a terminator, a signal peptide, an intron, a resistance gene, a marker gene, a multiple cloning site, and a ribosome binding site.

[0045] According to a specific embodiment of the present application, an hTERT promoter is selected to utilize its tumor cell-specific expression, and a VHL ligand in a more mature E3 ligase is used to construct a PROTAC plasmid targeting the anti-apoptotic protein Bcl-xL (Figure 1), which is referred to as TERT-Bcl-xL-PROTAC (also referred to as D-PROTAC-1 herein). It includes a human-optimized PROTAC module targeting Bcl-xL, which is named DNA-PROTAC-1. An enhanced red fluorescent protein (m-cherry) fluorescent sequence is inserted into the sequence, and a P2A element is used for self-cleavage, thereby achieving simultaneous and separate expression of the marker protein and p-PROTAC-1, and minimizing interference with the therapeutic effect.

[0046] Based on the design concept of DNA-PROTAC-1 single target degradation, the present application includes ERa and PI3K proteins in the degradation range of the DNA-PROTAC model, expands the method of the present application, and realizes double target degradation. Specifically, the design principle of DNA-PROTAC-2 is as follows: The p-PROTAC molecule of the gene coding model is replaced and designed, as shown in Figure 3, and a p-PROTAC polypeptide sequence targeting the classical tumor-related proteins ERa and PI3K is constructed. In other aspects of the design, the hTERT promoter, VHL E3 ligase ligand and red fluorescent protein (m-cherry) are still selected. After the design is completed and human optimization is performed, DNA-PROTAC-2 is obtained, and a PROTAC plasmid is further constructed (Figure 4), which is denoted as TERT-Dual-PROTAC (also referred to as D-PROTAC-2 herein).

[0047] On the basis of the above-mentioned model, the targeting protein ligand sequence or the E3 ligase ligand sequence can be replaced, thereby extending the effect to other overexpressed proteins of disease targets, and the present application also verifies the PROTAC system for RAD51 protein and USP30 protein; The specific start module can also be changed to other tissue-specific promoters, thereby extending the application range of the model. The above changes are within the protection scope of the present application.

[0048] The action principle of D-PROTAC in the embodiment of the present application is shown in Figure 5, and D-PROTAC can synthesize p-PROTAC and m-Cherry / EGFP for expression verification by using the translation mechanism in cells, and p-PROTAC can mediate the interaction between the target protein and VHL E3 ubiquitin ligase, thereby causing the degradation of the target protein.

[0049] The present application also provides any of the following applications of the above-mentioned DNA molecules and the above-mentioned biological materials:

[0050] M1) preparing a product for treating and / or preventing a disease / lesion mediated by the target protein;

[0051] M2) treating and / or preventing a disease / lesion mediated by the target protein;

[0052] M3) preparing a product for inhibiting cell proliferation;

[0053] M4) inhibiting cell proliferation;

[0054] M5) preparing a product for promoting apoptosis;

[0055] M6) promoting apoptosis.

[0056] The present application is suitable for various diseases mediated by the protein of interest, including but not limited to cancer, immune diseases, metabolic diseases, neurodegenerative diseases or inflammatory diseases;

[0057] According to a specific embodiment, the disease mediated by the protein of interest is cancer, preferably leukemia cancer, 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, gastric cancer, colon cancer, bladder cancer or rectal cancer. Accordingly, the cell is preferably a cancer cell.

[0058] In one embodiment of the present application, the cancer is breast cancer, the cell is a breast cancer cell, and the breast cancer cell is an MCF-7 cell. Specifically, in one example, the breast cancer cell is an MDA-MB-231 cell. In another example, the breast cancer cell is an MDA-MB-453 cell.

[0059] In another embodiment of the present application, the cancer is liver cancer, the cell is a liver cancer cell, and the liver cancer cell is an HepG2 cell.

[0060] In still another embodiment of the present application, the cancer is colon cancer, the cell is a colon cancer cell, and the colon cancer cell is an HCT116 cell.

[0061] In the present application, the product can be various conventional prophylactic or therapeutic products, preferably a vaccine or a drug.

[0062] The present application successfully constructs a gene therapy specific PROTAC molecular model with good anti-tumor effect, overcomes the problem of non-tumor specific toxicity, further proves the great potential of gene therapy in the research and application of PROTAC anti-cancer new drugs, innovates the action mode of PROTAC technology, and expands new treatment application scenarios for gene therapy.

[0063] Specifically, the application regulates expression by means of a tumor tissue / cell specific human telomerase reverse transcriptase (hTERT) promoter, so that the therapeutic polypeptide (p-PROTAC) is highly expressed in tumor cells, and the m-Cherry red fluorescent protein is introduced, and the specific expression of the target sequence is verified by flow cytometry analysis and confocal cell imaging technology; the therapeutic part is designed as a polypeptide mode, and the Western blot experiment proves that the model presents a concentration gradient, a time gradient, and a ubiquitination pathway to target degrade B lymphoma protein (Bcl-xL). The D-PROTAC-1 molecule targeting Bcl-xL can effectively inhibit the proliferation of breast cancer cells (MDA-MB-231), but does not affect the survival of normal breast epithelial cells (MCF-10A). At the same time, D-PROTAC-1 exhibits good protein degradation effect in 4T1 mouse cells, providing preliminary verification and data support for clinical experiments. The application also explores the diversity of therapeutic molecules, designs and synthesizes D-PROTAC-2, realizes the simultaneous degradation of estrogen receptor ERa and intracellular phosphatidylinositol kinase PI3K, and specifically affects the proliferation function and cell cycle of human breast cancer cells (MCF-7), verifying the flexibility and clinical application potential of the model. In addition, the application also designs and synthesizes D-PROTAC-3 and D-PROTAC-4, which respectively realize the degradation of RAD51 protein and USP30 protein, further verifying the universality of the model of the application.

[0064] The series of PROTACs can design therapeutic polypeptide ligand sequences according to target protein sequences, and specifically express and play a regulatory role in tumor cells by gene therapy, and target degrade target proteins. The application can also extend from single target to multi-target synergistic effect, and can also explore from cancer to immune, neurodegenerative and metabolic diseases. It can make up for the current non-tumor specific toxicity problem of PROTAC, and the administration mode of plasmid DNA can effectively reduce the molecular weight of the drug and enhance the effective drug concentration. The drug mode of the final polypeptide of the PROTAC also provides convenience and possibility for developing protein targeting chimeras for other protein degradation based on this model, which will further promote the construction and biological application of new PROTAC modes.

[0065] The D-PROTAC designed in the application has the following advantages:

[0066] (1) Based on plasmid DNA gene therapy, the plasmid DNA is designed to express and translate the target protein, and the therapeutic polypeptide is generated, which has better biocompatibility, higher efficiency and lower cost compared with traditional polypeptide PROTAC technology.

[0067] (2) Based on the controllable gene or protein element system regulation, the non-tumor specific toxicity is effectively reduced by means of tumor specific promoter, and a new idea is provided for controllable PROTAC.

[0068] (3) The multi-target degradation treatment mode is combined with drugs, two or more target proteins can be degraded at the same time, the drug resistance can be effectively reduced, the side effects and tissue toxicity of single target treatment drugs can be overcome, the curative effect can be improved, and the life quality of patients can be improved.

