Disordered region of pglyrp2 protein, fusion protein comprising same and use thereof

Abstract

Provided are a disordered region of a PGLYRP2 protein, a fusion protein comprising same, and the use thereof. The disordered region comprises an amino acid sequence as shown in SEQ ID NO: 1; or the amino acid sequence comprised in the disordered region has at least 80% identity to SEQ ID NO: 1. The fusion protein comprises the disordered region. Also provided is the use of the disordered region and the fusion protein in the preparation of a drug for preventing and / or treating a disease related to hepatitis B virus infections. The present disclosure breaks through the limitations of the prior art, provides an innovative, efficient and safe gene therapy method for the treatment of hepatitis B, and has a broad clinical application value and commercialization potential.

Description

A disordered region of the PGLYRP2 protein, a fusion protein containing it, and its applications.

[0001] This application claims priority to Chinese patent application 2025100781146, filed on January 17, 2025. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedical technology, specifically to the field of gene therapy and antiviral therapy, and more specifically to a disordered region of the PGLYRP2 protein, a fusion protein containing it, and its applications. Background Technology

[0003] Hepatitis B virus (HBV) infection is a major public health problem worldwide, causing acute and chronic hepatitis B, which can progress to cirrhosis, liver failure, and even hepatocellular carcinoma (HCC) in severe cases. Currently, treatments for hepatitis B mainly include nucleoside (acid) analogs and interferon-alpha, but these treatments have significant limitations. For example, they cannot completely eliminate the virus, only suppress HBV replication, and long-term treatment easily leads to drug resistance and adherence issues. Furthermore, the persistent presence of HBV's covalently closed circular DNA (cccDNA) in the hepatocyte nucleus makes the virus persistent, posing a significant challenge to treatment.

[0004] In recent years, with the development of gene therapy technology, novel strategies targeting HBV DNA and its replication process have gradually attracted attention. However, existing gene therapy drugs still suffer from poor efficacy in inhibiting HBV replication and clearing the virus, failing to effectively capture and eliminate viral DNA. Furthermore, the HBV infection environment is complex, and there is a lack of treatment methods that can simultaneously target DNA and enhance antiviral efficacy.

[0005] Liquid-liquid phase separation (LLPS) has become a research hotspot in recent years. It participates in important biological processes such as cell metabolism and signal transduction by forming dynamic intracellular membrane-independent structures. Previous studies have shown that the intrinsically disordered regions (IDRs) of certain proteins have the ability to mediate LLPS. They can form highly aggregated regions through phase separation, thereby improving the efficiency of intermolecular reactions. Traditionally, it is known in this field that disordered regions with liquid-liquid phase separation properties can improve the transcription efficiency of DNA through phase separation ((1) Cell. 2018 Dec 13; 175(7):1842-1855.e16.doi:10.1016 / j.cell.2018.10.042.(2) Nat Cell Biol. 2022 Apr; 24(4):513-525.doi:10.1038 / s41556-022-00872-5.). However, without experimental verification, the determination of the disordered region of a specific protein and whether it can ultimately produce the technical effect and specific function of mediating LLPS are unknown to those skilled in the art, and require creative exploration and research.

[0006] As a key protein involved in the immune response, the location of the intrinsic disorder region (IDR) of PGLYRP2 and the specific function of this region in the antiviral process are still scientific questions that have not yet been clarified in this field. Summary of the Invention

[0007] To address the shortcomings of existing technologies that have failed to resolve the IDR region of PGLYRP2 and explore its specific functions, as well as the lack of an innovative, efficient, and safe gene therapy strategy that effectively inhibits HBV replication and promotes its clearance, this invention provides a disordered region of the PGLYRP2 protein, a fusion protein containing it, and its applications.

[0008] This invention relates to the field of biomedical technology and discloses a gene therapy drug based on the intrinsically disordered region (IDR) of the PGLYRP2 protein (PGLYRP2-IDR) for enhancing the clearance of hepatitis B virus (HBV). The core of this invention lies in the fact that PGLYRP2-IDR can mediate liquid-liquid phase separation (LLPS), synergistically capturing and isolating HBV DNA through the HBV DNA binding domain (HBV-DBD), thereby more efficiently inhibiting HBV replication. This invention significantly improves the HBV clearance efficiency of hepatocytes through this unique mechanism, overcoming the technical bottleneck of existing gene therapy drugs' poor efficacy in inhibiting HBV replication and clearing the virus. This gene therapy drug provides a novel strategy for the treatment of hepatitis B and has broad clinical application value and development prospects.

[0009] This invention discovers that the disordered region of PGLYRP2 (PGLYRP2) IDR The PGLYRP2IDR-(209~377)-NLS composition formed by these components isolates HBV DNA through liquid-liquid phase separation, thereby blocking HBV transcription. This result indicates that its mechanism of action on HBV DNA is not, in the traditional sense, to enhance HBV DNA transcription efficiency through phase separation, but rather to inhibit HBV transcription through physical isolation.

[0010] To solve the above-mentioned technical problems, one of the technical solutions provided by the present invention is: a disordered region of a PGLYRP2 protein, wherein the disordered region contains an amino acid sequence as shown in SEQ ID NO:1; or the amino acid sequence contained in the disordered region has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:1.

[0011] As is known in the art, disordered regions of a protein refer to protein regions that lack a fixed three-dimensional structure under physiological conditions. These regions lack fixed secondary structures (such as α-helices, β-sheets, etc.) and do not form a stable three-dimensional conformation. Generally, the length of disordered regions of a protein can be approximately 30 to 100 amino acid residues.

