Application of miRNA (micro Ribonucleic Acid) gene cluster in preparation of medicine for treating hepatic fibrosis

Abstract

The invention discloses an application of a miRNA (micro Ribonucleic Acid) gene cluster in preparation of a medicine for treating hepatic fibrosis, which comprises the following steps: constructing an expression cassette by taking human miR-23b-27b-24-1 and a flanking sequence as core sequences, transferring the expression cassette into an adeno-associated virus vector to construct a recombinant adeno-associated virus vector, packaging the vector into a virus, and reducing a hepatic fibrosis target gene Col1a1, the compound can be used for preparing drugs for treating hepatic fibrosis and drugs for inhibiting expression of hepatic fibrosis target genes and / or target proteins. The recombinant adeno-associated virus is constructed by using human miR-23b-27b-24-1 and intercepting part of flanking sequences, and the recombinant adeno-associated virus is more suitable for preparing medicines for treating hepatic fibrosis diseases, and is higher in safety coefficient and more remarkable in effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology, and particularly relates to the application of a miRNA gene cluster in the preparation of a medicament for treating liver fibrosis. Background Art

[0002] Hepatic fibrosis refers to the excessive deposition of extracellular matrix (especially collagen) diffusely in the liver. It is not an independent disease, but many chronic liver diseases can cause hepatic fibrosis. Its etiologies can be roughly divided into infectious (chronic hepatitis B, C and D virus hepatitis, schistosomiasis, etc.), congenital metabolic defects (Wilson's disease, hemochromatosis, α1-antitrypsin deficiency, etc.) and chemical metabolic defects (chronic alcoholic liver disease, chronic drug-induced liver disease), as well as autoimmune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis, etc. In addition, various inflammatory factors and oxidative stress also continuously promote the process of hepatic fibrosis. If early hepatic fibrosis is not treated, it will eventually lead to cirrhosis and even liver cancer.

[0003] MicroRNA (miRNA) is a group of endogenous non-coding RNAs that regulate gene expression. miRNA can inhibit the expression of target genes by regulating transcription or post-transcriptional translation. Changes in miRNA expression lead to changes in genes involved in a series of biological processes, which can cause many human diseases. Therefore, miRNA has become a potential target for the treatment of many diseases. And due to its high stability in body fluids, circulating miRNA is considered a promising biomarker for diagnosis and disease prognosis.

[0004] The miR-23b-27b-24-1 gene cluster includes miR-23b, miR27b, and miR24-1, which are located within 881 bp on chromosome 9. The previous research by the Wu Jiangfeng team showed that the miR-23b-27b-24-1 gene cluster directly targets and downregulates the expression of several profibrotic genes (TGFβ2, Gremlin1, LOX, ITGα2, ITGα5). Mechanistically, the miR-23b-27b-24-1 cluster directly targets messenger RNA, thereby reducing the protein expression of five secreted profibrotic genes (TGF-b2, Gremlin1, LOX, Itga2, and Itga5) in HSCs. By downregulating TGF-b2, Itga2, and Itga5, the TGF-b signaling pathway is inhibited, and by inhibiting Gremlin1, the bone morphogenetic protein signaling pathway is activated, thereby reducing the extracellular matrix secretion of HSCs. miR-23b-27b-24-1 plays an antifibrotic role by targeting and downregulating the expression of five liver fibrosis-related genes, TGFβ2, Gremlin1, ITGα2, ITGα5, and LOX, in hepatic stellate cells, thereby inhibiting the activation of hepatic stellate cells.

[0005] With the increasing research on miRNAs in liver fibrosis diseases, miRNAs have been proven to be one of the important means for the diagnosis and treatment of liver fibrosis. However, currently, when diagnosing and treating liver fibrosis with miR-23b-27b-24-1, a murine gene cluster is generally used, and only the simple miR-23b-27b-24-1 sequence is delivered into cells using an AAV viral vector for diagnosis and treatment. Although the murine miR-23b-27b-24-1 has good effects in mouse models, there are uncertainties in the diagnosis and treatment of human liver fibrosis disease models. The expression of murine miR-23b-27b-24-1 in human cells may be limited or there may be other adverse effects. In addition, how to improve the tissue specificity and safety of the delivery vector is also particularly important for the diagnosis and treatment of liver fibrosis. Summary of the Invention

[0006] To address the above technical problems, the present invention provides an application of a miRNA gene cluster in the preparation of a drug for treating liver fibrosis. An expression cassette is constructed with the human miR-23b-27b-24-1 + flanking sequence as the core sequence and transferred into an adeno-associated virus vector to construct a recombinant adeno-associated virus vector. This vector can downregulate liver fibrosis target genes and target proteins and is used for the preparation of a drug for treating liver fibrosis.

