Pharmaceutical compound for preventing or treating hepatic fibrosis

Abstract

The present invention provides a compound useful as a single compound or as a combination of at least two compounds for the prevention and / or treatment of hepatic fibrosis and / or cirrhosis, for example as a pharmaceutically active compound for the prevention or treatment of hepatic fibrosis, which compound is a hyaluronate synthase inhibitor or integrin alpha-6 inhibitor.

Description

[0001] The present invention relates to a pharmaceutical compound, preferably to a combination of pharmaceutical compounds for preventing and treating liver fibrosis and / or liver cirrhosis, wherein the liver fibrosis may be, for example, pericentral or periportal liver fibrosis.

[0002] Compounds have been found to inhibit the activation of quiescent hepatic stellate cells (qHSCs) and thus inhibit one of the key steps in fibrogenesis involving the accumulation of extracellular matrix.

[0003] The activity of the pharmaceutical compounds for the prevention and / or treatment of liver fibrosis and / or cirrhosis is shown in in vitro assays in a murine liver fibrosis model and a human liver bud model as well as in in vivo assays.

[0004] Current state of the art

[0005] Song et al., Cell Stem Cell 18, 797-808 (2016) describe the generation of viral vectors AAV8 and AAV2p75.

[0006] Purpose of the Invention

[0007] The object of the present invention is to provide compounds suitable for preventing and treating liver fibrosis and / or liver cirrhosis. Preferably, the compounds should act on the pathway molecules involved in the transformation of healthy hepatocytes into myofibroblasts.

[0008] Description of the invention

[0009] The present invention achieves the purpose by providing a compound, which is used as a single compound or as a combination of at least two compounds for medical treatment, especially for preventing and / or treating liver fibrosis and / or liver cirrhosis, for example, as a pharmaceutically active compound for preventing or treating liver fibrosis, the compound is a hyaluronan synthase (HAS2, such as SEQ ID NO: 1 shows isoform Alpha-6X1X2B, the identifier is P23229-1 in UniProtKB) inhibitor or integrin α-6 (ITGA6, such as SEQ ID NO: 2 shows, the identifier is Q92819-1 in UniProtKB) inhibitor, or a combination of a hyaluronan synthase (HAS2) inhibitor and an integrin α-6 (ITGA6) inhibitor. The present invention also provides a hyaluronan synthase (HAS2) inhibitor, an integrin α-6 (ITGA6) inhibitor and a combination of these, which are used for medical treatment or for preventing disease.

[0010] The inhibitor can be an inhibitor of HAS2 expression or an inhibitor of ITGA6 expression, such as inhibitory RNA, especially microRNA (miR). Alternatively, the inhibitor can be a compound that inactivates HAS2 or ITGA6. Exemplary HAS2 inhibitors for preventing and / or treating liver fibrosis and / or cirrhosis are 4-methylumbelliferone (4-MU or MU), 4-methylesculetin (ME), brefeldin A, or a combination of at least two of these.

[0011] It is speculated that HAS2 inhibitors and ITGA6 inhibitors alone or in combination lead to a reduction in the formation of low molecular weight hyaluronic acid (LMW-HA) from hyaluronic acid (HA), which is a high molecular weight polymer of the extracellular matrix and a biomarker of chronic liver fibrosis. The formation of HA in liver tissue in liver disease is increased, and in liver injury, HA is especially degraded to LMW-HA in liver tissue, wherein LMW-HA can be activated by myofibroblasts and inflammation to aggravate damage. It has been found that the inhibition of HAS2 can lead to a reduction in HA formation, thereby leading to a reduction in the formation of LMW-HA, and leading to a reduction in the expression of fibrosis marker proteins COL1A1, ACTA2 and S100A6 in liver tissue. In addition, it is speculated that inhibition of ITGA6 (e.g., in primary human myofibroblasts (MF)) can lead to a reduction in the level of membrane-associated AREG, such as by reducing the association of ITGA6 with AREG and subsequently reducing the signaling of its associates. It is speculated that AREG-ITGA6 associates increase the motility, invasion, and activation of HSCs, whereas inhibition of ITGA6 results in decreased levels of AREG-ITGA6 associates, leading to decreased HSC activation and decreased MF generation or activation, as measured by decreased expression of the fibrotic markers COL1A1, ACTA2, and S100A6 in MFs.

[0012] Preferred inhibitors are microRNAs, particularly miR-296-3p (SEQ ID NO: 4) as an inhibitor of integrin alpha-6 (ITGA6) and miR-190b-5p (SEQ ID NO: 3) as an inhibitor of hyaluronan synthase (HAS2).

[0013] The microRNA forming the inhibitor can be expressed from an expression cassette encoding the microRNA, which is introduced into hepatocytes, for example, as a viral vector particle. Preferably, the coding sequence encoding the microRNA in the expression cassette is controlled by a promoter specifically expressed in profibrotic myofibroblasts (MF), and the promoter is preferably a Pdgfrb promoter (nucleotides 1 to 1346 of SEQ ID NO:5). Alternatively, the microRNA can be expressed under the control of a constitutive promoter, such as a CMV promoter (nucleotides 381 to 584 of SEQ ID NO:8), which is preferably before a CMV enhancer (e.g., nucleotides 1 to 380 of SEQ ID NO:8).

[0014] The expression cassette can be contained in a viral vector particle, for example based on adeno-associated virus (AAV). An exemplary viral vector for producing miR-190b-5p can include a nucleic acid sequence of SEQ ID NO: 5 (AAV-Pdgfrb-miR-190b-5p), which contains an expression cassette of miR-190b-5p under the control of the Pdgfrb promoter. An exemplary viral vector for producing miR-296-3p can include a nucleic acid sequence of SEQ ID NO: 6 (AAV-Pdgfrb-296-3p), which contains an expression cassette of miR-296-3p under the control of the Pdgfrb promoter (nucleotides 1 to 1346 of SEQID NO: 6).

[0015] An exemplary viral vector for producing miR-296-3p may comprise a nucleic acid sequence of SEQ ID NO: 7 (AAV-Cmv-296-3p), which contains an expression cassette of miR-296-3p (nucleotides 656 to 809 of SEQ ID NO: 7) under the control of a CMV promoter (nucleotides 381 to 584 of SEQ ID NO: 7), preferably preceded by a CMV enhancer (nucleotides 1 to 380 of SEQ ID NO: 7). An exemplary viral vector for producing miR-190b-5p may comprise a nucleic acid sequence of SEQ ID NO: 8 (AAV-Cmv-miR-190b-5p), which contains an expression cassette of miR-190b-5p (nucleotides 656 to 807 of SEQ ID NO: 8) under the control of a CMV promoter.

[0016] According to the efficacy of miR-190b-5p and miR-296-3p alone or in combination, the present invention relates to the use of miR-190b-5p and miR-296-3p alone or in combination for preventing or treating liver fibrosis, for example, miR-190b-5p and miR-296-3p alone or in combination as pharmaceutically active compounds for preventing or treating liver fibrosis.