[0012] In a specific embodiment of the present invention, the disordered region contains 30 to 100 amino acid residues, 35 to 55 amino acid residues, 40 to 50 amino acid residues, or 42 to 48 amino acid residues.

[0013] In a specific embodiment of the present invention, the disordered region contains 45 amino acid residues.

[0014] In a specific embodiment of the present invention, the amino acid sequence of the disordered region is as shown in SEQ ID NO:1; or the amino acid sequence of the disordered region has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:1.

[0015] To solve the above-mentioned technical problems, the second technical solution provided by the present invention is: a fusion protein, wherein the fusion protein includes the disordered region as described in the first technical solution.

[0016] In a specific embodiment of the present invention, the disordered region is located at the N-terminus of the fusion protein.

[0017] In a specific embodiment of the present invention, the fusion protein further comprises an HBV DNA binding domain.

[0018] In a specific embodiment of the present invention, the fusion protein further comprises the HBV DNA binding domain of the PGLYRP2 protein.

[0019] In a specific embodiment of the present invention, the fusion protein further includes the nuclear localization signal region of the PGLYRP2 protein.

[0020] In a specific embodiment of the present invention, the fusion protein satisfies one or more of the following conditions:

[0021] (1) The HBV DNA binding domain comprises an amino acid sequence as shown in SEQ ID NO:2, or the amino acid sequence comprising the HBV DNA binding domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:2.

[0022] (2) The nuclear localization signal region contains an amino acid sequence as shown in SEQ ID NO:3, or the amino acid sequence contained in the nuclear localization signal region has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:3; and,

[0023] (3) The fusion protein consists of the following components from the N-terminus to the C-terminus: the disordered region, the HBV DNA binding domain, and the nuclear localization signal region.

[0024] In a specific embodiment of the present invention, the amino acid sequence of the HBV DNA binding domain is as shown in SEQ ID NO:2, or the amino acid sequence of the HBV DNA binding domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:2.

[0025] In a specific embodiment of the present invention, the amino acid sequence of the nuclear localization signal region is as shown in SEQ ID NO:3, or the amino acid sequence of the nuclear localization signal region has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:3.

[0026] As illustrated in the embodiments of the present invention, PGLYRP2 IDR The region is the HBV DNA binding domain (PGLYRP2) of the PGLYRP2 protein. 209-377 ) and Nuclear Localization Signal (PGLYRP2) functional domain NLS The fusion protein PGLYRP2IDR-(209~377)-NLS is formed, which can efficiently capture and isolate HBV DNA, thereby significantly inhibiting viral replication.

[0027] To solve the above-mentioned technical problems, the third technical solution provided by the present invention is: a protein composition, wherein the composition comprises a disordered region as described in the first technical solution or a fusion protein as described in the second technical solution, as well as other functional proteins.

[0028] In a specific embodiment of the present invention, the other functional proteins include HBV nucleocapsid-binding proteins.

[0029] In a specific embodiment of the present invention, the HBV nucleocapsid-binding protein includes the HBV nucleocapsid-binding domain of PGLYRP2.

[0030] In a specific embodiment of the present invention, the HBV nucleocapsid-binding protein further includes the secretion signal peptide of PGLYRP2.

[0031] In a specific embodiment of the present invention, the HBV nucleocapsid-binding protein satisfies one or more of the following conditions:

[0032] (1) The HBV nucleocapsid binding domain contains an amino acid sequence as shown in SEQ ID NO:5, or the amino acid sequence contained in the nucleocapsid binding domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:5.

[0033] (2) The secretion signal peptide comprises the amino acid sequence shown in SEQ ID NO:4, or the amino acid sequence comprising the secretion signal peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:4; and,

[0034] (3) The HBV nucleocapsid binding domain is connected to the C-terminus of the secretion signal peptide.

[0035] In a specific embodiment of the present invention, the amino acid sequence of the HBV nucleocapsid binding domain is as shown in SEQ ID NO:5, or the amino acid sequence of the nucleocapsid binding domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:5.

[0036] In a specific embodiment of the present invention, the amino acid sequence of the secretory signal peptide is as shown in SEQ ID NO:4, or the amino acid sequence of the secretory signal peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:4.

[0037] In a specific embodiment of the present invention, the other functional protein is PGLYRP2.

[0038] In a specific embodiment of the present invention, the amino acid sequence of PGLYRP2 is shown as the sequence with NCBI Reference Sequence NP_443122.3.

[0039] To solve the above-mentioned technical problems, the fourth technical solution provided by the present invention is: an isolated nucleic acid, wherein the nucleic acid encodes the disordered region as described in the first technical solution, the fusion protein as described in the second technical solution, or the composition as described in the third technical solution.

[0040] In a specific embodiment of the present invention, the nucleic acid satisfies one or more of the following conditions:

[0041] (1) The nucleic acid also contains a promoter;

[0042] (2) The nucleic acid also contains a negative regulatory element of the PGLYRP2 protein; and,

[0043] (3) The nucleic acid also contains IRES.

[0044] In a specific embodiment of the present invention, the promoter comprises a nucleotide sequence as shown in SEQ ID NO:6, or the nucleotide sequence comprised by the promoter has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:6.

[0045] In a specific embodiment of the present invention, the nucleotide sequence of the promoter is as shown in SEQ ID NO:6, or the nucleotide sequence of the promoter has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:6.

[0046] In a specific embodiment of the present invention, the negative regulatory element comprises a nucleotide sequence as shown in SEQ ID NO:7, or the nucleotide sequence comprising the negative regulatory element has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:7.

[0047] In a specific embodiment of the present invention, the nucleotide sequence of the negative regulatory element is as shown in SEQ ID NO:7, or the nucleotide sequence of the negative regulatory element has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:7.

