DNA molecule for targeted degradation of target protein and application

Abstract

The invention belongs to the field of gene therapy, and relates to a DNA (deoxyribonucleic acid) molecule for targeted degradation of target protein and application. The DNA molecule comprises at least one group of D-PROTAC units, each group of D-PROTAC units comprises at least one first DNA chain segment for coding a ubiquitin ligase targeting peptide and at least one second DNA chain segment for coding a target protein targeting peptide, and a third DNA chain segment for coding a connecting peptide is optionally arranged between the adjacent first DNA chain segment and second DNA chain segment. The gene therapy specific PROTAC molecular model is successfully constructed, the huge potential of a gene therapy method in the aspects of research, development and application of new PROTAC anticancer drugs is proved, the action mode of the PROTAC technology is innovated, and a new treatment application scene is expanded for gene therapy.

Description

Technical Field

[0001] The present invention belongs to the field of gene therapy, and in particular, relates to a DNA molecule for targeted degradation of a target protein, a biomaterial comprising the DNA molecule, and applications of the DNA molecule and the biomaterial. Background Art

[0002] In countries around the world, cancer is the leading cause of death and a major obstacle to increasing the average human life expectancy. According to estimates by the World Health Organization (WHO) of 183 countries in 2019, cancer is the first or second leading cause of death for people under the age of 70 in 112 of these countries, and ranks third or fourth in another 23 countries. There are currently a variety of treatment strategies for cancer, mainly chemotherapy, surgery, and radiotherapy. Among them, chemotherapy has remained the main treatment method for cancer since the 1940s, and anticancer drugs are currently the focus of drug research and development. However, chemotherapy is often accompanied by some toxic side effects, which are usually caused by the adverse effects of anticancer drugs on hematopoietic cells in the bone marrow, hair follicles, and cells in the digestive tract and reproductive system. Therefore, the research and development of anticancer drugs is committed to improving nonspecificity 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. The overexpression of proteins has a causal relationship with tumor formation. Overexpressed proteins in tumor cells can be used as tumor markers for accurate diagnosis of early cancer, and can also be used as targets for anti-tumor drugs.

[0004] The goal of modern drug development is to develop highly effective, selective, and target-specific drugs. In the past few decades, the one-target theory has achieved great success in guiding the development of drugs for single-cause diseases, especially antibiotics targeting specific single pathogen protein targets. However, cancer or inflammatory diseases are usually the result of the interaction of multiple factors, involving the synergistic effects of multiple mediators or the increase of multiple receptors, and the signaling network of crosstalk between the two. In cancer, tumor cells often elevate different growth-promoting factors, which can act independently and promote each other through signaling networks within the cell. Tumor cells are prone to acquire drug resistance by increasing alternative factors or switching signaling pathways that promote proliferation; therefore, treatment targeting only a single target is limited. In addition to drug resistance, single-target therapeutic drugs can also lead to reduced efficacy and quality of life for patients due to side effects, tissue toxicity, etc. In order to overcome the shortcomings of single-target drugs, combined use of two different methods related to disease development can achieve additive or synergistic effects, reduce drug resistance, and reduce side effects due to the reduction in the dose of each single drug. This has become a recognized effective method.

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

[0006] Research on PROTAC technology is very popular in the field of new drug development. It uses the ubiquitin-proteasome system to induce target protein degradation in an "event-driven" manner, catalyzes degradation patterns, has high activity and selectivity, and can target more "undruggable" targets. It shows great potential in new drug development. Currently, some drugs have entered the clinical stage. In-depth research on PROTAC plays an important role in the development of new anti-cancer drugs.

[0007] However, there are still some problems that need to be solved for the successfully developed PROTACs, such as large molecular weight, poor membrane permeability, inconvenience for oral administration, off-target toxicity, non-cancer specific toxicity, difficulty in forming triple complexes, etc. Among them, when PROTACs are working, there is a situation where tumor cells and normal cells are attacked indiscriminately, and the off-target effects and non-cancer specific toxicity caused by them are the biggest concerns about the safety of PROTAC technology. Summary of the invention

[0008] The present invention provides a DNA molecule for targeted degradation of a target protein, wherein the DNA molecule is a PROTAC molecule in DNA form.

[0009] In one embodiment, the DNA PROTAC molecule of the present invention has tumor specificity, which can solve the problems of poor specificity and weak controllability of tumor cells. Specifically, the PROTAC molecule is designed to be a DNA form including a specific expression part and a therapeutic part. Among them, the specific expression part adopts a disease (such as a tumor) specific promoter, which can be specifically expressed in disease (such as a tumor) cells to reduce non-target side effects; the therapeutic part adopts the action form of gene therapy to translate the polypeptide, overcome the problems of transmembrane effect concentration, and use unique physicochemical properties to target small molecule PROTAC drug targets that are difficult to target, improve drug resistance, reduce toxicity, good biosafety, and achieve the function of targeted protein degradation. At the same time, the present invention also explores the modular replacement of different therapeutic parts-the dual-target degradation of PI3K and ERα combined medication, which provides a new action model for the development of new anti-cancer drugs.

[0010] In order to achieve the above-mentioned object, the present invention provides a DNA molecule for targeted degradation of a target protein, wherein the DNA molecule comprises at least one group of D-PROTAC units, 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 connecting peptide is optionally arranged between adjacent first DNA segments and second DNA segments.

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

[0012] According to one specific embodiment of the present invention, the D-PROTAC can induce the single-target degradation of B lymphocytoma protein (Bcl-xL); according to another specific embodiment of the present invention, the D-PROTAC can induce the single-target degradation of RAD51 protein; according to another specific embodiment of the present invention, the D-PROTAC can induce the single-target degradation of USP30 protein.

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

[0014] The present invention enriches the means of PROTAC regulation by designing PROTACs that can function in cells at the DNA level. For example, a very effective design is to design a promoter upstream of all D-PROTAC units, and 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, and 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 responsive promoter, a low pH / high pH responsive promoter, a lactate promoter and a H2O2 responsive promoter. Due to the organ, tissue, cell selectivity or environmental 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 may 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 may be selected from muscle tissue, adrenal tissue, appendix tissue, bladder tissue, bone, intestinal tissue, brain tissue, breast tissue, bronchial tube, coronary tissue, ear tissue, esophageal tissue, eye tissue, gallbladder tissue, genital tissue, heart tissue, hypothalamus 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, ureteral tissue, urethral tissue, soft and connective tissue, peritoneal tissue, vascular tissue, adipose tissue or a combination thereof;

[0017] The cell can be selected from hepatocytes, epithelial cells, endothelial cells, cardiomyocytes, skeletal muscle cells, sinusoidal cells, afferent neurons, efferent neurons, interneurons, glial cells, astrocytes, oligodendrocytes, microglia, ependymal cells, lung epithelial cells, Schwann cells, satellite cells, photoreceptor cells, retinal ganglion cells, T cells, B cells, NK cells, macrophages, dendritic cells 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 thyroxine transporter (TTR) promoter, a human α-1 antitrypsin promoter, a HBV promoter and an Afp promoter; a heart-specific promoter selected from at least one of cTNT, α-MHC, MLC-2v, Desmin, ANF, Tnnt2, MYH6, MYL2 and SERCA2a. a muscle-specific promoter selected from at least one of a phosphoglycerate 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 α promoter, an actin β promoter, an actin γ 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, S100β promoter and EAAT2 / GLT-1 promoter, NG2 promoter, CD68 promoter, F4 / 80 promoter, Slc1a3 promoter and Rlbp1 promoter; cone cell and rod cell specific promoter, selected from at least one of hGRK1p, RedO promoter, SynP136 promoter, GNAT2 promoter, SynPVI promoter and mCAR promoter; pluripotent stem cell specific promoter, selected from mEtnAhC3p, mEtnAhS4p, mN at least one of ANOGp; a neuron-specific promoter selected from at least one of Syn promoter, huc promoter, and CaMKIIa promoter; endothelial cell-specific promoter K18; chondrocyte-specific promoter Col2a1; macrophage-specific promoter CD11b or CD68 gene; B cell-specific promoter ProA; plasma cell-specific promoter ProB; and an erythroid-specific promoter selected from at least one of α-spectrin promoter, ankyrin-1 promoter, γ-globin promoter, and β-globin promoter.

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

[0020] The ubiquitin ligase targeting peptide is an essential component of p-PROTAC, but the selection of its specific type can be flexible, and the present invention is not particularly limited to this. For example, it can be at least one of the VHL E3 ubiquitin ligase targeting peptide, CRBN E3 ubiquitin ligase targeting peptide, MDM2 E3 ubiquitin ligase targeting peptide, IAPs E3 ubiquitin ligase targeting peptide, DCAF E3 ubiquitin ligase targeting peptide and RNF E3 ubiquitin ligase targeting peptide of E3 ubiquitin ligase.