[0017] A hyaluronan synthase inhibitor or an integrin α-6 inhibitor, preferably a combination thereof, is suitable for use in a method for preventing or treating liver fibrosis.

[0018] The figure shows

[0019] - Figure 1 Micrographs of SMA immunocytochemical staining of primary human myofibroblasts after transfection with A) control miR (Scramble), B) miR-190b-5p, or C) miR-296-3p.

[0020] - Figure 2 Figure 1 Quantitative analysis of SMA staining,

[0021] - Figure 3 Quantitative analysis of ACTA2 expression in primary human myofibroblasts after transfection with control miR (Scramble), with miR-190b-5p, or with miR-296-3p.

[0022] - Figure 4 A) Human miR-190b-5p and B) Human miR-296-3p levels,

[0023] - Figure 5 Schematic diagram of the measurement,

[0024] - Figure 6 The expression level of the reporter gene dTomato,

[0025] - Figure 7 A) Schematic diagram of in vivo administration of miRNA in a CCl4-induced fibrosis model and determination of Figure 7 B) miR-190b-5p and Figure 7 C) expression level of miR-296-3p,

[0026] - Figure 8 A) miR-190b-5p and B) miR-296-3p expression levels in the liver of virus-transduced animals,

[0027] - Fig. 9 The expression level of hydroxyproline in the liver of virus-transduced animals,

[0028] - Fig.10 Expression levels of Col1a1 and Acta2 in the liver of virus-transduced animals,

[0029] - Fig.11A), B), C) images of livers of animals transduced with control, AAV8-Pdgfrb-190b-5p, or AAV8-Pdgfrb-296-3p viruses, D), E), F) HE staining, G), H), I) Sirius red staining, J), K), L) SMA staining, M), N), O) Desmin staining,

[0030] - Fig.12 A) fibrosis area, B) DEMIN and C) SMA levels in the livers of virally transduced animals.

[0031] - Fig.13 A) Schematic diagram of in vivo administration of miRNA in the DDC-induced fibrosis model and expression levels of B) miR-190b-5p and C) miR-296-3p in the liver of virally transduced animals,

[0032] - Fig.14 Hydroxyproline levels in livers of virus-transduced animals,

[0033] - Fig.15 The expression levels of A) Acta2, B) fibrosis area, C) SMA level and D) DESMIN level in the liver of virus-transduced animals.

[0034] - Fig.16 A), B), C) HE staining, D), E), F) Sirius red staining, G), H), I) SMA immunostaining, and J), K), L) Desmin immunostaining of livers of animals transduced with control, AAV8-Pdgfrb-190b-5p, or AAV8-Pdgfrb-296-3p viruses.

[0035] - Fig.17 Flow cytometry results of HAS2 protein levels in cells transfected with miR-190b-5p or miR-296-3p,

[0036] - Fig.18 Flow cytometry results of ITGA6 protein levels in cells transfected with miR-190b-5p or miR-296-3p,

[0037] - Fig.19 Expression levels of luciferase reporter genes for A) HAS2 and B) ITGA6 in transduced primary human MF

[0038] - Fig. 20 A), B) staining of HAS2 and C), D) staining of ITGA6 in primary human MFs transfected with miR-190b-5p1, miR-296-3p or Scramble,

[0039] - Fig.21 A), B) Has2 mRNA and C), D) Itga6 mRNA expression levels in liver tissues of mice transduced with the corresponding AAVs in pericentral and periportal liver fibrosis,

[0040] - Fig. 22 Western blot of A) protein levels of HAS2 and BITGA6 in mouse liver when overexpressing miR-190b-5p and miR-296-3p,

[0041] - Fig.23 Immunohistochemical analysis of formalin-fixed sections of human liver at different stages of fibrosis, targeting HAS2 in A), B), C), D) and ITGA6 in E), F), G), H).

[0042] - Fig.24A ), B), C), D), E) levels of A) TIMP1 and B) CTGF secretion measured by ELISA, and expression levels of fibrosis markers C) ACTA2, D) COL1A1, and E) S100A6 in co-transfected human MF cells by qRT-PCR,

[0043] - Fig.25 Immunocytochemical staining of SMA in transfected MFs A) transfected with Scramble, B) transfected with miR-296-3p, C) overexpressing ITGA6 and transfected with miR-296-3p, D) transfected with miR-190b-5p, or E) overexpressing HAS2 and transfected with miR-190b-5p.

[0044] - Fig.26 LMW-HA levels in transfected primary human MFs,

[0045] - Fig. 27 Immunocytochemical staining of SMA in MF treated with A) Scramble, B) LMW-HA or C) AREG.

[0046] - Fig.28 Levels of membrane-associated AREG in transfected primary human MFs,

[0047] - Fig.29 The expression level of ACTA2 in transfected primary human MFs,

[0048] - Fig.30 Expression levels of COL1A1 in transfected primary human MFs,

[0049] - Fig.31 Expression levels of S100A6 in transfected primary human MFs,

[0050] - Fig.32 Schematic diagram of in vivo assay,

[0051] - Fig.33 A) miR-190b-5p and B) miR-296-3p expression levels in hepatic stellate cells of virally transduced animals,

[0052] - Fig.34 The expression level of Has2 in hepatic stellate cells in the liver of virus-transduced animals by RT-PCR,

[0053] - Fig.35 The expression level of Itga6 in hepatic stellate cells in the liver of virus-transduced animals by RT-PCR,

[0054] - Fig.36 A) Fibrotic areas and B) SMA levels in livers of virally transduced animals.

[0055] - Fig.37 A), B), C) HE staining, D, E), F) Sirius red staining, and G), H), I) SMA immunostaining of livers of animals transduced with control, AAV2p75-Pdgfrb-190b-5p / AAV8-Pdgfrb-HAS2, or AAV2p75-Pdgfrb-296-3p / AAV8-Pdgfrb-ITGA6 viruses.

[0056] - Fig.38 Western blot of A) protein levels of HAS2 and BITGA6 in mouse liver when overexpressing miR-190b-5p and miR-296-3p,

[0057] - Fig.39 A), B) immunocytochemical staining of HAS2 and C), D) immunocytochemical staining of ITGA6 in MFs overexpressing HAS2 or ITGA6 transfected with control, miR-190b-5p or miR-296-3p,

[0058] - Fig.40 Schematic representation of the in vitro assay using human liver buds,

[0059] - Fig.41 Expression level of TIMP1 in model human liver buds by ELISA,

[0060] - Fig.42 Secretion levels of CCL2 by ELISA from model human liver buds,

[0061] - Fig.43 The expression level of ALB in the model human liver buds,

[0062] - Fig.44 The expression level of HNF4A in the model human liver buds,

[0063] - Fig.45 The expression level of TTR in the model human liver bud,

[0064] - Fig.46 Schematic diagram of in vivo assay,

[0065] - Fig.47 Volcano plot of proteins from transduced fibrotic MFs from mouse liver,

[0066] - Fig.48 Volcano plot of proteins from transduced fibrotic MFs from mouse liver,

[0067] - Fig.49 Schematic diagram of the putative signal transduction pathway.