[0007] To achieve the above object, the present invention provides a miRNA gene cluster, and the miRNA gene cluster is wu-miR-02 or wu-Mir-02; the sequence of wu-miR-02 is SEQ ID NO:1, and the sequence of wu-Mir-02 is SEQ ID NO:2.

[0008] Preferably, the wu-miR-02 is human miR-23b-27b-24-1 containing flanking sequences; the wu-Mir-02 is murine miR-23b-27b-24-1 containing flanking sequences.

[0009] The present invention also provides an adeno-associated virus expression plasmid carrying a miRNA gene cluster, which contains an adeno-associated virus genome with a CMV promoter and the miRNA gene cluster.

[0010] Preferably, the miRNA gene cluster is wu-miR-02 or wu-Mir-02; the sequence of wu-miR-02 is SEQID NO:1, and the sequence of wu-Mir-02 is SEQ ID NO:2.

[0011] The present invention also provides a method for constructing an adeno-associated virus expression plasmid carrying a miRNA gene cluster, which includes the following steps: (1) Double-digest the pAAV-GFP plasmid with restriction endonucleases Hind Ⅲ and Xol Ⅰ to obtain a linearized vector; amplify wu-miR-02 or wu-Mir-02 and add restriction sites, and double-digest the amplified PCR fragment with HindⅢ and XolⅠ to obtain the wu-miR-02 or wu-Mir-02 gene fragment; (2) Use a homologous recombinase to ligate the wu-miR-02 or wu-Mir-02 gene fragment with the linearized vector, and transform and culture; (3) Verify by double digestion and sequencing to obtain the recombinant adeno-associated virus vector AAV-GFP-wu-miR-02 or AAV-GFP-wu Mir-02.

[0012] The present invention also provides a gene therapy drug based on an AAV vector, which includes: a recombinant adeno-associated virus, and the recombinant adeno-associated virus has an AAV capsid, an AAV vector genome containing a CMV promoter packaged in the AAV capsid, and a miRNA gene cluster.

[0013] Preferably, the miRNA gene cluster is wu-miR-02 or wu-Mir-02; the sequence of wu-miR-02 is the nucleotide sequence shown in SEQID NO:1; the sequence of wu-Mir-02 is the nucleotide sequence shown in SEQ ID NO:2.

[0014] The present invention also provides a preparation method of a gene therapy drug based on an AAV vector, comprising the following steps: (1) Mixing and infecting 293T cells with the recombinant adeno-associated virus vector containing the miRNA gene cluster, AAV-CMV-GFP plasmid, and pAdDeltaF6 plasmid; (2) Collecting and lysing the cells, and purifying with iodixanol to obtain rAAV-GFP-miRNA gene cluster and the gene therapy drug based on the AAV vector.

[0015] Preferably, the mass ratio of the recombinant adeno-associated virus vector containing the miRNA gene cluster, AAV-CMV-GFP plasmid, and pAdDeltaF6 plasmid in step (1) is 7:20:7.

[0016] Preferably, the recombinant adeno-associated virus vector containing the miRNA gene cluster is AAV-wu-miR-02 or AAV-wu-Mir-02.

[0017] The present invention also provides an application of the gene therapy drug based on the AAV vector, and the application is for the treatment of liver fibrosis.

[0018] The beneficial effects of the present invention are as follows: 1. Using the human miR-23b-27b-24-1 + flanking sequence as the core sequence, constructing an expression cassette with CMV as the promoter, and further constructing a recombinant adeno-associated virus vector with an AAV adeno-associated virus vector as the backbone. After packaging the recombinant vector into a recombinant adeno-associated virus, it can efficiently infect the LX-2 cell line in vitro, and at the same time has a high infectivity for the 293T cell line and T6 cell line.