[0021] The widely used VHL E3 ubiquitin ligase targeting peptide is selected in the embodiment of the present invention, and its amino acid sequence is PIYPALA (SEQ ID NO: 1). 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, those skilled in the art can determine that the target protein can be selected arbitrarily, depending on the degradation target. Specifically, the target protein includes but is not limited to B lymphocyte tumor protein (Bcl-xL, Human B-cell lymphoma XL), estrogen receptor α (Erα, Estrogen Receptor Alpha), intracellular phosphatidylinositol kinase (PI3K, Phosphatidylinositide3-kinases), estrogen receptor 1 (ESR1, Estrogen Receptor 1), RAD51 protein, USP30 protein (Ubiquitin specific peptidase 30, ubiquitin specific protease 30), androgen receptor (AR, Androgen receptor), BTK tyrosine protein kinase (BTK, Tyrosine-proteinkinaseBTK), interleukin-1 receptor-associated kinase 4 (IRAK4, Interleukin-1receptor-associatedkinase 4), RACα-serine / threonine protein kinase (AKT1, RAC-alpha serine / threonine-proteinkinase), RACβ serine / threonine protein kinase (AKT2, RAC-beta serine / threonine-protein kinase), bromodomain-containing protein 4 (BRD4), cyclin-dependent kinase 9 (CDK9), receptor-interacting serine / threonine-protein kinase 1 (RIPK1 ， At least one of the following: receptor-interacting serine / threonine-protein kinase1), transcription intermediary factor 1-alpha (TRIM24), epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (MET), and mast / stem cell growth factor receptor (KIT).

[0023] Some embodiments of the present invention use several commonly expressed proteins in tumor cells as examples, such as B lymphocytoma protein (Bcl-xL), estrogen receptor α (ERα), 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 Bcl-xL targeting peptide is GQVGRQLAIIGDAINR (SEQ ID NO: 3), the sequence of ERα targeting peptide is HKILHRLLQ (SEQ ID NO: 4), the sequence of PI3K targeting peptide is PGGDYAAMGACPASEQGYEEMRA (SEQ ID NO: 5), the sequence of RAD51 protein targeting peptide is LLGFHTASGKKVKIAK (SEQ ID NO: 6), and the sequence of USP30 protein targeting peptide is GIYVIWGPITERKKRRKG (SEQID NO: 7). The DNA coding sequences of the five target protein targeting peptides can be determined based on the above amino acid sequences, and this technical means is very routine for those skilled in the art.

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

[0025] In the present invention, the connecting peptide is used to separate the polypeptides at both ends to prevent them from affecting each other, so any sequence that meets this function can be used. There are many known connecting peptide sequences in the art, which can be used in the present invention, such as (G) m , (GS) n Wherein m and n can be integers of 2-20, specifically 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 invention, the D-PROTAC unit can be one or more groups. When it is a group, the D-PROTAC formed is a single target, and when it is multiple groups, the D-PROTAC formed is a multi-target. Generally, the number of the D-PROTAC units is 1-6 groups, for example, 1 group, 2 groups, 3 groups, 4 groups, 5 groups, 6 groups, and the D-PROTACs in each group can be the same or different, that is, the entire DNA molecule can be expressed as multiple identical p-PROTACs, or can be expressed as multiple different p-PROTACs.

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

[0028] According to a specific embodiment of the present invention, 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, and its sequence is shown in SEQ ID NO: 8:

[0029] 5'-CCCATCTATCCTGCTCTGGCTGGAGGAGGGGGCGGCGGCGGA CAGGTGGGAAGACAGCTGGCTATCATTGGCGATGCTATTAATAGA-3'

[0030] (SEQ ID NO: 8).

[0031] According to another specific embodiment of the present invention, 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, and its sequence is shown in SEQ ID NO: 9:

[0032] 5'-CTGCTGGGCTTTCACAACCGCCTCTGGCAAAAAAGTGAAGATTG CCAAAAGCGGCTCCGGCGCCCTGGCCCCATACATCCCC-3' (SEQ ID NO: 9).

[0033] According to another specific embodiment of the present invention, the DNA molecule includes a group of D-PROTAC units, including a USP30 protein targeting peptide coding sequence, a connecting peptide coding sequence, and a VHL targeting peptide coding sequence, and its sequence is shown in SEQ ID NO: 10:

[0034] 5'-GGCATCTACGTGATTTGGGGCCCCATCACCGAGAGGAAAAAG AGGAGAAAGGGAAGCGGCAGCGGCGCCCTGGCTCCTTACATCCCT-3' (SEQ ID NO: 10).

[0035] According to another specific embodiment of the present invention, the DNA molecule comprises two groups of D-PROTAC units, the first group of D-PROTAC units comprises an ERα targeting peptide coding sequence, a connecting peptide coding sequence, and a VHL targeting peptide coding sequence, and the second group of D-PROTAC units comprises a PI3K targeting peptide coding sequence, a connecting peptide coding sequence, and 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 thereof is shown in SEQ ID NO: 11:

[0036] 5'-ATCTACCCAGCCCTGGCTTCTGGATCTGGCCATAAAATTCTGCA TAGACTGCTGCAGGGCCTGTTTGGCGGACCCGGCGGCGATTACGCTG CCATGGGAGCCTGTCCTGCCTCTGAGCAGGGCTATGAAGAGATGAGAGCTTCCGGCTCCGGTGCCCTGGCCCCTTACATCCCATAG-3' (SEQ ID NO: 11).

[0037] In the case of verifying the system of the present invention or having other needs, the DNA molecule further includes a reporter protein encoding gene, and a spacer is provided between the reporter protein encoding gene and the D-PROTAC unit, wherein the spacer is a sequence encoding a self-cleaving peptide or an intracellular protease recognition site, and the self-cleaving peptide or intracellular protease recognition site is used to separate the functional protein to which it is connected and self-cleaves or is cleaved under appropriate conditions, so that the functional proteins to which they are connected (i.e., the reporter protein and p-PROTAC) exist independently of each other.

[0038] According to a specific embodiment of the present invention, the reporter protein encoding gene is an m-Cherry encoding gene or an EGFP encoding gene, and the spacer region is a coding sequence of a P2A peptide. Those skilled in the art can select other reporter proteins and self-cleaving peptides as needed, and the present invention is not particularly limited thereto.

[0039] The present invention also provides a biomaterial, which is any one of the following A1) to A5):

[0040] A1) an expression cassette containing the above-mentioned DNA molecule;

[0041] A2) a recombinant vector containing the above-mentioned DNA molecule;

[0042] A3) a recombinant microorganism containing the above-mentioned DNA molecule, or a recombinant microorganism containing the expression cassette described in A1), or a recombinant microorganism containing the recombinant vector described in A2);

[0043] A4) cells transfected with the above-mentioned DNA molecule, or the expression cassette described in A1), or the recombinant vector described in A2), or the recombinant microorganism described in A3);

[0044] A5) A virus transfected with the above-mentioned DNA molecule, or the expression cassette described in A1), or the recombinant vector described in A2).

[0045] According to the present invention, the recombinant vector also includes functional elements, which can be selected from one or more of replication origin, enhancer, Kozak sequence, terminator, signal peptide, intron, resistance gene, marker gene, multiple cloning site and ribosome binding site.

[0046] According to a specific embodiment of the present invention, hTERT promoter was selected to utilize its tumor cell-specific expression, and VHL, a more mature E3 ligase, was used to connect the ligand to construct a PROTAC plasmid targeting the anti-apoptotic protein Bcl-xL ( Figure 1 ), denoted as TERT-Bcl-xL-PROTAC (also referred to as D-PROTAC-1 in this article). It includes a human-optimized PROTAC module targeting Bcl-xL, named DNA-PROTAC-1. The enhanced red fluorescent protein (m-cherry) fluorescent sequence is inserted into the sequence, and the P2A element is used for self-cleavage, so as to achieve the simultaneous expression of the marker protein and p-PROTAC-1, minimizing the interference with the therapeutic effect.

[0047] Based on the design concept of DNA-PROTAC-1 single-target degradation, the present invention includes ERα and PI3K proteins in the degradation range of the DNA-PROTAC model, expands the method of the present invention, and achieves dual-target degradation. Specifically, the design principle of DNA-PROTAC-2 is as follows: the p-PROTAC molecule of the gene encoding model is replaced and designed, such as Figure 3 As shown in the figure, a p-PROTAC peptide sequence targeting both classic tumor-related proteins ERα and PI3K was constructed. In other aspects of the design, hTERT promoter, VHL E3 ligase ligand and red fluorescent protein (m-cherry) were still selected. After the design was completed and optimized by humanization, DNA-PROTAC-2 was obtained, and the PROTAC plasmid ( Figure 4 ), denoted as TERT-Dual-PROTAC (also referred to as D-PROTAC-2 herein).

