PROTAC capable of achieving efficient degradation through phase separation and delivery mode of PROTAC

Abstract

The invention discloses PROTAC capable of efficiently degrading a target protein and a preparation and delivery method of the PROTAC, a phase separation sequence and a designed double-targeting molecule are fused to respectively target the target protein and a proteasome, and phase separation of the PROTAC and the target protein is realized, so that efficient degradation of the target protein is realized. In addition, the delivery of PROTAC is also realized by encapsulating mRNA with LNP, and the target protein can also be efficiently degraded in cells, so that a way is also provided for the delivery of peptide or protein PROTAC into the human body.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, and specifically, to a system that can undergo phase separation to achieve efficient degradation of target proteins, and a preparation method and application thereof. Background Art

[0002] Protein-protein or protein-nucleic acid complexes will spontaneously separate into two phases under specific conditions, forming a high-concentration phase and a low-concentration phase, and this process is called liquid-liquid phase separation (LLPS) [Reference 1]. LLPS is involved in biological processes such as the formation of membraneless organelles in cells, RNA metabolism, DNA damage signal complexes, the cytoskeleton, and many other supramolecular assemblies. The occurrence of phase separation is mediated by weak interactions between proteins-proteins or proteins-nucleic acids. For example, the phase separation of FUS is mediated by π-π interactions and π-cation interactions between residues in its intrinsically disordered region IDR. Phase separation can provide a local high concentration, so it is used to promote enzymatic reactions, and in addition, it is also used for the delivery of macromolecules in cells [Reference 2].

[0003] The degradation of most proteins in cells depends on the proteasome. After a protein is ubiquitinated and labeled by an E3 ubiquitin ligase, it is recognized by the proteasome and then degraded. The occurrence of some diseases is related to protein imbalance, and the specific degradation of these proteins has become a feasible strategy for treating these diseases. Proteolytic targeting chimera (PROTAC) [Reference 3] is a dual-targeting molecule with a ligand targeting the target protein at one end and a ligand targeting the E3 ligase at the other end, so that the target protein can be targeted to the ubiquitin-proteasome degradation pathway to achieve the specific degradation of the target protein. With the continuous progress of PROTAC technology, LYTAC [Reference 4], AUTAC [Reference 5], etc. that degrade target proteins according to the lysosomal degradation pathway have been developed. It has become a feasible strategy for drug development, and small molecule ligands of available E3 ligases have also been continuously developed, such as thalidomide.

[0004] Peptide PROTACs do not depend on the "binding pocket" of the target protein, so the selectivity of the targeted protein is stronger. However, the poor cell permeability of peptide PROTACs has affected their further development [Reference 6]. Since mRNA can express proteins without entering the nucleus, it has become a transfection strategy superior to DNA. Due to the progress of liposome encapsulation technology, the mRNA-LNP technology has been widely used in the development of cancer immunotherapy, cancer and infectious disease vaccines, and is a safe, non-inflammatory, and non-integrative delivery method [Reference 7].

[0005] References:

[0006] [1] SCHNEIDER N, WIELAND F G, KONG D, et al. Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice[J]. Sci Adv, 2021, 7(1): eabd3568.

[0007] [2] SUN Y, LAU S Y, LIM Z W, et al. Phase-separating peptides for direct cytosolic delivery and redox-activated release of macromolecular therapeutics[J]. Nat Chem, 2022, 14(3): 274-283.

[0008] [3] Wang Ruifeng, Yang Bowen, Zhao Dongmei, et al. Research progress of proteolysis-targeting chimeras (PROTAC)[J]. Chinese Journal of Medicinal Chemistry, 2019, 29(3): 7.

[0009] [4] BANIK S M, PEDRAM K, WISNOVSKY S, et al. Lysosome-targeting chimaeras for degradation of extracellular proteins[J]. Nature, 2020, 584(7820): 291-297.

[0010] [5] TAKAHASHI D, MORIYAMA J, NAKAMURA T, et al. AUTACs: Cargo-Specific Degraders Using Selective Autophagy[J]. Mol Cell, 2019, 76(5): 797-810.e710.

[0011] [6]JIN J,WU Y,CHEN J,et al.The peptide PROTAC modality:a novel strategy for targeted protein ubiquitination[J].Theranostics,2020,10(22):10141-10153.

[0012] [7]RIZVI F,EVERTON E,SMITH A R,et al.Murine liver repair via transient activation of regenerative pathways in hepatocytes using lipid nanoparticle-complexed nucleoside-modified mRNA[J].Nat Commun,2021,12(1):613. Summary of the Invention

[0013] In the embodiments of the present invention, a polypeptide with phase separation ability is introduced into the fusion protein of a proteolytic targeting chimera (PROTAC) to form a phase separation enhanced proteolytic targeting chimera (PSETAC). One end of the fusion protein targets the target protein, and the other end targets the proteasome. The fusion protein and the target protein are co-phase separated by the inserted phase separation polypeptide, realizing the recruitment and local concentration of the target protein, and then targeting the proteasome to achieve the purpose of efficient degradation.

[0014] The present invention also provides a technical solution in which the fusion protein mRNA is encapsulated by a lipid nanoparticle (LNP), and the effect of delivering the fusion protein into cells is achieved through the LNP, overcoming the disadvantage of poor cell permeability of peptide PROTAC.

[0015] Based on the above work, the inventors completed this application and proposed the following technical solutions.

[0016] The first aspect of the present invention is to provide the use of a polypeptide with phase separation function in the preparation of a proteolytic targeting chimera.

[0017] According to the present invention, the proteolytic targeting chimera is a hetero-bifunctional molecule, one end of which is a part targeting the target protein, and the other end is a part targeting the proteasome, and the polypeptide with phase separation function connects the above two parts.

[0018] As described above, due to the presence of a polypeptide with phase separation function, the formed proteasome-targeting chimera can undergo phase separation behavior in the system, forming a local high concentration of the proteasome-targeting chimera in the system, achieving the recruitment and local concentration of the target protein, and improving the degradation efficiency of the proteasome.

[0019] Therefore, the present invention also provides the use of a polypeptide with phase separation function in improving the degradation efficiency of proteasome-targeting chimeras.

[0020] According to the present invention, the part targeting the target protein is located at the N-terminus or C-terminus of the polypeptide with phase separation function, while at the same time, the part targeting the proteasome is correspondingly located at the C-terminus or N-terminus of the polypeptide with phase separation function.

[0021] According to the present invention, between the part targeting the target protein and the polypeptide with phase separation function, and / or, between the part targeting the proteasome and the polypeptide with phase separation function, there may or may not be a linker structure.

[0022] According to the present invention, various domains or polypeptides that can cause phase separation of heterologous proteins can be used in the present invention, including but not limited to IDR region fragments of naturally occurring self-assembling proteins, such as the LC domain of the FUS protein, the IDR region of hnRNPA1 (MASASSSQRGRSGSG

[0023] NFGGGRGGGFGGNDNFGRGGNFSGRGGFGGSRGGGGYGGSGDGYNGFGNDGSNFGGGGSY NDFGNYNNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGYGGSSSSSSYGSGRRF), the IDR domain of LAF-1; or, artificially synthesized phase separation peptides, such as P5 (SGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSRGG), pepHBP (GHGVYGHGVYGHGPYGHGPYGHGLYW), etc.

[0024] In one embodiment of the present invention, the polypeptide with phase separation function is the IDR domain of LAF-1 or its homolog. In a specific embodiment of the present invention, the amino acid sequence of this polypeptide has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No.2. In a specific embodiment of the present invention, the amino acid sequence of this polypeptide is as shown in SEQ ID No.2.

[0025] In one embodiment of the present invention, the polypeptide with phase separation function is the LC domain of the FUS protein or its homolog. In a specific embodiment of the present invention, the amino acid sequence of this polypeptide has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No.10. In a specific embodiment of the present invention, the amino acid sequence of this polypeptide is as shown in SEQ ID No.10.

[0026] Various misexpressed, mutated or overactivated proteins that can cause diseases can be used as the target proteins of the present invention, including but not limited to mouse double minute 2 homolog (MDM2), MetAP-2, Aβ, TDP-43, α-synuclein, tau protein, androgen receptor, bromodomain and extra terminal (BET) protein family, c-MYC, estrogen receptor α (ERα), cyclin-dependent kinases (CDK), BCR-ABL fusion protein (breakpoint cluster region-cabl, BCR-ABL), B-cell lymphoma 6 protein (BCL6), anaplastic lymphoma kinase (ALK), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor-2 (HER2), c-mesenchymal epithelial transition factor (c-Met), Brutons tyrosine kinase (BTK), etc.