[0068] - Fig.50 The relative expression levels of markers in MF cells and LX2 cells,

[0069] - Fig.51 B), C), D) micrographs of MF cells and A) LX2 cells stained for SMA,

[0070] - Fig.52A , 52B , 52C TIMP1 levels in three MF cultures in the presence of miRNA mimics, and

[0071] - Fig.53A , 53B , 53C CTGF expression level.

[0072] Primers used for analysis of mouse genes in Sybr green qRT-PCR:

[0073]

[0074]

[0075] Primers used for analysis of human genes in Sybr green qRT-PCR:

[0076]

[0077] In the figures, data are ± sem, * indicates P < 0.05, ** indicates P < 0.005, and *** indicates P < 0.0005.

[0078] Scramble refers to any miRNA species, excluding miR-296-3p and miR-190b-5p.

[0079] For qRT-PCR, total RNA was extracted with TRIzol reagent (Qiagen) according to the manufacturer's instructions. cDNA was generated using TaqMan reverse transcription reagent (ThermoFisher) or iScript cDNA kit (Bio Rad). For qRT-PCR, TaqMan (Invitrogen) or SYBR Green (ThermoFisher) probes and kits were used. Relative mRNA expression was determined after normalization of expression relative to GAPDH, gapdh, ACTB, actb, or rpI4 as housekeeping genes. For miRNA reverse transcription and qRT-PCR, total RNA was extracted with TRIzol reagent (Qiagen) according to the manufacturer's protocol. cDNA of miRNA was generated using TaqMan MiRNA reverse transcription reagent (ThermoFisher) and miRNA-specific RT primers. Relative miRNA expression was determined after normalization relative to U6 or sno.

[0080] Virus titer was determined by quantitative polymerase chain reaction (qRT-PCR) using primers targeting miRNA or Pdgfrb. Primary mouse HSC isolation was performed according to Maschmeyer et al., J Vis Exp 2710 (2011). In brief, mouse livers were perfused with pronase E solution and collagenase P solution at a flow rate of 6.5 mL / min at 37°C for 4.5 minutes. All perfused mouse livers were collected and digested in vitro for 20 minutes at 37°C with 50 ml pronase E-collagenase P solution and 1 ml DNase I. The cell suspension was filtered through a 100 μm cell strainer and centrifuged at 600 x g for 10 minutes at 4°C. After discarding the supernatant, the cells were resuspended in Gey's balanced salt solution-B (GBSS-B), and 150 μL DNase 1 solution was added to the cell suspension. The cell suspension was centrifuged at 600 x g for 10 minutes at 4°C. After carefully discarding the supernatant, the cells were suspended with 36 mL of GBSS-B buffer and 150 μL of DNase 1 solution was added to the solution. 14 ml of Nycodenz solution (8 g Nycodenz and 28 ml of GBSS-A buffer) was added to the cell suspension to achieve an 8% Nycodenz gradient. The gradient was centrifuged at 1500 x g for 15 minutes at 4°C. Primary HSCs were harvested from the interphase of the gradient.

[0081] For flow cytometry, antibodies specific for ITGA6 (Invitrogen, 25-0495-82), HAS2 (sc-514737AF488) and AREG (sc-74501) were used. Prior to ITGA6 and AREG staining, cells were fixed with 4% PFA, whereas HAS2 staining was performed on fixed and permeabilized cells (4% PFA / 0.5% Tween20).

[0082] Blood was collected retro-orbitally, serum was prepared and stored at -20°C, and AST, ALT and bilirubin were measured in serum. TIMP1 and CCL2 (Abcam), CTGF (Abbexa), AREG (SigmaAldrich), LMW-HA (R&D Systems) were quantified according to the manufacturer's protocol.

[0083] For histology, immunohistochemistry and immunofluorescence, liver tissue was fixed with 4% formalin, embedded in paraffin, and cut into 2-5 μm thick sections. For Sirius red staining, after dewaxing, the sections were stained with Direct Red 80 (Merck) solution in picric acid (Merck) for 1 h at room temperature. Afterwards, the sections were washed in acetic acid water, and the slides were dehydrated and sealed. According to the manufacturer's instructions, Vectastain ABC-HRP (Biozol, VEC-PK-6101) kit was used to perform immunohistochemistry of HAS2 (Invitrogen, PA5-115388), SMA (ab5694) and DESMIN (ab15200). ITGA6 immunohistochemical staining was performed using ITGA6 antibody (Invitrogen, 14-0495-82). Quantification of immunohistochemical staining was performed using ImageJ software.

[0084] For HAS2 and ITGA6 western blots, proteins were isolated from liver tissue using the Mem-PERTM Plus membrane protein extraction kit (Thermo Scientific) according to the manufacturer's instructions. In brief, 20-40 mg of liver was washed with 4 mL of cell washing solution. The wash solution was discarded and 1 mL of permeabilization buffer was added to the tissue. The tissue was homogenized and the mixture was incubated on ice for 10 minutes with continuous stirring. The mixture was then centrifuged at 16000 x g for 15 minutes at 4 ° C. The supernatant containing cytoplasmic proteins was transferred to a new tube and kept on ice. The pellet was resuspended in 1 mL of dissolution buffer. The mixture was homogenized via pipetting and incubated at 4 ° C for 30 minutes with continuous stirring. The mixture was then centrifuged at 16000 x g for 15 minutes at 4 ° C. The supernatant containing membrane-associated proteins was transferred to a new tube and kept on ice. The fraction containing soluble proteins and the fraction containing membrane-associated proteins were mixed in a 1: 1 ratio, and the mixture was used for analysis.

[0085] Liver biopsy samples were obtained by percutaneous liver biopsy under local anesthesia. Approximately 5 mm of the needle biopsy was immediately stored in Allprotect tissue reagent (QIAGEN) and then stored at -80°C. The remaining cylinder was fixed in 4% neutral buffered formalin and embedded in paraffin. Histological examination and fibrosis stage scoring (Ishak score) were performed by an experienced liver pathologist.

[0086] Example 1: Anti-fibrotic activity of miR-296-3p and miR-190b-5p in MF

[0087] The expression of known fibrotic markers DES, ACTA2, LOX2 and COL1A was evaluated in primary human myofibroblasts (MF, purchased from Innoprot (Bizkaia, Spain) for in vitro assays and compared with SMALX2 cells (LX2), an established human HSC cell line. 96-well plates (TRP) were coated with gelatin for 10 min at 37°C. After discarding the gelatin solution, 9600 primary human myofibroblasts were seeded and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, 2-mercaptoethanol and non-essential amino acids.