[0048] On the basis of the above model, the targeting protein ligand sequence or E3 ligase ligand sequence can be replaced to extend the effect to overexpressed proteins of other disease targets. The present invention also verifies the PROTAC system for RAD51 protein and USP30 protein; the specific promoter module can also be changed to other tissue-specific promoters to extend the scope of application of the model. The above changes are all within the scope of protection of the present invention.

[0049] The working principle of D-PROTAC in the embodiment of the present invention is as follows Figure 5 As shown, D-PROTAC can use the translation machinery in the cell to synthesize the functional p-PROTAC and m-Cherry / EGFP as expression verification. p-PROTAC can mediate the interaction between the target protein and VHL E3 ubiquitin ligase, causing the degradation of the target protein.

[0050] The present invention also provides any of the following applications of the above-mentioned DNA molecules and the above-mentioned biological materials:

[0051] M1) preparing a product for treating and / or preventing a disease / lesion mediated by the target protein;

[0052] M2) treating and / or preventing diseases / lesions mediated by the target protein;

[0053] M3) preparing products for inhibiting cell proliferation;

[0054] M4) inhibits cell proliferation;

[0055] M5) preparing products that promote cell apoptosis;

[0056] M6) promotes cell apoptosis.

[0057] The present invention is applicable to various diseases mediated by the target protein, including but not limited to cancer, immune diseases, metabolic diseases, neurodegenerative diseases or inflammatory diseases;

[0058] According to a specific embodiment, the disease mediated by the target protein is cancer, and the cancer is preferably leukemia, lymphoma, lung cancer, breast cancer, ovarian cancer, cervical cancer, human brain glioma, melanoma, glioblastoma, nasopharyngeal carcinoma, 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.

[0059] In one embodiment of the present invention, the cancer is breast cancer, the cells are breast cancer cells, and the breast cancer cells are MCF-7 cells. Specifically, in one example, the breast cancer cells are MDA-MB-231 cells. In another example, the breast cancer cells are MDA-MB-453 cells.

[0060] In another embodiment of the present invention, the cancer is liver cancer, the cells are liver cancer cells, and the liver cancer cells are HepG2 cells.

[0061] In still another embodiment of the present invention, the cancer is colon cancer, the cells are colon cancer cells, and the colon cancer cells are HCT116 cells.

[0062] In the present invention, the product can be any conventional preventive or therapeutic product, preferably a vaccine or a medicine.

[0063] The present invention successfully constructed a gene therapy-specific PROTAC molecular model with good anti-tumor effect, overcame the problem of non-tumor specific toxicity, further demonstrated the great potential of gene therapy in the research and development and application of new PROTAC anti-cancer drugs, innovated the action mode of PROTAC technology, and expanded new therapeutic application scenarios for gene therapy.

[0064] Specifically, the present invention uses the tumor tissue / cell-specific human telomerase reverse transcriptase (hTERT) promoter to regulate expression, so that the therapeutic polypeptide (p-PROTAC) is highly expressed in tumor cells, and m-Cherry red fluorescent protein is introduced, and flow cytometry and confocal cell imaging techniques are used to verify the specific expression of the target sequence; the treatment part is designed as a polypeptide action mode, and immunoblotting experiments have shown that the model targets and degrades B lymphocyte tumor protein (Bcl-xL) by concentration gradient, time gradient, and ubiquitination pathway. 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 mammary epithelial cells (MCF-10A). At the same time, D-PROTAC-1 showed good protein degradation effects in 4T1 mouse cells, providing preliminary verification and data support for the conduct of clinical experiments. The present invention also explored the diversity of therapeutic molecules, designed and synthesized D-PROTAC-2, achieved the simultaneous degradation of the dual targets of estrogen receptor ERα and intracellular phosphatidylinositol kinase PI3K, and specifically affected 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 present invention also designed and synthesized D-PROTAC-3 and D-PROTAC-4, which respectively achieved the degradation of RAD51 protein and USP30 protein, further verifying the versatility of the model of the present invention.

[0065] This series of PROTACs can design therapeutic polypeptide ligand sequences according to the target protein sequence and specifically express and regulate in tumor cells through gene therapy, targeting and degrading target proteins. The present invention can also be extended from a single target to a multi-target synergistic effect, and can also explore the extension from cancer to immune, neurodegenerative, and metabolic-related diseases. It can make up for the widespread non-tumor-specific toxicity problem of current PROTACs, and the administration of plasmid DNA can effectively reduce the molecular weight of the drug and enhance the effective drug concentration. The drug-making model of the final PROTAC polypeptide also provides convenience and possibility for the development of protein-targeted chimeras for the degradation of other proteins based on this model, which will further promote the construction and biological application of new PROTAC models.

[0066] The D-PROTAC designed in the present invention has the following advantages:

[0067] (1) Based on plasmid DNA gene therapy, plasmid DNA is designed to express and translate the target protein to produce therapeutic peptides. Compared with traditional peptide PROTAC technology, it has better biocompatibility, higher efficiency and lower cost.

[0068] (2) Based on the systematic regulation of controllable genes or protein elements and with the help of tumor-specific promoters, non-tumor-specific toxicity is effectively reduced, providing new ideas for controllable PROTACs.

[0069] (3) The combination of multi-target degradation therapy can degrade two or more target proteins at the same time, which can effectively reduce drug resistance, overcome the side effects and tissue toxicity of single-target therapeutic drugs, improve efficacy, and enhance the quality of life of patients.

[0070] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0072] Figure 1 This is the plasmid map of TERT-Bcl-xL-PROTAC.

[0073] Figure 2 Schematic diagram of the structure of TERT-Bcl-xL-PROTAC.

[0074] Figure 3 Schematic diagram of the TERT-Dual-PROTAC structure.

[0075] Figure 4 This is the TERT-Dual-PROTAC plasmid map.

[0076] Figure 5 Schematic diagram of the working principle of D-PROTAC.

[0077] Figure 6 The results of agarose gel electrophoresis of (a) TERT-Bcl-xL-PROTAC plasmid and (b) TERT-Dual-PROTAC plasmid were identified by enzyme digestion.

[0078] Figure 7 The protein level of Bcl-xL after 24 hours of D-PROTAC-1 treatment in (a) MDA-MB-231 cells and (b) 4T1 cells is shown; the grayscale quantitative analysis bar graph of Bcl-xL protein level after 24 hours of D-PROTAC-1 treatment in (c) MDA-MB-231 cells and (d) 4T1 cells is shown. The statistical data are the mean ± SEM of three repeated experiments.

[0079] Figure 8The figure shows (a) Bcl-xL protein level after 2 μg / mL D-PROTAC-1 acts on MDA-MB-231 cells for different time periods; (b) quantitative analysis bar graph of Bcl-xL protein level grayscale, and the statistical data are the mean ± SEM of three repeated experiments.

[0080] Fig. 9 The results of cell viability analysis after 24 h of treatment of MDA-MB-231 cells with different concentrations of D-PROTAC-1 are shown. The statistical data are the mean ± SEM of three repeated experiments.

[0081] Fig.10 The results of cell apoptosis analysis of MDA-MB-231 cells after 24 hours of treatment with different concentrations of D-PROTAC-1 are shown: (a) flow cytometry results; (b) total quantitative analysis ratio results; (c) late apoptosis quantitative analysis ratio results; (d) early apoptosis quantitative analysis ratio results. The statistical data are the mean ± SEM of three repeated experiments.

[0082] Fig.11 The levels of apoptotic factors in MDA-MB-231 cells after treatment with different concentrations of D-PROTAC-1 for 24 h are shown.

[0083] Fig.12 The results of cycle analysis after treating MDA-MB-231 cells with different concentrations of D-PROTAC-1 are shown: (ae) Cycle flow cytometry results after 24 hours of treatment of cells with different concentrations of D-PROTAC-1; (f) Quantitative analysis ratio result diagram, the statistical data are the mean ± SEM of three repeated experiments.

[0084] Fig.13 The results of the cell invasion experiment after treating MDA-MB-231 cells with different concentrations of D-PROTAC-1 are shown.

[0085] Fig.14 The results of cell biological function analysis after treating MCF-10A cells with different concentrations of D-PROTAC-1 are shown. (a) Cell apoptosis results; (b) Cell viability results. The statistical data are the mean ± SEM of three repeated experiments.

[0086] Fig.15 The ERα and PI3K protein levels of MCF-7 cells treated with D-PROTAC-2 at concentrations of 0, 0.5, 1, 1.5, and 2 μg / mL for 24 h are shown: (a) immunoblotting results; (b) grayscale quantitative analysis bar graph, the statistical data are the mean ± SEM of three repeated experiments.