[0019] 2. Transferring the recombinant adeno-associated virus containing the human miR-23b-27b-24-1 + flanking sequence into TGFβ1-stimulated LX-2 cells can significantly down-regulate the expression of liver fibrosis-related target genes TGFβ-2 and LOX their expression.

[0020] 3. When the recombinant adeno-associated virus containing the human miR-23b-27b-24-1 + flanking sequence is injected into the mouse liver fibrosis model through the tail vein, the recombinant adeno-associated virus has a tropism for liver tissue, making the liver surface of the liver fibrosis mouse model return to a smooth state without obvious fine granular nodules and sharp edges, and at the same time significantly reducing the stem cell vacuolization and necrosis, and reducing the fibrotic septum; in addition, it can also significantly reduce the expression of ALT and AST in the mouse serum, reduce LOX,[[]]END]] TGFβ-2Expression levels of genes, TIMP1 protein, and a-SMA protein.

[0021] 4. The present invention selects the adeno-associated virus vector AAV5, which enhances the delivery efficiency and safety of the recombinant adeno-associated virus, also has good liver tissue tropism, enhances the targeting of the drug to fibrotic liver tissue, and can further reduce the dosage of the drug for treating liver fibrosis and lower the cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a graph showing the detection results of fluorescence intensity in Example 2.

[0023] Figure 2 It is a bar graph showing the expression of miR-23b-27b-24-1 3p and miR-23b-27b-24-1 5p after transfection in Example 2. In the figure, A is miR-23b, B is miR-24-1, and C is miR-27b.

[0024] Figure 3 It shows the effect of the flanking sequence on the expression level of miR-23b-27b-24-1 in LX-2 cells in Example 3.

[0025] Figure 4 It shows the effect of the flanking sequence on the expression level of miR-23b-27b-24-1 in T6 cells in Example 3.

[0026] Figure 5 It is the fluorescence intensity after the infection of LX-2 cells with AAV-wu-miR-02 in Example 4.

[0027] Figure 6 It is the fluorescence intensity after the infection of HEK-293T cells with AAV-wu-miR-02 in Example 4.

[0028] Figure 7 It is a graph showing the expression of wu-miR-02 under the stimulation of TGFβ1 in Example 5.

[0029] Figure 8 It is a graph showing the distribution results of the virus in mouse organs in Example 6.

[0030] Figure 9 It is a gross view of the liver of the mouse in Example 7.

[0031] Figure 10 It is a graph showing the staining results of the liver tissue of the mouse in Example 7.

[0032] Figure 11 It is a bar graph showing the expression levels of serological detection indicators in Example 8.

[0033] Figure 12Bar chart of the expression levels of mature 3p and 5p in Example 9. In the figure, A is 23-b, B is 27-b, and C is 24-1.

[0034] Figure 13 Bar chart of the expression levels of mature 3p and 5p in experimental group 2 of Example 9. In the figure, A is 23-b, B is 27-b, and C is 24-1.

[0035] Figure 14 Bar chart of the expression levels of TIMP1 gene and α-SMA gene in Example 9.

[0036] Figure 15 Diagram of the expression of TIMP1 protein and α-SMA protein in Example 9. Detailed implementation methods

[0037] The technical solutions of the present invention will be further explained and illustrated below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the following embodiments are only the preferred embodiments of the present invention and should not be construed as limitations on the present invention. The protection scope of the present invention shall be subject to the content recorded in the claims. Modifications and substitutions made by those skilled in the art to the technical solutions of the present invention without creative efforts all fall within the protection scope of the present invention.

[0038] Experimental animals, cells and plasmids Specific pathogen free (SPF) male C57BL / 6 mice: purchased from the Experimental Animal Center of China Three Gorges University and caged and fed in the Experimental Animal Center of China Three Gorges University; the initial body weight of the experimental mice was 18-20 g; the animal breeding room was maintained at a constant temperature of (22±2)°C and a constant humidity of (60±5)%; during the feeding period, the experimental mice were allowed to eat and drink freely; all animal feedings were approved by the Animal Experiment Ethics Committee, and the feeding of the experimental mice was carried out in accordance with the "Guidelines for the Care and Use of Laboratory Animals of China Three Gorges University"; HEK-293T cell line: donated by Professor Chayunhong of Yichang Central People's Hospital.