[0088] The results showed that the expression of all these markers was significantly higher in MF than in SMALX2 cells. In addition, immunocytochemical staining of α-smooth muscle actin (SMA) confirmed the profibrotic characteristics of MF.

[0089] The antifibrotic potential of miRNAs was independently evaluated for separate batches of primary human MFs. Integrin α6 (ITGA6) inhibitor miR-296-3p and hyaluronan synthase inhibitor miR-190b-5p were identified based on the regulation of TIMP1 secretion and the measurement of connective tissue growth factor (CTGF) levels in the culture medium collected from MFs transfected with the comparison miRNAs. In addition, the secretion of CTGF was assessed in the culture supernatants of miRNA-transfected myofibroblasts, using scramble-transfected cells as a control in the CTGF ELISA. CTGF, also known as CCN2, is a member of the CCN family of extracellular matrix-associated heparin-binding proteins that can act as an agonist of the TGF-β signaling pathway, one of the most potent pathways in fibrosis. CTGF secretion analysis also confirmed that hsa-miR-190b-5p and hsa-miR-296-3p significantly and consistently regulated the secretion of CTGF in all three batches of primary cells, demonstrating the antifibrotic activity of these miRNAs.

[0090] The antifibrotic activity of miR-296-3p and miR-190b-5p was demonstrated in immunocytochemical staining of SMA on MFs transfected with scramble, with miR-190b-5p, or with miR-296-3p. Micrographs are depicted in Figure 1 Compared with the control (Scramble), these miRNAs significantly reduced SMA levels.

[0091] Expression of platelet-derived growth factor receptor β (PDGFRb) on the surface of MF transfected with miR-190b-5p, miR-296-3p or scramble (control). PDGFRb is another characteristic fibrosis marker, and its expression on the surface of activated MF2122 was upregulated by FACS. It was observed that the surface expression of PDGFRb was reduced to 22% after transfection with miR-190b-5p, reduced to 28.5% after transfection with miR-296-3p, and reduced to only 58% in the scramble control. This shows that the presence of miR-190b-5p and miR-296-3p leads to a significantly stronger reduction in the surface expression of PDGFRb.

[0092] qRT-PCR analysis ( Figure 3 Data depicted in ) showed that the expression of LOX2 and ACTA2 was lower in MFs transfected with miR-190b-5p or miR-296-3p compared with control-transfected cells.

[0093] In addition, in human fibrotic liver biopsies with fibrosis stage 3 (Ishak score) Figure 4, fibrosis) and human healthy liver tissue ( Figure 4 In the fibrotic tissues (n=24), the expression of miR-190b-5p and miR-296-3p was analyzed by qPCR. The expression of miR-190b-5p and miR-296-3p in fibrotic tissues was significantly downregulated compared with that in non-fibrotic tissues.

[0094] These results identified hsa-miR-296-3p and hsa-miR-190b-5p as regulators of fibrosis.

[0095] Example 2: Inhibition of HAS2 or ITGA6 in vivo for the treatment of liver fibrosis

[0096] MiR-190b-5p of HAS2 was used as an exemplary inhibitor, and miR-296-3p was used as an exemplary inhibitor of ITGA6.

[0097] Since primary human MF is considered to be the most important contributor to liver fibrosis, miR-190b-5p and miR-296-3p were preferentially transfected into myofibroblasts in vivo. For preferential expression in MF, the most abundant cells in the liver, which are hepatocytes, were bypassed by expressing miR-190b-5p or miR-296-3p under the control of the MF-specific Pdgfrb promoter in vivo. It has been previously reported that the activity of the Pdgfrb promoter is upregulated upon HSC23 activation. The coding sequences of miR-190b-5p or miR-296-3p were cloned downstream of the Pdgfrb promoter in an adeno-associated virus (AAV) vector. AAV8-Pdgfrb-296-3p (SEQ ID NO: 6) contains an expression cassette of miR296-3p under the control of the Pdgfrb promoter, and AAV8-Pdgfrb190b-5p (SEQ ID NO: 5) contains an expression cassette of miR-190b-5p under the control of the Pdgfrb promoter.

[0098] After intravenous administration of AAV control, AAV8-Pdgfrb190b-5p or AAV8-Pdgfrb-296-3p into Lrat-Cre-dTomato mice (8-12 week old male BALB / c mice), the expression of miR-190b-5p or miR-296-3p in myofibroblasts after AAV in vivo administration was analyzed. In this mouse model, CredTomato expression is driven by the phosphatidylcholine retinol acyltransferase (trat) promoter, which is preferentially active in HSC and HSC-derived MF. AAV8 and AAV2p75 were generated as described in Song et al., Cell Stem Cell 18, 797-808 (2016).

[0099] like Figure 5 As schematically depicted in , mice were treated with a single dose of 8 μl / g 10% CCl4 / olive oil prior to AAV8 injection. Six days after AAV8 administration, hepatocytes and all non-parenchymal cells were isolated, of which dTomato-positive (HSCs and HSC-derived MFs) and dTomato-negative cell populations were sorted. Figure 6 The results depict that 6.12% of the detected dTomato-positive cells (dTomato+) were dTomato-negative cells (dTomato-). In the dTomato-positive cell population, the expression of miR-190b5p and miR-296-3p was determined by qRT-PCR. Figure 7 Depicted in Figure 4. The expression of both miRNAs in dTomato-positive cells isolated from mice injected with the corresponding AAV8-Pdgfrb miRNA was significantly higher than that in control mice. These data in Lrat-Cre-dTomato mice demonstrate that delivery of AAV8-Pdgfrb-190b-5p or AAV8-Pdgfrb-296-3p results in upregulation of the corresponding miRNAs in HSCs and HSC-derived MFs.

[0100] When the antifibrotic activity of miR-190b-5p and / or miR-296-3p was analyzed in vivo, it was found that overexpression of miR-190b-5p and miR-296-3p in myofibroblasts ameliorated pericentral liver fibrosis. A mouse model of pericentral liver fibrosis was induced by biweekly injection of CCl4 for 8 weeks. At the 8th week of CCl4 injection, a high titer adeno-associated virus (AAV) vector serotype 8 (1x10 10 The mice were injected with AAV and CCl4 was continued for another 2 weeks before being killed. The overexpression of each miRNA in liver tissue was confirmed by qRT-PCR. Figure 8 Depicted, expression levels are shown.

[0101] The hydroxyproline content in the liver was measured as an unbiased parameter related to the amount of collagen in the tissue. The results are depicted in Fig. 9 In the study, we showed that the concentration of hydroxyproline was reduced in the liver of mice injected with AAV8-Pdgfrb-190b-5p or AAV8-Pdgfrb-2963p compared with control mice. In addition, the expression levels of the fibrosis genes Col1a1 and Acta2 were measured. Fig.10As depicted, both Col1a1 and Acta2 were significantly downregulated after overexpression of miR-190b-5p or miR-296-3p.