[0087] Fig.16The ERα and PI3K protein levels of MCF-7 cells treated with 2 μg / mL D-PROTAC-2 for 0, 6, 12, 24, and 48 hours are shown: (a) immunoblotting results; (b) grayscale quantitative analysis bar graph, the statistical data are the mean ± SEM of three repeated experiments.

[0088] Fig.17 The results of immunoblot analysis showing that cathepsin B inhibitors inhibited target protein degradation.

[0089] Fig.18 The cell viability of MCF-7 cells treated with different concentrations of D-PROTAC-2 (a) after 24 h, (b) after 72 h, and (c) apoptotic factor levels are shown.

[0090] Fig.19 The results of cell apoptosis analysis after MCF-7 cells were treated with different concentrations of D-PROTAC-2 for 24 hours are shown: (a) flow cytometry results; (b) quantitative analysis ratio results; (c) quantitative analysis results of early apoptosis ratio; (d) quantitative analysis results of late apoptosis ratio. The statistical data are the mean ± SEM of three repeated experiments.

[0091] Fig. 20 The results of cell cycle analysis after treatment of MCF-7 cells with different concentrations of D-PROTAC-2 are shown: (a) cell cycle flow cytometry results; (b) quantitative analysis ratio results, the statistical data are the mean ± SEM of three repeated experiments.

[0092] Fig.21 The results of cell proliferation and apoptosis analysis after treating MCF-10A cells with different concentrations of D-PROTAC-2 are shown: (a) cell cycle result graph of flow cytometry analysis; (b) cell apoptosis result graph of flow cytometry analysis; (c) MTT method cell proliferation inhibition result graph. The statistical data are the mean ± SEM of three repeated experiments.

[0093] Fig. 22 The anti-tumor activity of D-PROTAC-1 in the 4T1 xenograft animal model is shown. (a) In vivo animal experiment; (b) Physical picture of the change of tumor volume over time in the 4T1 mouse model; (c) Curve chart of the change of tumor volume over time; (d) Mouse weight change chart.

[0094] Fig.23 The anti-tumor activity of D-PROTAC-1 in different tumor cells is shown.

[0095] Fig.24(A) RAD51 protein level after D-PROTAC-3 acted on MDA-MB-231 cells at different concentrations for 72 hours; (B) quantitative analysis bar graph of RAD51 protein level grayscale, statistical data are the mean ± SEM of three repeated experiments.

[0096] Fig.25 (A) RAD51 protein level after 2 μg / mL D-PROTAC-3 acted on MDA-MB-231 cells for different time periods; (B) quantitative analysis bar graph of RAD51 protein level grayscale, statistical data are the mean ± SEM of three repeated experiments.

[0097] Fig.26 (A) RAD51 protein level after 2 μg / mL D-PROTAC-3 acted on MDA-MB-231 cells for 72 h in the presence of 10 μM MG132; (B) quantitative analysis bar graph of RAD51 protein level grayscale, statistical data are the mean ± SEM of three repeated experiments.

[0098] Fig. 27 The RAD51 protein level after 72 h of treatment of MDA-MB-436 cells with 2 μg / mL D-PROTAC-3 is shown.

[0099] Fig.28 The results showing D-PROTAC-3 inhibited tumor cell proliferation.

[0100] Fig.29 (A) and (C) USP30 protein levels after D-PROTAC-4 acted on HepG2 cells and HCT116 cells at different concentrations for 72 hours; (B) and (D) quantitative analysis bar graphs of the grayscale of USP30 protein levels. The statistical data are the mean ± SEM of three repeated experiments.

[0101] Fig.30 (A) and (C) USP30 protein levels after 2 μg / mL D-PROTAC-4 acted on HepG2 cells and HCT116 cells for different time periods; (B) and (D) quantitative analysis bar graphs of the grayscale of USP30 protein levels. The statistical data are the mean ± SEM of three repeated experiments.

[0102] Fig.31 (A) USP30 protein level after 2 μg / mL D-PROTAC-4 acted on HepG2 cells for 72 h in the presence of 10 μM MG132; (B) quantitative analysis bar graph of USP30 protein level grayscale, statistical data are the mean ± SEM of three repeated experiments. DETAILED DESCRIPTION

[0103] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0104] If the specific conditions are not specified in the examples, all the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, all of them are conventional products that can be purchased from the market.

[0105] The experimental operations involved in the following examples are as follows:

[0106] 1. Plasmid extraction: After the plasmid is transformed into competent cells, it is cultured overnight in liquid culture medium. The next day, plating, single clone picking and expansion experiments are performed. After E. coli is expanded overnight in 100mL LB culture medium, the plasmid is extracted according to the kit. The plasmid concentration is generally >100ng / μL.

[0107] 2. Cell culture: breast cancer MDA-MB-231 cells, MDA-MB-453 cells, MCF-7 cells, HepG2 cells, HCT116 cells (all from ATCC). All cells were cultured in high-glucose DMEM, and all culture media were supplemented with 10% FBS, 100U / mL penicillin and 100μg / mL streptomycin. The cells were cultured in an incubator at 37°C and 5% CO2.

[0108] 3. Protein degradation detection: Culture the cells in a 12-well plate, use a transfection reagent to transfect a specific concentration (such as 2 μg / mL) of D-PROTAC plasmid into the cells, incubate at 37°C for 4 hours, wash with PBS and replace with complete culture medium, collect the cells after a specific time, and perform a western blot experiment to detect the expression level of the target protein in the cells.

[0109] 4. Cytotoxicity experiment (cell viability test): The cytotoxicity experiment uses the MTT method. To facilitate subsequent experiments, the 250mg Methylthiazolyldiphenyl-tetrazoliumbromide (MTT) powder of Yisheng Company was selected. Methylthiazolyldiphenyl-tetrazoliumbromide, also known as thiazolyl blue tetrazolium bromide, has been widely used to detect cell viability, cell proliferation and cytotoxicity analysis. The specific method is as follows:

[0110] (1) Preparation for cell plating: Transfer an appropriate amount of cells to a 96-well plate approximately 12 hours in advance. The cell density, state, and distribution should be appropriate.

[0111] (2) Administration: After administration, the cells were placed in a cell culture incubator at 37°C and 5% CO2 for 72 hours.

[0112] (3) MTT action: 10 μL of 0.5% MTT solution was added to each well, incubated in the dark for 4 h, and then the culture was terminated.

[0113] (4) Result determination: discard the staining solution, add 100 μL DMSO, mix thoroughly and dissolve the crystals for 10 minutes, and measure the absorbance at OD490 nm using a microplate reader.

[0114] (5) Data processing: In Excel, the relative absorbance value was obtained by subtracting the absorbance of the blank well from the absorbance of each well. The relative value of cell death was obtained by subtracting the absorbance of the control group from the absorbance of the blank well. After processing the data of each well, the corresponding IC50 and error value were obtained by processing with Graphpad Prism 8.

[0115] 5. Cell apoptosis analysis: Phosphatidylserine will appear on the membrane surface during cell apoptosis, which can be recognized and stained by Annexin V-FITC, showing the green signal of FITC fluorescent probe. Cell apoptosis is often accompanied by the rupture and loss of integrity of the cell membrane. Propidium iodide can stain cells with incomplete cell membranes, including mechanical necrotic cells and late apoptotic cells, showing red signals. Therefore, this experiment mainly distinguishes different states of cells by different cell staining under different cell states. The specific steps are as follows:

[0116] (1) Cell preparation: 12 h in advance, transfer an appropriate amount of cells to a 12-well plate. The cell density, state, and distribution should be appropriate. Perform drug treatment according to the experimental plan and terminate the culture after a certain period of time.

[0117] (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, pay attention to observe the cell digestion status under the microscope to avoid insufficient or excessive digestion affecting the experimental results, suspend the obtained cells in the original culture medium, mix them with the washed PBS, add them to the pre-prepared centrifuge tube, and centrifuge.

[0118] (3) Removal of cell debris: discard the supernatant, wash the collected cells in the tube with 1.5 mL of pre-cooled PBS, and centrifuge to discard any cell debris.

[0119] (4) Cell staining: Add 1.5 mL of pre-cooled PBS, wash twice to remove any possible pancreatic enzyme residues, centrifuge for 5 minutes, remove the supernatant, add the corresponding staining reagents, including buffer and two staining solutions, to each experimental group, and mix well. It is important to prepare a group of blanks (no staining), a group of Annexin V-FITC single-stained cells, and a group of propidium iodide single-stained cells to facilitate subsequent instrument adjustment, gate drawing, and area division.

[0120] (5) Incubation at room temperature: Wrap in aluminum foil and react in a dark room for 10-20 minutes. Mix several times during the reaction. Place at 4°C and immediately test on an instrument.

[0121] (6) Instrument detection: After pretreatment, the instrument draws gates, divides regions, and analyzes the fluorescence content in the cells. The experiment is repeated three times, and at least 10,000 gated events are collected and analyzed each time.

