WO2023151624A1

WO2023151624A1 - Uses of asgr1 inhibitor in promoting cholesterol efflux and treating non-alcoholic fatty liver disease

Info

Publication number: WO2023151624A1
Application number: PCT/CN2023/075224
Authority: WO
Inventors: 宋保亮; 王菊琼
Original assignee: 武汉大学
Priority date: 2022-02-11
Filing date: 2023-02-09
Publication date: 2023-08-17
Also published as: CN116617390A

Abstract

The use of an ASGR1 inhibitor in treating non-alcoholic fatty liver disease (NAFLD), the use of an ASGR1 inhibitor in promoting cholesterol efflux, the use of the combination of an ASGR1 inhibitor and a second lipid-lowering drug in treating non-alcoholic fatty liver disease (NAFLD), an anti-ASGR1 monoclonal antibody and the use thereof.

Description

Application of ASGR1 inhibitors in promoting cholesterol efflux and treating nonalcoholic fatty liver disease

technical field

The present invention relates to the application of ASGR1 inhibitor in promoting cholesterol efflux and treating non-alcoholic fatty liver disease. The present invention also relates to anti-ASGR1 monoclonal antibodies and the combined use of ASGR1 inhibitors and another lipid-lowering drug.

Background technique

Non-alcoholic fatty liver disease (NAFLD) is a type of chronic liver disease, excluding liver steatosis caused by alcohol, drugs, genetics and other clear liver injury factors (defined as the presence of > 5% fat transgender), most common in Western countries. According to reports, the prevalence of NAFLD worldwide is 25%, and it shows a gradual upward trend. The incidence of NAFLD in obese and diabetic patients is significantly higher than that in the general population. As an important part of metabolic syndrome, NAFLD is the manifestation of metabolic syndrome in the liver and can be divided into simple non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (non-alcoholic steatohepatitis, NASH). NAFL and NASH are considered major fatty liver diseases because they account for the largest proportion of individuals with elevated hepatic lipids. The severity evaluation indicators of NAFL/NASH include the presence of lipids, inflammatory cell infiltration, vacuolar degeneration of hepatocytes, and degree of fibrosis.

In the early stage of NAFLD, it mainly manifests as excessive accumulation of triglyceride (TG), cholesterol and cholesteryl ester in the liver in the form of lipid droplets, and there is no or only a small amount of inflammation or liver cell damage in the liver. The accumulation of a large number of lipid droplets in the liver leads to oxidative stress in the mitochondria, which induces the production of a large number of cytokines, such as tumor necrosis factor (TNFα) and interleukin 6 (IL-6), which makes NAFLD develop into liver fibrosis and eventually develop become cirrhosis and liver cancer.

Type II diabetes, obesity, hyperlipidemia, insulin resistance, cardiovascular and cerebrovascular diseases are closely related to the development of NAFLD, and these factors cooperate with each other to promote the occurrence and development of NAFLD. Since the onset and symptoms of NAFLD are extremely difficult to detect, even though more and more researchers are trying to reveal the mechanism behind NAFLD, there are still many obstacles.

At present, there are no drugs approved for the specific treatment of NAFLD at home and abroad. Weight control and improvement of insulin resistance are still the mainstream treatment for NAFLD. It should be pointed out that traditional lipid-lowering drugs such as statins cannot effectively treat fatty liver. On the one hand, lipid-lowering drugs can damage the liver and increase transaminases; Fatty liver, and the occurrence of fatty liver is not all caused by high blood lipids. If it can actively promote the efflux of cholesterol in liver cells and reduce the accumulation of excess lipids in the liver, it can prevent the occurrence of NAFLD at an early stage and prevent the transformation of NAFL into NASH.

Contents of the invention

The inventors found that lipid accumulation in the liver of Asgr1 systemic deficient mice was significantly alleviated, and further research found that the amount of total cholesterol efflux in the liver into bile and feces was significantly increased. Subsequently, it was identified that Asgr1-deficient mice significantly up-regulated the protein level of the transcription factor LXRα, which regulates cholesterol efflux, and promoted the increase of ABCG5/8 and ABCA1 cholesterol efflux transporters; at the same time, the activation of AMPK inhibited the transcription factor SREBP-1c of lipid synthesis in the liver It enters the nucleus and down-regulates the downstream target genes of lipid synthesis, thereby inhibiting the synthesis of lipids in the liver.

In a first aspect, the present invention provides the use of an ASGR1 inhibitor in the preparation of a medicament for promoting cholesterol efflux.

In a second aspect, the present invention provides the use of an ASGR1 inhibitor in the preparation of a medicament for treating NAFLD.

In a third aspect, the present invention provides an ASGR1 inhibitor for use in promoting cholesterol efflux.

In a fourth aspect, the present invention provides ASGR1 inhibitors for use in the treatment of NAFLD.

In a fifth aspect, the invention provides a method of promoting cholesterol efflux comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor.

In a sixth aspect, the invention provides a method of treating NAFLD comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor.

In the seventh aspect, the present invention provides a pharmaceutical composition for treating NAFLD or promoting cholesterol efflux, which comprises a therapeutically effective amount of an ASGR1 inhibitor and a pharmaceutically acceptable carrier.

The inventors also found that the combined administration of an ASGR1 inhibitor and another lipid-lowering drug produced a significant synergistic lipid-lowering effect, and both the total cholesterol level and the triglyceride level in the serum and liver were significantly reduced.

In an eighth aspect, the present invention provides a combination of an ASGR1 inhibitor and a second lipid-lowering drug for promoting cholesterol efflux, treating NAFLD, reducing blood and/or liver total cholesterol levels, and reducing blood and/or liver glycerol Triester levels, or the use of drugs to prevent cardiovascular disease.

In the ninth aspect, the present invention provides a combination of an ASGR1 inhibitor and a second lipid-lowering drug for promoting cholesterol efflux, treating NAFLD, reducing total cholesterol levels in blood, reducing triglyceride levels in blood, or preventing cardiovascular diseases. combination.

In a tenth aspect, the present invention provides a method for promoting cholesterol efflux, treating NAFLD, lowering blood and/or liver total cholesterol levels, lowering blood and/or liver triglyceride levels, or preventing cardiovascular disease, wherein The method includes administering to the subject a therapeutically effective amount of an ASGR1 inhibitor and a therapeutically effective amount of a second lipid-lowering agent.

In an eleventh aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an ASGR1 inhibitor and a therapeutically effective amount of a second lipid-lowering drug.