[0048] In a specific embodiment of the present invention, the nucleic acid encoding the composition is linked via the IRES to the nucleotide sequence encoding the fusion protein and the nucleotide sequence encoding the other functional protein.

[0049] In a specific embodiment of the present invention, the nucleic acid from the 5' end to the 3' end consists of: a nucleotide sequence encoding the fusion protein, IRES, and a nucleotide sequence encoding the other functional proteins.

[0050] As illustrated in the embodiments of the present invention, the fusion protein PGLYRP2IDR-(209~377)-NLS is combined with the secretory signal peptide of PGLYRP2 protein (PGLYRP2). SP) and HBV nucleocapsid binding domain (PGLYRP2) PGRP The fusion protein SP-PGLYRP2 formed by combining ) PGRP The two fusion proteins are co-expressed via an IRES (Internal Ribosome Entry Site) sequence. This design not only efficiently captures and isolates HBV DNA but also promotes the secretion of the HBV nucleocapsid while preventing viral entry into the nucleus. Through synergistic action of multiple mechanisms, the two fusion proteins significantly improve the efficiency of hepatocyte clearance of HBV, providing an innovative and efficient strategy for gene therapy of hepatitis B.

[0051] To solve the above-mentioned technical problems, the fifth technical solution provided by the present invention is: a recombinant expression vector, wherein the recombinant expression vector comprises the nucleic acid as described in the fourth technical solution.

[0052] In a specific embodiment of the present invention, the backbone of the recombinant expression vector is an adeno-associated virus vector, an adenovirus vector, a lentiviral vector, or a retroviral vector.

[0053] In a specific embodiment of the present invention, the adeno-associated virus vector is an adeno-associated virus vector of type AAV2, AAV5, AAV7, AAV8, AAV9 or AAV-DJ.

[0054] In a specific embodiment of the present invention, the backbone of the recombinant expression vector is pAAV-EnII-EnCMV-M.

[0055] To solve the above-mentioned technical problems, the sixth technical solution provided by the present invention is: a transformant, wherein the transformant comprises the nucleic acid as described in the fourth technical solution or the recombinant expression vector as described in the fifth technical solution; the transformant is a non-plant or non-animal variety.

[0056] In a specific embodiment of the present invention, the host cell of the transformant is a eukaryotic cell or a prokaryotic cell.

[0057] In a specific embodiment of the present invention, the prokaryotic cell is Escherichia coli; and / or, the eukaryotic cell is HEK 293T cell.

[0058] To solve the above-mentioned technical problems, the seventh technical solution provided by the present invention is: a method for preparing the disordered region as described in the first technical solution, the fusion protein as described in the second technical solution, the composition as described in the third technical solution, or the recombinant expression vector as described in the fifth technical solution, the method comprising culturing the transformant as described in the sixth technical solution and isolating the target product from the culture.

[0059] To solve the above-mentioned technical problems, the eighth technical solution provided by the present invention is: a pharmaceutical composition comprising a disordered region as described in the first technical solution, a fusion protein as described in the second technical solution, a composition as described in the third technical solution, a nucleic acid as described in the fourth technical solution, a recombinant expression vector as described in the fifth technical solution or a transformant as described in the sixth technical solution, and a pharmaceutically acceptable vector.

[0060] To solve the above-mentioned technical problems, the ninth technical solution provided by the present invention is: a method for mediating liquid-liquid phase separation, the method including the use of disordered regions as described in one of the technical solutions.

[0061] In a specific embodiment of the present invention, the method includes linking the disordered region to a target protein to be phase-separated; and / or, the method is for non-diagnostic and / or therapeutic purposes.

[0062] The disordered regions of this invention can be linked to any target protein to be separated to achieve the technical effect of mediating liquid-liquid phase separation, as has been verified in Examples 1 and 2 of this application.

[0063] To solve the above-mentioned technical problems, the tenth technical solution provided by the present invention is: a method for isolating and / or capturing DNA or inhibiting DNA transcription in vivo or in vitro, the method comprising contacting the DNA with the disordered region as described in the first technical solution, the fusion protein as described in the second technical solution, or the composition as described in the third technical solution.

[0064] In a specific embodiment of the present invention, the method is not for diagnostic and / or therapeutic purposes; and / or, the DNA is hepatitis B virus DNA.

[0065] The HBV DNA-binding domain of the PGLYRP2 protein is PGLYRP2. 209-377 It recognizes and binds to the Enhancer II region of the HBV DNA core promoter. This HBV Enhancer II region is directly involved in regulating HBV transcription and replication, which are essential for viral survival. Therefore, it is highly conserved in genotypes A-J of hepatitis B virus (J Gen Virol, 2001 Mar; 82(Pt 3):531-535. doi:10.1099 / 0022-1317-82-3-531.).

[0066] In a specific embodiment of the present invention, the hepatitis B virus is selected from any one or more of the hepatitis B virus genotypes A to J (for example, hepatitis B virus genotype D).

[0067] To solve the above-mentioned technical problems, the eleventh technical solution provided by the present invention is: a method for inhibiting hepatitis B virus in vivo or in vitro, the method comprising using the disordered region as described in technical solution one, the fusion protein as described in technical solution two, the composition as described in technical solution three, the nucleic acid as described in technical solution four, the recombinant expression vector as described in technical solution five, the transformant as described in technical solution six, or the pharmaceutical composition as described in technical solution eight.

[0068] In a specific embodiment of the present invention, the method is not for diagnostic and / or therapeutic purposes; and / or, the hepatitis B virus is selected from any one or more of the A to J genotype hepatitis B viruses (e.g., the D genotype hepatitis B virus).