[0122] 6. Cell cycle analysis: The present invention adopts propidium iodide staining, which is a classic method for analyzing cell cycle and cell apoptosis. The specific steps are as follows:

[0123] (1) Cell preparation: Transfer cells to a 5 cm small culture dish 12 h in advance. The cell density, state, and distribution should be appropriate. Perform drug treatment according to the experimental plan and terminate the culture after a certain period of time.

[0124] (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, pay attention to observe the cell digestion status under the microscope to avoid insufficient or excessive digestion affecting the experimental results, suspend the obtained cells in the original culture medium, mix them with the washed PBS, add them to the pre-prepared centrifuge tube, and centrifuge.

[0125] (3) Washing and counting: discard the supernatant, use 1.5 mL of pre-cooled PBS to wash the collected cells in the tube, count the cells, take 100,000 to 1,000,000 cells of the suspension into a centrifuge tube, centrifuge and discard the supernatant to remove any cell debris that may be present in the collected sample.

[0126] (4) Cell fixation: Add 1.5 mL of pre-cooled PBS and wash again. Residual trypsin may affect staining and centrifugation. Discard the supernatant and add 1 mL of pre-cooled 70% ethanol. Resuspend evenly and fix at 4°C overnight.

[0127] (5) Cell staining: Take the fixed cells and centrifuge to discard the ethanol, wash with PBS, centrifuge and discard the supernatant. Prepare propidium iodide staining solution (for use on the same day). Add 0.5 mL of propidium iodide dye to each experimental group, gently blow the cells with a gun, wrap them in tin foil at room temperature and stain them in a dark room for 30 minutes, and store them in an ice bath at 4°C away from light.

[0128] (6) Instrument detection: After pretreatment, the red fluorescence was detected by instrument gate drawing and 488 nm wavelength. The experiment was repeated three times, and at least 10,000 gated events were collected and analyzed each time.

[0129] Example 1: Construction and extraction verification of D-PROTAC

[0130] 1. Design and plasmid construction of D-PROTAC-1:

[0131] (1) Design of p-PROTAC: p-PROTAC was designed based on the peptide PROTAC sequence targeting Bcl-xL, specifically: PIYPALA GGGGGG GQVGRQLAIIGDAINR (SEQ ID NO: 12), denoted as p-PROTAC-1. The double-underlined sequence is the VHL ligand (derived from the HIF fragment), the unmarked sequence in the middle is the linker, and the single-underlined sequence is the target protein Bcl-xL targeting peptide.

[0132] (2) Design of D-PROTAC: A DNA base sequence was designed according to the codon sequence corresponding to the p-PROTAC-1 polypeptide sequence, and humanized optimization was performed. The sequence of the designed DNA-PROTAC-1 is shown in SEQ ID NO: 8.

[0133] (3) hTERT promoter was selected to utilize its tumor cell-specific expression to construct a PROTAC plasmid targeting the anti-apoptotic protein Bcl-xL ( 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 enhanced red fluorescent protein (m-cherry) fluorescent sequence is inserted into the sequence, and the P2A element is used for self-cleavage, thereby achieving the simultaneous expression of the marker protein and p-PROTAC-1, minimizing the interference with the therapeutic effect. The schematic diagram of the expressed polypeptide sequence is shown in Figure 2 .

[0134] 2. Design and plasmid construction of D-PROTAC-2:

[0135] According to the design ideas of D-PROTAC-1 above, the p-PROTAC molecules of the gene encoding model were replaced and designed, such as Figure 3 As shown, the present invention constructs a p-PROTAC polypeptide sequence that simultaneously targets the classic tumor-related proteins ERα and PI3K. In the design of other aspects, hTERT promoter, VHL E3 ligase ligand and red fluorescent protein (m-cherry) are still selected. The polypeptide sequence is: PIYPALASGSGHKILHRLLQGLFGGPGGDYAAMGACPASEQGYEEMRASGSGALAPYIP (SEQ ID NO: 13), recorded as p-PROTAC-2, which degrades ERα and PI3K simultaneously.

[0136] The p-PROTAC part that degrades ERα is: PIYPALA SGSG HKILHRLLQ, where the double-underlined sequence is the VHL ligand, the unmarked sequence in the middle is the linker, and the single-underlined sequence is the ERα targeting peptide. The p-PROTAC part that degrades PI3K is: PGGDYAAMGACPASEQGYEEMRA SGSG ALAPYIP , where the double-underlined sequence is the VHL ligand, the unmarked sequence in the middle is the linker, and the single-underlined sequence is the PI3K targeting peptide. The p-PROTAC that degrades ERα and the p-PROTAC that degrades PI3K are connected by the cathepsin B sequence GLFGG, which can be recognized and cut by highly expressed cathepsin B in tumor cells. Therefore, the two segments of the targeting peptide can be separated to avoid the influence of protein steric hindrance on the degradation effect.

[0137] After sequence design was completed and human bases were optimized, DNA-PROTAC-2 was obtained, and its sequence is shown in SEQ ID NO: 11. PROTAC plasmid was constructed according to the above method ( Figure 4 ), denoted as TERT-Dual-PROTAC (D-PROTAC-2), and the sequence is shown in SEQ ID NO: 17.

[0138] 3. Design and plasmid construction of D-PROTAC-3:

[0139] According to the design ideas of D-PROTAC-1 above, the p-PROTAC molecule of the gene encoding model was replaced and designed, mainly including: replacing the Bcl-xL targeting peptide with the RAD51 protein targeting peptide. The sequence of the designed p-PROTAC-3 is: LLGFHTASGKKVKIAK SGSG ALAPYIP (SEQ ID NO: 14), wherein the double-underlined sequence is the VHL ligand, the unmarked sequence in the middle is the linker, the single-underlined sequence is the target protein RAD51 protein targeting polypeptide, and m-cherry is replaced by EGFP.

[0140] After sequence design was completed and human bases were optimized, DNA-PROTAC-3 was obtained, and its sequence is shown in SEQ ID NO: 9. According to the above method, a PROTAC plasmid was constructed, which was named RAD51-PROTAC (D-PROTAC-3), and its sequence is shown in SEQ ID NO: 18.

[0141] 4. Design and plasmid construction of D-PROTAC-4:

[0142] According to the design ideas of D-PROTAC-3 above, the p-PROTAC molecule of the gene encoding model was replaced and designed, mainly including: replacing the RAD51 protein targeting peptide with the USP30 protein targeting peptide. The sequence of the designed p-PROTAC-4 is: GIYVIWGPITERKKRRKG SGSG ALAPYIP (SEQ ID NO: 15), wherein the double-underlined sequence is the VHL ligand, the unmarked sequence in the middle is the linker, and the single-underlined sequence is the target protein USP30 protein targeting polypeptide.

[0143] After sequence design was completed and human bases were optimized, DNA-PROTAC-4 was obtained, and its sequence is shown in SEQ ID NO: 10. According to the above method, a PROTAC plasmid was constructed, which was recorded as USP30-PROTAC (D-PROTAC-4), and its sequence is shown in SEQ ID NO: 19.

[0144] 5. Plasmid verification:

[0145] After the plasmid is constructed, it is amplified using bacteria using the plasmid amplification method, and then the target plasmid is extracted. The plasmid extracted after amplification is treated with double enzyme digestion overnight to determine that the enzyme digestion efficiency is basically 100%. The plasmid after enzyme digestion is subjected to agarose gel electrophoresis to identify the molecular weight of the plasmid and whether the construction is correct. The results are as follows Figure 6 As shown in the figure, the corresponding molecular weights are consistent and there is less tailing, which proves that the purity of the extracted plasmid is relatively high. The molecular weight of the DNA fragment obtained after double enzyme digestion is also consistent with expectations. The results of enzyme digestion analysis and agarose gel electrophoresis confirm that the preparation and extraction of DNA are successful and can be used in the next step.

[0146] Example 2: D-PROTAC-1 induces degradation of Bcl-xL protein

[0147] (1) D-PROTAC-1 induces Bcl-xL protein degradation in a concentration-dependent manner

[0148] The present invention treats MDA-MB-231 and 4T1 cells with different concentrations of D-PROTAC-1 at 0, 0.5, 1, 1.5, and 2 μg / mL for 24 hours. Then, the relative protein level of Bcl-xL was analyzed by protein immunoblotting, 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 acts on cells is quantified as 1, and the relative protein levels of other groups are calculated based on this, and all subsequent protein level analyses adopt this method.

[0149] For example, protein immunoblotting ( Figure 7 a, 7b) and grayscale analysis ( Figure 7 c, 7d) As shown in the results, after different concentrations of D-PROTAC-1 acted on MDA-MB-231 and 4T1 cells, the efficiency of degrading Bcl-xL increased with the increase of concentration, and the degradation efficiency could reach more than 50%, among which DC50 (the concentration at which the drug causes 50% protein degradation) was even less than 1 μg / mL in MDA-MB-231 cells, and the addition of transfection reagent also eliminated the interference that affected protein degradation. Among them, the good protein degradation effect of D-PROTAC-1 in 4T1 mouse cells provided preliminary verification and data support for the in vivo experiment.