[0102] like Fig.11 As shown, for control livers and livers transduced with AAV8-Pdgfrb-190b-5p or AAV8-Pdgfrb-2963p, the degree of fibrosis was determined by HE and Sirius red staining as well as SMA and DESMIN staining. Histological analysis as well as SMA and DESMIN immunohistochemistry showed that liver fibrosis was alleviated in mice of the AAV8-Pdgfrb-190b-5p and AAV8-Pdgfrb-296-3p groups compared with the control group, as shown Fig.12 Depicted.

[0103] Additionally, ALT, AST, bilirubin, and albumin levels were measured in sera collected from mice injected with control virus, AAV8-Pdgfrb-190b-5p, or AAV8-Pdgfrb-296-3p. Fig.13 The results showed that AAV8-mediated overexpression of miR-190b-5p or miR-296-3p in myofibroblasts can improve CCl4-induced central pericentral liver fibrosis. In addition, AAV8 viral vectors were generated containing the coding sequences of miR-190b-5p (AAV8-Cmv-190b-5p) or miR-296-3p (AAV8-Cmv-296-3p) cloned downstream of the constitutively active CMV promoter. These AAV viral vectors were administered at week 8 of CCl4 injection, and two weeks after AAV administration, mice were sacrificed for analysis. Fig.14 qRT-PCR data demonstrating overexpression of these miRNAs from AAV vectors in collected liver tissues are depicted. Subsequently, it was found that hydroxyproline content was reduced in livers overexpressing miR-190b-5p (AAV8-Cmv-190b-5p) or miR-296-3p (AAV8-Cmv-296-3p), as shown in Figure 1. Fig.15 Depicted. Measurement of fibrosis markers Col1a1 and Acta2 levels ( Fig.16 ) showed that both Col1a1 and Acta2 were decreased in livers overexpressing miR-190b-5p (AAV8-Cmv-190b-5p) or miR-296-3p (AAV8-Cmv-296-3p).

[0104] The degree of liver fibrosis was determined by HE and Sirius red staining as well as SMA and DESMIN staining. Fig.17The data showed that liver fibrosis was reduced in both AAV8-Cmv-190b-5p and AAV8-Cmv-296-3p groups compared with the control group. In general, CMV-driven overexpression of miR-190b-5p and miR-296-3p can improve CCl4-induced liver fibrosis.

[0105] Example 3: Inhibition of HAS2 or ITGA6 in vivo for the treatment of liver fibrosis

[0106] Using miR-190b-5p as an exemplary inhibitor of HAS2 and miR-296-3p as an exemplary inhibitor of ITGA6 for the treatment of periportal liver fibrosis, it was found that overexpression of miR-190b-5p and miR-296-3p alleviated periportal liver fibrosis.

[0107] As a model, liver fibrosis was induced by feeding mice with a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for 5 weeks. At week 5, AAV control, AAV8-Pdgfrb-190b-5p or AAV8Pdgfrb-296-3p were administered intravenously. The mice were sacrificed two weeks later, during which time the DDC-containing diet was continued. Since the degree of fibrosis in this model was higher than in the central-peripheral model of Example 2, it was sought to address whether the AAV8-Pdgfrb system could also preferentially express miRNAs in myofibroblasts in this model. Myofibroblasts were isolated from injected mice via in vivo perfusion. Fig.18 As shown in the data, the overexpression of miR-190b-5p and miR296-3p in myofibroblasts isolated from mice injected with the corresponding AAV was confirmed. In addition, the expression of Acta2 and Desmin in isolated cells was measured by qRT-PCR, and the data are shown in Fig.19 The data showed that even in cholestasis-induced liver fibrosis, AAV8-Pdgfrb-miRNA entered myofibroblasts and achieved overexpression of miRNA, thereby suppressing HAS2 and ITGA6.

[0108] Furthermore, overexpression of miR-190b-5p and miR-296-3p suppressed the levels of fibrotic markers in myofibroblasts. The extent of fibrosis in liver tissue was analyzed by measuring the hydroxyproline content in liver tissue of mice injected with AAV control, AAV8-Pdgfrb190b-5p, or AAV8-Pdgfrb-296-3p. Fig. 20 The depicted results show that miR-190b-5p or miR-296-3p leads to a decrease in hydroxyproline.

[0109] The expression of fibrosis markers Tgfbr1 and Acta2 was analyzed by qRT-PCR ( Fig.21 ) showed that the total collagen content and mRNA levels of fibrosis markers determined by hydroxyproline assay were significantly downregulated after overexpression of miR-190b-5p or miR-296-3p compared with the control group. The anti-fibrotic activity of these miRNAs was further confirmed by HE and Sirius red staining ( Fig. 22 , Fig.23 ).

[0110] In addition, significantly fewer SMA- and DESMIN-positive cells were observed in liver sections of mice injected with AAV8-Pdgfrb-190b-5p or AAV8-Pdgfrb296-3p compared with the control group ( Fig. 22 , Fig.23 These results suggest that HAS2 or ITGA6 inhibitors (e.g., overexpression of miR-190b-5p and miR-296-3p) in MF suppress periportal liver fibrosis.

[0111] In addition, CMV-driven overexpression of miR-190b-5p and miR296-3p was found to reduce periportal liver fibrosis. Among them, AAV control, AAV8-Cmv-190b-5p or AAV8-Cmv-296-3p were administered intravenously in the 5th week of the DDC-containing diet. The DDC-containing diet was continued for another 2 weeks, after which the mice were sacrificed. The overexpression of these miRNAs in liver tissue was confirmed by qRT-PCR (Figure 24). Hydroxyproline content ( Fig.25 ) and the expression of fibrosis markers Tgfbr1 and Acta2 ( Fig.26 ) also showed the anti-fibrotic activity of miR-190b-5p and miR296-3p.

[0112] HE, Sirius red staining, and SMA and DESMIN immunohistochemical staining ( Fig. 27 , Fig.28 ) confirmed the anti-fibrotic activity of these inhibitors of HAS2 and ITGA6.

[0113] Based on hydroxyproline content, expression of fibrosis markers, and immunohistochemical staining, the data showed that AAV8-Cmv-190b-5p or AAV8-Cmv-296-3p administration resulted in improvement of periportal liver fibrosis compared with the control group.

[0114] Example 4: Analysis of the mechanism of action of inhibiting HAS2 and ITGA6

[0115] When analyzing the post-transcriptional regulation of HAS2 by miR-190b-5p and ITGA6 by miR-296-3p, hyaluronan synthase 2 (HAS2) and integrin alpha-6 (ITGA6) were identified as targets of miR-190b-5p and miR-296-3p, respectively. For example, gene set enrichment analysis (GSEA) showed that several signaling pathways were downregulated in the miRNA-regulated group compared with scramble. The downregulated signaling pathways included TGFβ receptor complex signaling, NGF signaling, and growth hormone receptor signaling.

[0116] Computer analysis confirmed the anti-fibrotic mechanism of miR-190b-5p and miR-296-3p by downregulating or inhibiting HAS2 and ITGA6, respectively, and the analysis showed that the 3'UTR of HAS2 and ITGA6 contained binding sites for miR190b-5p and miR-296-3p.