[0039] HSC-T6 cell line (rat hepatic stellate cells): donated by Professor Song Yuhu of Tongji Hospital Affiliated to Huazhong University of Science and Technology.

[0040] LX-2 cell line (human hepatic stellate cells): donated by Professor Liuchangbai of the Basic Medical College of China Three Gorges University.

[0041] AAV-CMV-GFP plasmid: purchased from the Addgene official website.

[0042] pAAV5 plasmid, pAAV8 plasmid: purchased from the Addgene official website.

[0043] pAdDeltaF6 plasmid: donated by Teacher Li Botao from the School of Health Sciences, China Three Gorges University Example 1 Construction of recombinant adeno-associated virus vector (1) Obtain the human miR-23b-27b-24-1 and its flanking sequences, and the murine miR-23b-27b-24-1 and its flanking sequences on the NCBI website, design primers for PCR amplification. Among them, the human miR-23b-27b-24-1 and the flanking sequences (denoted as wu-miR-02) are SEQ ID NO: 1, and the murine miR-23b-27b-24-1 and the flanking sequences (denoted as wu-Mir-02) are SEQ ID NO: 2, and design amplification primers. The primer sequences are shown in Table 1; (2) Use a double digestion system composed of HindⅢ, Xhol Ⅰ, EcoRⅠ and 10×Tango buffer to perform double digestion on AAV-CMV-GFP and AAV-CMV respectively to obtain linearized vectors; (3) By homologous recombination method, mix and ligate the wu-miR-02 sequence or the linearized vector with the wu-Mir-02 sequence at a molar ratio of 10:1, then transform, screen, and perform sequencing detection to obtain the successfully constructed recombinant adeno-associated virus vectors AAV-GFP-wu-miR-02, AAV-wu-miR-02, AAV-GFP-wu-Mir-02, and AAV-wu-Mir-02, and extract the recombinant adenovirus plasmid vector plasmids for standby.

[0044] Table 1 Amplification primers for wu-miR-02

[0045] Example 2 Functional verification of recombinant adeno-associated virus vector Take the AAV-GFP-wu-miR-02 and AAV-wu-miR-02 recombinant adeno-associated virus vectors as examples. After transfection, observe whether wu-miR-02 can be successfully expressed and whether GFP will affect the expression of wu-miR-02. The specific method is as follows: (1) Experimental group (AAV-GFP-wu-miR-02): Transfect the AAV-GFP-wu-miR-02 recombinant vector into 293T cells through a transfection reagent; Control group (AAV-wu-miR-02): Transfect the AAV-wu-miR-02 recombinant vector into 293T cells through a transfection reagent; Blank control group (AAV-GFP): Transfect the AAV-GFP plasmid into 293T cells through a transfection reagent; (2) Observe the fluorescence intensity of transfected cells by fluorescence microscopy 48 h after transfection; (3) Extract the total RNA of cells in each group, and detect the expression levels of miR-23b-27b-24-1 3p and miR-23b-27b-24-1 5p by qPCR.

[0046] The results showed that: 48 h after transfection, the fluorescence intensity of the experimental group was significantly higher than that of the control group, indicating that wu-miR-02 could be successfully expressed ( Figure 1 ), and GFP did not affect the expression of wu-miR-02 ( Figure 2 ).

