GPR110 overexpression adeno-associated virus and application thereof in preparation of medicine for treating metabolism-related steatohepatitis

Abstract

The invention discloses an adeno-associated virus overexpressing GPR110 and application of the adeno-associated virus in preparation of a medicine for treating metabolism-related steatohepatitis. The adeno-associated virus comprises a GPR110 gene sequence encoded and operably connected to a liver specific promoter TBG, an in-vivo experiment is carried out in a constructed MASH mouse model, and the result shows that the expression of the GPR110 gene in the liver tissue of the MASH mouse model is reduced, so that the GPR110 gene can be used as a biomarker and a therapeutic target of MASH, and the application of the GPR110 gene in the MASH mouse model is proven. According to the present invention, the MASH-related symptoms can be significantly improved through the AAV adeno-associated virus targeting liver specific overexpression GPR110, wherein the MASH-related symptoms comprise liver triglyceride content reducing, liver injury relieving, liver inflammation improving and liver fibrosis improving; the AAV-mediated GPR110 overexpression can effectively improve the MASH pathological process, and new intervention drugs and means can be provided for MASH treatment.

Description

Technical Field

[0001] This application relates to adeno-associated virus (AAV) overexpressing GPR110 and its application in the preparation of drugs for treating metabolic-associated steatohepatitis, belonging to the field of biomedical technology. Background Technology

[0002] Metabolic dysfunction-associated steatohepatitis (MASH) is one of the leading chronic liver diseases with a continuously rising global incidence. [1,2] Its characteristics include excessive triglyceride deposition in hepatocytes, chronic inflammation, and persistent hepatocyte damage, which in turn activate hepatic stellate cells and promote collagen deposition, leading to liver fibrosis. Liver fibrosis is irreversible and can further develop into cirrhosis and hepatocellular carcinoma. [2] Epidemiological studies show that MASH has become one of the fastest-growing causes of liver cancer in the past decade. [3] Currently, the US FDA has approved two drugs for the treatment of MASH: Resmetirom (approved in 2024) and Semaglutide (approved in 2025). Resmetirom is a thyroid hormone receptor β (THRβ) agonist, but its clinical response rate is low, only about 25%–30%, indicating limited efficacy. [4] Semaglutide, a GLP-1 receptor agonist, primarily improves liver metabolism through weight loss mechanisms, but its efficacy remains unsatisfactory in non-obese patients with MASH or those with severe liver fibrosis. Therefore, current treatment for MASH lacks effective drugs that can directly target liver metabolic disorders and inflammatory responses, necessitating the development of novel treatment strategies and molecular targets.

[0003] G protein-coupled receptor 110 (GPR110, also known as ADGRF1) is a member of the adhesive G protein-coupled receptor family and possesses a typical seven-transmembrane domain. Previous research has primarily focused on the function of GPR110 in the nervous system, finding its involvement in processes such as neurite growth and synapse formation. [4] However, there are no reports, either domestically or internationally, on the role of GPR110 in MASH, or whether targeting GPR110 can be used as an intervention for MASH.

[0004] References: 1. Miao L, Targher G, Byrne CD, Cao YY, Zheng MH. Current status and future trends of the global burden of MASLD. Trends Endocrinol Metab. 2024;35(8):697-707. 2. Feng G, Valenti L, Wong VW, Fouad YM, Yilmaz Y, Kim W, et al. Recompensation in cirrhosis: unravelling the evolving natural history ofnonalcoholic fatty liver disease. Nat Rev Gastroenterol Hepatol. 2024;21(1):46-56. 3. Huang DQ, Singal AG, Kono Y, Tan DJH, El-Serag HB, Loomba R. Changing global epidemiology of liver cancer from 2010 to 2019: NASH is the fastest growing cause of liver cancer. Cell Metab. 2022; 34(7):969-977. 4. Kwon H, Kevala K, Xin H, Patnaik S, Marugan J, Kim HY. Ligand-induced GPR110 activation facilitates axon growth after injury. Int J MolSci. 2021; 22(7):3386. Summary of the Invention The purpose of this invention is to address the shortcomings of the prior art by providing an adeno-associated virus overexpressing GPR110 and its application in the preparation of a drug for treating metabolic-associated steatohepatitis.

[0005] To achieve the above objectives, this application adopts the following technical solution: This application provides the use of adeno-associated virus overexpressing GPR110 in the preparation of a medicament for treating metabolic-associated steatohepatitis, wherein the adeno-associated virus comprises a GPR110 gene sequence operatively linked to the liver-specific promoter TBG. In some implementations, the adeno-associated virus is adeno-associated virus type 9 (AAV9).