In a twelfth aspect, the present invention provides a kit comprising a first pharmaceutical composition and a second pharmaceutical composition, the first pharmaceutical composition comprising a therapeutically effective amount of an ASGR1 inhibitor and a pharmaceutically acceptable carrier , the second pharmaceutical composition includes a therapeutically effective amount of the second lipid-lowering drug and a pharmaceutically acceptable carrier.

In any of the above aspects, the NAFLD is NAFL, NASH, NAFLD with liver fibrosis, NAFLD with liver cirrhosis, or NAFLD with hepatocellular carcinoma.

In any of the above aspects, the ASGR1 inhibitor may include: (1) an inhibitor binding to ASGR1; (2) an inhibitor binding to a ligand of ASGR1 (such as asialoglycoprotein); (3) reducing or Inhibitors that block binding of ASGR1 to its ligand; (4) inhibitors that reduce or block endocytosis of ASGR1; (5) inhibitors that reduce protein levels of ASGR1; (6) inhibitors that reduce or block protein activity of ASGR1 and (7) an inhibitor that reduces or blocks expression of a gene encoding ASGR1.

In any of the above aspects, the ASGR1 inhibitor can be a small molecule compound, antisense oligonucleotide (ASO), interfering nucleic acid (such as shRNA, siRNA, gRNA), nucleic acid aptamer targeting ASGR1, anti-ASGR1 antibody or a combination of them.

In some embodiments, the ASGR1 inhibitors of the invention are ASGR1 antibodies. In some embodiments, the ASGR1 antibody is an ASGR1 monoclonal antibody or an antigen-binding fragment thereof. In some embodiments, the ASGR1 monoclonal antibody binds to human ASGR1 comprising the sequence shown in SEQ ID NO: 1, and inhibits the binding of human ASGR1 to its natural ligand (such as asialoglycoprotein) and/or human ASGR1 endocytosis. In some embodiments, the ASGR1 monoclonal antibody binds to the carbohydrate binding region of ASGR1 and inhibits the binding of human ASGR1 to its natural ligand (eg, asialoglycoprotein) and/or the endocytosis of human ASGR1. In some embodiments, the carbohydrate binding region of ASGR1 comprises, consists essentially of, or consists of the sequence of SEQ ID NO:2. In some embodiments, the carbohydrate binding region of ASGR1 comprises, consists essentially of, or consists of the sequence of SEQ ID NO:3. In some embodiments, the epitope to which the ASGR1 monoclonal antibody binds includes Containing one or more of Q240, D242, W244, E253, N265, D266, D267, R237, N209, H257, T259 and Y273 in SEQ ID NO:1. In some embodiments, the epitope to which the ASGR1 monoclonal antibody binds comprises one or more of Q240, D242, W244, E253, N265 and D266 in SEQ ID NO:1. In some embodiments, the epitope to which the ASGR1 monoclonal antibody binds comprises Q240, D242, W244, E253, N265 and D266 in SEQ ID NO:1.

In some embodiments, an ASGR1 inhibitor of the invention is a nucleic acid that targets the DNA or mRNA of ASGR1 and inhibits the expression of ASGR1. In some embodiments, the nucleic acid is selected from antisense oligonucleotides (ASO), siRNA, shRNA and gRNA. In some embodiments, the nucleic acid is ASO, and the ASO can be modified on the backbone, sugar groups or bases to resist degradation in vivo. Suitable modifications include, but are not limited to, phosphorothioation (PSP), phosphorodiamidate morpholino oligonucleotides (PMO), 2'-O-methoxyethyl modification (2'-MOE), 5-methylcytosine (5mC). In some embodiments, the nucleic acid is siRNA or shRNA, and the siRNA can be delivered by a suitable carrier. Such suitable carriers are eg GalNAc, LNP (lipid nanoparticles) or AAV. In some embodiments, the nucleic acid targets the sequence shown in SEQ ID NO: 36 and inhibits the expression of the gene encoding ASGR1. In some embodiments, the nucleic acid is gRNA, and the gRNA and Crispr/Cas enzyme (such as Crispr/Cas9) constitute a gene editing system to inhibit or block the expression of the gene encoding ASGR1. In some embodiments, the nucleic acid targets the sequence shown in SEQ ID NO: 36 and inhibits the expression of the gene encoding ASGR1.

In some embodiments, the ASGR1 inhibitors of the invention are aptamers targeting ASGR1. In some embodiments, the nucleic acid aptamer binds to human ASGR1 and inhibits the binding of human ASGR1 to its natural ligand (eg, asialoglycoprotein) and/or the endocytosis of human ASGR1.

In any aspect above, the second lipid-lowering drug is preferably an HMGCR inhibitor, an NPC1L1 inhibitor and/or a PCSK9 inhibitor. In some embodiments, the HMGCR inhibitor is a statin. In some embodiments, the statin is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin, and pravastatin. In some embodiments, the statin is atorvastatin. In some embodiments, the NPC1L1 inhibitor is ezetimibe. In some embodiments, the PCSK9 inhibitor is an anti-PCSK9 antibody or an interfering nucleic acid (such as siRNA or shRNA) targeting a gene encoding PCSK9. In some embodiments, the PCSK9 inhibitor is Evolocumab, Alirocumab, or Inclisiran.

In a thirteenth aspect, the present invention provides an anti-ASGR1 monoclonal antibody or antigen-binding fragment thereof that inhibits the binding of human ASGR1 to its natural ligand and/or the endocytosis of human ASGR1. In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region comprises LCDR1, LCDR2, LCDR3, respectively comprising SEQ ID NO: 4-6 The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, respectively comprising the sequences shown in SEQ ID NO: 7-9. In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region comprises LCDR1, LCDR2, LCDR3, respectively comprising SEQ ID NO: 10-12 The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, respectively comprising the sequences shown in SEQ ID NO: 13-15. In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region comprises LCDR1, LCDR2, LCDR3, respectively comprising SEQ ID NO: 16-18 The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively comprising the sequences shown in SEQ ID NO: 19-21. In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region comprises LCDR1, LCDR2, LCDR3, respectively comprising SEQ ID NO:22-24 The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, respectively comprising the sequences shown in SEQ ID NO: 25-27. In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO: 28, and the heavy chain variable region Contains the sequence shown in SEQ ID NO:29. In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises a sequence shown in SEQ ID NO: 30, and the heavy chain variable region Contains the sequence shown in SEQ ID NO:31. In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO: 32, and the heavy chain variable region Contains the sequence shown in SEQ ID NO:33. In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO: 34, and the heavy chain variable region Contains the sequence shown in SEQ ID NO:35.

Other aspects and advantages of the invention will be apparent from the following detailed description of the invention.