[0027] In some embodiments of the present invention, the target protein is Aβ.

[0028] In some embodiments of the present invention, the target protein is α-synuclein.

[0029] In some embodiments of the present invention, the target protein is MDM2.

[0030] According to the present invention, the portion targeting the target protein can be a small molecule compound or a polypeptide. For example: the small molecule compound targeting ATR disclosed in CN117024413A, the small molecule compound targeting α-synuclein disclosed in CN115650957A, the ligand of estrogen receptor disclosed in CN115835886A, the polypeptide targeting α-synuclein, etc.

[0031] In some embodiments of the present invention, the target protein is Aβ. The ligands or target molecule sequences targeting Aβ include but are not limited to Aβ antibodies, the affinity peptide VDNKFNKEMASAGGEIVYLPNLNPDQLCAFIHSLHDDPSQSANLLAEAKKLNDAQAPK of Aβ and its homologous sequences.

[0032] In some embodiments of the present invention, the target protein is α-synuclein. The ligands or target molecule sequences targeting α-synuclein, for example, are the affinity peptide VDNKFNKEMASADGEIFYLPNLNPDQLCAFFHSVHDDPSQSANLLA EAKKLNDAQAPK of α-synuclein and its homologous sequences. In some other embodiments of the present invention, the polypeptide targeting α-synuclein is the fragment GVLYVGSKTR of β-synuclein or its homologues, etc. In one embodiment of the present invention, the amino acid sequence of the polypeptide has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with GVLYVGSKTR. In a specific embodiment of the present invention, the amino acid sequence of the polypeptide is GVLYVGSKTR.

[0033] In some embodiments of the present invention, the target protein is MDM2. Ligands or target molecule sequences targeting MDM2 include but are not limited to small molecule inhibitors of MDM2 such as Nutlin-3a, RG7112, Idasanutlin, SAR405838, Milademetan, APG-115, AMG232, NVP-CGM097, Siremadlin, and MK-8242. In some other embodiments of the present invention, the polypeptide targeting MDM2 is a fragment of the p53 protein or its homolog. In one embodiment of the present invention, the amino acid sequence of the polypeptide has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No.9. In one specific embodiment of the present invention, the amino acid sequence of the polypeptide is as shown in SEQ ID No.9.

[0034] According to the present invention, the part targeting the proteasome is a small molecule compound or a polypeptide, for example: small molecule compounds or polypeptides targeting the ubiquitin ligase E3, such as: cereblon, lenalidomide, pomalidomide, iberdomide, (S,R,S)-AHPC, thalidomide, VH-298, CC-122, CC-885, E3 ligase ligand 8, TD-106, VL285, VH032, VH101, VH298, VHL ligand 4, VHL ligand 7, VHL-2 ligand 3, E3 ligase ligand 3, E3 ligase ligand 2, BC-1215, the C-terminal of mouse ornithine decarboxylase (cODC), PEST sequences (a large class of sequences rich in proline, glutamate, serine, and threonine), unstable N-termini containing arginine, tyrosine, phenylalanine (such as: HKSGAWKLPVSLVK), etc.

[0035] In some embodiments of the present invention, the part targeting the proteasome is a polypeptide, and the polypeptide is the C-terminal of mouse ornithine decarboxylase (cODC) or its homolog. In one specific embodiment of the present invention, the amino acid sequence of the polypeptide has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No.3. In one specific embodiment of the present invention, the amino acid sequence of the polypeptide is as shown in SEQ ID No.3.

[0036] According to the present invention, the linker structure can be a commonly used linker polypeptide in the art for connecting two polypeptides, such as a flexible linker, including but not limited to GnSm linker sequence, (Gly)n linker sequence, DDK linker sequence, or a combination of these linker sequences. The linker structure can also be a linker structure for small molecule PROTACs in the art, such as a polyethylene glycol fragment, etc.

[0037] The present invention further provides a fusion protein, which is a phase separation enhanced proteasome targeting chimera (PSETAC) with heterobifunctional targeting.

[0038] The fusion protein comprises three parts: polypeptide A targeting a target protein, phase separation polypeptide B, and polypeptide C targeting the proteasome. The N-terminus of polypeptide B is polypeptide A and the C-terminus is polypeptide C, or the N-terminus of polypeptide B is polypeptide C and the C-terminus is polypeptide A. There may or may not be a linker sequence between polypeptide A and polypeptide B, and / or between polypeptide B and polypeptide C.

[0039] According to the present invention, polypeptide A is an amino acid sequence capable of specifically binding to a target protein in the cell. Any protein whose misexpression, mutation, or overactivation can cause diseases can be used as the target protein of the present invention, as listed above.

[0040] Polypeptide A can be, for example, an antibody, and the antibody can bind to the target protein. More preferably, for example, a monoclonal antibody or its recognition fragment or polypeptide. Those skilled in the art can understand that the antibody is from a mammal, preferably human, mouse, rabbit, goat, etc. Polypeptide A can also be, for example, a ligand of the target protein or a fragment of the ligand.

[0041] In some embodiments of the present invention, the target protein is eGFP, and polypeptide A is an amino acid sequence capable of binding to eGFP. In one embodiment of the present invention, this polypeptide A is GFP nanobody or its homolog. In one embodiment of the present invention, the amino acid sequence of this polypeptide A has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No.1. In a specific embodiment of the present invention, the amino acid sequence of this polypeptide A is as shown in SEQ ID No.1.

[0042] In some embodiments of the present invention, the target protein is α-synuclein, and the polypeptide A is an amino acid sequence capable of binding to α-synuclein. In one embodiment of the present invention, the polypeptide A is the fragment GVLYVGSKTR of β-synuclein or its homolog. In one embodiment of the present invention, the amino acid sequence of the polypeptide A has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with GVLYVGSKTR. In a specific embodiment of the present invention, the amino acid sequence of the polypeptide A is GVLYVGSKTR.

[0043] In some embodiments of the present invention, the target protein is MDM2, and the polypeptide A is an amino acid sequence capable of binding to MDM2. In one embodiment of the present invention, the polypeptide A is a fragment of the p53 protein or its homolog. In one embodiment of the present invention, the amino acid sequence of the polypeptide A has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No.9. In a specific embodiment of the present invention, the amino acid sequence of the polypeptide A is as shown in SEQ ID No.9.

[0044] According to the present invention, various domains or polypeptides capable of causing phase separation of heterologous proteins can be used as polypeptide B, as listed above.

[0045] In one embodiment of the present invention, the polypeptide B is the IDR domain of LAF-1 or its homolog. In a specific embodiment of the present invention, the amino acid sequence of the polypeptide B has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No.2. In a specific embodiment of the present invention, the amino acid sequence of the polypeptide B is as shown in SEQ ID No.2.

[0046] In one embodiment of the present invention, the polypeptide B is the LC domain of the FUS protein or its homolog. In a specific embodiment of the present invention, the amino acid sequence of the polypeptide B has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No.10. In a specific embodiment of the present invention, the amino acid sequence of the polypeptide B is as shown in SEQ ID No.10.

[0047] According to the present invention, the polypeptide C is an amino acid sequence that can be recognized by the proteasome, including but not limited to an amino acid sequence that can target the ubiquitin ligase E3 and ubiquitinate the target protein, as listed above.

[0048] In one embodiment of the present invention, the polypeptide C is the C-terminus of mouse ornithine decarboxylase (cODC) or its homolog. In a specific embodiment of the present invention, the amino acid sequence of the polypeptide C has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No.3. In a specific embodiment of the present invention, the amino acid sequence of the polypeptide C is as shown in SEQ ID No.3.

[0049] According to the present invention, polypeptide A, polypeptide B and polypeptide C can be directly coupled or coupled through a linker sequence. The linker can be a commonly used linker polypeptide in the art, such as a flexible linker, including but not limited to the GnSm linker sequence, (Gly)n linker sequence, DDK linker sequence, or a combination of these linker sequences.

[0050] In some embodiments of the present invention, the amino acid sequence of the linker is GGGSGS.