[0069] To solve the above-mentioned technical problems, the twelfth technical solution provided by the present invention is: the use of the disordered region as described in technical solution one, the fusion protein as described in technical solution two, the composition as described in technical solution three, the nucleic acid as described in technical solution four, the recombinant expression vector as described in technical solution five, the transformant as described in technical solution six, or the pharmaceutical composition as described in technical solution eight in the preparation of a drug for the prevention and / or treatment of diseases related to hepatitis B virus infection.

[0070] In a specific embodiment of the present invention, the disease is acute or chronic hepatitis B, cirrhosis, liver cancer, liver fibrosis, hepatic encephalopathy, extrahepatic complications, or hepatic diabetes; and / or, the hepatitis B virus is selected from any one or more of genotype A to J hepatitis B viruses (e.g., genotype D hepatitis B virus).

[0071] To address the aforementioned technical problems, the thirteenth technical solution provided by this invention is: a method for preventing and / or treating diseases related to hepatitis B virus infection, the method comprising administering to a subject in need an effective amount of the disordered region as described in technical solution one, the fusion protein as described in technical solution two, the composition as described in technical solution three, the nucleic acid as described in technical solution four, the recombinant expression vector as described in technical solution five, the transformant as described in technical solution six, or the pharmaceutical composition as described in technical solution eight.

[0072] In a specific embodiment of the present invention, the disease is acute or chronic hepatitis B, cirrhosis, liver cancer, liver fibrosis, hepatic encephalopathy, extrahepatic complications, or hepatic diabetes; and / or, the hepatitis B virus is selected from any one or more of genotype A to J hepatitis B viruses (e.g., genotype D hepatitis B virus).

[0073] To solve the above-mentioned technical problems, the fourteenth technical solution provided by the present invention is: the disordered region as described in technical solution one, the fusion protein as described in technical solution two, the composition as described in technical solution three, the nucleic acid as described in technical solution four, the recombinant expression vector as described in technical solution five, the transformant as described in technical solution six, or the pharmaceutical composition as described in technical solution eight, which are used for the prevention and / or treatment of diseases related to hepatitis B virus infection.

[0074] In a specific embodiment of the present invention, the disease is acute or chronic hepatitis B, cirrhosis, liver cancer, liver fibrosis, hepatic encephalopathy, extrahepatic complications, or hepatic diabetes; and / or, the hepatitis B virus is selected from any one or more of genotype A to J hepatitis B viruses (e.g., genotype D hepatitis B virus).

[0075] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0076] The reagents and raw materials used in this invention are all commercially available.

[0077] The positive and progressive effects of this invention are as follows:

[0078] 1. Effectively blocks HBV replication:

[0079] By binding to the HBV DNA binding domain for specific HBV DNA recognition and PGLYRP2 IDR By leveraging the aggregation effect, this invention overcomes the technical challenges of existing gene therapy drugs in viral recognition and clearance. Through PGLYRP2... IDR The phase separation effect and the precise capture capability of the HBV DNA binding domain significantly improve the binding and isolation efficiency of HBV DNA, thereby blocking the viral replication process.

[0080] 2. Innovative treatment mechanism:

[0081] This invention is the first to apply the liquid-liquid phase separation (LLPS) mechanism to gene therapy for HBV, forming a novel antiviral treatment strategy that overcomes the limitations of traditional treatment methods. By combining PGLYRP2... IDR By combining the liquid-liquid phase separation (LLPS) properties of HBV with the precise targeting capability of HBV-DBD, an innovative gene therapy drug has been developed to efficiently inhibit HBV replication and promote viral clearance. This invention overcomes the limitations of existing technologies, providing an innovative, efficient, and safe gene therapy method for the treatment of hepatitis B, with broad clinical application value and commercial potential. Attached Figure Description

[0082] Figure 1 shows the disordered region of the PGLYRP2 protein predicted by PONDR.

[0083] Figure 2 shows PGLYRP2 labeled with DsRed. IDR Expression and phase separation analysis.

[0084] Figure 3 shows the expression and phase separation analysis of DsRed-labeled PGLYRP2IDR / 209-377 and its FAM-Enh II colocalization analysis with HBV DNA.

[0085] Figure 4 shows the dynamic changes of fluorescently labeled HBV DNA FAM-Enh II in PGLYRP2IDR / 209-377 aggregates as analyzed by fluorescence recovery photobleaching.

[0086] Figure 5 shows the quantitative analysis of FRAP fluorescence intensity.

[0087] Figure 6 shows PGLYRP2 IDR The mediated phase separation model serves as a platform for capturing HBV DNA.

[0088] Figure 7 is a schematic diagram of the full-length PGLYRP2 fusion protein with different functional domains.

[0089] Figure 8 shows the HBV DNA level in the HepAD38 cell line, which stably expresses the full-length PGLYRP2 fusion protein with different functional domains, analyzed by real-time quantitative PCR.

[0090] Figure 9 shows the HBsAg level in the supernatant of HepAD38 cell lines that stably express full-length PGLYRP2 fusion proteins with different functional domains, as detected by ELISA.

[0091] Figure 10 shows the disordered regions of PGLYRP2 predicted by the AIUPred tool (https: / / iupred2a.elte.hu / ).

[0092] Figure 11 is a schematic diagram of the mouse modeling and treatment process.

[0093] Figure 12 shows the results of ELISA detection of HBsAg levels in serum.

[0094] Figure 13 shows the results of RT-PCR detection of HBV cccDNA expression levels in serum.