[0150] (2) D-PROTAC-1 induces Bcl-xL protein degradation in a time-dependent manner

[0151] The present invention also explored the time gradient of D-PROTAC-1-mediated Bcl-xL protein degradation. The experiment used 2 μg / mL of D-PROTAC-1 at the same concentration to treat cells for 6 h, 12 h, 24 h, and 48 h, respectively, and each group of transfection reagents was 4 μL lipo6000.

[0152] Then, each group of experiments was subjected to protein immunoblotting and grayscale analysis ( Figure 8 ). The quantitative results of grayscale analysis showed that under the action of D-PROTAC-1 molecules at different times, the protein level of Bcl-xL in MDA-MB-231 breast cancer cells continued to decrease, and showed a good time gradient. At 12h of action time, the average degradation efficiency exceeded 50%. Under the action of D-PROTAC-1 molecules at 24h and 48h, the degradation effect exceeded 60%, and the degradation efficiency was as high as 80%. This result also proves that over time, the concentration of effective p-PROTAC-1 molecules expressed by plasmids is also increasing. Compared with traditional peptide PROTACs, the generation of molecules is more reliable and the biocompatibility is better. The results show that D-PROTAC-1 not only has the advantage of targeting tumors, but also has the therapeutic ability to reduce the level of target proteins.

[0153] Example 3: Effect of D-PROTAC-1 on MDA-MB-231 cell activity

[0154] (1) D-PROTAC-1 effectively reduces the vitality of tumor cells

[0155] The MTT method was used to determine the cytotoxicity of D-PROTAC-1 to MDA-MB-231 at different times and concentrations. The experimental results showed that D-PROTAC-1 had a significant growth inhibitory effect on MDA-MB-231 cells, and the inhibitory effect became more obvious over time. Among them, at a 24h action time, the half-inhibitory concentration value was 1.67μg / mL ( Fig. 9 ).

[0156] Then, a cell apoptosis assay was performed to detect the effect of D-PROTAC-1 on cancer cell cytotoxicity. After treating MDA-MB-231 cells with 0, 0.5, 1, and 2 μg / mL D-PROTAC-1 for 24 h, the cells were collected and stained with Annexin V and Propidium Iodide (PI).

[0157] like Fig.10 As shown, the results of flow cytometry showed that with the increase of drug concentration, the apoptosis rate of MDA-MB-231 cells also increased, especially the proportion of late apoptosis of cells. When the drug concentration increased from 1μg / mL to 2μg / mL, the late apoptosis rate 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 was roughly similar. The total apoptosis rate of MDA-MB-231 cells at 2μg / mL D-PROTAC-1 was 64.45%, while the total apoptosis rate of MDA-MB-231 cells at 1μg / mL D-PROTAC-1 was only 16.54%, indicating that MDA-MB-231 cell apoptosis was dose-dependent. In addition, under the action of D-PROTAC-1, the trend of cell apoptosis was obvious, proving that D-PROTAC-1 can effectively reduce the vitality of tumor cells and achieve a good tumor inhibition effect.

[0158] In addition, the present invention further detects intracellular apoptosis-related factors, such as Fig.11 As shown in Figure 2, with the increase in D-PROTAC-1 concentration, the levels of intracellular apoptosis-related factors such as Caspase-3 and Cleaved-PARP protein increased, which further verified the effect of D-PROTAC-1 on tumor cell proliferation and apoptosis.

[0159] (2) D-PROTAC-1 causes tumor cell cycle arrest

[0160] Next, PI staining and flow cytometry analysis of 0, 0.5, 1, and 2 μg / mL D-PROTAC-1 treatment for 24 hours verified the effect of the cell cycle. As the concentration of D-PROTAC-1 increased, the cycle of MDA-MB-231 cells showed G2 / M phase arrest at low concentrations; and G0 / GI phase arrest under the action of high concentrations of D-PROTAC-2. The proportions of the S phase and G2 / M phase decreased with concentration, indicating that D-PROTAC-1 inhibits proliferation by inducing tumor cell cycle arrest ( Fig.12 ).

[0161] (3) D-PROTAC-1 effectively inhibits the migration and invasion of triple-negative breast tumor cells

[0162] Triple-negative breast cancer cells often have tumor metastasis problems in the late clinical stage due to their strong migration and invasion abilities. Therefore, it is very important to improve the quality of life of patients with triple-negative breast cancer by reducing the migration and invasion abilities of MDA-MB-231 cells through drug action. In order to test the inhibitory ability of D-PROTAC-1, the present invention detected the effects of different concentrations of D-PROTAC-1 on the migration and invasion of MDA-MB-231 cells after 24 hours through the Transwell assay. Fig.13 As shown, under a 5×10 microscope magnification, as the concentration of D-PROTAC-1 acting on MDA-MB-231 cells increased, the proliferation and migration of cells were more severely inhibited.

[0163] Example 4: D-PROTAC-1 has no effect on proliferation and apoptosis of normal breast epithelial cells MCF-10A

[0164] To test the specific selectivity of D-PROTAC-1, the present invention detected the effects of different concentrations of D-PROTAC-1 on cell proliferation and apoptosis of MCF-10A cells.

[0165] like Fig.14 As shown, the present invention conducted apoptosis and cell proliferation inhibition detection experiments to detect the effect of D-PROTAC-1 on normal cell proliferation and apoptosis. After treating MCF-10A cells with 0, 1 and 2 μg / mL D-PROTAC-1 for 24 hours, the cells were collected and stained with annexin V (Annexin V) and propidium iodide (PI). The results of flow cytometry showed that different concentrations of D-PROTAC-1 had no effect on the state of cell apoptosis, and there was no significant change in the proportion of early apoptosis and late apoptosis. Under the action of three concentrations of D-PROTAC-1, the cell apoptosis rate was kept below 4%.

[0166] After treating MCF-10A cells with 0, 1, and 2 μg / mL D-PROTAC-1 for 72 h, the results of the MTT assay showed that it had no inhibitory effect on cell proliferation. As the concentration increased, the number of proliferating cells was even higher than that in the control group.

[0167] For the treatment of MCF-10A cells, no obvious effect of D-PROTAC-1 on normal cells was found, whether in terms of cell proliferation or apoptosis. These results confirm that D-PROTAC-1 can selectively and effectively kill breast cancer cells without affecting normal breast epithelial cells, demonstrating that the DNA-PROTAC model designed in the present invention can effectively reduce non-tumor specific toxicity.

[0168] Example 5: D-PROTAC-2 induces degradation of dual targets of ERα and PI3K proteins

[0169] (1) D-PROTAC-2 induces degradation of both ERα and PI3K proteins in a concentration-dependent manner:

[0170] The transfection reagent used in this embodiment is lipo6000, and the transfection method is to select a medium without dual antibodies and culture for 4-6 hours, then change the medium to complete medium and continue to culture for 24 hours. Different concentrations of D-PROTAC-2 plasmids 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 is used to digest and collect cells, the target protein may be destroyed, affecting the experimental results), the whole protein in the cells was lysed and extracted, and immunoblot analysis was performed to detect the protein levels of ERα and PI3K dual targets in the cells after the drug action. Then western blot detection and grayscale analysis were performed on it, and the protein level results are shown in Fig.15 It can be found that compared with the drug-free group, 1μg / mL of D-PROTAC-2 can cause a certain level of degradation of ERα and PI3K proteins in the cell, and as the concentration increases, the efficiency of ERα and PI3K degradation increases, which reflects the dose dependence of protein degradation in this model. It also indirectly shows that with the increase of plasmid concentration, the expression and translation of p-PROTAC-2 in MCF-7 cells gradually increases, thereby causing an increase in degradation efficiency. At a concentration of 2μg / mL, the degradation efficiency can reach 85% and 65%, respectively, further proving the feasibility of the DNA-PROTAC targeted protein degradation model to expand the therapeutic module strategy.

[0171] (2) D-PROTAC-2 induces degradation of ERα and PI3K proteins in a time-dependent manner

[0172] In the time gradient verification part of this embodiment, 2 μg / mL of PROTAC-2 and 4 μL / mL of transfection reagent were used to treat MCF-7 cells for 6h, 12h, 24h, and 48h respectively. After the same concentration of D-PROTAC acted on MCF-7 cells at different times, the present invention performed western blot detection and grayscale analysis on the drug-treated MCF-7 cells ( Fig.16 ), the results showed that under the action of D-PROTAC-2 at different times, the protein levels of ERα and PI3K in MCF-7 cells continued to decrease with time, and the degradation efficiency at 48h was 92% and 89%, respectively, showing a good time gradient. The reason may be that the increase in the plasmid action time has caused the p-PROTAC-2 expressed by the plasmid to accumulate to a certain extent, and the effective concentration has increased, thereby acting on more target proteins, and then triggering the effective degradation of the two target proteins. Compared with the difference in protein degradation between 12h and 24h, the protein degradation between 24h and 48h is not much different, which also indirectly proves that the plasmid gene can basically reach an effective concentration after 24h of cell action, which provides certain time selection support for subsequent drug action experiments and other gene therapy methods.