[0051] According to the present invention, in some embodiments of the present invention, the target protein of the fusion protein is MDM2, the polypeptide A that can be recognized by MDM2 and specifically bind thereto is a p53 fragment or its homolog, and the polypeptide B is the IDR region of the FUS protein or its homolog; the polypeptide C is the C-terminal cODC of mouse ornithine decarboxylase or its homolog. In one embodiment of the present invention, the N-terminus of polypeptide B is polypeptide A and the C-terminus is polypeptide C, polypeptide A is directly coupled to polypeptide B or coupled through a linker sequence, and polypeptide C is directly coupled to polypeptide B or coupled through a linker sequence. In one embodiment of the present invention, the N-terminus of polypeptide B is polypeptide C and the C-terminus is polypeptide A, polypeptide A is directly coupled to polypeptide B or coupled through a linker sequence, and polypeptide C is directly coupled to polypeptide B or coupled through a linker sequence. In one embodiment of the present invention, the N-terminus of polypeptide B is polypeptide A and the C-terminus is polypeptide C, polypeptide A is coupled to polypeptide B through a linker sequence, and polypeptide C is directly coupled to polypeptide B.

[0052] In a preferred embodiment, the amino acid sequence of polypeptide A has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No.9. In a specific embodiment of the present invention, the amino acid sequence of this polypeptide A is as shown in SEQ ID No.9.

[0053] In a preferred embodiment, the amino acid sequence of polypeptide B has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No.10. In a specific embodiment of the present invention, the amino acid sequence of this polypeptide B is as shown in SEQ ID No.10.

[0054] In a preferred embodiment, the amino acid sequence of polypeptide C has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No.3. In a specific embodiment of the present invention, the amino acid sequence of this polypeptide C is as shown in SEQ ID No.3.

[0055] In one embodiment of the present invention, the amino acid sequence of the fusion protein has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No. 8. In a specific embodiment of the present invention, the amino acid sequence of the fusion protein is as shown in SEQ ID No. 8.

[0056] According to the present invention, in some embodiments of the present invention, the target protein of the fusion protein is α-synuclein, the polypeptide A that can be recognized by α-synuclein and specifically bind to it is a fragment of β-synuclein or its homolog, and the polypeptide B is the IDR domain of LAF-1 or its homolog; the polypeptide C is the C-terminal cODC of mouse ornithine decarboxylase or its homolog. In one embodiment of the present invention, the N-terminus of polypeptide B is polypeptide A and the C-terminus is polypeptide C, polypeptide A is directly coupled to polypeptide B or through a linker sequence, and polypeptide C is directly coupled to polypeptide B or through a linker sequence. In one embodiment of the present invention, the N-terminus of polypeptide B is polypeptide C and the C-terminus is polypeptide A, polypeptide A is directly coupled to polypeptide B or through a linker sequence, and polypeptide C is directly coupled to polypeptide B or through a linker sequence. In one embodiment of the present invention, the N-terminus of polypeptide B is polypeptide A and the C-terminus is polypeptide C, polypeptide A is directly coupled to polypeptide B, and polypeptide C is directly coupled to polypeptide B.

[0057] In a preferred embodiment, the amino acid sequence of polypeptide A has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with GVLYVGSKTR. In a specific embodiment of the present invention, the amino acid sequence of this polypeptide A is GVLYVGSKTR.

[0058] In a preferred embodiment, the amino acid sequence of polypeptide B has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No. 2. In a specific embodiment of the present invention, the amino acid sequence of this polypeptide B is as shown in SEQ ID No. 2.

[0059] In a preferred embodiment, the amino acid sequence of polypeptide C has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No. 3. In a specific embodiment of the present invention, the amino acid sequence of polypeptide C is as shown in SEQ ID No. 3.

[0060] In one embodiment of the present invention, the amino acid sequence of the fusion protein has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No. 6. In a specific embodiment of the present invention, the amino acid sequence of the fusion protein is as shown in SEQ ID No. 6.

[0061] The second aspect of the present invention is to provide a polynucleotide encoding the fusion protein described in the first aspect.

[0062] According to the present invention, the polynucleotide is DNA.

[0063] According to the present invention, the polynucleotide is mRNA. The mRNA contains an open reading frame (ORF) encoding the fusion protein described in the first aspect. The mRNA may further contain a 5' cap structure, 5' UTR, 3' UTR and poly-A tail, etc. from the 5' end to the 3' end.

[0064] In one embodiment of the present invention, the mRNA is the mRNA encoding the fusion protein with the amino acid sequence shown in SEQ ID No. 8 or SEQ ID No. 6. In a specific embodiment of the present invention, the ORF of the mRNA has a nucleotide sequence with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the nucleotide sequence shown in SEQ ID No. 13. In a specific embodiment of the present invention, the nucleotide sequence of the ORF of the mRNA is as shown in SEQ ID No. 13.

[0065] The third aspect of the present invention is to provide an expression vector, which contains the polynucleotide described in the second aspect of the present invention.

[0066] According to the present invention, the expression vector includes but is not limited to plasmid vectors or viral vectors, such as: pcDNA3.1 plasmid, pDsRed N1 plasmid, pCMV-NEO-BAN plasmid, pMRX-puro plasmid, pLVX-IRES-zsGreeen1, pLVX-IRES-puromycin, pLVX-P2A / T2A-zsGreeen1, pLVX-P2A / T2A-puromycin, etc.

[0067] The present invention also provides a host cell, which contains the above-mentioned expression vector or the polynucleotide as described above is integrated into its genome. In some embodiments, the host cell is a prokaryotic cell, such as Escherichia coli. In some embodiments, the host cell is a eukaryotic cell.

[0068] The fourth aspect of the present invention is to provide a composition, which contains the fusion protein described in the first aspect of the present invention or the polynucleotide described in the second aspect.

[0069] According to the present invention, in some embodiments, the composition is a pharmaceutical composition, which contains the fusion protein described in the first aspect of the present invention, and optionally contains a pharmaceutically acceptable excipient.

[0070] In other embodiments, the composition contains lipid nanoparticles (LNP), and the mRNA described in the second aspect of the present invention is contained in the lipid nanoparticles. The composition may further optionally contain a pharmaceutically acceptable excipient.

[0071] According to the present invention, the LNP further contains lipid molecules, such as phospholipids, cholesterol lipid molecules, PEGylated lipid molecules, ionizable cationic lipid molecules, etc.

[0072] In some embodiments, the phospholipid is a phospholipid commonly used in the art for forming LNP, for example, it may be a phosphatidylcholine compound selected from the formula E The phosphatidylethanolamine compound shown in formula F wherein Ra, Rb, Rc, Rd are independently selected from straight-chain or branched-chain C10-30 alkyl groups, straight-chain or branched-chain C10-30 alkenyl groups, preferably CH3(CH2) 17 CH2-, CH3(CH2) 15 CH2-, CH3(CH2) 13 CH2-, CH3(CH2) 11CH2-, CH3(CH2)9CH2-, CH3(CH2)7CH2-, CH3(CH2)7-CH=CH-(CH2)7-, CH3(CH2)4CH=CHCH2CH=CH(CH2)7-, CH3(CH2)7-CH=CH-(CH2)9-. For example: dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-docosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyl oleoyl phosphatidylcholine (POPC), lysophosphatidylcholine, dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof.

[0073] Cholesterol-like lipid molecules refer to sterols and lipids containing a sterol moiety, including but not limited to cholesterol, 5-heptadecylresorcinol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, α-tocopherol and mixtures thereof, cholesterol hemisuccinate. In one embodiment, the cholesterol-like lipid molecule is cholesterol (CHOL).

[0074] The PEGylated lipid molecule comprises a lipid moiety and a PEG-based polymer moiety, represented as "lipid moiety-PEG-number average molecular weight". The lipid moiety can be diacylglycerol or diacylglycerol amide, selected from dilauroylglycerol, dimyristoylglycerol, dipalmitoylglycerol, distearoylglycerol, dilaurylglyceramide, dimyristylglyceramide, dipalmitoylglyceramide, distearoylglyceramide, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; the number average molecular weight of PEG can be about 130 to about 50,000, such as about 150 to about 30,000, about 150 to about 20,000, about 150 to about 15,000, about 150 to about 10,000, about 150 to about 6,000, about 150 to about 5,000, about 150 to about 4,000, about 150 to about 3,000, about 300 to about 3,000, about 1,000 to about 3,000, about 1,500 to about 2,500, such as about 2000. For example: PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), PEG-dilaurylglyceramide, PEG-dimyristylglyceramide, PEG-dipalmitoylglyceramide and PEG-distearoylglyceramide, PEG-cholesterol (1-[8'-(cholest-5-en-3[β]-yloxy)carbamoyl-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-di-tetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMG-PEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), 1,2-distearoyl-sn-glycero-methoxypolyethylene glycol (DSG-PEG2000), poly(ethylene glycol)-2000-dimethacrylate (DMA-PEG2000) and 1,2-distearoyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (DSA-PEG2000).