Description of drawings

Figure 1. Loss of ASGR1 increases LXR protein levels, promotes cholesterol efflux, and reduces lipid levels. (a) GSEA analysis showed that target genes of the LXR pathway were significantly enriched. Knock down ASGR1 in human liver cancer cells Huh7 by small interfering RNA (siRNA), extract total RNA for transcriptome sequencing and perform GSEA analysis; (b) Huh7 cells were transfected with designated small interfering RNA, 72 hours later, lysed Total RNA was extracted from the cells, and real-time quantitative PCR analysis was performed to detect the expression of LXR target genes; (c) Huh7 cells were transfected with designated small interfering RNA, lysed after 72 hours, total intracellular protein was extracted, and the protein expression levels of LXRα, LXRβ and ASGR1 were detected , Actin as an internal reference; (d) Using CRISPR/Cas9 gene editing technology to construct an ASGR1 knockout cell line in Huh7 cells, Western blot detection was performed in wild-type (WT) cells and ASGR1 knockout cell lines (ASGR1 KO-A,- B) Changes in the expression of LXRα, LXRβ and ASGR1 proteins, Actin was used as an internal reference; (e) Huh7 cells were transfected with a plasmid expressing ASGR1 protein to construct a cell line stably expressing ASGR1. Western blotting detection of LXRα, LXRβ and ASGR1 protein expression changes in wild-type (WT) cells and ASGR1 stable expression cell lines (ASGR1OE-A, -B), Actin was used as an internal reference; (f) real-time quantitative PCR detection of ASGR1 stable expression cells Changes in LXR downstream target genes in strains. Eight-week-old ASGR1 heterozygous mice, homozygous mice and male wild-type mice were randomly divided into groups (6 in each group), free to drink water, high in fat, high cholesterol and cholate (HF/HC/ BS) diet (60% fat, 1.25% cholesterol and 0.5% cholate) for 4 weeks. All mice were starved for 4 hours before sampling, and all data were expressed as mean ± SEM. Statistical significance was calculated with a paired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. (g) total cholesterol in serum; (h) triglyceride in serum; (i) total cholesterol in liver; (j) triglyceride in liver; (k) hematoxylin-eosin staining in liver (left) and Oil red O staining (right); (l) gallbladder volume; (m) cholesterol concentration in bile; (n) total bile acid concentration in bile; (o) total amount of total cholesterol in bile; (p) total bile acid concentration in bile The total amount of bile acids; (q) representative pictures of bile; (r) Western blot analysis of liver samples; (s) real-time quantitative PCR analysis of mouse liver and cholesterol efflux, cholesterol synthesis and absorption, fatty acid synthesis genes, bile Acid metabolism-related genes and genes of other pathways, and cyclophilin (Cyclophilin) was used as an internal reference.

Figure 2. ASGR1 knockout strategy and the effect of ASGR1 on LDLR. (a) Distribution of mouse ASGR1 protein in various tissues; (b) Construction strategy of Asgr1 knockout mice; (c) Identification of Asgr1 ^+/+ , Asgr1 ^+/- , Asgr1 by deoxynucleotide agarose gel electrophoresis ^-/- , the wild-type band is 369bp, and the homozygous mouse is 600bp. If two bands appear at the same time, it is heterozygous; (d) For Asgr1 ^+/+ , Asgr1 ^+/- , Asgr1 ^-/- The liver tissues of the genotype mice were taken for Western blot analysis, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference; the mice were from the same batch as gs in Figure 1; (e) body weight; (f) Daily food intake; (g) ratio of liver to body weight; (h) blood sugar; (i) aspartate aminotransferase in serum; (j) alanine aminotransferase in serum; (k) phospholipid in bile; (l) ratio of cholesterol to phospholipid in bile ; (m) Huh7 cells were transfected with the specified small interfering RNA. After 56 hours, the cells were rinsed with 1×PBS, and fresh complete medium (DMEM+10% fetal bovine serum) or cholesterol-deficient culture was replaced as shown in the figure. Base (DMEM + 5% lipoprotein-free serum + 1 μM lovastatin + 10 μM mevalonate). After 16 hours, the cells were lysed and analyzed by western blot; (n) Huh7 cells were transfected with the indicated plasmids, and after 32 hours, the cells were washed with 1×PBS, Fresh complete medium (DMEM + 10% fetal bovine serum) or cholesterol deficient medium (DMEM + 5% lipoprotein-free serum + 1 μM lovastatin + 10 μM mevalonate) was replaced as indicated in the figure. After 16 hours, cells were lysed and analyzed by Western blot; (o) Huh7 cells were seeded in 12-well plates at 2.5×10 ⁴ cells/well on day 0, and cultured with complete medium. The indicated small interfering RNAs were transfected on the first day. After 72 hours, the cells were rinsed with 1×PBS, replaced with cholesterol-deficient medium and incubated at 4°C for 30 minutes. Subsequently, it was replaced with fresh cholesterol-deficient medium supplemented with 10 μg/ml DiI-LDL, and cultured at 4° C. for 1 hour. Then, wash the cells twice with 1×PBS, place the cells in a 37°C incubator to start endocytosis, fix at the specified time, and take pictures; (p) Quantitative statistics of endocytosed DiI-LDL, wild-type Huh7 cells The endocytosed LDL at hour 0 was defined as 0 (45 cells per group); (q) Huh7 cells were transfected with the indicated small interfering RNA, and the cells were cultured in complete medium or cholesterol curve medium for 12 hours. Subsequently, the cells were treated with the specified concentration of PCSK9 protein in the original medium for 4 hours. Cells were lysed for western blot analysis.

Figure 3. Phenotype of Asgr1 ^+/- heterozygous mice, cholesterol efflux genes are highly expressed in Asgr1 knockout heterozygotes. Eight-week-old Asgr1 ^+/- mice and littermate wild-type mice were randomly divided into 2 groups as shown in the figure, with 6 mice in each group. Free drinking water, high fat high cholesterol and cholate (HF/HC/BS) diet (60% fat, 1.25% cholesterol and 0.5% cholate) feeding for 4 weeks. The mice were uniformly starved for 4 hours before killing. All data are presented as mean ± SEM. Statistical significance was calculated with an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. (a) western blot analysis of liver samples; (b) real-time quantitative PCR analysis of genes related to cholesterol efflux, cholesterol synthesis and absorption, lipid synthesis genes, bile acid metabolism and other pathways in mouse liver, cyclophilic element as an internal reference.