[0047] Example 3 Effect of flanking sequences on the expression of miR gene cluster (1) Construct a recombinant adeno-associated virus vector without flanking sequences according to the method described in Example 1: AAV5-GFP-miR-23b-27b-24-1. The sequence of miR-23b-27b-24-1 without flanking sequences is SEQ ID NO:7, and the primer sequences are shown in Table 2; (2) Take the recombinant adeno-associated virus vectors AAV-GFP-wu-miR-02 and AAV-GFP-wu-Mir-02 containing flanking sequences in Example 1 and the recombinant adeno-associated virus vector AAV-GFP-miR-23b-27b-24-1 constructed in step (1) and transfect them into LX-2 and T6 cells respectively; at the same time, transfect AAV-GFP (NC group) into LX-2 as the control group; (3) Observe the fluorescence intensity of transfected cells by fluorescence inverted microscopy 48 h after transfection; (4) Extract the RNA of transfected cells and detect the expression levels of miR-23b-27b-24-1 3p and miR-23b / 27b-24-1 5p by qPCR; Table 2 Amplification primers for miR-23b-27b-24-1

[0048] The results showed that: in LX-2 cells, the expression level of wu-miR-02 containing flanking sequences was significantly higher than that of miR-23b-27b-24-1 without flanking sequences ( Figure 3 ); in T6 cells, the expression level of wu-miR-02 containing flanking sequences was significantly higher than that of miR-23b-27b-24-1 without flanking sequences ( Figure 4 ), and the difference in expression level was higher than that in LX-2 cells.

[0049] Example 4 Exploration of the infectivity of recombinant adeno-associated virus (1) The AAV-GFP-wu-miR-02 and AAV-GFP-wu-Mir-02 constructed in Example 1 were transfected into 293T cells respectively, packaged using the virus packaging system (Table 4), and then purified with iodixanol to obtain AAV viruses. (2) The AAV viruses obtained in step (2) were used to infect the LX-2 cell line and HEK-293T cell line in vitro respectively. (3) On the 4th day after infection, the fluorescence intensity was observed through an inverted fluorescence microscope to determine the virus infection situation.

[0050] Table 3 Virus packaging system

[0051] The results showed that after being packaged into AAV viruses, AAV-GFP-wu-miR-02 and AAV-GFP-wu-Mir-02 could infect the LX-2 cell line ( Figure 5 ), HEK-293T cell line ( Figure 6 ).

[0052] Example 5 Effect of TGFβ1 stimulation on the function of recombinant adeno-associated virus TGFβ1 is a transforming growth factor and one of the most effective pro-fibrotic factors in the liver at present. Therefore, a liver fibrosis cell activation model can be constructed by stimulating cells with TGFβ1, and then the liver fibrosis cell activation model can be infected with recombinant adeno-associated virus to detect the expression of wu-miR-02 and the expression of liver fibrosis-related target genes. The specific method is as follows: (1) 293T cells were stimulated with TGFβ1 for 24 h to construct a liver fibrosis activation model. Experimental group (TGFβ1-rAAV5-GFP-wu-miR-02): The AAV-GFP-wu-miR-02 recombinant AAV virus (rAAV5-GFP-wu-miR-02) constructed and packaged in Example 4 was used to infect the liver fibrosis activation model constructed in step (1). Control group 1 (TGFβ1): A 1×PBS solution with the same volume as the recombinant AAV virus was added to the liver fibrosis activation model without infected cells. Control group 2 (TGFβ1-rAAV-GFP): The AAV5-GFP virus was used to infect the liver fibrosis activation model. Blank control group (MOCK): A 1×PBS solution with the same volume as the recombinant AAV virus was added to the unstimulated LX-2 cells. (3) At 48 h after virus infection, total RNA was extracted from the cells of each group, and the expression levels of the mature 3p and 5p of miR-23b-27b-24-1 were detected by qPCR. At the same time, the expression levels of the liver fibrosis-related target genes TGFβ2 and LOX were detected using U6 and GAPDH.

[0053] The results were as Figure 7 shown. The expression levels of TGFβ2 and LOX in experimental group 1 were significantly lower than those in control group 1 and control 2, indicating that rAAV5-GFP-wu-miR-02 could significantly reduce the stimulation of TGFβ1 on 293T cells and alleviate liver fibrosis.