[0075] Ionizable positive lipid molecules such as 3(didodecylamino)N1,N1,4-tri-dodecyl-1-piperazineethanamine (KL10), N1[2(didodecylamino)ethyl]N1,N4,N4-tridodecyl-1,4-piperazinediethylamine (KL22), 14,25-di-tridecyl-15,18,21,24-tetraaza-tritriacontane (KL25), 1,2-di-linoleoyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-di-linoleoyl-4-dimethylaminomethyl[1,3]dioxolane (DLin-K-DMA), tritriaconta-6,9,28,31-tetraene-19-yl 4(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-di-linoleoyl-4(2-dimethylaminoethyl)[1,3]dioxolane (DLin-KC2-DMA), 1,2-dioleoyloxy-N,N-dimethylaminopropane (DODMA), 2({8[(3β-cholest-5-en-3-yloxy]octyl}oxy)N,N-dimethyl-3[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2({8[(3β)-cholest-5-en-3-yloxy)]octyl}oxy)N,N-dimethyl-3[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)) and (2S)-2({8[(3β)-cholest-5-en-3-yloxy]octyl}oxy)N,N-dimethyl[[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)), etc.

[0076] In some embodiments, the LNP may further contain quaternary amine compounds, such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazoline chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristoyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-(1,2-dioleoyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-dilinoleoyl-3-trimethylammonium-propane (DLTAP), 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), etc.

[0077] The fifth aspect of the present invention is to provide the use of the fusion protein described in the first aspect of the present invention, or the polynucleotide described in the second aspect, or the composition described in the fourth aspect in the preparation of a drug.

[0078] In some embodiments of the present invention, the drug is used for treating tumors.

[0079] In some embodiments of the present invention, the drug is used for treating nervous system diseases.

[0080] The sixth aspect of the present invention is to provide a method for treating a disease, which comprises administering a therapeutically effective amount of the fusion protein described in the first aspect of the present invention, or the polynucleotide described in the second aspect, or the composition described in the fourth aspect to a patient in need.

[0081] In some embodiments of the present invention, the disease is a tumor.

[0082] In some embodiments of the present invention, the disease is a nervous system disease.

[0083] The seventh aspect of the present invention is to provide the fusion protein described in the first aspect of the present invention, or the polynucleotide described in the second aspect, or the composition described in the fourth aspect for use as a drug.

[0084] In some embodiments of the present invention, the drug is used for treating tumors.

[0085] In some embodiments of the present invention, the drug is used for treating nervous system diseases.

[0086] The eighth aspect of the present invention is to provide the use of the fusion protein described in the first aspect, or the polynucleotide described in the second aspect, or the composition described in the fourth aspect of the present invention in the treatment of diseases.

[0087] In some embodiments of the present invention, the disease is a tumor.

[0088] In some embodiments of the present invention, the disease is a nervous system disease.

[0089] According to the present invention, the tumors include but are not limited to leukemia (such as acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute granulocytic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia, heavy chain disease, solid tumors such as sarcoma and cancer (such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial sarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, bronchial carcinoma, medullary carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma), esophageal cancer, gallbladder cancer, kidney cancer, multiple myeloma; preferably, the "tumors" include but are not limited to: pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer and glioma, and any combination thereof.

[0090] According to the present invention, the nervous system diseases include but are not limited to Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, chronic hyperkinetic dyskinesia, etc.

[0091] The beneficial effects of the present invention:

[0092] Introduce the phase separation polypeptide into the proteasome-targeting chimera, and utilize phase separation to achieve efficient degradation of the target protein.

[0093] In addition, mRNA-LNP is used to achieve the delivery of peptides or proteins, overcoming the disadvantage of their poor cell permeability.

[0094] The polypeptides involved in the present invention and their sequences are as follows:

[0095] SEQ ID No.1: Amino acid sequence of GFP nanobody

[0096] MAQVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS

[0097] SEQ ID No.2: Amino acid sequence of LAF-1 1-168

[0098] MESNQSNNGGSGNAALNRGGRYVPPHLRGGDGGAAAAASAGGDDRRGGAGGGGYRRGGGNSGGGGGGGYDRGYNDNRDDRDNRGGSGGYGRDRNYEDRGYNGGGGGGGNRGYNNNRGGGGGGYNRQDRGDGGSSNFSRGGYNNRDEGSDNRGSGRSYNNDRRDNGGDG

[0099] SEQ ID No.3: The terminal 37 amino acids of mouse ornithine decarboxylase (cODC)

[0100] FPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV

[0101] SEQ ID No.4: PSETAC LAF-1 - Amino acid sequence of E(GFP nanobody-LAF-1-cODC)

[0102] MAQVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSSGSMESNQSNNGGSGNAALNRGGRYVPPHLRGGDGGAAAAASAGGDDRRGGAGGGGYRRGGGNSGGGGGGGYDRGYNDNRDDRDNRGGSGGYGRDRNYEDRGYNGGGGGGGNRGYNNNRGGGGGGYNRQDRGDGGSSNFSRGGYNNRDEGSDNRGSGRSYNNDRRDNGGDGEFFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV

[0103] SEQ ID No.5: Amino acid sequence of PROTAC-E(GFPnanobody-cODC)

[0104] MAQVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSSGSGSGSEFFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV

[0105] SEQ ID No.6: PSETAC LAF-1 - Amino acid sequence of α(β-Syn(36 - 45)-LAF-1(1 - 168)-cODC)

[0106] MGVLYVGSKTRMESNQSNNGGSGNAALNRGGRYVPPHLRGGDGGAAAAASAGGDDRRGGAGGGGYRRGGGNSGGGGGGGYDRGYNDNRDDRDNRGGSGGYGRDRNYEDRGYNGGGGGGGNRGYNNNRGGGGGGYNRQDRGDGGSSNFSRGGYNNRDEGSDNRGSGRSYNNDRRDNGGDGEFFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV

[0107] SEQ ID No.7: Amino acid sequence of PROTAC-α(β-Syn(36 - 45)-cODC)

[0108] MGVLYVGSKTRGSGSGSEFFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV

[0109] SEQ ID No.8: Amino acid sequence of PSETAC-M (p53(17 - 26)-FUS(1 - 214)-cODC)

[0110] METFSDLWKLLGGGSGSMASNDYTQQATQSYGAYPTQPGQGYSQQSSQPYGQQSYSGYSQSTDTSGYGQSSYSSYGQSQNTGYGTQSTPQGYGSTGGYGSSQSSQSSYGQQSSYPGYGQQPAPSSTSGSYGSSSQSSSYGQPQSGSYSQQPSYGGQQQSYGQQQSYNPPQGYGQQNQYNSSSGGGGGGGGGGNYGQDQSSMSSGGGSGGGYGNQDQSGGGGSGGYGQQDRGEFFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINVSEQ ID No.9: Amino acids 17 - 26 of p53 (p53(17 - 26))

[0111] ETFSDLWKLL

[0112] SEQ ID No.10: Amino acids 1 - 214 of FUS (FUS(1 - 214))

[0113] MASNDYTQQATQSYGAYPTQPGQGYSQQSSQPYGQQSYSGYSQSTDTSGYGQSSYSSYGQSQNTGYGTQSTPQGYGSTGGYGSSQSSQSSYGQQSSYPGYGQQPAPSSTSGSYGSSSQSSSYGQPQSGSYSQQPSYGGQQQSYGQQQSYNPPQGYGQQNQYNSSSGGGGGGGGGGNYGQDQSSMSSGGGSGGGYGNQDQSGGGGSGGYGQQDRG

[0114] SEQ ID No.11: NLS sequence

[0115] RKRKLLRKRKLLRKRKLL

[0116] SEQ ID No.12: Amino acid sequence of PROTAC-M (p53(17 - 26)-NLS-cODC)