[0006] In some embodiments, the liver-specific promoter includes any one selected from albumin promoter, α1-antitrypsin (AAT) promoter, thyroid-binding globulin promoter, alpha-fetoprotein promoter, alcohol dehydrogenase promoter, apolipoprotein E (ApoE) promoter, low-density lipoprotein promoter, pyruvate kinase promoter, phosphoenolpyruvate carboxykinase promoter, phenylalanine hydroxylase promoter, lecithin-cholesterol acyltransferase (LCAT) promoter, apolipoprotein H (ApoH) promoter, apolipoprotein A-II promoter (APOA2), transferrin promoter, transthyretin promoter, and alpha-fetoprotein (AFP) promoter.

[0007] In some specific implementation schemes, the liver-specific promoter is the thyroid-binding globulin promoter.

[0008] In some embodiments, the drug comprises an active ingredient and pharmaceutically acceptable excipients, the active ingredient comprising an adeno-associated virus overexpressing GPR110. In some embodiments, the pharmaceutically acceptable excipients include one or more of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, cosolvents, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, coating materials, antioxidants, and buffers.

[0009] In some implementations, the drug is delivered to the liver via intravenous injection or local targeted delivery.

[0010] Compared with the prior art, this application has the following beneficial effects: The adeno-associated virus (AAV) overexpressing GPR110 provided in this application is an AAV9 that specifically overexpresses GPR110 in the liver. Animal experiments have shown that liver-specific GPR110 overexpression mediated by the adeno-associated virus serotype 9-thyroxine-binding globulin promoter (AAV9-TBG) can effectively improve MASH-related pathological features such as hepatic lipid deposition, inflammatory response, and fibrosis, thus providing a new gene intervention strategy for MASH treatment. Attached Figure Description

[0011] Figure 1 The mRNA expression (A) and protein expression (B) of the liver GPR110 gene were significantly reduced in MASH mice induced by a choline-deficient high-fat diet (CD-HFD).

[0012] Figure 2 MASH mice induced by a choline-deficient high-fat diet (CD-HFD) were injected via the tail vein with either control AAV-GFP or AAV-GPR110. The AAV-GPR110 group showed significant overexpression of the GPR110 gene in the liver and significantly improved liver indicators: (A) Detection of the abundance of GPR110 protein expression in the liver; (BD) Compared with the control group, the liver weight (B), liver weight to body weight ratio (C), and liver triglyceride content (D) of the AAV-GPR110 group were significantly reduced.

[0013] Figure 3 Eosin-hematoxylin staining and Oil Red O staining showed that, compared with the control group, the AAV-GPR110 group of mice had reduced lipid deposition and ballooning degeneration in the liver.

[0014] Figure 4 Compared with the control group, the plasma alanine aminotransferase (ALT) and plasma aspartate aminotransferase (AST) levels in the AAV-GPR110 group mice were significantly reduced.

[0015] Figure 5 Immunohistochemical staining (A) and quantitative statistical analysis (B) of F4 / 80 showed that, compared with the control group mice, the AAV-GPR110 group mice had reduced activation and infiltration of inflammatory cells in the liver.

[0016] Figure 6 Compared with the control group, the AAV-GPR110 group of mice showed improved collagen fiber deposition in the liver: (A) Sirius red staining indicates collagen fiber deposition; (B) Statistical analysis of Sirius red staining in the livers of the two groups of mice.

[0017] Figure 7 Compared with the control group, the AAV-GPR110 group mice showed significantly reduced mRNA expression of liver inflammation-related genes and fibrosis-related genes. Detailed Implementation

[0018] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0019] Unless otherwise specified, the experimental or testing methods described in the following examples are conventional methods; the reagents and materials described are obtained from conventional commercial sources unless otherwise specified.