[0054] Example 6 Distribution of Recombinant Adeno-Associated Virus in Mice Recombinant adeno-associated virus was injected into the mouse liver fibrosis model to explore the tissue distribution of the recombinant adeno-associated virus in mice and observe the tissue tropism of the recombinant adeno-associated virus. Experimental group (CCL4 + rAAV5-GFP-wu-miR-02): Mice after 4 weeks of modeling with 20% CCl4 were injected with rAAV-GFP-wu-miR-02 recombinant adeno-associated virus via the tail vein, and the injection dose was 2 × 10^11 v.p / mouse. Control group (CCL4): Mice after 4 weeks of modeling with 20% CCl4 were injected with an equal volume of 1×PBS solution (the solvent of the virus) via the tail vein. Blank control group (MOCK): Normal 4-week-old mice were injected with an equal volume of 1×PBS solution via the tail vein. (2) Four weeks after virus infection, the mice were sacrificed, and the heart, liver, spleen, lung, and kidney of the mice were taken out and subjected to small animal tissue imaging at an excitation wavelength of 475 nm.

[0055] The results were as Figure 8 shown. rAAV5-GFP-wu-miR-02 was mainly distributed in the liver, with a small amount also distributed in the lungs and kidneys, and no distribution in the heart and spleen. It can be seen that this virus has liver tropism.

[0056] Example 7 Effect of Recombinant Adeno-Associated Virus on Liver Fibrosis in Mice Experimental group 1 (CCL4 + rAAV5-GFP-wu-miR-02): Mice after 4 weeks of modeling with 20% CCl4 were injected with rAAV-GFP-wu-miR-02 via the tail vein, and the injection dose was 2 × 10^11 v.p / mouse. Experimental group 2 (CCL4 + rAAV5-GFP-wu-Mir-02): Mice after 4 weeks of modeling with 20% CCl4 were injected with rAAV-GFP-wu-Mir-02 via the tail vein, and the injection dose was 2 × 10^11 v.p / mouse. Control group 1 (CCL4 + rAAV5-GFP): Mice that had been modeled with 20% CCl4 for 4 weeks were intravenously injected with an equal amount of AAV5-GFP via the tail vein. Control group 2 (CCL4): Mice that had been modeled with 20% CCl4 for 4 weeks were intravenously injected with an equal volume of 1×PBS solution via the tail vein. Blank control group (MOCK): Normal mice were intravenously injected with an equal volume of 1×PBS solution via the tail vein. (2) Four weeks after virus infection, the mouse liver was perfused with normal saline, and then liver tissue was taken for gross photography of the liver to observe the liver fibrosis situation. (3) Liver tissues from mice in each group were collected and made into paraffin sections, and then HE staining and Masson staining were performed to observe the liver fibrosis situation in mouse liver tissues.

[0057] The results were as Figure 9 shown in Figure 10 Figure A and Figure 9 Figure B. The surface of the liver tissue in the MOCK group mice was smooth, the edges were sharp, and no nodules were formed. The surfaces of the livers in the CCl4 group and CCl4 + AAV-GFP group mice were relatively rough and covered with granular nodules, and the edges were blunt. While the surface of the liver in the experimental group (CCL4 + rAAV-GFP-wu-miR-02) mice was smoother and no obvious fine granular nodules were seen, and the edges were also sharper, indicating that injection of rAAV5-GFP-wu-miR-02 could improve liver fibrosis in mice. As

[0058] Example 8 Effect of recombinant adeno-associated virus on serological indexes in mice Mice in the four groups after 8 weeks of infection in Example 7 were taken, and orbital venous blood was collected for serological detection. The detection results showed that among the three serological detection indexes ALT and AST, the expression levels in the MOCK group were the lowest, and the expression levels in the CCl4 group and CCl4 + AAV5-GFP group were relatively high; the expressions of ALT and AST in the serum of mice in the CCl4 + AAV5-wu-miR-02 group were significantly decreased ( Figure 11 ).

[0059] Example 9 Expression of recombinant adeno-associated virus in mouse liver and regulation of target genes Mice in the four groups after 4 weeks of infection in Example 7 were sacrificed, and then total liver RNA and proteins were extracted. The expression levels of the mature 3p and 5p of miR-23b-27b-24-1 were detected respectively, and the expression levels of liver fibrosis target genes were also detected. In addition, β-actin protein was used as an internal reference, and the expression situations of target proteins TIMP1 protein and α-SMA protein were detected by Western Blot experiment.