Figure 4. Alterations of metabolic syndrome by ASGR1 deletion are dependent on LXRα. All mice were bred from Asgr1 ^+/- Lxrα ^+/- mice. Eight-week-old mice were randomly divided into 4 groups as shown in the figure, with 6 mice in each group. Free drinking water, high fat high cholesterol and cholate (HF/HC/BS) diet (60% fat, 1.25% cholesterol and 0.5% cholate) feeding for 6 weeks. All mice were starved for 4 hours before sampling. All data are presented as mean ± SEM. Statistical significance was calculated with an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. (a) Western blot analysis of liver samples; (b) Real-time quantitative PCR analysis of genes related to cholesterol efflux, lipid synthesis, bile acid synthesis and other pathways in mouse liver, cyclophilin was used as an internal reference; (c ) ratio of liver to body weight; (d) hematoxylin-eosin staining (top) and oil red O staining (bottom) in liver; (e) total cholesterol in serum; (f) triglyceride in serum; (g ) total cholesterol in the liver; (h) triglycerides in the liver; (i) fast liquid chromatography (FPLC) analysis of the distribution of lipoprotein components in the serum of wild-type and Asgr1 knockout mice: VLDL: very low Density lipoprotein; LDL: low-density lipoprotein; HDL: high-density lipoprotein; (j) rapid liquid chromatography analysis of the distribution of lipoproteins in mouse serum; (k) the total amount of total cholesterol in bile; (l ) the total amount of total cholesterol in feces, each mouse was continuously collected feces for 3 days; (m) body weight; (n) daily food intake; (o) blood sugar; (p) alanine aminotransferase in serum; grass transaminase.

Figure 5. Phenotype of female Asgr1 ^-/- mice. Eight-week-old Asgr1 ^-/- female mice and littermate wild-type mice were randomly divided into 2 groups as shown in the figure, with 6 mice in each group. Free drinking water and high fat high cholesterol and cholate (HF/HC/BS) diet (60% fat, 1.25% cholesterol and 0.5% cholate) were fed for 4 weeks. The mice were uniformly starved for 4 hours before killing. All data are presented as mean ± SEM. Statistical significance was calculated with an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. (a) Western blot analysis of liver samples; (b) real-time quantitative PCR analysis of genes related to cholesterol efflux, cholesterol synthesis and absorption, lipid synthesis, bile acid synthesis and other pathways in mouse liver, cyclophilin As an internal reference; (c) body weight; (d) ratio of liver to body weight; (e) total cholesterol in serum; (f) triglyceride in serum; (g) total cholesterol in liver; (h) triglyceride in liver (i) alanine aminotransferase in serum; (j) aspartate aminotransferase in serum; (k) volume of gallbladder; (l) concentration of total cholesterol in bile; (m) total amount of total cholesterol in bile; (n) concentration of total bile acids in bile; (o) total amount of total bile acids in bile.

Figure 6. ASGR1 regulates the degradation of LXRa. (a) Huh7 cells were transfected with the indicated plasmids. After 43 hours, the medium was replaced with or without 10 μM MG132 for 5 hours. Cells were lysed and detected by Western blot; (b) wild-type Huh7 and cells stably expressing ASGR1 (ASGR1OE-A and -B) were cultured in an incubator for 43 hours. Then it was replaced with complete medium supplemented with 10 μM MG132 for 5 hours. The cell lysate was co-immunoprecipitated with LXRα antibody specific for LXRα protein in the lysate. Ubiquitin, LXRα, and ASGR1 were detected in the supernatant and pellet, respectively; (c) wild-type Huh7 and ASGR1 knockout cells (ASGR1 KO-A and -B) were cultured in an incubator for 43 hours. Then it was replaced with complete medium supplemented with 10 μM MG132 for 5 hours. The cell lysate was co-immunoprecipitated with LXRα antibody specific for LXRα protein in the lysate. Ubiquitin, LXRα and ASGR1 were detected in supernatant and pellet respectively; (d) Huh7 cells were transfected with designated small interfering RNA. After 72 hours, the cells were harvested and lysed for protein co-immunoprecipitation analysis; (e) A simple schematic diagram of the mouse ASGR1-ligand binding complex. Mouse ASGR1 is a single transmembrane protein, the 1-39th amino acid is located in the cell, the 40-60th amino acid is the transmembrane segment, the 61-140th amino acid is the stem segment, and the 141-284th amino acid is Carbohydrate identification section. Among them, the six amino acids Q239, D241, W243, E252, N264 and D265 are very necessary for ligand binding; (f) Huh7 cells were transfected with the designated plasmid, and after 8 hours of transfection, the cells were rinsed with 1×PBS, and then replaced Treat with fresh medium containing 10% fetal bovine serum or no fetal bovine serum for 40 hours, lyse the cells, and perform Western blot analysis; (g) Huh7 cells are transfected with the indicated plasmids, and after 35 hours, the cells are treated with 1× phosphate Rinse once with buffer solution (PBS), replace with MEM medium and incubate for 6 hours. Then it was replaced by adding different concentrations of desialylated acidic protein for 8 hours. Finally, the cells were collected and lysed for Western blot analysis; (h) Huh7 cells were transfected with the indicated plasmids, in which 6A mutations were Q239, D241, W243, E252, N264 and D265 were all mutated to alanine. 48 hours after transfection, the cells were collected, lysed, and analyzed by Western blot; (i) Huh7 cells were transfected with the designated plasmids, and after 35 hours, the cells were washed once with 1× phosphate buffered saline (PBS) and replaced with MEM culture Incubate for 6 hours. Then it was replaced by adding different concentrations of desialylated acidic protein for 8 hours. Finally, the cells were collected and lysed for western blot analysis; (j) Huh7 cells were transfected with designated small interfering RNAs to specifically knock down clathrin heavy chain (CHC). After 12 hours, the cells were re-transfected with the designated plasmid. After 48 hours, the cells were collected and lysed for protein co-immunoprecipitation analysis; (k) Huh7 cells were transfected with the designated plasmid. After 46 hours, it was replaced with addition or no addition The fresh complete medium of 20nM Bafilomycin (Bafilomycin) A1 was treated for 2 hours, then the cells were collected and lysed, and the protein immunoassay was carried out; (1) Huh7 cells were transfected with the designated plasmid, and after 43 hours, it was replaced with addition or no Add fresh complete medium with 100 μM A769662 for 5 hours, then collect and lyse the cells for protein immunoassay.