[0069] Other features and advantages of the present application will be described in detail in the following specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0070] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which:

[0071] Fig. 1 is a TERT-Bcl-xL-PROTAC plasmid map.

[0072] Fig. 2 is a schematic diagram of TERT-Bcl-xL-PROTAC.

[0073] Fig. 3 is a schematic diagram of TERT-Dual-PROTAC.

[0074] Fig. 4 is a TERT-Dual-PROTAC plasmid map.

[0075] Fig. 5 is a D-PROTAC action principle diagram.

[0076] Fig. 6 is the enzyme digestion agarose gel electrophoresis identification result of (a) TERT-Bcl-xL-PROTAC plasmid and (b) TERT-Dual-PROTAC plasmid.

[0077] Fig. 7 shows the protein level of Bcl-xL after D-PROTAC-1 acts on (a) MDA-MB-231 cells and (b) 4T1 cells for 24h; the quantitative analysis column chart of the gray scale of the protein level of Bcl-xL after D-PROTAC-1 acts on (c) MDA-MB-231 cells and (d) 4T1 cells for 24h, the statistical data is the average value±SEM of three repeated experiments.

[0078] Fig. 8 shows the protein level of (a) Bcl-xL after 2μg / mL D-PROTAC-1 acts on MDA-MB-231 cells for different times; (b) the quantitative analysis column chart of the gray scale of the protein level of Bcl-xL, the statistical data is the average value±SEM of three repeated experiments.

[0079] Figure 9 shows the results of cell viability analysis after MDA-MB-231 cells were treated with different concentrations of D-PROTAC-1 for 24 h. The statistical data is the average value ± SEM of three repeated experiments.

[0080] Figure 10 shows the results of apoptosis analysis after MDA-MB-231 cells were treated with different concentrations of D-PROTAC-1 for 24 h: (a) flow cytometry result graph; (b) total quantitative analysis proportion result graph; (c) late apoptosis quantitative analysis proportion result graph; (d) early apoptosis quantitative analysis proportion result graph. The statistical data is the average value ± SEM of three repeated experiments.

[0081] Figure 11 shows the levels of apoptosis factors after MDA-MB-231 cells were treated with different concentrations of D-PROTAC-1 for 24 h.

[0082] Figure 12 shows the results of cycle analysis after MDA-MB-231 cells were treated with different concentrations of D-PROTAC-1: (a-e) cycle flow cytometry result graphs after cells were treated with different concentrations of D-PROTAC-1 for 24 h; (f) quantitative analysis proportion result graph. The statistical data is the average value ± SEM of three repeated experiments.

[0083] Figure 13 shows the results of cell invasion experiments after MDA-MB-231 cells were treated with different concentrations of D-PROTAC-1.

[0084] Figure 14 shows the results of cell biological function analysis after MCF-10A cells were treated with different concentrations of D-PROTAC-1: (a) apoptosis results; (b) cell viability results. The statistical data is the average value ± SEM of three repeated experiments.

[0085] Figure 15 shows the levels of ERa and PI3K proteins after MCF-7 cells were treated with D-PROTAC-2 at concentrations of 0, 0.5, 1, 1.5, and 2 μg / mL for 24 h: (a) immunoblotting experiment results; (b) gray scale quantitative analysis column chart. The statistical data is the average value ± SEM of three repeated experiments.

[0086] Figure 16 shows the levels of ERa and PI3K proteins after MCF-7 cells were treated with 2 μg / mL of D-PROTAC-2 for 0, 6, 12, 24, and 48 h: (a) immunoblotting experiment results; (b) gray scale quantitative analysis column chart. The statistical data is the average value ± SEM of three repeated experiments.

[0087] Figure 17 shows the results of immunoblotting analysis of cathepsin B inhibitor inhibiting the degradation of target proteins.

[0088] Figure 18 shows the cell viability after 24h (a) and 72h (b) and the level of apoptosis factor after 24h (c) of MCF-7 cells treated with different concentrations of D-PROTAC-2.

[0089] Figure 19 shows the results of apoptosis analysis of MCF-7 cells treated with different concentrations of D-PROTAC-2 for 24h: (a) flow cytometry results; (b) quantification of the proportion of results; (c) quantification of the proportion of early apoptosis; (d) quantification of the proportion of late apoptosis, statistical data is the mean ± SEM of three repeated experiments.

[0090] Figure 20 shows the results of cycle analysis of MCF-7 cells treated with different concentrations of D-PROTAC-2: (a) flow cytometry results; (b) quantification of the proportion of results, statistical data is the mean ± SEM of three repeated experiments.

[0091] Figure 21 shows the results of cell proliferation and apoptosis analysis of MCF-10A cells treated with different concentrations of D-PROTAC-2: (a) flow cytometry analysis of cycle results; (b) flow cytometry analysis of apoptosis results; (c) MTT method cell proliferation inhibition results, statistical data is the mean ± SEM of three repeated experiments.

[0092] Figure 22 shows the anti-tumor activity of D-PROTAC-1 in 4T1 xenograft animal models. (a) in vivo animal experiment; (b) actual figure of tumor volume change over time in 4T1 mouse model; (c) curve figure of tumor volume change over time; (d) mouse weight change figure.

[0093] Figure 23 shows the anti-tumor activity of D-PROTAC-1 in different tumor cells.

[0094] Figure 24 shows the level of (A) RAD51 protein and (B) quantification of the gray level of RAD51 protein after MDA-MB-231 cells were treated with D-PROTAC-3 at different concentrations for 72h, statistical data is the mean ± SEM of three repeated experiments.

[0095] Figure 25 shows the level of (A) RAD51 protein and (B) quantification of the gray level of RAD51 protein after MDA-MB-231 cells were treated with 2μg / mL D-PROTAC-3 for different times, statistical data is the mean ± SEM of three repeated experiments.

[0096] Figure 26 shows (A) RAD51 protein levels after 2 pg / mL D-PROTAC-3 was applied to MDA-MB-231 cells for 72 h in the presence of 10 mM MG132; (B) Quantitative analysis column chart of the gray scale of RAD51 protein levels, the statistical data is the average value ± SEM of three repeated experiments.

[0097] Figure 27 shows RAD51 protein levels after 2 pg / mL D-PROTAC-3 was applied to MDA-MB-436 cells for 72 h.

[0098] Figure 28 shows the results of D-PROTAC-3 inhibiting tumor cell proliferation.

[0099] Figure 29 shows (A), (C) USP30 protein levels after D-PROTAC-4 was applied to HepG2 cells and HCT116 cells for 72 h at different concentrations; (B), (D) Quantitative analysis column chart of the gray scale of USP30 protein levels, the statistical data is the average value ± SEM of three repeated experiments.

[0100] Figure 30 shows (A), (C) USP30 protein levels after 2 pg / mL D-PROTAC-4 was applied to HepG2 cells and HCT116 cells for different times; (B), (D) Quantitative analysis column chart of the gray scale of USP30 protein levels, the statistical data is the average value ± SEM of three repeated experiments.