[0095] Figure 14 shows the results of RT-PCR detection of HBV rcDNA expression levels in serum. Detailed Implementation

[0096] This invention proposes an innovative gene therapy drug based on the intrinsically disordered region (IDR) of the PGLYRP2 protein, aiming to enhance the ability to clear hepatitis B virus (HBV), belonging to the field of biomedical technology. This invention utilizes PGLYRP2... IDR Its unique structure and functional properties, through mediating liquid-liquid phase separation (LLPS), synergize with the HBV DNA binding domain (PGLYRP2) of the PGLYRP2 protein. 209-377 ) and Nuclear Localization Signal (PGLYRP2) functional domain NLS It can efficiently capture and isolate HBV DNA, thereby significantly inhibiting viral replication and improving the efficiency of hepatocytes in clearing the virus.

[0097] Unless otherwise defined herein, the scientific and technical terms used in this application will have the meanings commonly understood by one of ordinary skill in the art. Generally, the nomenclature and techniques used herein in relation to chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry are those well-known and frequently used in the art.

[0098] All publications, patents, and published patent applications cited in this application are expressly incorporated herein by reference. In case of conflict, this specification (including its specific definitions) shall prevail.

[0099] Each embodiment of the invention described herein can be used alone or in combination with one or more other embodiments of the invention.

[0100] Unless otherwise stated, the following definitions are provided for the specific terms used in the above written description.

[0101] In this invention, the letters in the amino acid sequence represent single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0102] Those skilled in the art can perform sequence alignment using techniques known in the art, such as publicly available software like BLAST, BLAST2, or Align. See, for example, Altschul et al., Methods in Enzymology 266:460-480 (1996); Pearson et al., Genomics-46:24-36, 1997, and the alignment program at molbiol.soton.ac.uk / compute / align. The percentage sequence identity according to the invention can be determined using these programs and their default settings. More generally, those skilled in the art can readily determine suitable parameters for determining the alignment, including any algorithm required to achieve maximum alignment over the full-length sequences being compared.

[0103] In this invention, nucleic acid refers to a nucleotide chain of any length and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate capable of being incorporated into the chain by DNA or RNA polymerases.

[0104] The recombinant expression vector of the present invention can be any suitable recombinant expression vector that can be used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably express the genes or sequences in the host cell.

[0105] In this invention, the pharmaceutical composition may comprise a suitable pharmaceutically acceptable carrier, such as pharmaceutical excipients, including buffers, as known in the art. "Pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextran, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions comprising the invention can be prepared by mixing the proteins or polypeptides of the invention, having the desired purity, with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Preferably, the composition is in the form of a lyophilized formulation or an aqueous solution.

[0106] In this invention, "non-diagnostic and / or therapeutic" applications include, but are not limited to, research in the laboratory for scientific purposes, such as in vitro mediating liquid-liquid phase separation, isolating and / or capturing DNA, inhibiting DNA transcription or inhibiting hepatitis B virus, or as a positive control to screen other small molecule drugs, proteins or peptides that can mediate liquid-liquid phase separation, isolating and / or capturing DNA, inhibiting DNA transcription or inhibiting hepatitis B virus.

[0107] As is known in this art, diseases associated with hepatitis B virus infection include the following: Acute or chronic hepatitis B: Acute hepatitis B is liver inflammation caused by the hepatitis B virus that lasts from several weeks to 6 months; chronic hepatitis B is defined as hepatitis B infection lasting more than 6 months and persistently positive for hepatitis B surface antigen in the serum. Cirrhosis: Long-term hepatocellular damage and inflammation can lead to liver fibrosis. Over time, these fibrous tissues gradually replace normal hepatocellular cells, leading to cirrhosis. Liver cancer: Primary liver cancer may develop on the basis of cirrhosis or chronic hepatitis. Liver fibrosis: Hepatitis B virus-related chronic liver diseases include a series of diseases that progress from hepatitis to liver fibrosis, cirrhosis, and even liver cancer. Hepatic encephalopathy: A neuropsychiatric syndrome caused by liver dysfunction, which can occur in severe hepatitis and cirrhosis. Extrahepatic complications: Hepatitis B virus infection can also cause a variety of extrahepatic diseases, such as skin diseases and arthritis. Hepatic diabetes mellitus: Clinical manifestations are similar to type 2 diabetes mellitus, the difference being that hepatic diabetes mellitus presents with significantly elevated fasting insulin levels while C-peptide levels are normal. This application has demonstrated PGLYRP2 IDR The functional domains enhance the hepatitis B virus clearance effect of PGLYRP2-related gene therapy drugs. Based on this, those skilled in the art can reasonably expect that the protein or polypeptide of the present invention has the potential to effectively prevent and / or treat diseases related to hepatitis B virus infection.

[0108] In this invention, the term "effective amount" refers to the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of a disease or condition, compared to a corresponding subject who did not receive that amount. Within its scope, the term also includes amounts that effectively enhance normal physiological function.