[0117] In addition, the protein levels of HAS2 and ITGA6 in primary human MFs transfected with scramble, miR-190b-5p, or miR-296-3p were measured by flow cytometry, which showed that the protein levels of HAS2 and ITGA6 were decreased in cells transfected with miR-190b-5p or miR-296-3p compared with those in cells transfected with scramble (data not shown in Figure 5A). Fig.29 Targeting HAS2 and Fig.30 Shown for ITGA6.

[0118] The human 3'UTR of HAS2 / ITGA6 is cloned into the dual luciferase pmiRGLO vector and luciferase reporter gene assay is performed, confirming the downregulation of miR-190b-5p or miR-296-3p.For luciferase reporter gene assay, the 3'UTR of ITGA6 and HAS2 is amplified from HEK293 gDNA by PCR.The amplicon is cloned into the miRGLO vector (Promega).Primary human myofibroblasts are transfected with 100ng miRGLO-3'UTR plasmid and 25nM miRNA.After 48h, double GLO luciferase reagent is added and incubated for 45 minutes, and then the firefly luminescence is measured.Subsequently, StopGLO reagent is added and incubated for 30 minutes, and then the sea renilla luminescence is read.Luminescence intensity is calculated using the firefly / sea renilla ratio. Primary human MFs were co-transfected with pmiRGLO-HAS2 and miR-190b-5p1Scramble or pmiRGLO-ITGA6 and miR-296-3p1Scramble. Fig.31The result data depicted in Figure 2 show that miR-190b-5p binds to the HAS2 3'UTR, while miR-296-3p binds to the ITGA6 3'UTR by reducing the expression of a luciferase reporter gene.

[0119] In addition, if Fig.32 As depicted, the downregulation of HAS2 and ITGA6 expression by miR-190b-5p and miR-296-3p was further confirmed by immunocytochemistry, showing that overexpression of miR190b-5p significantly reduced the expression of HAS2, while overexpression of miR296-3p significantly reduced the expression of ITGA6 compared with overexpression of scramble (control).

[0120] In conclusion, miR-190b-5p and miR-296-3p seed sequences were found to bind to the 3'UTR of HAS2 and ITGA6 in vitro, which resulted in lower protein levels of HAS2 and ITGA6. HAS2 was identified as a target of miR-190b-5p, and ITGA6 was identified as a target of miR-296-3p, identifying inhibitors of HAS2 and / or ITGA6 for the treatment of liver fibrosis.

[0121] The study found that the target genes HAS2 and ITGA6 were downregulated in vivo by AAV8-Pdgfrb-190b-5p or AAV8-Pdgfrb-296-3p administration. In the liver tissues of mice injected with AAV in the pericentral and periportal models of liver fibrosis, the levels of Has2 mRNA and Itga6 mRNA detected by qRT-PCR showed strong downregulation, as shown in the following results: Fig.33 As depicted. The mRNA levels of Has2 and Itga6 in the miRNA regulation group were lower than those in the control group. In addition, Western blotting ( Fig.34 ) and immunohistochemistry ( Fig.35 ) Analysis of the protein levels of HAS2 and ITGA6 showed that the protein levels of HAS2 and ITGA6 in the liver of mice were lower in vivo after overexpression of miR-190b-5p and miR-296-3p, respectively, compared with those of control mice.

[0122] Furthermore, it was confirmed by immunohistochemistry that these targets were predominantly expressed in MF in human fibrotic liver biopsies in the absence of their inhibitors, as shown in Fig.35As shown, HAS2 or ITGA6 immunostaining was performed on formalin-fixed liver sections without fibrosis (FO), fibrosis stage 3 (F3), fibrosis stage 5 (F5) or fibrosis stage 6 (F6). The data show that the expression of both HAS2 and ITGA6 in FO is low, and as fibrosis progresses to F3 and F5, the expression of HAS2 and ITGA6 increases. Interestingly, in F6, the expression of HAS2 and ITGA6 is lower than that observed in F5.

[0123] In addition, overexpression of ITGA6 and HAS2 in myofibroblasts in vitro showed that they are associated with the development of liver fibrosis, because overexpression of HAS2 and ITGA6 restored the profibrotic activity of myofibroblasts. Primary human MFs were co-transfected with miR-190b-5p and HAS2 expression vectors (pCMV6-XL6-HAS2), or co-transfected with miR-296-3p and ITGA6 expression vectors (pCMV6-XL4-ITGA6), where both pCMV6-XL6-HAS2 and pCMV6-XL4-ITGA6 did not contain 3'UTR sequences. The culture medium was collected 48h after transfection, where the secretion of TIMP1 and CTGF was measured by ELISA. In addition, the levels of fibrosis markers ACTA2, COL1A1 and S100A6 in co-transfected cells were evaluated by qRT-PCR ( Fig.36 AE). TIMP1 levels were significantly higher in cells co-transfected with miR-190b-5p / HAS2 or co-transfected with miR-296-3p / ITGA6 than in cells transfected with miR-190b-5p or miR-296-3p alone. mRNA levels of fibrosis markers and SMA protein levels were also higher in co-transfected cells ( Fig.37 ).

[0124] Taken together, these results suggest that HAS2 and ITGA6 contribute to the activation of MF and its profibrotic features, and that inhibition of HAS2 and / or ITGA6 reduces the development of liver fibrosis.

[0125] HAS2 is the necessary enzyme for synthesizing hyaluronic acid. In addition, it also regulates cell migration, proliferation and transdifferentiation. Hyaluronic acid (HA) is a high molecular weight polymer, which is a biomarker for chronic liver fibrosis. Its formation increases in liver disease, and it is a component of ECM. In liver injury, it is degraded into low molecular weight hyaluronic acid (LMW-HA) and medium molecular weight (MMW-HA) hyaluronic acid. LMW-HA is formed in an enzymatic or non-enzymatic manner, and it further aggravates damage via myofibroblast activation and inflammation.

[0126] Currently, the mechanism of action of HAS2 and ITGA6 in promoting fibrosis is speculated to be reduced through miR-190b-5p-mediated downregulation of HAS2, resulting in reduced HA formation and, in turn, reduced LMW-HA amounts. To test this hypothesis, LMW-HA ( Fig.38 ). The amount of LMW-HA was significantly reduced after miR-190b-5p transfection, while the amount of LMW-HA was restored after HAS2 overexpression. As an additional validation, primary human MFs were treated with 100 pg / mL LMW-HA for 48 hours, after which COL1A1, ACTA2, and S100A6 were measured by qRT-PCR. Treatment of cells with LMW-HA increased the expression of fibrotic markers, and immunocytochemistry showed that SMA protein levels were also increased ( Fig.39 ). Thus, it was found that miR-190b-5p-mediated HAS2 inhibition resulted in the improvement of liver fibrosis due to the lower formation of LMW-HA.