[0101] Figure 31 shows (A) USP30 protein levels after 2 pg / mL D-PROTAC-4 was applied to HepG2 cells for 72 h in the presence of 10 mM MG132; (B) Quantitative analysis column chart of the gray scale of USP30 protein levels, the statistical data is the average value ± SEM of three repeated experiments. DETAILED DESCRIPTION

[0102] The preferred embodiments of the present application will be described in more detail below. Although the preferred embodiments of the present application are described below, it is understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein.

[0103] Unless otherwise specified, the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are conventional products that can be obtained by commercial purchase.

[0104] The experimental operations involved in the following examples are as follows:

[0105] 1. Plasmid extraction: After plasmid transformation into competent cells, the cells were cultured in liquid medium overnight, and then plated, single colonies were picked and expanded the next day. E. coli was expanded in 100 mL LB medium overnight, and then the plasmid was extracted according to the kit. The concentration of the plasmid was generally > 100 ng / μL.

[0106] 2. Cell culture: Breast cancer MDA-MB-231 cells, MDA-MB-453 cells, MCF-7 cells, HepG2 cells, HCT116 cells (all from ATCC). Cells were cultured in high-sugar DMEM, all culture media were supplemented with 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin. Cells were cultured at 37°C in a culture box containing 5% CO2.

[0107] 3. Protein degradation detection: Cells were cultured in 12-well plates, and a specific concentration (e.g. 2 μg / mL) of D-PROTAC plasmid was transfected into cells using transfection reagent. After 4 h of incubation at 37°C, the cells were washed with PBS and replaced with complete medium. After a certain period of time, the cells were collected and subjected to western blot to detect the expression level of the target protein in the cells.

[0108] 4. Cytotoxicity experiment (cell viability test): The MTT method was used for cytotoxicity experiment. For convenience of subsequent experiments, 250 mg of Methylthiazolyldiphenyl-tetrazolium bromide (MTT) powder from Sigma was selected. Thiazolyl blue tetrazolium bromide, also known as thiazolyl blue tetrazolium bromide, has been widely used for cell viability detection, cell proliferation and cytotoxicity analysis. The specific method is as follows:

[0109] (1) Cell plating preparation: about 12 h in advance, an appropriate amount of cells were transferred to 96-well plates, and the cell density, state and distribution should be appropriate.

[0110] (2) Drug administration: after drug administration, the cells were placed in a 37°C, 5% CO2 cell incubator for 72 h.

[0111] (3) MTT action: add 10 μL of 0.5% MTT solution to each well, avoid light incubation for 4 h, and then terminate the culture.

[0112] (4) Result determination: discard the staining solution, add 100 μL of DMSO, mix thoroughly and dissolve the crystals for 10 minutes, and detect the absorbance at OD490 nm using a microplate reader.

[0113] (5) Data processing: In Excel, subtract the absorbance of the blank wells from the A490 absorbance of each well to get the relative absorbance value. Divide the absorbance of the control group by the value of the blank wells to get the relative value of cell death. After processing the data of each well, the corresponding IC50 and error value are obtained by Graphpad Prism 8 processing.

[0114] 5. Apoptosis analysis: Phosphatidylserine appears on the membrane surface during apoptosis and can be recognized and stained by Annexin V-FITC, showing a green signal of FITC fluorescent probe. Late apoptosis is often accompanied by cell membrane rupture and loss of integrity, and propidium iodide can stain cells with incomplete cell membranes, including mechanically necrotic cells and late apoptotic cells, showing a red signal. Therefore, this experiment mainly distinguishes the different states of cells by the different staining of cells in different states, and the specific steps are as follows:

[0115] (1) Cell preparation: Transfer an appropriate amount of cells to a 12-well plate 12 h in advance, and the cell density, state and distribution should be appropriate. According to the experimental scheme, the drug treatment is carried out, and the culture is terminated after the corresponding time.

[0116] (2) Cell collection: Collect the original culture medium, add 200 μL PBS to each well for washing and collection, add 200 μL trypsin to digest the cells, and observe the cell digestion state under a microscope to avoid under-digestion or over-digestion affecting the experimental results. The cells obtained by suspending the original culture medium are mixed with the washed PBS and added to the pre-prepared centrifuge tube, and centrifuged.

[0117] (3) Remove cell debris: Discard the supernatant, wash the collected cells in the tube with 1.5 mL pre-cooled PBS, and centrifuge to discard possible cell debris.

[0118] (4) Cell staining: Add 1.5 mL pre-cooled PBS, wash twice to remove possible residual trypsin, centrifuge for 5 minutes to remove the supernatant, and add corresponding staining reagents to each experimental group, including buffer and two staining solutions, and mix well. It should be noted to prepare a blank group (without staining), a group of Annexin V-FITC single-stained cells, and a group of propidium iodide single-stained cells to facilitate subsequent instrument adjustment, door drawing and regional division.

[0119] (5) Incubation at room temperature: Aluminum foil is wrapped in a dark room for 10-20 minutes, and the mixture should be mixed several times during this period. Store at 4°C and immediately detect on the machine.

[0120] (6) Instrument detection: After pretreatment, draw the door, divide the region and analyze the fluorescence content in the cells by the instrument. The experiment is repeated three times, and at least 10,000 gated events are collected and analyzed each time.

[0121] 6. Cell cycle analysis: The present application adopts propidium iodide staining method, which is a classical cell cycle and apoptosis analysis method. The specific steps are as follows:

[0122] (1) Cell preparation: 12h in advance, transfer the cells to a 5cm petri dish, the cell density, state and distribution should be appropriate, according to the experimental scheme for drug treatment, and terminate the culture after the corresponding time.

[0123] (2) Cell collection: collect the original culture medium, add 200μL PBS to each well for washing and collection, add 200μL trypsin to digest the cells, and observe the digestion state under the microscope, avoid under-digestion or over-digestion affecting the experimental results, suspend the cells in the original culture medium, mix them with the washed PBS, and add them to the prepared centrifuge tube, and centrifuge.

[0124] (3) Washing and counting: discard the supernatant, wash the collected cells in the tube with 1.5mL pre-cooled PBS, count the cells, take 10-100 million cell suspension in the centrifuge tube, centrifuge and discard the supernatant, and remove the cell fragments that may exist in the collected sample.

[0125] (4) Cell fixation: add 1.5mL pre-cooled PBS, wash again, discard the supernatant, add 1mL pre-cooled 70% ethanol, suspend, and fix at 4°C overnight.

[0126] (5) Cell staining: centrifuge the fixed cells to discard the ethanol, wash with PBS, centrifuge and discard the supernatant. Prepare propidium iodide staining solution (for use on the same day). Add 0.5mL propidium iodide dye to each experimental group, gently blow the cells with a gun, stain in a dark room for 30 minutes, and store at 4°C in an ice bath in the dark.

[0127] (6) Instrument detection: after pretreatment, draw the door, and detect the red fluorescence at 488nm wavelength. The experiment is repeated three times, and at least 10000 gated events are collected and analyzed each time.

[0128] Example 1: Construction and extraction verification of D-PROTAC

[0129] 1. Design and plasmid construction of D-PROTAC-1:

[0130] (1) Design of p-PROTAC: design p-PROTAC based on the polypeptide PROTAC sequence targeting Bcl-xL, specifically: marked as p-PROTAC-1. Among them, the double underlined sequence is the VHL ligand (derived from the fragment of HIF), the middle unmarked sequence is the linker, and the single underlined sequence is the target protein Bcl-xL targeting polypeptide.