[0117] METFSDLWKLLGGGSGSRKRKLLRKRKLLRKRKLLEFFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV

[0118] SEQ ID No.13: mRNA sequence of PSETAC-M

[0119] auggagaccuucagcgaccuguggaagcugcugggcggcggcagcggcagcauggccuccaacgacuacacccagcaggccacacagagcuacggcgccuaccccacccagccuggccaaggcuacucucagcagagcucccagcccuauggccaacaguccuacagcggguacagccaguccaccgauacaagcggcuacggccaguccagcuacagcucuuacgggcaaagccagaacaccggcuacggcacccagagcaccccucagggcuacggcuccaccggcggcuacggcagcucccagagcagccagucuagcuacggccagcaguccagcuacccuggcuacggccagcagcccgcccccagcucuacaagcggcuccuacggcagcagcagccagagcagcuccuauggccagccucagagcggcagcuacagccagcagcccagcuacggcggccaacagcagagcuacggccagcagcagagcuacaacccuccccagggcuacggccagcagaaucaguacaacucuagcucuggagguggaggaggaggcggcggaggcggcaacuacggccaggaccaguccagcaugagcagcggaggcggcuccggcggaggcuacggcaaccaggaucaaucuggaggcggcggcagcggcggcuacggccagcaggacagaggcgaguuuuucccucccgaaguggaggagcaggacgauggcacccugccuauguccugcgcucaggaaagcggaauggaccggcacccagccgccugugccagcgcuagaaucaacgugugauaauag

[0120] Definition:

[0121] “And / or” shall be regarded as a specific disclosure of each of two designated features or components with or without the other. Thus, the term “and / or” used in a phrase such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” used in a phrase such as “A, B and / or C” is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0122] “Comprising” and “including” have the same meaning and are intended to be open-ended and to permit, but not require, the inclusion of additional elements or steps. When the terms “comprising” or “including” are used herein, the terms “consisting of” and / or “consisting essentially of” are thus also included and disclosed.

[0123] In this specification and the claims, nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise specified, nucleotide sequences are written from left to right in the 5' to 3' direction. A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil. Those skilled in the art will understand that the T base in the codons disclosed herein is present in DNA, and the T base will be replaced by the U base in the corresponding RNA. For example, for a codon-nucleotide sequence in the form of DNA disclosed herein, such as a vector or an in vitro translation (IVT) template, its T base is transcribed into a U base in its corresponding transcribed mRNA. In this regard, both the codon-optimized DNA sequences (containing T) and their corresponding mRNA sequences (containing U) are considered to be the codon-optimized nucleotide sequences of the present disclosure. Those skilled in the art will also understand that equivalent codon maps can be generated by replacing one or more bases with non-natural bases.

[0124] The terms “nucleic acid sequence”, “nucleotide sequence” or “polynucleotide sequence” are used interchangeably and refer to a continuous nucleic acid sequence. The sequence can be single-stranded or double-stranded DNA or RNA, such as mRNA.

[0125] “The nucleotide sequence encoding...” refers to the coding sequence of a nucleic acid (e.g., an mRNA or DNA molecule) that encodes a polypeptide. The coding sequence can further include start and stop signals operably linked to regulatory elements, which include a promoter and a polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.

[0126] In this specification and the claims, the conventional one-letter or three-letter codes for amino acid residues are used. Unless otherwise specified, amino acid sequences are written from left to right in the amino to carboxyl orientation.

[0127] "About": The term "about" as used in connection with a numerical value throughout the specification and claims means an interval of accuracy that is familiar and acceptable to those skilled in the art. Generally, such an interval of precision is ±10%.

[0128] Homology: As used herein, the term "homology" refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (such as DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Generally, the term "homology" implies an evolutionary relationship between two molecules. Thus, two homologous molecules will have a common evolutionary ancestor. In the context of the present disclosure, the term homology includes identity and similarity.

[0129] In some embodiments, polymeric molecules are considered to be "homologous" to each other if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the monomers are the same (identical monomers) or similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0130] The terms "coding region" and "coding area" refer to the open reading frame (ORF) in a polynucleotide that, when expressed, gives rise to a polypeptide or protein.

[0131] "Operably linked" refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties, etc.

[0132] Domain: As used herein, when referring to a polypeptide, the term "domain" refers to a motif of a polypeptide that has one or more recognizable structural or functional features or properties (e.g., binding ability, serving as a site for protein-protein interaction).

[0133] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an mRNA template from a DNA sequence (e.g., by transcription); (2) processing of the mRNA transcript (e.g., by splicing, editing, 5' capping and / or 3' end processing); (3) translation of the mRNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.

[0134] "Antibody" generally refers to a polypeptide of the immunoglobulin family or a polypeptide comprising an immunoglobulin fragment capable of binding a corresponding antigen non-covalently, reversibly, and in a specific manner. As used herein, the term "antibody" means to encompass all variants of antibodies and their fragments having one or more specific binding specificities, including full-length antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies (ScFv, camelids), Fab, Fab', multimeric forms of these fragments (e.g., F(ab')2), single-domain antibodies (sdAB, VHH fragments), heavy-chain antibodies (HCAb), nanobodies, diabodies, and minibodies. An antibody can have more than one binding specificity, for example, be bispecific.

[0135] "Pharmaceutically acceptable excipient" refers to any component other than the proteins or mRNAs described herein and having substantially non-toxic and non-inflammatory properties in the human body, including but not limited to any and all solvents, dispersion media, or other liquid carriers, dispersion or suspension aids, diluents, granulating and / or dispersing agents, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, binders, lubricants, coloring agents, sweetening or flavoring agents, stabilizers, antioxidants, antimicrobial or antifungal agents, osmolarity regulators, pH regulators, buffers, chelating agents, cryoprotectants, and / or fillers, as suitable for the particular dosage form desired. The various excipients used in formulating pharmaceutical compositions and the techniques for preparing the compositions are known in the art. Exemplary antimicrobial or antifungal agents include but are not limited to benzalkonium chloride, benzethonium chloride, methylparaben, ethylparaben, propylparaben, butylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate or sodium benzoate, potassium sorbate or sodium sorbate, sodium propionate, sorbic acid, etc., and combinations thereof. Exemplary preservatives include but are not limited to vitamin A, vitamin C, vitamin E, β-carotene, citric acid, ascorbic acid, butylated hydroxyanisole, ethylenediamine, sodium dodecyl sulfate (SLS), sodium laureth sulfate (SLES), etc., and combinations thereof. Exemplary buffers for controlling pH can include but are not limited to sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, etc., and / or combinations thereof. Exemplary cryoprotectants include but are not limited to mannitol, sucrose, trehalose, lactose, glycerol, dextrose, etc., and combinations thereof. Exemplary fillers can include but are not limited to sucrose, trehalose, mannitol, glycine, lactose, raffinose, and combinations thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0136] Figure 1 : Schematic diagram of the principle for achieving efficient degradation of target proteins using PSETAC

[0137] Figure 2 : The fusion protein PSETAC of the present inventionLAF-1 - Schematic diagram of the structure of E

[0138] Figure 3 : Transfect PSETAC LAF-1 - Confocal images of E transfected into cells

[0139] Figure 4 : Characterization of PSETAC by flow cytometry LAF-1 - Degradation efficiency of E on eGFP

[0140] Figure 5 : Transfect PSETAC LAF-1 - Confocal images of α transfected into cells

[0141] Figure 6 : Characterization of PSETAC by flow cytometry LAF-1 - Degradation efficiency of α on α-Syn

[0142] Figure 7 : Schematic diagram of the structure of the fusion protein PSETAC-M of the present invention

[0143] Figure 8 : Confocal images of PSETAC-M transfected into cells

[0144] Figure 9 : Characterization of the degradation efficiency of PSETAC-M on MDM2 by Western blot

[0145] Figure 10 : Characterization of the degradation efficiency of PSETAC-M mRNA-LNP on MDM2 by Western blot

[0146] Figure 11 : Characterization of the effect of different concentrations of PSETAC-M mRNA-LNP on the degradation efficiency of MDM2 by Western blot Detailed implementation method

[0147] The following examples are used to further describe the present invention. It should be noted that the examples cannot be used to limit the protection scope of the present invention. Those skilled in the art understand that any improvements and changes made on the basis of the present invention are within the protection scope of the present invention.

[0148] All the conventional reagents used in the following examples can be obtained commercially. The biological experiments carried out are all conventional biological experiments in the art and can be carried out according to the corresponding experimental manuals or kit instructions.