[0109] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0110] The sequences of the proteins or polypeptides involved in this invention are as follows:

[0111] Full-length sequence of PGLYRP2 protein:

[0112] NCBI Reference Sequence:NP_443122.3

[0113] The disordered region of the PGLYRP2 protein (PGLYRP2) IDR The amino acid sequence of ) (SEQ ID NO:1):

[0114] HBV DNA binding domain of PGLYRP2 (PGLYRP2) 209-377 The amino acid sequence of ) (SEQ ID NO:2):

[0115] The amino acid sequence of the nuclear localization signal NLS of PGLYRP2 (SEQ ID NO:3):

[0116] The amino acid sequence of the secretion signal peptide SP of PGLYRP2 (SEQ ID NO:4):

[0117] The nucleocapsid binding domain of PGLYRP2 PGRP The amino acid sequence (SEQ ID NO:5):

[0118] Nucleotide sequence of the truncated M of the PGLYRP2 gene promoter (SEQ ID NO:6):

[0119] The nucleotide sequence of EnII, the negative regulatory element of the PGLYRP2 protein (SEQ ID NO:7):

[0120] Example 1: Analysis and identification of disordered regions of PGLYRP2 protein

[0121] Currently, there are various IDR sequence prediction algorithms and tools in this field. Different algorithms or tools can produce significant differences in the IDR sequence prediction of the same protein (for example, when using the AIUPred tool (https: / / iupred2a.elte.hu / ) to predict the IDR sequence of PGLYRP2, the IUPred2(long) algorithm is similar to the IDR predicted by the PONDR tool, but the IDR obtained by the ANCHOR2 algorithm is quite different from that in Figure 1 (see Figure 10)). After obtaining the prediction results using existing prediction tools, inventors still need to put in creative effort to analyze and verify the results in order to confirm the technical effect. That is, they need to explore and select the IDR sequence with better function in mediating liquid-liquid phase separation through experiments, and explore whether the IDR sequence may have other application value and development prospects that have not been revealed by existing technologies.

[0122] After comparison and analysis, this invention ultimately selected the PONDR (Predictor of Natural Disordered Regions, https: / / www.pondr.com / ) tool to predict the intrinsically disordered regions (IDRs) in the PGLYRP2 protein. The inventors conducted in-depth research, analysis, and screening of the predicted fragments, ultimately defining the 164-208 amino acid sequence as the disordered region of the PGLYRP2 protein. IDR (Figure 1), its sequence is shown in SEQ ID NO:1.

[0123] Subsequently, the present invention expressed DsRed-PGLYRP2 in vitro in Escherichia coli. IDR DsRed-PGLYRP2 was obtained after purification by Ni chelation chromatography and molecular sieve. IDR Protein. Analysis of DsRed-PGLYRP2 using confocal microscopy. IDR The protein expression and phase separation are shown in Figure 2. (DsRed-PGLYRP2) IDR The protein forms aggregates in vitro, indicating the presence of disordered regions in the PGLYRP2 protein and its phase separation function.

[0124] Example 2: PGLYRP2 IDR Analysis of the role of mediated phase separation in the isolation and capture of HBV DNA

[0125] In this invention, the DsRed-PGLYRP2IDR-209-377 protein was expressed in vitro in Escherichia coli. After purification by Ni chelation chromatography and molecular sieve, the DsRed-PGLYRP2IDR-209-377 protein was obtained. Then, the DsRed-PGLYRP2IDR-209-377 protein was incubated with FAM-Enh II of HBV DNA fragment (FAM-labeled HBV DNA Enh II). The co-localization was analyzed by confocal microscopy (Figure 3). The results showed that the DsRed-PGLYRP2IDR-209-377 protein and the FAM-Enh II of HBV DNA fragment had a clear co-localization.

[0126] Next, this invention utilizes fluorescence recovery after photobleaching (FRAP) technology to analyze the dynamic changes of HBV DNA FAM-Enh II in the PGLYRP2IDR / 209-377 aggregate (Figure 4), and quantitative analysis of FRAP fluorescence intensity (Figure 5). The results show that the HBV DNA FAM-Enh II fluorescence signal in the photobleached region gradually recovers, indicating that there is dynamic exchange of HBV DNA in the PGLYRP2IDR-209-377 aggregate.

[0127] These results further confirm the effective isolation and capture of HBV DNA by the PGLYRP2IDR / 209-377-mediated phase separation process. This invention summarizes the PGLYRP2... IDR The phase separation properties of PGLYRP2 protein facilitate the precise capture and isolation of HBV DNA (Figure 6).

[0128] Example 3: PGLYRP2 IDR Analysis of the hepatitis B virus clearance effect of gene therapy drugs with enhanced functional domain PGLYRP2

[0129] This invention relates to the full length of PGLYRP2, PGLYRP2ΔIDR, and whether or not PGLYRP2 is present. IDRThe gene coding sequences of the fusion proteins with different functional domains (see Figure 7 for a schematic diagram) were inserted into the pAAV-EnII-EnCMV-M(rAAV2 / 8) vector (i.e., the gene therapy delivery vector pAAV-EnII-EnCMV-M described in Example 1 of patent application publication number CN117904201A, which contains a truncated PGLYRP2 gene promoter M and the PGLYRP2 protein negative regulatory element EnII) according to the following adeno-associated virus gene therapy vector preparation method). Adeno-associated virus packaging was completed in HEK 293T cells, and the AAV virus was concentrated by ultracentrifugation.

[0130] (1) pAAV-EnII-EnCMV-M(rAAV2 / 8) was double-digested with restriction endonucleases ClaI and BamHI and ligated with the PGLYRP2IDR-(209~377)-NLS fragment, and transformed to obtain the rAAV2 / 8-PGLYRP2IDR-(209~377)-NLS recombinant;

[0131] (2) The rAAV2 / 8-PGLYRP2IDR-(209~377)-NLS recombinant was double-digested with restriction endonucleases BamHI and SalI and ligated with the IRES fragment. After transformation, the rAAV2 / 8-PGLYRP2IDR-(209~377)-NLS-IRES recombinant was obtained.

[0132] (3) The rAAV2 / 8-PGLYRP2IDR-(209~377)-NLS-IRES recombinant was then double-digested with restriction endonucleases SalI and BglII and ligated with SP-PGLYRP2 PGRP The fragment was converted to obtain rAAV2 / 8-PGLYRP2IDR-(209~377)-NLS-IRES-SP-PGLYRP2 PGRP Recombinant.