[0131] (2) Design of D-PROTAC: The DNA base sequence was designed according to the codon sequence corresponding to the p-PROTAC-1 polypeptide sequence, and human source optimization was performed. The sequence of the designed DNA-PROTAC-1 is shown in SEQ ID NO: 8.

[0132] (3) The hTERT promoter was selected to utilize its tumor cell-specific expression, and a PROTAC plasmid targeting the anti-apoptotic protein Bcl-xL was constructed (Figure 1), denoted as TERT-Bcl-xL-PROTAC (D-PROTAC-1), and the sequence is shown in SEQ ID NO: 16. In order to verify the transfection efficiency and specific expression of the promoter, the present application inserts the enhanced red fluorescent protein (m-cherry) fluorescent sequence into the sequence, and utilizes the P2A element for self-cleavage, thereby realizing the simultaneous and separate expression of the marker protein and p-PROTAC-1, and minimizing the interference with the treatment effect. The polypeptide sequence after expression is shown in Figure 2.

[0133] 2. Design and plasmid construction of D-PROTAC-2:

[0134] According to the design idea of D-PROTAC-1 above, the p-PROTAC molecule of the gene coding model was replaced and designed, as shown in Figure 3, and the present application constructed a p-PROTAC polypeptide sequence simultaneously targeting the classical tumor-related proteins ERa and PI3K. In other aspects of the design, the hTERT promoter, VHL E3 ligase ligand and red fluorescent protein (m-cherry) were still selected. The polypeptide sequence is: PIYPALASGSGHKILHRLLQGLFGGPGGDYAAMGACPASEQGYEEMRASGSGALAPYI P (SEQ ID NO: 13), denoted as p-PROTAC-2, which simultaneously degrades ERa and PI3K.

[0135] Among them, the p-PROTAC part degrading ERa is: Among them, the sequence marked with double underlines is the VHL ligand, the sequence without marks in the middle is the linker, and the sequence marked with single underlines is the ERa targeting peptide. The p-PROTAC part degrading PI3K is: Among them, the sequence marked with double underlines is the VHL ligand, the sequence without marks in the middle is the linker, and the sequence marked with single underlines is the PI3K targeting peptide. The p-PROTAC degrading ERa and the p-PROTAC degrading PI3K are connected by the sequence GLFGG of cathepsin B, which can be recognized and cleaved by the highly expressed cathepsin B in tumor cells, so as to separate the two targeting polypeptides and avoid the influence of protein steric hindrance on the degradation effect.

[0136] After the sequence design is completed and optimized by human base, DNA-PROTAC-2 is obtained, and the sequence is shown in SEQ ID NO: 11. According to the method above, the PROTAC plasmid is constructed (Figure 4), which is recorded as TERT-Dual-PROTAC (D-PROTAC-2), and the sequence is shown in SEQ ID NO: 17.

[0137] 3. Design and plasmid construction of D-PROTAC-3:

[0138] According to the design idea of D-PROTAC-1 above, the p-PROTAC molecule of the gene coding model is designed by replacement, mainly including: the Bcl-xL targeting peptide is replaced by the RAD51 protein targeting peptide, and the sequence of the designed p-PROTAC-3 is: Wherein, the sequence marked with double underlines is the VHL ligand, the sequence in the middle is the linker, and the sequence marked with single underlines is the target protein RAD51 protein targeting peptide. Replace m-cherry with EGFP.

[0139] After the sequence design is completed and optimized by human base, DNA-PROTAC-3 is obtained, and the sequence is shown in SEQ ID NO: 9. According to the method above, the PROTAC plasmid is constructed, which is recorded as RAD51-PROTAC (D-PROTAC-3), and the sequence is shown in SEQ ID NO: 18.

[0140] 4. Design and plasmid construction of D-PROTAC-4:

[0141] According to the design idea of D-PROTAC-3 above, the p-PROTAC molecule of the gene coding model is designed by replacement, mainly including: the RAD51 protein targeting peptide is replaced by the USP30 protein targeting peptide, and the sequence of the designed p-PROTAC-4 is: Wherein, the sequence marked with double underlines is the VHL ligand, the sequence in the middle is the linker, and the sequence marked with single underlines is the target protein USP30 protein targeting peptide.

[0142] After the sequence design is completed and optimized by human base, DNA-PROTAC-4 is obtained, and the sequence is shown in SEQ ID NO: 10. According to the method above, the PROTAC plasmid is constructed, which is recorded as USP30-PROTAC (D-PROTAC-4), and the sequence is shown in SEQ ID NO: 19.

[0143] 5. Plasmid verification:

[0144] After the plasmid construction, the bacteria were used for amplification by plasmid amplification method, and then the target plasmid was extracted. The extracted plasmid after amplification was treated with double enzyme cutting overnight to determine that the enzyme cutting efficiency basically reached 100%. The plasmid after enzyme cutting was subjected to agarose gel electrophoresis to identify the molecular weight of the plasmid and whether the construction was correct, and the results are shown in Figure 6. The corresponding molecular weight is consistent, and the tailing is less, proving that the purity of the extracted plasmid is relatively high. The molecular weight of the DNA fragment obtained after double enzyme cutting is also consistent with the expected value. The enzyme cutting analysis and agarose gel electrophoresis results confirm that the preparation and extraction of DNA are successful, and the next step application can be carried out.

[0145] Example 2: D-PROTAC-1 induces degradation of Bcl-xL protein

[0146] (1) D-PROTAC-1 induces degradation of Bcl-xL protein in a concentration-dependent manner

[0147] The present application uses 0, 0.5, 1, 1.5, and 2 μg / mL of D-PROTAC-1 of different concentrations to treat MDA-MB-231 and 4T1 cells for 24 h. Then, by Western blotting, the relative protein level of Bcl-xL was analyzed with the expression level of Actin protein in each group as a reference. The gray value ratio of Bcl-xL protein to Actin protein when 0 μg / mL of the cells was quantified as 1, and the relative protein level of other groups was calculated based on this. All subsequent protein level analysis uses this method.

[0148] As shown in the results of Western blotting detection (Figures 7a and 7b) and gray scale analysis (Figures 7c and 7d), after different concentrations of D-PROTAC-1 were used to treat MDA-MB-231 and 4T1 cells, the degradation efficiency of Bcl-xL increased with the increase of concentration, and the degradation efficiency could reach more than 50%. The DC50 (the concentration of the drug that causes 50% protein degradation) in MDA-MB-231 cells was even less than 1 μg / mL, and the addition of transfection reagent also ruled out the interference of protein degradation. Among them, D-PROTAC-1 showed good protein degradation effect in 4T1 mouse cells, which provided preliminary verification and data support for the in vivo experiment.

[0149] (2) D-PROTAC-1 induces degradation of Bcl-xL protein in a time-dependent manner

[0150] The present application also explores the time gradient of D-PROTAC-1 mediated degradation of Bcl-xL protein. The experiment uses 2 μg / mL of D-PROTAC-1 of the same concentration to treat cells for 6 h, 12 h, 24 h, and 48 h, respectively, and the transfection reagent in each group is 4 μL lipo6000.