[0149] Example 1 Design and characterization of PSETAC (PSETAC LAF-1 -E) targeting eGFP

[0150] (1) The gene sequences of the single-domain peptide GFPnanobody (SEQ ID No.1) from camelid animals that specifically binds to eGFP, the gene sequence of the LAF-1 protein IDR (1-168) sequence (SEQ ID No.2), the gene sequence of mouse guanosine decarboxylase cODC (SEQ ID No.3), the ribosome skipping sequence IRES gene sequence, and the eGFP gene sequence were cloned into the pcDNA3.1 plasmid. The gene sequence synthesis and molecular cloning were completed by General Biosystems Co., Ltd. This plasmid can express the GFPnanobody-LAF-1(1-168)-cODC fusion protein in eukaryotic cells, hereinafter referred to as PSETAC LAF-1 -E (SEQ ID No.4) and eGFP( Figure 2 ), were transformed into Escherichia coli Mach T1 (purchased from Bomed Biotechnology Co., Ltd.) and can be stored at -80 °C in 15% glycerol. Using the same preparation method, a plasmid that can express the amino acid sequence of PROTAC-E (SEQ ID No.5, without the phase separation polypeptide) was prepared as a control reagent in subsequent experiments.

[0151] (2) Plasmid transfection and expression of PSETAC LAF-1 -E

[0152] Transfection was carried out using the lipo8000 liposome transfection reagent purchased from Beyotime. The transfection reagent was configured according to the volume of the cells plated, for example, the volume required for a 6-well plate, 125 μL of OptiMEM medium, 4 μL of transfection reagent, and 2.5 μg of plasmid; gently mix. The cells were plated into the wells according to the required number of cells; cultured in a 37 °C incubator for 24 h until the required cell density; the transfection reagent was prepared; the original medium was aspirated, and the cells were gently washed once with PBS; fresh DMEM medium containing 10% FBS was added; the mixed transfection reagent was dropped into the wells and gently shaken to mix. The cells were placed in a 37 °C constant temperature incubator, and the protein situation was detected after 24 h.

[0153] The plasmid expressing PSETAC LAF-1 -E (SEQ ID No.4) and eGFP was transfected into HEK293T cells, and the cells were cultured for another 24 h after transfection to allow the plasmid to express. The cells on the cell slides were fixed with 4% paraformaldehyde and blocked with a PBS solution containing 3% BSA. After blocking, a monoclonal mouse antibody against the 20S subunit PSMC2 of the proteasome (purchased from Sino Biological) was used; then incubated with a Cy3-labeled goat anti-mouse secondary antibody at room temperature for 2 h; after DAPI staining, the slides were sealed. Observation was carried out using a confocal microscope.

[0154] The results are as Figure 3As shown, the proteasome is in red, eGFP is in green, and the nucleus is in blue. The merged imaging shows that PSETAC LAF-1 -E forms droplets with eGFP in cells and co-localizes with a part of the proteasome. This indicates that PSETAC LAF-1 -E phase-separates in cells and can recruit the proteasome.

[0155] (3) Characterization of PSETAC LAF-1 -E's degradation efficiency of eGFP

[0156] After adding the protein synthesis inhibitor CHX to the transfected cells for 8 h, the cells were digested and resuspended in 1 mL of PBS, and the fluorescence decline rate was characterized using a flow cytometer after adding the synthesis inhibitor for a period of time.

[0157] The results are as Figure 4 shown. For the cells transfected with PSETAC LAF-1 -E, the fluorescence decline was obvious after adding the inhibitor, which was 57% of the group without the inhibitor; while for the control group GFPnanobody-cODC(PROTAC-E)(SEQ ID No.5) transfected without phase separation, the fluorescence decline was less, which was 71% of the group without the inhibitor. This shows that phase separation promotes the degradation of the target protein.

[0158] Example 2 Design and characterization of PSETAC (PSETAC LAF-1 -α) targeting α-synuclein (α-Synuclein, α-Syn)

[0159] (1) Construction of the PROTAC-α vector

[0160] Synthesize the upstream and downstream primers of the PBD sequence (β-Syn(36-45)):

[0161] Upstream primer: 5’-CTAGCATGGGCGTGCTGTACGTGGGCAGCAAGACCAGAG-3’;

[0162] Downstream primer: 5’-GATCctCTGGTCTTGCTGCCCACGTACAGCACGCCCATG-3’; After annealing, the PBD sequence is obtained, which has NheI / BamHI restriction sites;

[0163] Connect with the PROTAC-E plasmid digested with NheI / BamHI to obtain the plasmid pcDNA3.1-PBD-cODC-eGFP;

[0164] The α-Syn-dsRED fragment was obtained by PCR from the plasmid already existing in the laboratory. The plasmid pcDNA3.1-PBD-cODC-eGFP and this fragment were respectively digested with BstxI / XhoI and then ligated to obtain pcDNA3.1-PBD-cODC-α-Syn-dsRED, namely PROTAC-α.

[0165] (2) PSETAC LAF-1 - Construction of the α vector

[0166] Primers were designed according to the Gibson recombination method to amplify the PBD-LAF-1-cODC sequence;

[0167] Forward primer: 5’-TCACTATAGGGAGACCCAAGCTGGCTAGCATGGGCGTGCTGTACGTGGGCAGCAAGACCAGAAT GGAAAGTAACCAATCGAACAATGGA-3’;

[0168] Reverse primer: 5’-GGAGGGAGAGGGGGCGGCCGCTTACACGTTGATCCTG-3’

[0169] The PROTAC-α plasmid was digested with NheI and NotI to obtain a linearized plasmid, which was Gibson recombined with the above-mentioned PBD-LAF-1-cODC fragment of PCR to obtain pcDNA3.1-PBD-LAF-1-cODC-α-Syn-dsRED, namely PSETAC LAF-1 -α.

[0170] (3) Transforming PROTAC-α and PSETAC LAF-1 -α into Escherichia coli, and storing the bacterial strain at -80 °C; After a large amount of plasmid extraction, cell transfection and confocal microscopy imaging were carried out according to the method described in Example 1 (the cells on the cell coverslips were fixed with 4% paraformaldehyde and blocked with PBS solution containing 3% BSA. After blocking, a monoclonal mouse antibody against the 20S subunit PSMC2 of the proteasome was used; then incubated with Alexa-488-labeled goat anti-mouse secondary antibody at room temperature for 2 h; After DAPI staining, the coverslips were sealed), and flow cytometry detection was performed.

[0171] The results showed that: PSETAC LAF-1 -α formed droplets with α-Syn in the cells and co-localized with a part of the proteasome. This indicates that PSETAC LAF-1 -α undergoes phase separation in the cells and can recruit the proteasome (see Figure 5, the red one is α-Syn, the green one is the proteasome, and the blue represents the cell nucleus). After flow cytometry detection with CHX added, the fluorescence in the phase separation group decreased more (see Figure 6 , transfected with PSETAC LAF-1 -α, the fluorescence decreased significantly after adding the inhibitor, which was 72% of the group without the inhibitor, and the decrease was significantly different; while the fluorescence of the transfected PROTAC-α without phase separation decreased less, which was 83% of the group without the inhibitor, and the decrease was not significantly different, indicating that phase separation promoted the degradation of the target protein).

[0172] Example 3 Design and Characterization of PSETAC (PSETAC-M) Against MDM2

[0173] (1) Construct PSETAC-M (SEQ ID No. 8), in which the specific binding sequence targeting MDM2 is the amino acid sequence at positions 17-26 of the p53 protein (SEQ ID No. 9), and the phase separation sequence is the amino acids at positions 1-214 from the FUS protein (SEQ ID No. 10). Its schematic diagram is as Figure 7 shown. The vector was constructed as follows:

[0174] Amplify the FUS (1-214) gene sequence

[0175] Design upstream and downstream primers, introduce BamHI and EcoRI restriction sites, and perform PCR using the full-length human FUS gene as a template:

[0176] Upstream: FUSn-F-BamHI: CGCGGATCCatggcctcaaacgattatacccaaca

[0177] Downstream: FUSn-R-EcoRI: CCGGAATTCtccacggtcctgctgtccatagccac

[0178] Clone the obtained PCR product into the vector PROTAC-E, and ligate the synthesized p53 (17-26) gene sequence (purchased from General Biosystems) to obtain p53 (17-26)-FUS-cODC-eGFP; Digest p53 (17-26)-FUS-cODC with NheI / NotI and clone it into the pDsRED-N1 plasmid to obtain the plasmid expressing PSETAC-M.

[0179] (2) Construct the PROTAC-M recombinant plasmid

[0180] The PSETAC-M plasmid was digested with BamH I and EcoR I, and ligated with the gene sequence aggaagaggaagctgctgaggaagaggaagctgctgaggaagaggaagctgct of the synthetic NLS (SEQ ID No.11) to obtain the PROTAC-M (SEQ ID No.12) plasmid as the control group in the subsequent experiments.