[0173] (3) D-PROTAC-2 relies on cathepsin B-specific cleavage to achieve selective degradation

[0174] During the D-PROTAC-2 sequence design stage, the present invention introduced a cathepsin B sequence between two targeted degradation sequences. Previous studies have shown that this sequence can be recognized and cleaved by highly expressed cathepsin B in tumor cells. Therefore, the two targeted polypeptides can be separated to avoid the influence of protein steric hindrance on the degradation effect.

[0175] In order to further verify the specific recognition and cleavage of the sequence, and its effect on steric hindrance and protein degradation, this example uses a cathepsin B inhibitor for verification. In the presence of a cathepsin B inhibitor, the activity of cathepsin B is inhibited, so that it cannot specifically recognize and cleave the sequence GLFGG, and the two targeted degradation sequences cannot be separated.

[0176] In this example, three parallel groups were set up: the control group was not treated with drugs, and only DMSO and transfection reagents were added in the same amount as the experimental group; experimental group 1 was treated with 2μg / mL D-PROTAC-2 for 24h; experimental group 2 was treated with 2μg / mL D-PROTAC-2 and 10μM cathepsin B inhibitor for 24h. After 24h of drug action, the experimental cells were subjected to protein immunoblotting analysis.

[0177] The results of Western blot analysis were as follows Fig.17As shown, DMSO and transfection reagents have no effect on ERα and PI3K protein levels, D-PROTAC-2 can degrade ERα and PI3K protein levels to very low levels, and after adding cathepsin B inhibitors, the ubiquitin-proteasome pathway is inhibited and the degradation effect of D-PROTAC-2 is weakened. The above results prove that cathepsin B in D-PROTAC-2 can achieve the function of specific recognition.

[0178] Example 6: D-PROTAC-2 has a significant inhibitory effect on the proliferation of MCF-7 cells

[0179] (1) D-PROTAC-2 induces tumor cell apoptosis:

[0180] In this example, six concentration gradients of 0, 0.5, 1, 1.5, 2, and 4 μg / mL were set, the action time was 24 h, and three parallel measurements were performed. The analysis results are as follows: Fig.18 As shown. The results of the MTT assay showed that with increasing concentration, the inhibitory ability of D-PROTAC-2 on MCF-7 cells increased, and at a 72h action time, a very low D-PROTAC-2 concentration could cause strong cytotoxicity, which also indirectly proved that D-PROTAC-2 was time-dependent when it took effect. At a 24h action time, 4μg / mL of D-PROATC-2 molecules can cause nearly 60% of cell apoptosis, and the IC50 value is about 2.3μg / mL. At a 72h action time, low concentrations of drug molecules also showed good tumor suppression effects, with a 35% cell apoptosis rate and an IC50 of approximately 1.65μg / mL.

[0181] This example further detected intracellular apoptosis-related factors. As the concentration of D-PROTAC-2 increased, the levels of intracellular apoptosis-related factors such as Cleaved-Caspase-3 and Cleaved-PARP proteins increased, which further verified the effect of D-PROTAC-2 on tumor cell apoptosis.

[0182] In this example, a cell apoptosis detection experiment was performed to detect the effect of D-PROTAC-2 on cell cytotoxicity.

[0183] After treating MCF-7 cells with 0, 0.5, 1, and 2 μg / mL D-PROTAC-2 for 24 h, the cells were collected and stained with Annexin V and propidium iodide (PI). Fig.19The results showed that the apoptosis rate was 53.89% at 2 μg / mL D-PROTAC-2, while the apoptosis rate was only 19.1% at 1 μg / mL D-PROTAC-2, and cell apoptosis showed a clear concentration gradient. In addition, as the concentration of D-PROTAC-2 increased, the proportion of cells with early apoptosis characteristics increased significantly.

[0184] (2) D-PROTAC-2 causes tumor cell cycle arrest:

[0185] PI staining and flow cytometric analysis of 0, 0.5, 1, and 2 μg / mL D-PROTAC-2 treatment for 24 h verified the effect on the cell cycle. As the concentration of D-PROTAC-2 increased, more MCF-7 cells were arrested in the G0 / G1 phase, while the proportions of the S phase and G2 / M phase decreased with the concentration, indicating that D-PROTAC-2 inhibited proliferation by inducing G0 / G1 phase arrest of tumor cells ( Fig. 20 ).

[0186] Example 7: D-PROTAC-2 has no effect on proliferation and apoptosis of normal breast epithelial cells MCF-10A

[0187] To test the specific selectivity of D-PROTAC-2, this example detected the effects of D-PROTAC-2 at different concentrations on the proliferation and apoptosis of normal breast epithelial cells MCF-10A.

[0188] First, the effect on cell cycle. After 0, 0.5, 1, 1.5, and 2 μg / mL D-PROTAC-2 acted on human breast epithelial cells MCF-10A for 24 hours, the cell cycle was detected by flow cytometry by PI staining. The results are as follows: Fig.21 As shown in (a), there is no obvious cycle arrest effect, proving that D-PROTAC-2 has no obvious cycle effect on normal cells. Then, cell apoptosis and cell proliferation inhibition assays were performed to detect the effect of D-PROTAC-2 on the cytotoxicity of normal cells. Fig.21 As shown in (b), after treating MCF-7 cells with 0, 1, and 2 μg / mL D-PROTAC-2 for 24 hours, the cells were collected and stained with Annexin V and propidium iodide (PI). Flow cytometry results showed that different concentrations of D-PROTAC-2 had no effect on the state of cell apoptosis, and there was no significant change in the proportion of early and late apoptosis. Fig.21 (c) shows no significant inhibition of cell proliferation.

[0189] Example 8: D-PROTAC-1 inhibits tumor growth in vivo

[0190] 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 in situ injection of AAV-encapsulated PROTAC group (AAV-PROTAC) (n=5); ② in situ injection of PBS control group (n=5); ③ in situ injection of AAV control group (AAV) (n=5).

[0191] (1) Subcutaneous tumor: 4T1 cells were digested and centrifuged, resuspended in PBS, and counted. 1×10 7 The tumor volume was measured every 2 to 3 days using a vernier caliper.

[0192] (2) Administration: Select tumors with a volume of approximately 100 ± 50 mm 3 mice were injected orthotopically into 5-week-old BALB / c mice (1×10 12 The tumor volume was measured with a vernier caliper every 2 to 3 days.

[0193] (3) Tumor detachment: The tumor volume of mice in the control group reached 1500mm 3 The tumor volume was measured and photographed.

[0194] The results are as follows Fig. 22 As shown, the mice in the drug-treated group showed a stable body weight consistent with that of the control group throughout the experiment. In addition, the tumor volumes of the PBS group and the AAV control group were basically the same, and the tumor volume of the experimental group was significantly reduced, proving that D-PROTAC-1 has low toxicity and effectively inhibits tumor growth in vivo.

[0195] Example 9: D-PROTAC-1 has a significant inhibitory effect on the proliferation of other tumor cells

[0196] This example sets six concentration gradients of 0, 0.5, 1, 1.5, 2, and 4 μg / mL, with an action time of 72 h. The analysis results are as follows Fig.23 The results of the MTT assay showed that as the concentration increased, D-PROTAC-2's inhibitory ability 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 inhibitory effect on a variety of tumor cells.

[0197] Example 10: D-PROTAC-3 induces degradation of RAD51 protein

[0198] (1) D-PROTAC-3 induces degradation of RAD51 protein in a concentration-dependent manner

[0199] The present invention treated MDA-MB-231 cells with different concentrations of D-PROTAC-3 at 0, 0.5, 1, 1.5, and 2 μg / mL for 24 hours. 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.

[0200] Such as protein immunoblotting and grayscale analysis ( Fig.24 ) The results showed that after different concentrations of D-PROTAC-3 acted on MDA-MB-231 cells, the efficiency of degrading RAD51 protein increased with the increase of concentration, and the degradation efficiency could reach more than 50%.

[0201] (2) D-PROTAC-3 induces degradation of RAD51 protein in a time-dependent manner

[0202] The present invention also explored the time gradient of D-PROTAC-3-mediated RAD51 protein degradation. The experiment used 2 μg / mL D-PROTAC-3 to treat cells for 24h, 48h, and 72h, respectively, and each group of transfection reagents was jet prime.