[0151] Then, Western blotting and gray scale analysis were performed for each group of experiments (Figure 8). The results of gray scale analysis quantification showed that the protein level of Bcl-xL in MDA-MB-231 breast cancer cells under the action of D-PROTAC-1 molecules at different times was continuously decreasing, and showed a good time gradient. The average degradation efficiency was more than 50% at 12h, more than 60% at 24h and 48h, and the highest degradation efficiency could reach 80%. The results also proved that with the passage of time, the concentration of effective p-PROTAC-1 molecules expressed by plasmid was also increasing, compared with traditional polypeptide PROTAC, the generation of molecules was more reliable, and the biocompatibility was better. The results showed that D-PROTAC-1 not only had the advantage of targeting tumors, but also had the therapeutic ability of reducing the level of target proteins.

[0152] Example 3: Effect of D-PROTAC-1 on MDA-MB-231 cell activity

[0153] (1) D-PROTAC-1 effectively reduces the viability of tumor cells

[0154] The cytotoxicity of D-PROTAC-1 on MDA-MB-231 cells at different times and different concentrations was determined by MTT method. The experimental results showed that D-PROTAC-1 had a significant growth inhibition effect on MDA-MB-231 cells, and the inhibition effect became more and more obvious with the increase of time. Among them, the half-inhibitory concentration value was 1.67μg / mL at 24h (Figure 9).

[0155] Then, apoptosis detection experiments were carried out to detect the cytotoxicity of D-PROTAC-1 on cancer cells. After MDA-MB-231 cells were treated with 0, 0.5, 1, and 2μg / mL D-PROTAC-1 for 24h, the cells were collected and stained with Annexin V and Propidium Iodide (PI).

[0156] As shown in Figure 10, the flow cytometry results show that as the drug concentration increases, the proportion of apoptosis of MDA-MB-231 cells also increases, especially the proportion of late apoptosis. From 1 μg / mL drug concentration to 2 μg / mL concentration, the proportion of late adjustment of MDA-MB-231 cells increased from almost none to 26.07% and 42.16%, respectively. The total apoptosis rate of MDA-MB-231 cells showed similar results. The total apoptosis rate of MDA-MB-231 cells at 2 μg / mL D-PROTAC-1 was 64.45%, while the total apoptosis rate of MDA-MB-231 cells at 1 μg / mL D-PROTAC-1 was only 16.54%, indicating that the apoptosis of MDA-MB-231 cells was dose-dependent. And the apoptosis trend of cells under the action of D-PROTAC-1 was obvious, which proved that D-PROTAC-1 could effectively reduce the life activity of tumor cells and achieve good tumor inhibition effect.

[0157] In addition, the present application further detects the apoptosis-related factors in cells, as shown in Figure 11. With the increase of D-PROTAC-1 concentration, the levels of apoptosis-related factors such as Caspase-3 and Cleaved-PARP protein in cells increase, which further verifies the influence of D-PROTAC-1 on tumor cell proliferation and apoptosis.

[0158] (2) D-PROTAC-1 causes tumor cell cycle arrest

[0159] Next, by PI staining, flow cytometry was used to analyze the effect of cell cycle after 0, 0.5, 1, and 2 μg / mL D-PROTAC-1 treatment for 24 h. With the increase of D-PROTAC-1 concentration, the cycle of MDA-MB-231 cells showed G2 / M phase arrest at low concentration; under the action of high concentration D-PROTAC-2, it showed G0 / GI phase arrest. The proportion of S phase and G2 / M phase decreased with the decrease of concentration, indicating that D-PROTAC-1 inhibits proliferation by inducing tumor cell cycle arrest (Figure 12).

[0160] (3) D-PROTAC-1 effectively inhibits the migration and invasion of triple negative breast tumor cells

[0161] Triple negative breast cancer cells have strong migration and invasion ability, and tumor metastasis often occurs in the late stage of clinical treatment. Therefore, reducing the migration and invasion ability of MDA-MB-231 cells by drugs is crucial to improve the quality of life of triple negative breast cancer patients. In order to test the inhibitory ability of D-PROTAC-1, the present application detects the influence of different concentrations of D-PROTAC-1 on the migration and invasion of MDA-MB-231 cells after 24 hours by Transwell test. As shown in Figure 13, under 5x10 microscope magnification, as the concentration of D-PROTAC-1 acting on MDA-MB-231 cells increases, the proliferation and migration of cells are inhibited more severely.

[0162] Example 4: D-PROTAC-1 has no effect on the proliferation and apoptosis of normal breast epithelial cells MCF-10A

[0163] In order to test the specific selectivity of D-PROTAC-1, the present application detects the influence of different concentrations of D-PROTAC-1 on the proliferation and apoptosis of MCF-10A cells.

[0164] As shown in Figure 14, the present application carries out apoptosis and cell proliferation inhibition detection experiments to detect the influence of D-PROTAC-1 on the proliferation and apoptosis of normal cells. After MCF-10A cells are treated with 0, 1 and 2 μg / mL D-PROTAC-1 for 24 hours, the cells are collected and stained with Annexin V and PI. The results of flow cytometry show that different concentrations of D-PROTAC-1 have no effect on the apoptosis state of the cells, and the proportion of early and late apoptosis does not change significantly. Under the action of three concentrations of D-PROTAC-1, the cell apoptosis rate remains below 4%.

[0165] After MCF-10A cells are treated with 0, 1 and 2 μg / mL D-PROTAC-1 for 72 hours, the results of MTT experiment show that it has no inhibition on cell proliferation, and even the number of proliferating cells is higher than that of the control group as the concentration increases.

[0166] For MCF-10A cells, neither cell proliferation nor apoptosis shows obvious effect of D-PROTAC-1 on normal cells, which confirms that D-PROTAC-1 can selectively kill breast cancer cells without affecting normal breast epithelial cells, and proves that the DNA-PROTAC model designed in the present application can effectively reduce non-tumor specific toxicity.

[0167] Example 5: D-PROTAC-2 induces degradation of double targets of ERa and PI3K protein

[0168] (1) D-PROTAC-2 induced degradation of dual targets of ERa and PI3K protein in a concentration-dependent manner:

[0169] The transfection reagent of this example was lipo6000, and the transfection method was to culture in double antibody-free medium for 4-6 h, then change to complete medium for continuous culture for 24 h. Different concentrations of D-PROTAC-2 plasmid were transfected into MCF-7 cells, and the cells were collected with a cell scraper (because the measured PI3K protein is a membrane protein, if trypsin digestion is used to collect cells, the target protein may be damaged, affecting the experimental results), the whole cell protein was lysed and extracted, and immunoblotting analysis was performed to detect the protein levels of dual target proteins ERa and PI3K in cells after drug action. Then western blot detection and gray scale analysis were performed, and the protein level results are shown in Figure 15. It can be found that compared with the no drug group, 1 pg / mL of D-PROTAC-2 can cause a certain level of degradation of ERa and PI3K proteins in cells, and with the increase of concentration, the degradation efficiency of ERa and PI3K is improved, which reflects the dose-dependent degradation of the model protein, and also indicates that with the increase of plasmid concentration, the expression and translation of p-PROTAC-2 in MCF-7 cells gradually increase, thereby improving the degradation efficiency. And at a concentration of 2 pg / mL, the degradation efficiency can reach 85% and 65% respectively, further proving the feasibility of the DNA-PROTAC targeted protein degradation model expansion therapy module strategy.