[0181] The plasmid was extracted according to the plasmid extraction kit to make the plasmid concentration greater than 500 ng / μL and stored at -20 °C.

[0182] Culture of MCF-7 cells and plasmid transfection

[0183] MCF-7 cells were inoculated into a T25 culture flask, and the medium used was high-glucose DMEM medium supplemented with 10% FBS. It was placed in an incubator at 37 °C and 5% CO2 for culture. After 12 h, the cells adhered to the bottom of the culture flask. When the cell confluence reached about 70%-80%, transfection could be carried out. The lipo8000 liposome transfection reagent purchased from Beyotime was used for transfection according to the instructions of the kit.

[0184] (3) Phase separation of MDM2 was induced after transfection of PSETAC-M into cells

[0185] The above-mentioned plasmid PSETAC-M and the control group PROTAC-M were transfected into MCF-7 cells. After transfection, the cells were cultured for another 24 h to allow the plasmid to express. The cells on the cell slides were fixed with 4% paraformaldehyde and blocked with PBS solution containing 3% BSA. After blocking, they were incubated overnight with the anti-proteasome 20S subunit PSMB5 monoclonal mouse antibody (purchased from Santa Cruz) and the anti-MDM monoclonal rabbit polyclonal antibody (purchased from CST); then incubated with Cy3-labeled goat anti-rabbit secondary antibody and Alexa-488-labeled goat anti-mouse secondary antibody at room temperature for 2 h; after DAPI staining, the slides were sealed. Observation was carried out using a confocal microscope.

[0186] The results are as Figure 8 shown. The red color represents MDM2, the green color represents the proteasome, and the blue color represents the cell nucleus. The merged image shows that PSETAC-M forms droplets with MDM2 in the cells and co-localizes with a part of the proteasome. This indicates that PSETAC-M undergoes phase separation in the cells and can recruit the proteasome.

[0187] (4) Western blot semi-quantitative experiment to characterize the degradation efficiency of PSETAC

[0188] The effect of phase separation on degradation efficiency after transfection was analyzed by Western blot.

[0189] MCF-7 cells were evenly seeded into a six-well plate at a density of 250,000 cells per well. After culturing for 24 h, the cells were transfected with PSETAC-M plasmid and PROTAC-M plasmid respectively. Total cell proteins were harvested 24 h after transfection.

[0190] Prepare DMEM medium containing 10% FBS serum, PBS, and trypsin; Take out the cells and gently rinse the dead cells on the cell surface with PBS; Add 500 μL of trypsin to each well and digest for 2 min; Add 500 μL of medium to each well to terminate digestion and wash down the cells, then quickly transfer them to a 1.5 mL EP tube; Add 500 μL of DMEM medium to each well to rinse the remaining cells in the well and transfer them to the corresponding tube; Centrifuge at 3500 rpm for 5 min; Discard the supernatant, add 1 mL of PBS and gently flick to suspend the precipitate; Centrifuge again; Discard the supernatant; Spin dry for 1 min; Use a pipette to aspirate the remaining supernatant; Suspend the precipitate in 60 μL of RIPA (weak) containing protease inhibitor (purchased from Beyotime Biotechnology Co., Ltd.), gently blow until the solution is clear, and lyse on ice for 5 min - 10 min; After lysis, centrifuge at 12000 rpm and 4 °C for 10 min; Aspirate the supernatant, add 4× loading buffer, and boil the sample at 100 °C for 5 min to denature the protein.

[0191] Load the samples for SDS-PAGE; After electrophoresis at 120 V for 1 h, transfer the membrane using the semi-dry method; Transfer the membrane at a constant current of 300 mA for 1 h. The PVDF membrane with transferred protein was blocked with TBST solution containing 5% skim milk powder. After blocking, the membrane was cut according to the molecular weights of the internal reference GAPDH, p53, and MDM2; The membrane was incubated overnight at 4 °C with anti-GAPDH rabbit monoclonal antibody (purchased from Santa Cruz), anti-p53 mouse monoclonal antibody (purchased from Santa Cruz), and anti-MDM2 antibody respectively. After incubation with the primary antibody, wash with TBST for 5 min; Wash 3 times, wash once with TBS and then incubate with the secondary antibody. After incubation with the secondary antibody, repeat the TBST and TBS washes, add ECL hypersensitive luminescent solution to make the bands luminescent, and use a Qinxiang exposure instrument to expose and photograph the membrane.

[0192] The results are as Figure 9 shown.

[0193] The results showed that, when the internal reference GAPDH was basically the same, the level of MDM2 in the cells transfected with PSETAC-M decreased, and the decrease in the level of MDM2 was more significant compared to the control group of cells transfected with PROTAC-M without the phase separation sequence, reaching 65% of the negative control. Along with the decrease in MDM2, the level of its downstream protein p53 increased, and the increase in the level of p53 was more significant compared to the control group of cells transfected with PROTAC-M without the phase separation sequence, reaching 168% of the negative control. This indicates that phase separation can not only achieve the efficient degradation of target proteins in cells, but also regulate the protein levels in cells, which has a positive effect on the treatment of diseases caused by protein misfolding or overexpression.

[0194] (5) Characterization of the degradation efficiency of PSETAC-M mRNA-LNP by Western blot semi-quantitative experiment

[0195] The genes expressing PSETAC-M and PROTAC-M were respectively transcribed into mRNA in vitro and encapsulated with LNP (Yunzhou Biologics). Transfection of MCF-7 cells was carried out by mixing them into serum-free medium at a dose of 2 μg per well of a six-well plate. After 24 h, the cells were collected and lysed, and the protein levels of MDM2 and p53 in the cell lysate were detected by Western blot.

[0196] The results were as Figure 10 shown. After delivering PSETAC-M into cells using PSETAC-M mRNA-LNP, the protein level of MDM2 decreased significantly, and the decrease in the level of MDM2 was more significant compared to the control group delivering PROTAC-M mRNA-LNP without the phase separation sequence, reaching 67% of the negative control group. PSETAC-M mRNA-LNP also significantly increased the protein expression of the downstream protein p53 of MDM2, raising its level to 267% of the negative control group, and the protein level increased compared to the control group delivering PROTAC-M mRNA-LNP without the phase separation sequence.

[0197] In addition, different concentrations of PSETAC-M mRNA-LNP (0.1 μg, 0.5 μg, 1 μg, 2 μg) were added to the cell culture medium, mixed into serum-free medium for MCF-7 cell transfection, and after overnight incubation, the cells were collected and lysed. The protein levels of MDM2 and p53 in the cell lysate were detected by Western blot. It was observed that as the concentration of PSETAC-M mRNA-LNP increased, the level of MDM2 decreased successively, while the level of p53 increased successively. The results were as Figure 11 shown. This indicates that mRNA-LNP did successfully deliver PSETAC-M into cells and successfully achieved its function of degrading target proteins.

[0198] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a polypeptide with phase separation function in the preparation of a proteasome-targeted chimera, wherein one end of the proteasome-targeted chimera is a part targeting the target protein, the other end is a part targeting the proteasome, and the polypeptide with phase separation function connects the above two parts; the part targeting the target protein is located at the N-terminus or C-terminus of the polypeptide with phase separation function, and at the same time, the part targeting the proteasome is correspondingly located at the C-terminus or N-terminus of the polypeptide with phase separation function; there is or is not a linker structure between the part targeting the target protein and the polypeptide with phase separation function, and / or between the part targeting the proteasome and the polypeptide with phase separation function; Preferably, the part targeting the target protein is a small molecule compound or a polypeptide; or, Preferably, the part targeting the proteasome is a small molecule compound or a polypeptide.

2. A fusion protein, characterized in that, The fusion protein comprises three parts: polypeptide A targeting the target protein, phase separation polypeptide B, and polypeptide C targeting the proteasome; the N-terminus of polypeptide B is polypeptide A and the C-terminus is polypeptide C, or the N-terminus of polypeptide B is polypeptide C and the C-terminus is polypeptide A; there is or is not a linker sequence between polypeptide A and polypeptide B, and / or between polypeptide B and polypeptide C.