[0203] Then, each group of experiments was subjected to protein immunoblotting and grayscale analysis ( Fig.25 ). The grayscale analysis quantification results showed that as the D-PROTAC-3 molecule action time increased, the level of RAD51 protein in MDA-MB-231 breast cancer cells continued to decrease. The results showed that D-PROTAC-3 not only has the advantage of targeting tumors, but also has the therapeutic ability to reduce the level of target protein.

[0204] (3) MG132 inhibition experiment

[0205] The culture medium containing 10 μM MG132 was added to inhibit the ubiquitin degradation pathway of cellular proteins. The cells were collected at different time points (0, 3, 6, and 12 hours), and then the total protein was extracted and analyzed by western blotting. Fig.26 As shown, after inhibiting the ubiquitin proteasome degradation pathway of cellular proteins, RAD51 protein was not degraded, which verified that RAD51 protein degradation was achieved through the PROTAC pathway mediated by D-PROTAC-3.

[0206] Example 11: Degradation effect of D-PROTAC-3 in MDA-MB-436 cells

[0207] The present invention also verifies that D-PROTAC-3 induces degradation of RAD51 protein in MDA-MB-436 cells. MDA-MB-436 cells were treated with 2 μg / mL of D-PROTAC-3 for 72 hours. Fig. 27As shown. It can be seen that in MDA-MB-436 cells, D-PROTAC-3 can also significantly induce the degradation of RAD51 protein.

[0208] Example 12: D-PROTAC-3 inhibits tumor cell proliferation

[0209] 96-well plates were plated with 3000-4000 MDA-MB-231 cells per well in a volume of 100 μL. 24h, 48h, 72h, and 96h after transfection with 2 μg / mL RAD51-PROTAC plasmid, 10 μL CCK8 reagent was added. After 1-4h of reaction, the OD value was measured at a wavelength of 450nm. The results are shown in Fig.28 As shown. It can be seen that D-PROTAC-3 can significantly inhibit the proliferation of tumor cells.

[0210] Example 13: D-PROTAC-4 induces degradation of USP30 protein

[0211] (1) D-PROTAC-4 induces USP30 protein degradation in a concentration-dependent manner

[0212] The present invention treated HepG2 cells and HCT116 cells with D-PROTAC-4 at concentrations of 0, 0.5, 1, and 2 μg / mL for 24 hours, respectively. Then, the relative protein level of USP30 was analyzed by western blotting, with the expression level of GAPDH protein in each group as a reference.

[0213] Such as protein immunoblotting and grayscale analysis ( Fig.29 ) The results showed that after different concentrations of D-PROTAC-4 acted on HepG2 cells and HCT116 cells, the efficiency of degrading USP30 protein increased with the increase of concentration.

[0214] (2) D-PROTAC-4 induces USP30 protein degradation in a time-dependent manner

[0215] The present invention also explored the time gradient of D-PROTAC-4-mediated USP30 protein degradation. The experiment used 2 μg / mL of D-PROTAC-4 to treat cells for 24h, 48h, and 72h, respectively.

[0216] Then, each group of experiments was subjected to protein immunoblotting and grayscale analysis ( Fig.30 ). The grayscale analysis quantification results showed that as the D-PROTAC-4 molecule action time increased, the USP30 protein level in HepG2 cells and HCT116 cells continued to decrease. The results showed that D-PROTAC-4 has the therapeutic ability to reduce the level of the target protein.

[0217] (3) MG132 inhibition experiment

[0218] The culture medium containing 10 μM MG132 was added to inhibit the ubiquitin degradation pathway of cellular proteins. HepG2 cells were collected at different time points (0, 3, 6, and 12 hours), and then total protein was extracted and analyzed by western blotting. Fig.31 As shown, after inhibiting the ubiquitin proteasome degradation pathway of cellular proteins, USP30 protein was not degraded, which verified that USP30 protein degradation was achieved through the PROTAC pathway mediated by D-PROTAC-4.

[0219] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent 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 target protein, characterized in that: The DNA molecule includes at least one group of D-PROTAC units, each group of D-PROTAC units includes 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 connecting peptide is optionally arranged between adjacent first DNA segments and second DNA segments.

2. The DNA molecule for targeted degradation of target protein according to claim 1, characterized in that: The DNA molecule also includes a promoter, which is 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 / hyperoxygen 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 target protein according to claim 2, characterized in that: The tumor organ, tissue or cell specific promoter is selected from at least one of the following: human telomerase reverse transcriptase (hTERT) promoter, apoptosis inhibitory protein (Survivin) promoter, alpha-fetoprotein (AFP) promoter, osteocalcin (OC) promoter, neuron-specific enolase (NSE) promoter, carcinoembryonic antigen (CEA) promoter, cyclooxygenase-2 (COX-2) promoter, prostate-specific antigen (PSA) promoter, E2F-1 promoter, cholecystokinin promoter, C-erbB2 / neu oncogene promoter, CXCR4 promoter, HE4 promoter, type II hexokinase promoter, L-reticulin promoter, MUC1 promoter, TRP1 promoter, tyrosinase promoter, hypoxia-inducible factor-1 promoter, tumor antigen mucin promoter, secretory leukocyte protease inhibitor promoter, survivin promoter and MetR specific promoter.

4. The DNA molecule for targeted degradation of target protein according to claim 1, characterized in that: The ubiquitin ligase targeting peptide is 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; Preferably, the ubiquitin ligase targeting peptide is a VHL E3 ubiquitin ligase targeting peptide, and its amino acid sequence is shown in SEQ ID NO:

1.

5. The DNA molecule for targeted degradation of target protein according to claim 1, characterized in that: The target protein is at least one of B lymphocyte tumor protein, estrogen receptor α, intracellular phosphatidylinositol kinase, RAD51 protein, USP30 protein, estrogen receptor 1, androgen receptor, BTK tyrosine protein kinase, interleukin-1 receptor associated kinase 4, RACα-serine / threonine protein kinase, RACβ serine / threonine protein kinase, bromoprotein 4, cyclin-dependent kinase 9, receptor-interacting serine / threonine protein kinase 1, transcription intermediary factor 1-α, epidermal growth factor receptor, hepatocyte growth factor receptor and mast / stem cell growth factor receptor.

6. The DNA molecule for targeted degradation of target protein according to claim 5, characterized in that: When the target protein is B lymphocyte tumor protein, estrogen receptor α, intracellular phosphatidylinositol kinase RAD51 protein and USP30 protein, the sequences of the corresponding target protein targeting peptides are shown in SEQ ID NOs: 3 to 7, respectively.

7. The DNA molecule for targeted degradation of target protein according to claim 1, characterized in that: The number of the D-PROTAC units is 1-6 groups, and a coding sequence for a controllable cleavage peptide chain is provided between adjacent D-PROTAC units; the controllable cleavage peptide chain can achieve peptide chain cleavage under controllable conditions so that the translation products of each D-PROTAC unit exist independently; Preferably, the controllably cleavable peptide chain is an intracellular protease recognition site or a self-cleaving peptide, and the intracellular protease recognition site is preferably a disease cell-specific highly expressed protease recognition site, more preferably a cathepsin B recognition site.

8. The DNA molecule for targeted degradation of target protein according to claim 7, characterized in that: The DNA molecule comprises a set of D-PROTAC units, the sequence of which is shown in SEQ ID NO: 8; or The DNA molecule comprises a set of D-PROTAC units, the sequence of which is shown in SEQ ID NO: 9; or The DNA molecule comprises a set of D-PROTAC units, the sequence of which is shown in SEQ ID NO: 10; or The DNA molecule includes two groups of D-PROTAC units, and its sequence is shown in SEQ ID NO:

11.

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

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

11. The biomaterial according to claim 10, characterized in that The recombinant vector also includes functional elements, which are 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. Any of the following uses of the DNA molecule according to any one of claims 1 to 9, or the biomaterial according to claim 10 or 11: M1) preparing a product for treating and / or preventing a disease / lesion mediated by the target protein; M2) treating and / or preventing diseases / lesions mediated by the target protein; M3) preparing products for inhibiting cell proliferation; M4) inhibits cell proliferation; M5) preparing products that promote cell apoptosis; M6) promotes cell apoptosis; The disease mediated by the target protein is preferably cancer, immune disease, metabolic disease, neurodegenerative disease or inflammatory disease; The cell is preferably a cancer cell; The cancer is preferably leukemia, lymphoma, lung cancer, breast cancer, ovarian cancer, cervical cancer, human brain glioma, melanoma, glioblastoma, nasopharyngeal carcinoma, liver cancer, brain cancer, pancreatic cancer, uterine cancer, testicular cancer, skin cancer, stomach cancer, colon cancer, bladder cancer or rectal cancer; The product is preferably a vaccine or a medicine.

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