Figure 7. The ASGR1-AMPK axis regulates LXRα and SREBP proteins. (a) Huh7 cells were transfected with designated small interfering RNAs to specifically knock down BRCA1 and BARD1. After 12 hours, the cells were transfected with the plasmids shown in the figure. After 43 hours, the cells were replaced with complete cells containing 10 μM MG132 The medium was incubated for 5 hours. Finally, the cells were collected, lysed, and co-immunoprecipitated with anti-Myc magnetic beads. Western blot analysis; (b) Huh7 cells were transfected with the specified small interfering RNA to specifically knock down BRCA1 and BARD1, and after 12 hours, the cells were transfected with the plasmids shown in the figure. After 48 hours, the cells were lysed and analyzed by western blot; (c) ASGR1 knockout cell lines and wild-type cell lines were cultured in the incubator for 48 hours, and finally the cells were lysed for western blot analysis; (d) Huh7 cells After 48 hours of transfection with the designated plasmid, the cells were collected and lysed for Western blot analysis; (e) Huh7 cells were transfected with the designated plasmid, and after 35 hours, the cells were washed once with 1× phosphate buffered saline (PBS) and replaced with Incubate in MEM medium for 6 hours. Then replace it with different concentrations of desialylated fetuin A treatment for 8 hours. Finally, the cells were collected and lysed for western blot analysis; (f) Huh7 cells were transfected with the designated plasmids. After 35 hours, the cells were rinsed once with 1×PBS, replaced with MEM medium and incubated for 6 hours. Then it was replaced by adding or not adding desialylated fetuin A (1 μg/ml) for 3 hours, and finally adding 10 μM MG132 for 5 hours on this basis. Cells were lysed and analyzed by western blot; (g) Huh7 cells were transfected with the designated small interfering RNA to specifically knock down BRCA1 and BARD1. After 60 hours, the cells were washed once with 1×PBS, and replaced with MEM medium for incubation 6 hours. Then it was replaced by adding or not adding desialylated fetuin A (1 μg/ml) for 8 hours. Cells were collected and lysed for western blot analysis; (h) Huh7 cells were transfected with indicated small interfering RNAs to specifically knock down CHC. After 60 hours, the cells were rinsed once with 1×PBS, replaced with MEM medium and incubated for 6 hours. Then it was replaced by adding or not adding desialylated fetuin A (1 μg/ml) for 8 hours. Cells were collected and lysed for western blot analysis; (i) wild-type Huh7 cells and ASGR1 knockout cells (ASGR1 KO-A, -B) were cultured in an incubator for 46 hours, and then 20nM Bafilonium was added to the medium Incubate with prime A1 for 2 hours. Cells were collected and lysed for western blot analysis; (j) After wild-type Huh7 cells were cultured in an incubator for 48 hours, they were treated with AMPK agonist A-769662 at concentrations of 0, 30 and 100 μM for 5 hours. Then the cells were lysed, and the protein was analyzed by immunoblotting; (k) After the wild-type Huh7 cells were cultured in the incubator for 48 hours, they were rinsed once with 1×PBS, replaced with sugar-free DMEM and cultured for 4 hours, and then inhibited with AMPK Dorsomophin was treated for 5 hours according to the concentrations of 0, 3 and 10 μM. Then cells were lysed, and proteins were analyzed by Western blot; (l) ASGR1 knockout cell lines (ASGR1 KO-A, -B) and wild-type Huh7 cells were cultured in the incubator for 48 hours, and rinsed once with 1×PBS , replaced with sugar-free DMEM for 4 hours, and then treated with or without AMPK inhibitor Dorsomophin (10 μM) for 5 hours. Cells were then lysed and proteins were subjected to immunoblot analysis; (m) Simplified schematic diagram. After ASGR1 binds to asialoglycoprotein, it initiates endocytosis and enters the lysosome for degradation. Lysosome release of nutrients activates mTORC1 and inhibits phosphorylation of AMPK. Loss of Asgr1 inhibits mTORC1 and activates AMPK. AMPK phosphorylation reduces the protein stability of BRCA1/BARD1, further increases the protein level of LXR, and then transcriptionally activates the expression of ABCG5/8 and ABCA1. ABCA1 transports cholesterol to HDL, while ABCG5/8 help secrete cholesterol into bile and feces. At the same time, AMPK activation can also inhibit lipid synthesis by inhibiting the splicing of SREBP1 into the nucleus.

Figure 8. AAV shRNA-mediated Asgr1 silencing increases cholesterol efflux and improves metabolic syndrome. Eight-week-old male mice of the Balb/c strain were injected with AAV2/8-shAsgr1 or control AAV2/8-shControl with a titer of 1×10 ¹¹ viral genomes (viral genome, vg). They were fed with a high-fat/high cholesterol/cholate (HF/HC/BS) diet (60% fat, 1.25% cholesterol and 0.5% cholate) for 4 weeks, with free access to water for 4 weeks. The mice were uniformly starved for 4 hours before killing. All data are presented as mean ± SEM. Statistical significance was calculated with an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. (a) Western blot analysis of liver samples; (b) real-time quantitative PCR analysis of genes related to cholesterol efflux, cholesterol synthesis and absorption, lipid synthesis, bile acid synthesis and other pathways in mouse liver, cyclophilin As an internal reference; (c) total cholesterol in serum; (d) triglyceride in serum; (e) total cholesterol in liver; (f) triglyceride in liver; (g) hematoxylin-eosin staining of liver sections (h) Oil Red O staining of liver sections; (i) total amount of total cholesterol in bile; (j) total amount of total bile acids in bile; (k) body weight; (l) ratio of liver to body weight; ( m) alanine aminotransferase in serum; (n) aspartate aminotransferase in serum.

Figure 9. Preparation of ASGR1 neutralizing antibodies. (a) Steps for preparing monoclonal neutralization of ASGR1. The purified ASGR1 protein was used as an antigen to immunize rabbits. After the first round of screening, 4 B cell monoclonal strains were obtained as candidates. The coding region of the variable region of the antibody is then sequenced and constructed into an antibody repertoire vector and transfected into mammalian cells. Its effect was confirmed by western blot and real-time quantitative PCR. Finally, the rabbit-derived Fc fragment was replaced with the mouse-derived Fc fragment. Finally, the neutralizing antibody 4B9 was selected for large-scale production and used in subsequent experiments. (b) Coomassie Brilliant Blue staining of ASGR1 protein purified from HEK293T cells; (c) Huh7 cells were incubated with purified neutralizing antibody for 72 hours, and the protein expression of LXRα was analyzed by western blot; (d) protein expression of LXRα from 8-week-old wild type Isolation of primary liver cells from C57B/L6 mice. Primary hepatocytes were incubated with different monoclonal neutralizing antibodies at different concentrations for 72 hours, the cells were collected, lysed and analyzed by Western blot; (e) Liver was isolated from 8-week-old wild-type C57B/L6 mice Primary cells. Primary hepatocytes were incubated with ASGR1 neutralizing antibody 4B9 for 72 hours, the cells were collected, total RNA was extracted, and the target gene of LXR was detected by real-time quantitative PCR analysis; (f) Huh7 cells were transfected with the designated plasmids. 48 hours after transfection, cells were harvested, lysed, and the expression of each protein was analyzed by western blot.