3. The application according to claim 1 or the fusion protein according to claim 2, characterized in that The target protein is a protein whose misexpression, mutation or overactivation can cause diseases; Preferably, the target protein is selected from mouse double minute 2 homolog (MDM2), MetAP-2, Aβ, TDP-43, α-synuclein, tau protein, androgen receptor, bromodomain and extraterminal (BET) protein family, c-MYC, estrogen receptor α (ERα), cyclin-dependent kinases (CDK), BCR-ABL fusion protein (breakpoint cluster region-cabl, BCR-ABL), B-cell lymphoma 6 protein (BCL6), anaplastic lymphoma kinase (ALK), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor-2 (HER2), c-mesenchymal epithelial transition factor (c-Met), Bruton tyrosine kinase (BTK); More preferably, the target protein is mouse double minute 2 homolog (MDM2), α-synuclein or Aβ; Preferably, the polypeptide targeting the target protein is an Aβ antibody, the Aβ affinity peptide VDNKFNKEMASAGGEIVYLPNLNPDQLCAFIHSLHDDPSQSANLLAEAKKLNDAQAPK or its homologous sequence; Preferably, the polypeptide targeting the target protein is the α-synuclein affinity peptide VDNKFNKEMASADGEIFYLPNLNPDQLCAFFHSVHDDPSQSANLLA EAKKLNDAQAPK or its homologous sequence; Preferably, the polypeptide targeting the target protein is the β-synuclein fragment GVLYVGSKTR or its homolog; Preferably, the amino acid sequence of the polypeptide targeting the target protein has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with GVLYVGSKTR; Preferably, the amino acid sequence of the polypeptide targeting the target protein is GVLYVGSKTR; Preferably, the polypeptide targeting the target protein is a fragment of the p53 protein or its homolog; Preferably, the amino acid sequence of the polypeptide targeting the target protein has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No. 9; Preferably, the amino acid sequence of the polypeptide targeting the target protein is as shown in SEQ ID No.

9.

4. The application according to claim 1 or the fusion protein according to claim 2 or 3, characterized in that, The phase separation polypeptide is selected from fragments of the IDR region of a naturally occurring self-assembling protein or a synthetic phase separation polypeptide; Preferably, the phase separation polypeptide is selected from the LC domain of the FUS protein, the IDR region of hnRNPA1, the IDR domain of LAF-1, SGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSRGG, GHGVYGHGVYGHGPYGHGPYGHGLYW; Preferably, the phase separation polypeptide is the IDR domain of LAF-1 or its homolog; Preferably, the amino acid sequence of the phase separation polypeptide has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No. 2; Preferably, the amino acid sequence of the phase separation polypeptide is as shown in SEQ ID No. 2; Preferably, the phase separation polypeptide is the LC domain of the FUS protein or its homolog; Preferably, the amino acid sequence of the phase-separating polypeptide has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No. 10; Preferably, the amino acid sequence of the phase-separating polypeptide is as shown in SEQ ID No.

10.

5. The application according to claim 1 or the fusion protein according to any one of claims 2-4, characterized in that, The polypeptide or small molecule targeting the proteasome is selected from cereblon, lenalidomide, pomalidomide, iberdomide, (S,R,S)-AHPC, thalidomide, VH-298, CC-122, CC-885, E3 ligase ligand 8, TD-106, VL285, VH032, VH101, VH298, VHL ligand 4, VHL ligand 7, VHL-2 ligand 3, E3 ligase ligand 3, E3 ligase ligand 2, BC-1215, the C-terminus of murine ornithine decarboxylase (ODC) (cODC), PEST sequence, the labile N-terminus containing arginine, tyrosine, phenylalanine; Preferably, the polypeptide targeting the proteasome is selected from the C-terminus of murine ornithine decarboxylase (cODC) or its homolog, PEST sequence, the labile N-terminus containing arginine, tyrosine, phenylalanine; Preferably, the amino acid sequence of the polypeptide targeting the proteasome has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No. 3; Preferably, the amino acid sequence of the polypeptide targeting the proteasome is as shown in SEQ ID No. 3; Preferably, the polypeptide targeting the proteasome is HKSGAWKLPVSLVK.

6. The fusion protein according to any one of claims 2-5, characterized in that, The linker sequence is selected from the GnSm linker sequence, (Gly)n linker sequence, DDK linker sequence or a combination thereof; Preferably, the amino acid sequence of the linker is GGGSGS.

7. The fusion protein according to any one of claims 2-6, characterized in that, The amino acid sequence of the fusion protein has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No. 8; Preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID No. 8; Alternatively, the amino acid sequence of the fusion protein has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the amino acid sequence shown in SEQ ID No. 6; Preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID No.

6.

8. A polynucleotide, characterized in that, The polynucleotide encodes the fusion protein according to any one of claims 2-7; Preferably, the polynucleotide is DNA; Preferably, the polynucleotide is mRNA; Preferably, the mRNA is an mRNA encoding a fusion protein with the amino acid sequence shown in SEQ ID No. 8 or SEQ ID No. 6; Preferably, the ORF of the mRNA has a nucleotide sequence with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homology with the nucleotide sequence shown in SEQ ID No. 13; Preferably, the nucleotide sequence of the ORF of the mRNA is as shown in SEQ ID No.

13.

9. An expression vector, characterized in that, Containing the polynucleotide according to claim 8; Preferably, the expression vector is selected from plasmid vectors or viral vectors; Preferably, the expression vector is selected from pcDNA3.1 plasmid, pDsRed N1 plasmid, pCMV-NEO-BAN plasmid, pMRX-puro plasmid, pLVX-IRES-zsGreeen1, pLVX-IRES-puromycin, pLVX-P2A / T2A-zsGreeen1, pLVX-P2A / T2A-puromycin.

10. A host cell, characterized in that, The host cell contains the expression vector according to claim 9, or the polynucleotide according to claim 8 is integrated into its genome.

11. A composition, characterized in that, It contains the fusion protein according to any one of claims 2-7 or the polynucleotide according to claim 8; Preferably, the composition further optionally contains a pharmaceutically acceptable excipient; Preferably, the composition comprises lipid nanoparticles, and the lipid nanoparticles contain the mRNA according to claim 8; Preferably, the lipid nanoparticles further comprise lipid molecules; Preferably, the lipid molecule comprises a phospholipid, preferably selected from phosphatidylcholine compounds represented by Formula E Phosphatidylethanolamine compounds represented by Formula F wherein Ra, Rb, Rc, and Rd are independently selected from straight-chain or branched C10-30 alkyl groups, straight-chain or branched C10-30 alkenyl groups, preferably CH3(CH2) 17 CH2-, CH3(CH2) 15 CH2-, CH3(CH2) 13 CH2-, CH3(CH2) 11 CH2-, CH3(CH2)9CH2-, CH3(CH2)7CH2-, CH3(CH2)7-CH=CH-(CH2)7-, CH3(CH2)4CH=CHCH2CH=CH(CH2)7-, CH3(CH2)7-CH=CH-(CH2)9-; Preferably, the lipid molecules comprise cholesterol lipid molecules, preferably selected from cholesterol, 5-heptadecylresorcinol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, α-tocopherol and mixtures thereof, cholesterol hemisuccinate; Preferably, the lipid molecules comprise PEGylated lipid molecules "lipid moiety-PEG-number average molecular weight", preferably the lipid moiety is selected from diacylglycerol or diacylglyceramide, and the number average molecular weight of PEG is about 130 to about 50,000; Preferably, the lipid molecule comprises an ionizable positive lipid molecule, preferably selected from 3(didodecylamino)N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1[2(didodecylamino)ethyl]N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-di-tridecyl-15,18,21,24-tetraaza-38-ane (KL25), 1,2-di-linoleoyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-di-linoleoyl-4-dimethylaminomethyl[1,3]dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-di-linoleoyl-4(2-dimethylaminoethyl)[1,3]dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2({8[(3β-cholest-5-en-3-yloxy]octyl}oxy)N,N-dimethyl-3[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2({8[(3β-cholest-5-en-3-yloxy)]octyl}oxy)N,N-dimethyl-3[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)) and (2S)-2({8[(3β)-cholest-5-en-3-yloxy]octyl}oxy)N,N-dimethyl[[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)).

12. Use of the fusion protein according to any one of claims 2-7, the polynucleotide according to claim 8 or the composition according to claim 11 in the preparation of a medicament; Preferably, the medicament is used for treating tumors; Preferably, the medicament is used for treating nervous system diseases; Preferably, the tumor is selected from leukemia (such as acute leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, acute granulocytic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute leukemia, chronic leukemia, chronic granulocytic leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia, heavy chain disease, solid tumors such as sarcoma and cancer (such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endotheliosarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, bronchial carcinoma, medullary carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma), esophageal cancer, gallbladder cancer, kidney cancer, multiple myeloma; Preferably, the neurological disease is selected from Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, chronic hyperkinetic dyskinesia.

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