[0170] (2) D-PROTAC-2 induced degradation of dual targets of ERa and PI3K protein in a time-dependent manner

[0171] The same concentration of 2 pg / mL D-PROTAC-2 and 4 pL / mL transfection reagent were selected for the time gradient verification part of the embodiment to treat MCF-7 cells for 6 h, 12 h, 24 h, and 48 h, respectively. After the MCF-7 cells were treated with D-PROTAC at different times and the same concentration, the present application performed western blot detection and gray scale analysis on the MCF-7 cells treated with the drug (FIG. 16). The results showed that the protein levels of ERa and PI3K in the MCF-7 cells decreased continuously with the extension of time under the action of D-PROTAC-2 at different times, and the degradation efficiency reached 92% and 89% respectively at 48 h, showing a good time gradient. The reason may be that the increase of plasmid action time to a certain extent makes the accumulation of p-PROTAC-2 expressed by the plasmid, the effective concentration is increased, thereby more target proteins are acted on, and then the effective degradation of the two target proteins is triggered. Compared with the difference in protein degradation between 12 h and 24 h, the protein degradation between 24 h and 48 h is not much different, which also proves that the effective concentration of the plasmid gene can be basically reached after acting on the cells for 24 h, thereby providing certain time selection support for the drug action experiment and other gene therapy methods.

[0172] (3) D-PROTAC-2 realizes selective degradation in a cathepsin B specific cleavage dependent manner

[0173] In the sequence design stage of D-PROTAC-2, the present application introduced a cathepsin B sequence between the two target degradation sequences. Previous studies have shown that this sequence can be recognized and cleaved by the highly expressed cathepsin B in tumor cells, so as to separate the two target polypeptides and avoid the influence of spatial steric hindrance on the degradation effect.

[0174] To further verify the specific recognition and cleavage of the sequence, and its influence on spatial steric hindrance and protein degradation, the present embodiment uses a cathepsin B inhibitor for verification. In the presence of the cathepsin B inhibitor, the activity of cathepsin B is inhibited, so that it cannot specifically recognize and cleave the sequence GLFGG, and the two target degradation sequences cannot be separated.

[0175] Three groups of parallel experiments were set in the present embodiment: the control group was not treated with drugs, only the same amount of DMSO and transfection reagent as the experimental group were added; the experimental group 1 was treated with 2 pg / mL D-PROTAC-2 for 24 h; and the experimental group 2 was treated with 2 pg / mL D-PROTAC-2 and 10 pM cathepsin B inhibitor at the same time for 24 h. Western blot analysis was performed on the experimental cells after 24 h of drug action.

[0176] The results of Western blotting analysis are shown in Figure 17. DMSO and transfection reagent have no effect on the protein levels of ERa and PI3K. D-PROTAC-2 can degrade the protein levels of ERa and PI3K to a very low level. After adding cathepsin B inhibitor, the ubiquitin-proteasome pathway is inhibited, and the degradation effect of D-PROTAC-2 is weakened. The above results prove that the cathepsin B in D-PROTAC-2 can achieve the function of specific recognition.

[0177] Example 6: D-PROTAC-2 has a significant inhibitory effect on the proliferation of MCF-7 cells

[0178] (1) D-PROTAC-2 induces tumor cell apoptosis:

[0179] This example sets six concentration gradients of 0, 0.5, 1, 1.5, 2, and 4 pg / mL, 24 h of action time, and three parallel determinations. The analysis results are shown in Figure 18. The MTT method determination results show that as the concentration increases, the inhibitory ability of D-PROTAC-2 on MCF-7 cells is enhanced, and under 72 h of action time, very low D-PROTAC-2 concentration can cause very strong cytotoxicity, which also proves that there is a time dependence when D-PROTAC-2 acts. Under 24 h of action time, 4 pg / mL of D-PROTAC-2 molecules can cause nearly 60% of cell apoptosis, and the IC50 value is about 2.3 pg / mL. Under 72 h of action time, low-concentration drug molecules also show good tumor inhibition effect, with a cell apoptosis rate of 35%, and the IC50 is about 1.65 pg / mL.

[0180] This example further detects the apoptosis-related factors in the cells. With the increase of the concentration of D-PROTAC-2, the protein levels of apoptosis-related factors such as Cleaved-Caspase-3 and Cleaved-PARP in the cells increase, which further verifies the influence of D-PROTAC-2 on tumor cell apoptosis.

[0181] This example performs an apoptosis detection experiment to detect the influence of D-PROTAC-2 on cell cytotoxicity.

[0182] MCF-7 cells were treated with 0, 0.5, 1, and 2 pg / mL D-PROTAC-2 for 24 h, and then collected and stained with Annexin V and propidium iodide (PI). The flow cytometry results are shown in Figure 19. The apoptosis rate was 53.89% at 2 pg / mL D-PROTAC-2, while the apoptosis rate was only 19.1% at 1 pg / mL D-PROTAC-2, showing a clear concentration gradient in cell apoptosis. In addition, the proportion of cells with early apoptosis characteristics increased significantly with increasing D-PROTAC-2 concentration.

[0183] (2) D-PROTAC-2 causes tumor cell cycle arrest:

[0184] The effect on the cell cycle was verified by flow cytometry analysis of PI staining after treatment with 0, 0.5, 1, and 2 pg / mL D-PROTAC-2 for 24 h. With increasing D-PROTAC-2 concentration, more MCF-7 cells were arrested in the G0 / G1 phase, while the proportions of cells in the S and G2 / M phases decreased with concentration, indicating that D-PROTAC-2 inhibited proliferation by inducing G0 / G1 phase arrest in tumor cells (Figure 20).

[0185] Example 7: D-PROTAC-2 has no effect on normal breast epithelial cell MCF-10A proliferation and apoptosis

[0186] To test the specific selectivity of D-PROTAC-2, this example detected the effect of D-PROTAC-2 on normal breast epithelial cell MCF-10A proliferation and apoptosis at different concentrations.

[0187] First, the effect on the cell cycle, after 0, 0.5, 1, 1.5, 2 pg / mL D-PROTAC-2 was applied to human breast epithelial cells MCF-10A for 24 h, flow cytometry was used to detect the cell cycle after PI staining, and the results are shown in Figure 21(a). There was no obvious cycle arrest effect, proving that D-PROTAC-2 had no obvious cycle effect on normal cells. Then, cell apoptosis and cell proliferation inhibition detection experiments were carried out to detect the cytotoxicity of D-PROTAC-2 on normal cells. As shown in Figure 21(b), after MCF-7 cells were treated with 0, 1, and 2 pg / mL D-PROTAC-2 for 24 h, the cells were collected and stained with Annexin V and propidium iodide (PI). The flow cytometry results showed that different concentrations of D-PROTAC-2 had no effect on the apoptosis state of the cells, and the proportions of early and late apoptosis did not change significantly. And as shown in Figure 21(c), cell proliferation was not significantly inhibited.

[0188] Example 8: D-PROTAC-1 inhibits tumor growth in vivo

[0189] To verify the effect of D-PROTAC-1 in vivo, animal experiments were conducted. Fifteen female BALB / c mice were divided into three groups: ① AAV-PROTAC group (n=5); ② PBS control group (n=5); and ③ AAV control group (n=5).