Figure 10. ASGR1 neutralizing antibody increases cholesterol efflux and decreases blood and liver lipids. Eight-week-old Asgr1 knockout mice and littermate wild-type mice were randomly divided into 4 groups as shown in the figure, with 6 mice in each group. Free drinking water, high fat high cholesterol and cholate (HF/HC/BS) diet (60% fat, 1.25% cholesterol and 0.5% cholate) feeding. At the same time, the mice were intraperitoneally injected with the control antibody or ASGR1 neutralizing antibody 4B9 at a dose of 10 mg/kg/day every other day. After 14 days, the mice were uniformly starved for 4 hours before killing. All data are presented as mean ± SEM. Statistical significance was calculated with an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. (a) Western blot analysis of liver samples; (b) Real-time quantitative PCR analysis of genes related to cholesterol efflux, cholesterol synthesis and absorption, lipid synthesis, and bile acid metabolism in mouse liver, with cyclophilin as an internal reference; (c ) total cholesterol in serum; (d) triglycerides in serum; (e) total cholesterol in liver; (f) triglycerides in liver; (g) volume of gallbladder; (h) concentration of cholesterol in bile; ) amount of total cholesterol in bile; (j) representative pictures of gallbladder volume; (k) amount of total cholesterol in feces; (l) concentration of total bile acids in bile; (m) total amount of total bile acids in bile (n) body weight; (o) daily food intake; (p) ratio of liver to body weight; (q) blood sugar; (r) serum alanine aminotransferase; (s) serum aspartate aminotransferase.

Figure 11. The combination of ASGR1 neutralizing antibody and atorvastatin shows a synergistic lipid-lowering effect. Eight-week-old Asgr1 knockout mice and littermate wild-type mice were randomly divided into 8 groups as shown in the figure, with 6 mice in each group. Free drinking water, high fat, high cholesterol and cholate (HF/HC/BS) diet (60% fat, 1.25% cholesterol and 0.5% cholate) feeding. At the same time, the mice were intraperitoneally injected with the control antibody or ASGR1 neutralizing antibody 4B9 at a dose of 10 mg/kg/day every other day, and atorvastatin was administered orally at a dose of 30 mg/kg/day every day. After 14 days, mice were uniformly starved for 4 hours before killing. All data are presented as mean ± SEM. Statistical significance was calculated with an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. (a) Western blot analysis of liver samples; (b) total cholesterol in serum; (c) triglycerides in serum; (d) total cholesterol in liver; (e) triglycerides in liver; (g) total bile acids in bile; (h) total cholesterol in feces; (i) body weight; (j) daily food intake; (k) ratio of liver to body weight; (l) Blood glucose; (m) alanine aminotransferase in serum; (n) aspartate aminotransferase in serum; (o-p) real-time quantitative PCR analysis of genes related to cholesterol efflux, cholesterol synthesis and absorption, lipid synthesis, and bile acid synthesis in mouse liver and Genes of other pathways, cyclophilin as an internal reference; (q) hematoxylin-eosin staining of liver sections; (r) oil red O staining of liver sections.

Figure 12. Neutralizing antibody 4B9 synergizes with ezetimibe. Eight-week-old Asgr1 knockout mice and littermate wild-type mice were randomly divided into 8 groups as shown in the figure, with 6 mice in each group. Free drinking water, high fat high cholesterol and cholate (HF/HC/BS) diet (60% fat, 1.25% cholesterol and 0.5% cholate) feeding. At the same time, the mice were intraperitoneally injected with the control antibody or ASGR1 neutralizing antibody 4B9 at a dose of 10 mg/kg/day every other day, and gavaged with ezetimibe at a dose of 10 mg/kg/day every day. Eight days later, the mice were uniformly starved for 4 hours before killing. All data are presented as mean ± SEM. statistically significant Sex was calculated with unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. (a) Western blot analysis of liver samples; (b) total cholesterol in serum; (c) triglycerides in serum; (d) total cholesterol in liver; (e) triglycerides in liver; The amount of cholesterol; (g) the total amount of bile acids in bile; (h) body weight; (i) daily food intake; (j) liver weight/body weight; (k) blood sugar; (l) alanine aminotransferase in serum; ( m) aspartate aminotransferase in serum; (no) Real-time quantitative PCR analysis of genes related to cholesterol efflux, cholesterol synthesis and absorption, fatty acid synthesis, bile acid synthesis and other pathway genes in mouse liver, cyclophilin as an internal reference; ( p) Hematoxylin-eosin staining of liver sections; (q) Oil red O staining of liver sections.

Figure 13. AAV shRNA-mediated Asgr1 silencing alleviates atherosclerosis formation. 10-week-old Ldlr knockout mice and littermate wild-type mice were randomly divided into 8 groups as shown in the figure, with 8-16 mice in each group. Water was freely available and fed a high-fat, high-cholesterol and cholate (HF/HC/BS) diet (60% fat, 1.25% cholesterol and 0.5% cholate) or a normal diet. AAV2/8-shAsgr1 or control AAV2/8-shControl with a titer of 1×10 ¹¹ viral genomes (vg) were injected into groups as shown in the figure. After 8 weeks, the mice were uniformly starved for 4 hours before killing. All data are presented as mean ± SEM. Statistical significance was calculated with an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. (a) total cholesterol in serum; (b) total triglyceride in serum; (c) supernatant after centrifugation of mouse blood; (d) body weight; (e) liver/body weight; (f) blood glucose; (g) main Arterial staining; (h) quantification of aortic atherosclerotic plaques; (i) hematoxylin-eosin staining of liver sections.

Detailed ways

definition

In the present invention, the term "treatment" refers to therapeutic as well as prophylactic measures, which prevent or slow down an undesired physiological change or condition in a subject, such as the development or progression of fatty liver. Beneficial or desired clinical effects include, but are not limited to, relief of symptoms, reduction in extent of disease, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, alleviation or palliation of disease state, and partial or total Cure, regardless of whether the above effects are detectable. "Treatment" can also refer to prolonging survival compared to no treatment. Those in need of treatment include those already with the disease or disorder as well as those at risk of having the disease or disorder or those in which the disease or disorder is to be prevented.

"Subject" or "patient", "individual" refers to any subject, especially a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Mammals include humans, domestic animals, farm animals, zoo animals, sport animals, or pets, such as dogs, cats, pigs, rabbits, rats, mice, horses, cows, cows, and the like. The subject referred to herein is preferably a human being.