[0133] Subsequently, HepAD38 cells (human hepatocellular carcinoma cells expressing hepatitis B virus) were infected with the obtained AAV virus and cultured in Tet-off medium for 9 days after infection. The HBV DNA level in the cells was detected by real-time quantitative PCR (Figure 8), and the HBsAg level in the cell culture supernatant was detected by ELISA (Figure 9).

[0134] Experimental results show that, compared to the full-length AAV / PGLYRP2, the deletion of PGLYRP2... IDR The AAV / PGLYRP2ΔIDR's ability to clear HBV virus was significantly reduced. Furthermore, PGLYRP2... IDRThe functional domains all showed a promoting effect on the HBV clearance activity of the following proteins:

[0135] (1) PGLYRP2, which has HBV replication inhibitory effects 209-377 And PGLYRP2209-377-NLS;

[0136] (2) PGLYRP2 has the function of synergistically inhibiting HBV replication and preventing HBV nuclear entry. 209-377 -NLS-IRES-SP-PGLYRP2 PGRP .

[0137] These results further validate PGLYRP2. IDR The functional domain plays a key role in enhancing the hepatitis B virus clearance effect of PGLYRP2-related gene therapy drugs.

[0138] Example 4: Analysis of the hepatitis B virus clearance effect of PGLYRP2-related gene therapy drugs that enhance the PGLYRP2 IDR functional domain in mice

[0139] Experimental procedure:

[0140] First, C57BL / 6J mice were injected with AAV8-1.2HBV (dose 1×10⁻⁶). 10 Mice were administered HBV cccDNA levels (vg / mouse) for 5 weeks, and then randomly divided into two groups, receiving either PBS (blank control, n=5) or AAV2 / 8-CMV-IDR-PGLYRP2. 209-377 Intervention was performed using NLS-IRES-PGLYRP2 (vector backbone is the same as the pAAV-EnII-EnCMV-M(rAAV2 / 8) vector in Example 3, n=6). During the treatment period, blood was collected weekly from the mandibular vein of mice, and the expression levels of HBV cccDNA and rcDNA in the serum were detected by RT-PCR technology. At the same time, the HBsAg level was detected by ELISA. The specific modeling and treatment process is shown in Figure 11.

[0141] Experimental results: The test data of the treatment are shown in Figures 12-14:

[0142] 1. Serum HBsAg level: At week 9 of treatment, the AAV treatment group (AAV-CMV-IDR-PGLYRP2) 209-377 The expression level of HBsAg in the -NLS-IRES-PGLYRP2 group was significantly lower than that in the control group (PBS, blank control group);

[0143] 2. Serum HBV cccDNA level: At week 9 of treatment, the AAV treatment group (AAV-CMV-IDR-PGLYRP2) 209-377 The cccDNA expression level in the -NLS-IRES-PGLYRP2 group was significantly lower than that in the control group (PBS, blank control group), with the copy number decreasing from the initial 10. 7 / μL decreased to 10 6 / μL;

[0144] 3. Serum HBV rcDNA level: At week 9 of treatment, the AAV treatment group (AAV-CMV-IDR-PGLYRP2) 209- 377 The rcDNA expression level in the -NLS-IRES-PGLYRP2 group was significantly lower than that in the control group (PBS, blank control group), with the copy number decreasing from approximately 5 × 10⁻⁶ at the beginning. 9 / mL decreased to 1×10 8 / mL.

[0145] The above results suggest that by revealing and effectively utilizing the phase-separation ability of IDR sequences, their binding sequence with DNA (PGLYRP2) can be effectively linked. 209-377 By fusing the nuclear localization signal (NLS) with the PGLYRP2 IDR-(209~377)-NLS, the synergistic effect of the three can be fully utilized; this strategy can realize the core function of PGLYRP2 IDR-(209~377)-NLS and SP-PGLYRP2 PGRP The cytoplasmic functions are separate and do not interfere with each other—the latter acts as a nucleocapsid regulator in the cytoplasm, mainly playing a role in preventing viruses from entering the nucleus.

Claims

1. A disordered region of a PGLYRP2 protein, characterized in that, The disordered region contains an amino acid sequence as shown in SEQ ID NO:1; or the amino acid sequence contained in the disordered region has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO:

1.

2. A fusion protein, characterized in that, The fusion protein includes the disordered region as described in claim 1; preferably, the disordered region is located at the N-terminus of the fusion protein; Preferably, the fusion protein further comprises an HBV DNA binding domain, more preferably an HBV DNA binding domain of the PGLYRP2 protein; more preferably, it further comprises a nuclear localization signal region of the PGLYRP2 protein; More preferably, the fusion protein satisfies one or more of the following conditions: (1) The HBV DNA binding domain comprises an amino acid sequence as shown in SEQ ID NO:2, or the amino acid sequence comprising the HBV DNA binding domain has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO:

2. (2) The nuclear localization signal region contains the amino acid sequence shown in SEQ ID NO:3, or the amino acid sequence contained in the nuclear localization signal region has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO:3; and, (3) The fusion protein consists of the following components from the N-terminus to the C-terminus: the disordered region, the HBV DNA binding domain, and the nuclear localization signal region.