[0020] Example 1 I. Materials and Methods Mouse experiments: Eight-week-old male C57BL / 6 mice were purchased from Shanghai Silex Laboratory Animal Co., Ltd. Husbandry and experiments were conducted at the Department of Laboratory Animal Science, Shanghai Jiao Tong University School of Medicine. The normal diet was purchased from Shanghai Silex Laboratory Animal Co., Ltd., catalog number M01-F, consisting of 10 kcal% fat, 70 kcal% carbohydrates, and 20 kcal% protein. The choline-deficient high-fat diet was purchased from Research Diet Pharmaceuticals, USA, catalog number A06071302, consisting of 60 kcal% fat and 0.1% methionine. All mice were euthanized upon reaching the experimental endpoint. Whole blood was anticoagulated, centrifuged, and the supernatant was collected to obtain plasma, which was stored at -80℃ for subsequent experiments to detect plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. A portion of fresh liver tissue was collected, flash-frozen in liquid nitrogen, and stored at -80℃. In subsequent experiments, the frozen liver tissue was used for RNA extraction and real-time quantitative PCR experiments to detect the mRNA expression abundance of the target genes. Fresh liver tissue was collected, fixed in 4% paraformaldehyde for 48 hours, and then paraffin sections were prepared for eosin hematoxylin staining, F4 / 80 immunohistochemical staining, and Sirius red staining; frozen sections were also prepared for Oil Red O staining.

[0021] 2. AAV adeno-associated virus (AAV-GPR110) overexpressing the GPR110 gene was constructed by Shanghai Jikai Gene Chemical Technology Co., Ltd. 1) First, the target gene was obtained by PCR amplification. The target gene name is Adgrf1(NM_133776); the species is Mouse. 2) Construct an AVV recombinant plasmid overexpressing the Adgrf1 gene. The vector information is as follows: Vector name: GV632; Component order: TBGp-MCS-SV40 PolyA; Cloning site: BamHI / HindIII.

[0022] 3) Virus packaging and purification: GV632 recombinant plasmid + Rep / Cap9 plasmid + helper plasmid (pHelper) were co-transfected into HEK293 cells. Subsequent purification and titer determination were performed in the same manner as conventional AAV preparation.

[0023] 3. Triglyceride content detection: The detection was performed according to the instructions of the triglyceride quantitative detection kit provided by Beijing Pulilai Co., Ltd., China (catalog number E1013).

[0024] 4. Detection of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels: The tests were performed according to the instructions of the quantitative detection kits provided by Shanghai Kehua Co., Ltd., China.

[0025] 5. F4 / 80 Immunohistochemical Staining: F4 / 80 is a marker protein of inflammatory cells. Immunohistochemical staining of paraffin-embedded liver sections allows observation of the activation and infiltration of inflammatory cells in the liver. The F4 / 80 antibody was purchased from Abcam (UK), catalog number ab6640.

[0026] 6. Sirius Red Staining: Sirius red is an acidic dye that reacts with alkaline collagen to produce color, allowing observation of collagen fiber deposition in the liver. Sirius red dye was purchased from Shanghai Ruiyu Biotechnology Co., Ltd., catalog number Bry-0013.

[0027] 7. Detection of liver gene expression: The expression of the following genes was detected by real-time quantitative PCR: Adhesion G protein-coupled receptor F1 (Adgrf1, also known as Gpr110), tumor necrosis factor (Tnf), interleukin 1 beta (Il1b), interleukin 6 (Il6), interleukin 12 beta (Il1b), inducible nitric oxide synthase 2 (Nos2), chemokine ligand 2 (C-Cmotif chemokine ligand 2, Ccl2), chemokine 5 (CC motif chemokine ligand 5, Ccl5), and adhesion G protein-coupled receptor E1 (Adgre1) are hallmark genes of liver inflammation. Type I collagen alpha 1 (Col1a1), smooth muscle cell-specific alpha-actin (Actin alpha 2, smooth muscle, Acta2), transforming growth factor beta 1 (TGFb1), and matrix metalloproteinase 13 (Mmp13) are hallmark genes of liver fibrosis. The 36b4 gene was used as an internal control in real-time quantitative PCR experiments.

[0028] Statistical Analysis: All numerical variables in this study are expressed as mean ± standard error. A two-tailed Student's t-test was used to compare variables between the two groups. A p-value < 0.05 was considered statistically significant. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. II. Implementation Plan and Results 1. GPR110 gene expression was significantly downregulated in liver tissue of the MASH mouse model. To investigate the changes of GPR110 in the development and progression of MASH, a choline-deficient high-fat diet (CD-HFD) mouse model was induced. In the CD-HFD model, 8-week-old male C57BL / 6 mice were randomly divided into a normal diet group (ND) and a CD-HFD group, with 6 mice in each group. The mice were fed a normal diet for 10 weeks, and liver tissue was collected for analysis. The results showed that compared with the normal diet group, the mRNA expression of GPR110 in the liver of mice in the CD-HFD group was significantly reduced. Figure 1 A), protein levels were also simultaneously downregulated ( Figure 1 B). This finding suggests that GPR110 may play a key regulatory role in the occurrence and development of MASH, and has potential application value as a target for intervention and treatment.