Herein, "antigen-binding fragment" refers to a fragment comprising the portion of the corresponding antibody that specifically recognizes and binds its antigen, including but not limited to Fab, Fab', (Fab') ₂ , Fv and scFv. As used herein, when monoclonal antibodies are described, whether or not explicitly stated, the invention is intended to include antigen-binding fragments thereof.

As used herein, the term "therapeutically effective amount" or "effective amount" means that when the drug or pharmaceutical composition of the present invention is administered alone or in combination with another therapeutic agent to a cell, tissue or subject, it An amount effective to prevent or slow down the disease or condition being treated. A therapeutically effective dose further refers to an amount of the drug sufficient to cause symptomatic relief, such as treating, curing, preventing, or alleviating the associated medical condition, or increasing the rate of treatment, cure, prevention, or alleviation of said condition . When administering to a subject an active ingredient administered alone, a therapeutically effective amount refers to that ingredient alone. When administered in combination, a therapeutically effective amount refers to a combined amount of the active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

As used herein, a "pharmaceutically acceptable carrier" includes a material that, when combined with an active ingredient of a composition, allows that ingredient to retain biological activity and not cause a damaging reaction with the subject's immune system. These carriers may include stabilizers, preservatives, salts or sugar complexes or crystals and the like. "pharmaceutically acceptable" Refers to molecules and ingredients that do not produce an allergic reaction or similar undesired reaction when administered to the human body.

ASGR1 inhibitor

Asialoglycoprotein receptor 1 (Asialoglycoprotein receptor 1, ASGR1), mainly located in liver cells. ASGR1 is a single transmembrane protein that includes a cytoplasmic end, a transmembrane segment, a hinge region, and a carbohydrate-binding region. Glycoproteins in serum are processed by neuraminidase to form desialylated glycoproteins, which then bind to ASGR1 on the cell membrane to initiate endocytosis and enter the endosome pathway. The acidity of endosomes In the environment, receptors and ligands dissociate, these proteins are transported to lysosomes for degradation, and ASGR1 is recycled to the cell surface for reuse.

Human ASGR1 contains 291 amino acids, with a molecular weight of 33,186 Da, and its amino acid sequence is shown in SEQ ID NO: 1 (UniProtKB/Swiss-Prot: P07306.2). After ASGR1 binds to the ligand in the blood, that is, asialoglycoprotein, it enters into the lysosome for degradation through clathrin-mediated endocytosis. The cytoplasmic end of human ASGR1 is short (1-40aa), the transmembrane region is 40-60aa, and the extracellular region is divided into a stem region (62-141aa) and a carbohydrate binding region (142-291, SEQ ID NO:2 ). The gene ID of the human ASGR1 gene in NCBI is 432, and the sequence of the coding region is shown in SEQ ID NO: 36 (NCBI Reference Sequence: NM_001671.5).

Herein, "ASGR1 inhibitor" refers to a substance that can reduce or block the binding of ASGR1 to its natural ligand and/or ASGR1 endocytosis, as well as a substance that can reduce or block the expression of the gene encoding ASGR1.

In some embodiments, the ASGR1 inhibitors herein may include: (1) inhibitors that bind to ASGR1; (2) inhibitors that bind to a ligand of ASGR1 (such as asialoglycoprotein); (3) reduce or Inhibitors that block binding of ASGR1 to its ligand; (4) inhibitors that reduce or block endocytosis of ASGR1; (5) inhibitors that reduce protein levels of ASGR1; (6) inhibitors that reduce or block protein activity of ASGR1 and (7) an inhibitor that reduces or blocks expression of a gene encoding ASGR1.

In some embodiments, the ASGR1 inhibitor of the present invention may be a small molecule compound, a nucleic acid targeting a gene encoding ASGR1, a nucleic acid aptamer targeting ASGR1, an anti-ASGR1 antibody, or a combination thereof.

In some embodiments, the ASGR1 inhibitors of the invention are ASGR1 antibodies. In some embodiments, the ASGR1 antibody is an ASGR1 monoclonal antibody or an antigen-binding fragment thereof.

In some embodiments, the ASGR1 monoclonal antibody binds to human ASGR1 comprising the sequence shown in SEQ ID NO: 1, and inhibits the binding of human ASGR1 to its natural ligand (such as asialoglycoprotein) and/or human ASGR1 endocytosis.

In some embodiments, the ASGR1 monoclonal antibody binds to the carbohydrate binding region of ASGR1 and inhibits the binding of human ASGR1 to its natural ligand (eg, asialoglycoprotein) and/or the endocytosis of human ASGR1. In some embodiments, the carbohydrate binding region of ASGR1 comprises, or consists essentially of, or consists of the sequence of SEQ ID NO: 2. In some embodiments, the carbohydrate binding region of ASGR1 comprises, or consists essentially of, or consists of the sequence of SEQ ID NO: 3.

In some embodiments, the epitope to which the ASGR1 monoclonal antibody binds comprises one of Q240, D242, W244, E253, N265, D266, D267, R237, N209, H257, T259 and Y273 in SEQ ID NO:1 or more. In some embodiments, the epitope to which the ASGR1 monoclonal antibody binds comprises one or more of Q240, D242, W244, E253, N265 and D266 in SEQ ID NO:1. In some embodiments, the epitope to which the ASGR1 monoclonal antibody binds comprises Q240, D242, W244, E253, N265 and D266 in SEQ ID NO:1.

In some embodiments, the ASGR1 monoclonal antibody is a fully human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the ASGR1 monoclonal antibody is a fully human antibody or a humanized anti- body. In some embodiments, the ASGR1 monoclonal antibody is a chimeric antibody.

In some embodiments, the ASGR1 monoclonal antibody is of IgG type, such as IgG1, IgG2, IgG3 or IgG4 type. In some embodiments, the ASGR1 monoclonal antibody is IgG1.

In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region comprises LCDR1, LCDR2, LCDR3, respectively comprising SEQ ID NO:4-6 The sequence of the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, respectively comprising the sequences shown in SEQ ID NO: 7-9.

In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region comprises LCDR1, LCDR2, LCDR3, respectively comprising SEQ ID NO: 10-12 The sequence of the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, respectively comprising the sequences shown in SEQ ID NO: 13-15.

In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region comprises LCDR1, LCDR2, LCDR3, respectively comprising SEQ ID NO: 16-18 The sequence of the heavy chain variable region comprises HCDR1, HCDR2, HCDR3, respectively comprising the sequence shown in SEQ ID NO: 19-21.