[0060] The results showed that Figure 12 compared with control group 1, the levels of both mature 3p and 5p of miR-23b-27b-24-1 in experimental group 1 were significantly increased. The levels of mature 3p and 5p of miR-23b-27b-24-1 in experimental group 2 were also significantly increased ( Figure 13 ).

[0061] Detection of target gene levels found that the expression levels of TIMP1 gene and α-SMA gene were the highest in control group 1 and control group 2, significantly decreased in the experimental groups, and the lowest in the blank control group ( Figure 14 ), indicating that injection of rAAV5-GFP-wu-miR-02 could reduce the expression levels of liver fibrosis target genes and alleviate liver fibrosis.

[0062] The results of Western Blot experiment showed that the expression levels of TIMP1 protein and α-SMA protein were the highest in control group 1 and control group 2, significantly decreased in the experimental groups, and the lowest in the blank control group ( Figure 15 ), indicating that infection of mice with rAAV5-GFP-wu-miR-02 could reduce the expression levels of liver fibrosis target proteins and alleviate liver fibrosis.

Claims

1. A miRNA gene cluster, characterized in that: The miRNA gene cluster is wu-miR-02 or wu-Mir-02; the sequence of wu-miR-02 is SEQ ID NO:1, and the sequence of wu-Mir-02 is SEQ ID NO:

2.

2. An adeno-associated virus expression plasmid carrying a miRNA gene cluster, characterized in that: It contains an adeno-associated virus genome with a CMV promoter and the miRNA gene cluster.

3. The adeno-associated virus expression plasmid with miRNA gene cluster according to claim 2, characterized in that: The miRNA gene cluster is wu-miR-02 or wu-Mir-02 as described in claim 1; the sequence of wu-miR-02 is SEQ ID NO:1, and the sequence of wu-Mir-02 is SEQ ID NO:

2.

4. A method for constructing an adeno-associated virus expression plasmid carrying an miRNA gene cluster as described in claim 2, characterized in that: It includes the following steps: (1) Double-digest the pAAV-GFP plasmid with the restriction endonucleases Hind Ⅲ and Xol Ⅰ to obtain a linearized vector; amplify wu-miR-02 or wu-Mir-02 and add restriction sites, and double-digest the amplified PCR fragment with HindⅢ and XolⅠ to obtain the wu-miR-02 or wu-Mir-02 gene fragment; (2) Use a homologous recombinase to ligate the wu-miR-02 or wu-Mir-02 gene fragment with the linearized vector and transform and culture. (3) Verify by double digestion and sequencing to obtain the recombinant adeno-associated virus vector AAV-GFP-wu-miR-02 or AAV-GFP-wuMir-02.

5. A gene therapy drug based on AAV vector, characterized in that: It includes: A recombinant adeno-associated virus, which has an AAV capsid, an AAV vector genome containing a CMV promoter packaged in the AAV capsid, and a miRNA gene cluster.

6. The gene therapy drug according to claim 5, characterized in that: The miRNA gene cluster is wu-miR-02 or wu-Mir-02; the sequence of wu-miR-02 is the nucleotide sequence shown in SEQ ID NO:1; the sequence of wu-Mir-02 is the nucleotide sequence shown in SEQ ID NO:

2.

7. A method for preparing a gene therapy drug according to any one of claims 5 or 6, characterized in that: It includes the following steps: (1) Co-infect 293T cells with the recombinant adeno-associated virus vector containing the miRNA gene cluster, the AAV-CMV-GFP plasmid, and the pAdDeltaF6 plasmid; (2) Collect the cells and lyse them, and purify them with iodixanol to obtain rAAV-GFP-miRNA gene cluster and a gene therapy drug based on the AAV vector.

8. The preparation method according to claim 7, characterized in that: In step (1), the mass ratio of the recombinant adeno-associated virus vector containing the miRNA gene cluster, the AAV-CMV-GFP plasmid, and the pAdDeltaF6 plasmid is 7:20:

7.

9. The preparation method according to claim 7 or 8, characterized in that: The recombinant adeno-associated virus vector containing the miRNA gene cluster is AAV-wu-miR-02 or AAV-wu-Mir-02.

10. Use of a gene therapy drug based on an AAV vector, characterized in that: The application is for the treatment of liver fibrosis.

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