3. A protein composition, characterized in that, The composition comprises the disordered region as described in claim 1 or the fusion protein as described in claim 2, as well as other functional proteins; Preferably, the other functional protein includes an HBV nucleocapsid-binding protein, and more preferably, the HBV nucleocapsid-binding protein includes the HBV nucleocapsid-binding domain of PGLYRP2; more preferably, the HBV nucleocapsid-binding protein also includes a secretion signal peptide of PGLYRP2; and even more preferably, the other functional protein is PGLYRP2. More preferably, the HBV nucleocapsid-binding protein or the PGLYRP2 satisfies one or more of the following conditions: (1) The HBV nucleocapsid binding domain contains an amino acid sequence as shown in SEQ ID NO:5, or the amino acid sequence contained in the HBV nucleocapsid binding domain has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO:

5. (2) The secretion signal peptide comprises an amino acid sequence as shown in SEQ ID NO:4, or the amino acid sequence comprising the secretion signal peptide has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO:

4. (3) The HBV nucleocapsid binding domain is connected to the C-terminus of the secretion signal peptide; and, (4) The amino acid sequence of PGLYRP2 is shown in the NCBI Reference Sequence NP_443122.

3.

4. An isolated nucleic acid, characterized in that, The nucleic acid encodes the disordered region as described in claim 1, the fusion protein as described in claim 2, or the composition as described in claim 3; Preferably, the nucleic acid satisfies one or more of the following conditions: (1) The nucleic acid further comprises a promoter, preferably the promoter comprises a nucleotide sequence as shown in SEQ ID NO:6, or the nucleotide sequence comprising the promoter has at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity with SEQ ID NO:6; (2) The nucleic acid further comprises a negative regulatory element of the PGLYRP2 protein, preferably the negative regulatory element comprising a nucleotide sequence as shown in SEQ ID NO:7, or the nucleotide sequence comprising the negative regulatory element having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO:7; and, (3) The nucleic acid further comprises an IRES, preferably the nucleic acid encoding the composition connects the nucleotide sequence encoding the fusion protein to the nucleotide sequence encoding the other functional protein through the IRES, more preferably the nucleic acid from the 5' end to the 3' end is: the nucleotide sequence encoding the fusion protein, the IRES and the nucleotide sequence encoding the other functional protein.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 4; Preferably, the backbone of the recombinant expression vector is an adeno-associated virus vector, an adenovirus vector, a lentiviral vector, or a retroviral vector. More preferably, the adeno-associated virus vector is an adeno-associated virus vector of type AAV2, AAV5, AAV7, AAV8, AAV9, or AAV-DJ; More preferably, the backbone of the recombinant expression vector is pAAV-EnII-EnCMV-M.

6. A transformant, characterized in that, The transformant comprises the nucleic acid as described in claim 4 or the recombinant expression vector as described in claim 5; the transformant is a non-plant or non-animal species. Preferably, the host cell of the transformant is a eukaryotic cell or a prokaryotic cell; More preferably, the prokaryotic cell is Escherichia coli; and / or, the eukaryotic cell is HEK 293T cell.

7. A method for preparing the disordered region as described in claim 1, the fusion protein as described in claim 2, the composition as described in claim 3, or the recombinant expression vector as described in claim 5, characterized in that, The method includes culturing the transformant as described in claim 6 and isolating the target product from the culture.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the disordered region of claim 1, the fusion protein of claim 2, the composition of claim 3, the nucleic acid of claim 4, the recombinant expression vector of claim 5, or the transformant of claim 6, and a pharmaceutically acceptable vector.

9. A method for mediating liquid-liquid phase separation, characterized in that, The method includes using the disordered region as described in claim 1; Preferably, the method includes linking the disordered region to a target protein to be phase-separated; and / or, the method is for non-diagnostic and / or therapeutic purposes.

10. A method for isolating and / or capturing DNA or inhibiting DNA transcription in vivo or in vitro, characterized in that, The method includes contacting the DNA with the disordered region as described in claim 1, the fusion protein as described in claim 2, or the composition as described in claim 3. Preferably, the method is not for diagnostic and / or therapeutic purposes; and / or, the DNA is hepatitis B virus DNA.

11. A method for inhibiting hepatitis B virus in vivo or in vitro, characterized in that, The method includes using the disordered region as described in claim 1, the fusion protein as described in claim 2, the composition as described in claim 3, the nucleic acid as described in claim 4, the recombinant expression vector as described in claim 5, the transformant as described in claim 6, or the pharmaceutical composition as described in claim 8; Preferably, the method is for non-diagnostic and / or therapeutic purposes.

12. The use of the disordered region of claim 1, the fusion protein of claim 2, the composition of claim 3, the nucleic acid of claim 4, the recombinant expression vector of claim 5, the transformant of claim 6, or the pharmaceutical composition of claim 8 in the preparation of a medicament for the prevention and / or treatment of diseases associated with hepatitis B virus infection; Preferably, the disease is acute or chronic hepatitis B, cirrhosis, liver cancer, liver fibrosis, hepatic encephalopathy, extrahepatic complications, or hepatic diabetes.

13. A method for preventing and / or treating diseases associated with hepatitis B virus infection, characterized in that, The method includes administering to a subject in need an effective amount of the disordered region of claim 1, the fusion protein of claim 2, the composition of claim 3, the nucleic acid of claim 4, the recombinant expression vector of claim 5, the transformant of claim 6, or the pharmaceutical composition of claim 8; Preferably, the disease is acute or chronic hepatitis B, cirrhosis, liver cancer, liver fibrosis, hepatic encephalopathy, extrahepatic complications, or hepatic diabetes.

14. The disordered region of claim 1, the fusion protein of claim 2, the composition of claim 3, the nucleic acid of claim 4, the recombinant expression vector of claim 5, the transformant of claim 6, or the pharmaceutical composition of claim 8, for the prevention and / or treatment of diseases associated with hepatitis B virus infection; Preferably, the disease is acute or chronic hepatitis B, cirrhosis, liver cancer, liver fibrosis, hepatic encephalopathy, extrahepatic complications, or hepatic diabetes.

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