[0190] (1) Subcutaneous tumor-bearing: After 4T1 cells were digested and centrifuged, they were resuspended with PBS and counted. Each mouse was subcutaneously inoculated with 1×10 7 / 100 μL of 4T1 cells in the upper arm. The tumor volume was measured every 2-3 days using a vernier caliper.

[0191] (2) Drug administration: Mice with a tumor volume of about 100±50 mm 3 were selected, and 1×10 12 v.g. of D-PROTAC-1 was injected in situ into 5-week-old BALB / c mice. The tumor volume was measured every 2-3 days using a vernier caliper.

[0192] (3) Tumor dissection: The tumor was dissected when the tumor volume of the control group mice reached 1500 mm 3 . The tumor volume was measured and photographed.

[0193] The results are shown in Figure 22. The mice in the drug administration group showed consistent and stable body weight throughout the experiment, and the tumor volumes of the PBS and AAV control groups were basically consistent. The tumor volume of the experimental group was significantly reduced, demonstrating that D-PROTAC-1 has low toxicity and effectively inhibits tumor growth in vivo.

[0194] Example 9: D-PROTAC-1 significantly inhibits the proliferation of other tumor cells

[0195] This example sets six concentration gradients of 0, 0.5, 1, 1.5, 2, and 4 μg / mL, with a 72 h action time. The analysis results are shown in Figure 23. The MTT method showed that as the concentration increased, the inhibitory ability of D-PROTAC-2 on human liver cancer cells Huh7, human cervical cancer cells Hela, human lung cancer cells A549, and human breast cancer cells MCF-7 cells increased, indicating that this method has a significant tumor inhibition effect on a variety of tumor cells.

[0196] Example 10: D-PROTAC-3 induces degradation of RAD51 protein

[0197] (1) D-PROTAC-3 induces degradation of RAD51 protein in a concentration-dependent manner

[0198] The MDA-MB-231 cells were treated with 0, 0.5, 1, 1.5, and 2 μg / mL of D-PROTAC-3 for 24 h, respectively. Then, the relative protein level of RAD51 was analyzed by Western blotting with the expression level of TUBULIN protein in each group as a reference.

[0199] As shown in the results of Western blotting detection and gray scale analysis (FIG. 24), after the MDA-MB-231 cells were treated with different concentrations of D-PROTAC-3, the degradation efficiency of RAD51 protein increased with the increase of the concentration, and the degradation efficiency could reach more than 50%.

[0200] (2) D-PROTAC-3 induces degradation of RAD51 protein in a time-dependent manner

[0201] The time gradient of D-PROTAC-3-mediated degradation of RAD51 protein was also explored. In the experiment, the cells were treated with 2 μg / mL of D-PROTAC-3 for 24 h, 48 h, and 72 h, respectively, and the transfection reagent in each group was jet prime.

[0202] Then, Western blotting detection and gray scale analysis were performed for each group (FIG. 25). The gray scale analysis quantitative results showed that the level of RAD51 protein in MDA-MB-231 breast cancer cells decreased continuously with the increase of the action time of D-PROTAC-3 molecules. The results showed that D-PROTAC-3 not only has the advantage of targeting tumors, but also has the therapeutic ability of reducing the level of target proteins.

[0203] (3) MG132 inhibition experiment

[0204] The cell protein ubiquitin proteasome degradation pathway was inhibited by adding a culture medium containing 10 μM MG132, and the cells were collected at different time points (0, 3, 6, and 12 hours). Then, the total protein was extracted and analyzed by Western blotting. As shown in FIG. 26, after the cell protein ubiquitin proteasome degradation pathway was inhibited, the RAD51 protein was not degraded, which verified that the degradation of RAD51 protein was achieved through the PROTAC pathway mediated by D-PROTAC-3.

[0205] Example 11: Degradation effect of D-PROTAC-3 on MDA-MB-436 cells

[0206] The present application also verifies that D-PROTAC-3 induces degradation of RAD51 protein in MDA-MB-436 cells. MDA-MB-436 cells are treated with 2 μg / mL of D-PROTAC-3 for 72 hours. The results are shown in Figure 27. It can be seen that D-PROTAC-3 can also significantly induce degradation of RAD51 protein in MDA-MB-436 cells.

[0207] Example 12: D-PROTAC-3 inhibits tumor cell proliferation

[0208] 96-well plates are plated with MDA-MB-231 cells at 3000-4000 cells per well with a volume of 100 μL. 10 μL of CCK8 reagent is added after transfection with 2 μg / mL of RAD51-PROTAC plasmid for 24 h, 48 h, 72 h, and 96 h. The OD value is measured at a wavelength of 450 nm after 1-4 h of reaction. The results are shown in Figure 28. It can be seen that D-PROTAC-3 can significantly inhibit tumor cell proliferation.

[0209] Example 13: D-PROTAC-4 induces degradation of USP30 protein

[0210] (1) D-PROTAC-4 induces degradation of USP30 protein in a concentration-dependent manner

[0211] The present application uses 0, 0.5, 1, and 2 μg / mL concentrations of D-PROTAC-4 to treat HepG2 cells and HCT116 cells for 24 h. Then, the relative protein level of USP30 is analyzed by Western blotting, with the expression level of GAPDH protein in each group as a reference.

[0212] As shown in the results of Western blotting detection and gray scale analysis (Figure 29), after D-PROTAC-4 of different concentrations is used to treat HepG2 cells and HCT116 cells, the degradation efficiency of USP30 protein increases with increasing concentration.

[0213] (2) D-PROTAC-4 induces degradation of USP30 protein in a time-dependent manner

[0214] The present application also explores the time gradient of D-PROTAC-4-mediated degradation of USP30 protein. The experiment uses 2 μg / mL of D-PROTAC-4 to treat cells for 24 h, 48 h, and 72 h, respectively.

[0215] Then, Western blotting detection and gray scale analysis were performed for each group of experiments (Figure 30). The gray scale analysis quantification results showed that the USP30 protein levels in HepG2 cells and HCT116 cells were continuously reduced with the increase of the D-PROTAC-4 molecule action time. The results showed that D-PROTAC-4 had the therapeutic ability to reduce the target protein level.

[0216] (3) MG132 inhibition experiment

[0217] The cell protein ubiquitin proteasome degradation pathway was inhibited by adding a culture medium containing 10 μM MG132, and HepG2 cells were collected at different time points (0, 3, 6, 12 hours), and then total protein was extracted and analyzed by Western blotting. The results are shown in Figure 31. After the cell protein ubiquitin proteasome degradation pathway was inhibited, the USP30 protein was not degraded, which verified that the degradation of the USP30 protein was achieved through the D-PROTAC-4 mediated PROTAC pathway.

[0218] The above has described various embodiments of the present application, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes are obvious to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A DNA molecule for targeted degradation of a protein of interest, characterized in that, The DNA molecule comprises at least one group of D-PROTAC units, each group of D-PROTAC units comprising at least one first DNA segment encoding a ubiquitin ligase targeting peptide, at least one second DNA segment encoding a target protein targeting peptide, and optionally a third DNA segment encoding a linker peptide between adjacent first and second DNA segments.