[0127] Amphireguline (AREG), a protein previously identified as an important regulator of liver fibrosis, has been reported to interact with ITGA6. AREG is synthesized as a transmembrane protein (Pro-AREG) and then cleaved into AREG that binds to EGFR and the like. Binding of AREG leads to the formation and release of profibrotic and proinflammatory cytokines such as TGFβ and CCL239A. Therefore, it is speculated that the interaction between AREG and ITGA6 is an important pathway regulating the movement, invasion and activation of HSCs. AREG binds to membrane-localized ITGA6, which is involved in HSC activation. Measurement of the levels of membrane-associated AREG in primary human MFs transfected with miR-296-3p or scramble or co-transfected with miR-296-3p and ITGA6 expression plasmids showed that the level of membrane-associated AREG was lower in miR-2963p-transfected cells than in scramble-transfected cells, and this level was restored after ITGA6 overexpression ( Fig.40 In addition, primary human MFs were treated with 100 ng / mL AREG for 48 h, after which fibrotic markers were measured by qRT-PCR and immunocytochemistry. The expression of ACTA2, COL1A1, and S100A6 was upregulated compared with untreated cells ( Fig.36 The protein levels of CE and SMA were upregulated after AREG treatment, indicating the importance of AREG-ITGA6 signaling in HSC activation ( Fig.38 , 41 , 42 and 43).

[0128] Overexpression of HAS2 and ITGA6 in myofibroblasts in vivo showed the importance of HAS2 and ITGA6 in liver fibrosis in vivo. The ORF encoding one of HAS2 and ITGA6 was cloned downstream of the Pdgfrb promoter. AAV2p75-Pdgfrb-HAS2 contains the coding sequence of HAS2 under the control of the Pdgfrb promoter, and AAV2p75-Pdgfrb-ITGA6 contains the coding sequence of ITGA6 under the control of the Pdgfrb promoter. In addition, the viral vectors AAV2p75-Pdgfrb-190b-5p contained the coding sequence of 190b-5p under the control of the Pdgfrb promoter, AAV2p75-Pdgfrb-296-3p contained the coding sequence of 296-3p under the control of the Pdgfrb promoter, and as a control, AAV2p75-Pdgfrb-mCherry encoding the reporter gene mCherry under the control of the Pdgfrb promoter. AAV2p75-Pdgfrb-190b-5p, AAV2p75-Pdgfrb296-3p, or AAV2p75-Pdgfrb-mCherry was first administered intravenously to mice injected with CCl4 for 6 weeks, as described in the previous article. Fig.44 Four days after the initial AAV injection, one of AAV2p75-Pdgfrb-HAS2 or AAV2p75Pdgfrb-ITGA6 was administered as a second injection to mice injected with AAV2p75-Pdgfrb-190b-5p or AAV2p75-Pdgfrb-296-3p, respectively. Ten days after the second AAV injection, the mice were sacrificed and tissues were analyzed.

[0129] The overexpression of miR-190b-5p and miR296-3p was confirmed by qRT-PCR ( Fig.45 Then, the levels of Has2 and Itga6 were measured in the corresponding groups ( Fig.46 , 47 ). Hydroxyproline content and expression of fibrosis markers COL1A1 and Acta2 were determined ( Fig.48 , 49 HE and Sirius red staining and SMA immunohistochemistry are shown in Fig.50 The protein levels of HAS2 and ITGA6 were analyzed by Western blotting and immunohistochemistry ( Fig.51, 52). Unfortunately, in each group, one mouse was an outlier. Notably, these were the mice with the lowest expression of HAS2 and ITGA6. Based on these data, no significant differences in the expression of fibrosis markers and the extent of fibrosis were observed. Significant histological changes were observed in the ITGA6 overexpression group in the form of irregularly shaped cells that appeared to form and reside in their own niche, surrounded by collagen fibers.

[0130] The human liver bud model confirms that the inhibitors of HAS2 and ITGA6, miR-190b and miR-296, are suitable for treating and preventing fibrosis. In order to generate the human liver bud model, primary human MFs were transfected with miR-190b5p, miR-296-3p or Scramble. The transfected MFs were then aggregated with organoids derived from primary human hepatocytes, human Kupffer cells and endothelial cells, as schematically depicted in Figure 53. Afterwards 48h and 96h, culture medium was collected, wherein TIMP1 levels (Figure 54) and CCL2 levels were measured by ELISA. Consistent with previous findings, in the presence of miR-190b and miR-296, the levels of TIMP1 and CCL2 determined in the liver buds were lower than those of controls (scramble).

[0131] In addition, compared with the control, it was found that the pro-inflammatory cytokine CCL2 at a lower level in the liver buds regulated by miR-190b-5p and miR-296-3p was expressed. In addition, the expression of ALB (Figure 56), HNF4A (Figure 57) and TTR (Figure 58) was measured by qRT-PCR. It is worth noting that, compared with the control, the presence of miR-296-3p in the liver buds caused the expression of ALB and TTR to be significantly higher. This shows that miR-296-3p may also serve as a pro-hepatogenic miRNA. These results confirm that the inhibitors of HAS2 and / or ITGA6, as exemplified by miR-190b-5p and miR-296-3p, are suitable as anti-fibrotic drug compounds.

[0132] The protein changes that occurred at the single cell level in vivo after administration of AAV2p75-Pdgfrb-190b-5p or AAV2p75-Pdgfrb-296-3p in myofibroblasts were analyzed by single cell proteomics (ScopeMS). AAV was administered intravenously during the sixth CCl4 injection of Lrat-Cre-ZsGreen mice, as schematically depicted in Figure 59. Four days later, hepatocytes and all non-parenchymal cells were isolated, of which ZsGreen-positive cells (HSC and HSC-derived MF) and ZsGreen-negative cell populations were sorted by FACS. ScopeMs analysis was performed using ZsGreen-positive cells, and the MS results of ZsGreen+MF overexpressed by miRNA were then compared with the MS results of ZsGreen+MF injected with the control. Compared with the control group, five proteins were detected to be downregulated by 2 times in the AAV2p75-Pdgfrb-190b-5p group (Figure 60). These proteins are TPM1, SEC61A1, PSME3, TARS and OLA1. Of note, TPM1 was previously reported to be upregulated on the surface of activated HSCs and its expression correlated with that of SMA41. In addition, upon TGFβ1 treatment of MFs, the expression of Tpm1 was induced and this was necessary for the stable incorporation of SMA into stress fibers, as schematically depicted in FIG62 .

[0133] In the AAV2p75-Pdgfrb-296-3p group, 2 proteins were detected to be downregulated 2-fold compared to the control group (Figure 61). These two proteins are KARS and SEC61A1. In cancer cells, KARS can translocate into the membrane and interact with ITGA6. This interaction leads to the activation of the ERK / C-Jun pathway and the migration of cancer cells (Figure 62). It is noteworthy that this pathway also contributes to the activation of HSC48.