[0029] 2. Aims to improve liver pathological changes in MASH mice by AAV-mediated GPR110 overexpression. The aforementioned results showed that GPR110 gene expression was significantly downregulated in the liver tissue of the MASH mouse model, suggesting that GPR110 may play an important role in the occurrence and development of MASH. Therefore, an AAV-mediated GPR110 overexpression virus (AAV-GPR110) was further constructed to verify its protective effect in MASH mice. Eight-week-old male C57BL / 6 mice were fed a choline-deficient high-fat diet (CD-HFD) for 10 weeks to induce the MASH model, and then randomly divided into two groups: the first group was injected with the control virus AAV-GFP via tail vein (control group), and the second group was injected with AAV-GPR110 via tail vein (GPR110 overexpression group). After 4 weeks of continued feeding following injection, the mice were sacrificed, and liver and blood samples were collected for testing.

[0030] Protein expression detection results showed that GPR110 gene expression was increased in the liver of mice in the GPR110 overexpression group, confirming successful GPR110 overexpression. Figure 2 A). Liver-related indicators showed that, compared with the control group, the liver weight and liver / body weight ratio of mice in the GPR110 overexpression group were significantly decreased, and the liver triglyceride content was significantly reduced (A). Figure 2 B-2D). Eosin hematoxylin staining and Oil Red O staining results showed that GPR110 overexpression significantly improved hepatic lipid deposition and ballooning degeneration (B-2D). Figure 3 A-3B). The above data indicate that triglyceride deposition in the liver of mice overexpressing GPR110 was improved. Furthermore, the levels of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice overexpressing GPR110 were significantly reduced. Figure 4 (A-4B) indicates that GPR110 overexpression can alleviate liver damage. F4 / 80 staining shows a reduction in the activation and infiltration of inflammatory cells in the liver ( Figure 5A-5B), Sirius red staining indicates improved liver fibrosis ( Figure 6 A-6B). Further analysis showed that GPR110 overexpression could reduce the mRNA levels of inflammatory factors and fibrosis-related genes in liver tissue (A-6B). Figure 7 ).

[0031] In conclusion, AAV-mediated GPR110 overexpression can significantly reduce lipid deposition, inflammatory response, and fibrosis progression in MASH mice, and can serve as a targeted therapeutic agent for MASH.

[0032] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. Use of an adeno-associated virus overexpressing GPR110 in the manufacture of a medicament for the treatment of metabolic-associated steatohepatitis, characterized in that, The adeno-associated virus comprises a GPR110 gene sequence operably linked to a liver-specific promoter TBG.

2. Use according to claim 1, characterized in that, The adeno-associated virus is a type 9 adeno-associated virus (AAV9).

3. Use according to claim 1, characterized in that, The liver-specific promoter comprises any one selected from the group consisting of an albumin promoter, an alpha 1 -antitrypsin (AAT) promoter, a thyroxin-binding globulin promoter, an alpha-fetoprotein promoter, an alcohol dehydrogenase promoter, an apolipoprotein E (ApoE) promoter, a low-density lipoprotein promoter, a pyruvate kinase promoter, a phosphoenolpyruvate carboxykinase promoter, a phenylalanine hydroxylase promoter, a lecithin-cholesterol acyltransferase (LCAT) promoter, an apolipoprotein H (ApoH) promoter, an apolipoprotein A-II promoter (APOA2), a transferrin promoter, a transthyretin promoter, and an alpha-fetoprotein (AFP) promoter.

4. Use according to claim 1, characterized in that, The liver-specific promoter is a thyroxin-binding globulin promoter.

5. The use according to claim 1, characterized in that, The medicine comprises an active ingredient comprising an adeno-associated virus overexpressing GPR110 and a pharmaceutically acceptable excipient.

6. Use according to claim 5, characterized in that, The pharmaceutically acceptable excipient comprises one or more of a diluent, a binder, a wetting agent, a lubricant, a disintegrant, a solvent, an emulsifier, a cosolvent, a preservative, a pH adjuster, an osmotic pressure adjuster, a surfactant, a coating material, an antioxidant, and a buffer.

7. The use according to any one of claims 1 to 6, characterized in that, The medicine is delivered to the liver by intravenous injection or local targeted delivery.

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