2. The DNA molecule for targeted degradation of a protein of interest according to claim 1, wherein, The DNA molecule further comprises a promoter arranged upstream of all D-PROTAC units; The promoter is preferably an organ-, tissue- or cell-specific promoter or an environment-specific promoter; The organ-, tissue- or cell-specific promoter is preferably a disease organ-, tissue- or cell-specific promoter, more preferably a tumor organ-, tissue- or cell-specific promoter; The environment-specific promoter is selected from at least one of a hypoxia / hyperoxia response promoter, a low pH / high pH response promoter, a lactic acid promoter and a H2O2 response promoter.

3. The DNA molecule for targeted degradation of a protein of interest according to claim 2, wherein, The tumor organ-, tissue- or cell-specific promoter is selected from at least one of a human telomerase reverse transcriptase (hTERT) promoter, a survivin promoter, an alpha-fetoprotein (AFP) promoter, an osteocalcin (OC) promoter, a neuron-specific enolase (NSE) promoter, a carcinoembryonic antigen (CEA) promoter, a cyclooxygenase-2 (COX-2) promoter, a prostate-specific antigen (PSA) promoter, an E2F-1 promoter, a cholecystokinin promoter, a C-erbB2 / neu oncogene promoter, a CXCR4 promoter, a HE4 promoter, a hexokinase type II promoter, a L-lactoferrin promoter, a MUC1 promoter, a TRP1 promoter, a tyrosinase promoter, a hypoxia-inducible factor-1 promoter, a tumor-associated mucin promoter, a secretory leukocyte protease inhibitor promoter, a survivin promoter and a MetR-specific promoter. 4.The DNA molecule for targeted degradation of a protein of interest according to claim 1, wherein, The ubiquitin ligase targeting peptide is at least one of a VHL E3 ubiquitin ligase targeting peptide, a CRBN E3 ubiquitin ligase targeting peptide, a MDM2 E3 ubiquitin ligase targeting peptide, an IAPs E3 ubiquitin ligase targeting peptide, a DCAF E3 ubiquitin ligase targeting peptide and a RNF E3 ubiquitin ligase targeting peptide. Preferably, the ubiquitin ligase targeting peptide is a VHL E3 ubiquitin ligase targeting peptide, and the amino acid sequence thereof is shown in SEQ ID NO:

1. 5.The DNA molecule of claim 1, wherein, The target protein is at least one of a B-cell lymphoma protein, an estrogen receptor alpha, an intracellular phosphatidylinositol kinase, a RAD51 protein, a USP30 protein, an estrogen receptor 1, an androgen receptor, a BTK tyrosine protein kinase, an interleukin-1 receptor-associated kinase 4, a RAC alpha-serine / threonine protein kinase, a RAC beta-serine / threonine protein kinase, a bromodomain-containing protein 4, a cyclin-dependent kinase 9, a receptor-interacting serine / threonine protein kinase 1, a transcription intermediary factor 1-alpha, an epidermal growth factor receptor, a hepatocyte growth factor receptor and a hepatocyte growth factor receptor.

6. The DNA molecule for targeted degradation of a protein of interest according to claim 5, wherein, When the target protein is B lymphoma protein, estrogen receptor alpha, intracellular phosphatidylinositol kinase RAD51 protein and USP30 protein, the sequence of the corresponding target protein targeting peptide is shown in SEQ ID NO: 3-7.

7. The DNA molecule for targeted degradation of a protein of interest according to claim 1, wherein, The number of D-PROTAC units is 1-6 groups, and a coding sequence of a controllable cleavage peptide chain is arranged between adjacent D-PROTAC units; the controllable cleavage peptide chain can realize peptide chain cleavage under controllable conditions to make the translation products of each D-PROTAC unit exist independently; Preferably, the controllable cleavage peptide chain is an intracellular protease recognition site or a self-cleavage peptide, and the intracellular protease recognition site is preferably a disease cell-specific high-expression protease recognition site, and more preferably a cathepsin B recognition site.

8. The DNA molecule for targeted degradation of a protein of interest according to claim 7, wherein, The DNA molecule comprises a group of D-PROTAC units, and the sequence of the D-PROTAC units is shown in SEQ ID NO: 8; or The DNA molecule comprises a group of D-PROTAC units, and the sequence of the D-PROTAC units is shown in SEQ ID NO: 9; or The DNA molecule comprises a group of D-PROTAC units, and the sequence of the D-PROTAC units is shown in SEQ ID NO: 10; or The DNA molecule comprises two groups of D-PROTAC units, and the sequence of the D-PROTAC units is shown in SEQ ID NO:

11. 9.The DNA molecule for targeted degradation of a protein of interest according to claim 1, wherein, The DNA molecule further comprises a reporter protein coding gene, and a spacer is arranged between the reporter protein coding gene and the D-PROTAC unit, the spacer is a sequence coding a self-cleavage peptide or an intracellular protease recognition site, and the self-cleavage peptide or the intracellular protease recognition site is used to separate the functional proteins connected thereto and to self-cleave or be cleaved under suitable conditions, so that the functional proteins connected thereto exist independently; Preferably, the reporter protein coding gene is an m-Cherry coding gene or an EGFP coding gene, and the spacer is a coding sequence of a P2A peptide.

10. A biomaterial, characterized by, The biological material is any one of the following A1) to A5): A1) an expression cassette containing the DNA molecule of any one of claims 1-9; A2) a recombinant vector containing the DNA molecule of any one of claims 1-9; A3) a recombinant microorganism containing the DNA molecule of any one of claims 1-9, or an expression cassette of A1), or a recombinant vector of A2); A4) a cell transfected with the DNA molecule of any one of claims 1-9, or the expression cassette of A1), or the recombinant vector of A2), or the recombinant microorganism of A3); A5) a virus transfected with the DNA molecule of any one of claims 1-9, or the expression cassette of A1), or the recombinant vector of A2).

11. The biomaterial of claim 10, wherein, The recombinant vector further comprises a functional element selected from one or more of a replication origin, an enhancer, a Kozak sequence, a terminator, a signal peptide, an intron, a resistance gene, a marker gene, a multiple cloning site and a ribosome binding site.

12. Use of the DNA molecule according to any one of claims 1 to 9, or of the biological material according to claim 10 or 11, for any one of the following: M1) the preparation of a product for the treatment and / or prevention of a disease / pathology mediated by said protein of interest; M2) the treatment and / or prevention of a disease / pathology mediated by said protein of interest; M3) the preparation of a product for the inhibition of cell proliferation; M4) the inhibition of cell proliferation; M5) the preparation of a product for the promotion of apoptosis; M6) the promotion of apoptosis; the disease mediated by said protein of interest is preferably a cancer, an immune disease, a metabolic disease, a neurodegenerative disease or an inflammatory disease; the cell is preferably a cancer cell; the cancer is preferably a leukemia, a lymphoma, a lung cancer, a breast cancer, an ovarian cancer, a cervical cancer, a human glioma, a melanoma, a glioblastoma, a nasopharyngeal carcinoma, a liver cancer, a brain cancer, a pancreatic cancer, a uterine cancer, a testicular cancer, a skin cancer, a stomach cancer, a colon cancer, a bladder cancer or a rectal cancer; the product is preferably a vaccine or a drug.

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