[0134] Taking AAV8-Pdgfrb-miRNA or AAV2p75-PdgfrbmiRNA as examples, these results also suggest that miRNAs that are inhibitors of HAS2 and / or ITGA6 are best contained in AAV-based viral particles, which have been shown to preferentially deliver antifibrotic miRNAs to myofibroblasts in vivo, for example in central-peripheral models of liver fibrosis and in cholestasis models.

[0135] Comparative Example: Testing the anti-fibrotic activity of a large number of miRNAs in human primary fibroblasts

[0136] As described in Example 1, primary human myofibroblasts (MF) and SMALX2 cells (LX2) as a further comparison were used to test the anti-fibrotic activity of a number of microRNAs, analyzed by qPCR for the fibrotic markers DES, ACTA2, LOX2 and CO1A1, by ELISA to measure the regulation of TIMP1 secretion and by ELISA to measure CTGF levels. Analysis of these markers (Figure 63) showed that their expression in MF was significantly higher than that in LX2 cells. As shown in the micrographs of Figure 64, the pro-fibrotic characteristics of MF were confirmed by immunocytochemical staining for alpha smooth muscle actin (SMA).

[0137] MicroRNA is used in the form of microRNA mimics, which are non-natural double-stranded miRNA-like RNA fragments that have a motif complementary to the 3'UTR portion of the target gene at the 5' end. In cells, this RNA fragment simulating endogenous miRNA binds to its target gene for inhibition of translation. In this test, MF was used in three independent batches, and separate aliquots of MF were transfected with miRNA mimics. Figures 65a, 65b, and 65c show the TIMP1 levels in three MF cultures MF1, MF2, and MF3 in the presence of miRNA mimics, wherein the horizontal line indicates the TIMP1 level in the control (MF without miRNA mimics). The following table identifies miRNAs that cause a high degree of variation in TIMP1 expression:

[0138] miRNA TIMP1 change (%)

[0139] hsa-miR-27b-3p 52.94068 hsa-miR-195-3p 50.57661 hsa-miR-18a-5p 47.36129 hsa-miR-487b-3p 47.30115 hsa-miR-486-3p 47.28265 hsa-miR-103a-2-5p 34.80073 hsa-miR-1247-5p 34.23632 hsa-miR-190b-5p 31.95899 hsa-miR-543 31.95205 hsa-miR-296-3p 30.49051 hsa-miR-411-3p 27.44713 hsa-miR-423-3p 25.9586 hsa-miR-30c-1-3p 22.13955 hsa-miR-154-3p 39.57282 hsa-miR-433-5p 35.1107 hsa-miR-135b-5p 31.96593 hsa-miR-653-Sp 31.28316 hsa-miR-200b-3p 30.99324 hsa-miR-145-5p 30.8799 hsa-miR-455-5p 29.35782 hsa-miR-199a-3p 27.8797 hsa-miR-183-3p 25.72227 hsa-miR-744-5p 25.49828 hsa-miR-30la-3p 25.41848 hsa-miR-9-3p 24.70139 hsa-miR-124-3p 21.01303 hsa-miR-744-3p 20.23695 hsa-miR-494-3p 17.56292 hsa-miR-129-1-3p 12.87642 hsa-miR-17-5p 10.15265 hsa-let-7g-5p 25.0424 hsa-miR-30b-5p 28.0854 hsa-miR-328-3p 32.6469

[0140] Then, the effects of these miRNAs on the expression level of CTGF in MF were determined using their mimics as representatives. Figures 53A-53C The expression levels of CTGF in individual aliquots of MF batches MF1, MF2, MF3 are shown, wherein the horizontal line indicates the CTGF level in the control.

[0141] This assay showed that among the miRNA panel, only miR-190b-5p and miR-296-3p (a preferred combination) had a strong anti-fibrotic effect.

Claims

1. A compound for preventing or treating liver fibrosis, characterized in that The compound is miR-190b-5p, miR-296-3p or a combination of these.

2. A compound for use in medical treatment or prevention, characterized in that The compounds are hyaluronan synthase (HAS2) inhibitors and / or integrin alpha-6 (ITGA6) inhibitors.

3. The compound for use in medical treatment or prevention according to claim 2, wherein the medical treatment or prevention is the treatment or prevention of liver fibrosis.

4. The compound for use according to any one of claims 2 to 3, characterized in that The hyaluronan synthase (HAS2) inhibitor is miR-190b-5p.

5. The compound for use according to any one of claims 2 to 4, characterized in that The integrin alpha-6 (ITGA6) inhibitor is miR-296-3p.

6. Compound for use according to one of the preceding claims, characterized in that miR-190b-5p, miR-296-3p, or a combination of these is encoded in a nucleic acid construct under the control of a promoter for expression in profibrotic myofibroblasts.

7. Compound for use according to one of the preceding claims, characterized in that The miR-190b-5p and / or miR-296-3p are encoded in an expression cassette under the control of a promoter that is specifically expressed in profibrotic myofibroblasts (MF).

8. The compound used according to claim 6, characterized in that The promoter specifically expressed in profibrotic myofibroblasts is the Pdgfrb promoter.

9. Compound for use according to one of the preceding claims, characterized in that The miR-190b-5p, miR-296-3p or a combination of these are encoded in AAV-based viral particles.

10. The compound for use according to claim 3, characterized in that The hyaluronan synthase (HAS2) inhibitor is at least one of 4-methylumbelliferone (4-MU or MU), 4-methylesculetin (ME), and brefeldin A.

11. Compound for use according to one of the preceding claims, characterized in that Liver fibrosis is periportal and / or pericentral and / or cholestatic liver fibrosis.

12. Method for treating or preventing liver fibrosis by administering a compound according to any one of the preceding claims.

13. The method for treating or preventing liver fibrosis according to claim 12, which is carried out by administering the selected compound.

14. A method for treating or preventing liver fibrosis by administering a hyaluronan synthase (HAS2) inhibitor and / or an integrin alpha-6 (ITGA6) inhibitor.

15. The method for treating or preventing liver fibrosis according to claim 14, wherein the hyaluronan synthase inhibitor is a nucleic acid construct encoding miR-190b-5p, and the integrin α-6 inhibitor is a nucleic acid construct encoding miR-296-3p. 16 . The method for treating or preventing liver fibrosis according to claim 15 , wherein the nucleic acid construct encoding miR-190b-5p encodes miR-190b-5p under the control of Pdgfrb promoter. 17 . The method for treating or preventing liver fibrosis according to claim 15 , wherein the nucleic acid construct encoding miR-296-3p encodes miR-296-3p under the control of Pdgfrb promoter.

18. The method for treating or preventing liver fibrosis according to claim 15, wherein the nucleic acid construct encoding miR-190b-5p is contained in a viral particle.

19. The method for treating or preventing liver fibrosis according to claim 15, wherein the nucleic acid construct encoding miR-296-3p is contained in a viral particle.

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