In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region comprises LCDR1, LCDR2, LCDR3, respectively comprising SEQ ID NO:22-24 The sequence of the heavy chain variable region comprises HCDR1, HCDR2, HCDR3, respectively comprising the sequence shown in SEQ ID NO: 25-27.

In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO: 28, and the heavy chain variable region Comprising the sequence shown in SEQ ID NO:29.

In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO: 30, and the heavy chain variable region Comprising the sequence shown in SEQ ID NO:31.

In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO: 32, and the heavy chain variable region Comprising the sequence shown in SEQ ID NO:33.

In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO: 34, and the heavy chain variable region Comprising the sequence shown in SEQ ID NO:35.

Other exemplary ASGR1 monoclonal antibodies include those described in WO 2017058944A1 , WO2022006327A1 , or US 20210130473A1 , the entire disclosures of which are incorporated herein by reference.

In some embodiments, an ASGR1 inhibitor of the invention is a nucleic acid that targets the DNA or mRNA of ASGR1 and inhibits the expression of ASGR1.

In some embodiments, the nucleic acid is selected from antisense oligonucleotides (ASO), siRNA, shRNA and gRNA.

In some embodiments, the nucleic acid is ASO, and the ASO can be modified on the backbone, sugar groups or bases to resist degradation in vivo. Suitable modifications include, but are not limited to, phosphorothioation (PSP), phosphorodiamidate morpholino oligonucleotides (PMO), 2'-O-methoxyethyl modification (2'-MOE), 5-methylcytosine (5mC).

In some embodiments, the nucleic acid is siRNA or shRNA, and the siRNA can be delivered by a suitable carrier. Such suitable vectors are eg GalNAc, LNP or AAV.

In some embodiments, the nucleic acid targets the sequence shown in SEQ ID NO:36 and inhibits Expression of the gene encoding ASGR1.

In some embodiments, the nucleic acid is gRNA, and the gRNA and CRISPR/Cas enzyme (such as CRISPR/Cas9) constitute a gene editing system to inhibit or block the expression of the gene encoding ASGR1.

On the premise that the coding gene of ASGR1 is known, those skilled in the art can obtain and screen the above nucleic acid sequence through conventional techniques.

In some embodiments, the nucleic acid (such as ASO, siRNA, shRNA or gRNA) targets the sequence shown in SEQ ID NO: 36 and inhibits the expression of the gene encoding ASGR1.

In some embodiments, the ASGR1 inhibitor of the present invention is an aptamer targeting human ASGR1. In some embodiments, the nucleic acid aptamer binds to human ASGR1 and inhibits the binding of human ASGR1 to its natural ligand (eg, asialoglycoprotein) and/or the endocytosis of human ASGR1.

Therapeutic Methods and Applications

One aspect of the invention provides a method of promoting cholesterol efflux comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor described herein. In some embodiments, the promotion of cholesterol efflux includes promoting the efflux of total cholesterol in the liver into bile and feces.

Another aspect of the invention provides a method of reducing total cholesterol levels in the liver, the method comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor described herein.

Another aspect of the invention provides a method of reducing triglyceride levels in the liver, the method comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor described herein.

Accordingly, another aspect of the present invention provides the use of the ASGR1 inhibitor described herein in the preparation of a drug for promoting cholesterol efflux. In some embodiments, the promotion of cholesterol efflux includes promoting the efflux of total cholesterol in the liver into bile and feces.

Accordingly, another aspect of the present invention provides the use of the ASGR1 inhibitor described herein in the preparation of a medicament for reducing the total cholesterol level in the liver.

Accordingly, another aspect of the present invention provides the use of the ASGR1 inhibitors described herein in the preparation of a medicament for reducing triglyceride levels in the liver.

Accordingly, another aspect of the present invention provides an ASGR1 inhibitor as described herein for use in promoting cholesterol efflux. In some embodiments, the promotion of cholesterol efflux includes promoting the efflux of total cholesterol in the liver into bile and feces.

Accordingly, another aspect of the present invention provides an ASGR1 inhibitor as described herein for use in lowering total cholesterol levels in the liver.

Accordingly, another aspect of the present invention provides an ASGR1 inhibitor as described herein for use in reducing triglyceride levels in the liver.

Another aspect of the invention provides a method of treating non-alcoholic fatty liver disease (NAFLD), the method comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor described herein.

Accordingly, another aspect of the present invention provides the use of the ASGR1 inhibitor described herein in the preparation of a medicament for treating non-alcoholic fatty liver disease (NAFLD).

Accordingly, another aspect of the present invention provides an ASGR1 inhibitor as described herein for use in the treatment of non-alcoholic fatty liver disease (NAFLD).

In another aspect, the inventors found that the combined administration of ASGR1 inhibitors and lipid-lowering drugs produced a significant synergistic lipid-lowering effect, and the total cholesterol level and triglyceride level in serum and liver were significantly reduced.

Accordingly, in some embodiments, the invention provides a method of promoting cholesterol efflux comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor described herein and a therapeutically effective amount of a second lipid-lowering agent. In some embodiments, said promotion of cholesterol efflux includes promotion of total cholesterol in the liver Excreted in bile and feces.

In some embodiments, the invention provides a method of reducing total cholesterol levels in the liver comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor described herein and a therapeutically effective amount of a second lipid-lowering agent.

In some embodiments, the invention provides a method of reducing triglyceride levels in the liver comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor described herein and a therapeutically effective amount of a second lipid-lowering agent.

Accordingly, in some embodiments, the present invention provides the use of the combination of the ASGR1 inhibitor described herein and a second lipid-lowering drug in the preparation of a drug for promoting cholesterol efflux. In some embodiments, the promotion of cholesterol efflux includes promoting the efflux of total cholesterol in the liver into bile and feces.

Accordingly, in some embodiments, the present invention provides the use of the combination of an ASGR1 inhibitor described herein and a second lipid-lowering drug in the manufacture of a medicament for reducing total cholesterol levels in the liver.

Accordingly, in some embodiments, the present invention provides the use of the combination of an ASGR1 inhibitor described herein and a second lipid-lowering drug in the manufacture of a medicament for reducing triglyceride levels in the liver.

Accordingly, in some embodiments, the invention provides a combination of an ASGR1 inhibitor described herein and a second lipid-lowering agent for use in promoting cholesterol efflux. In some embodiments, the promotion of cholesterol efflux includes promoting the efflux of total cholesterol in the liver into bile and feces.

Accordingly, in some embodiments, the present invention provides a combination of an ASGR1 inhibitor described herein and a second lipid-lowering agent for use in lowering total cholesterol levels in the liver.

Accordingly, in some embodiments, the present invention provides a combination of an ASGR1 inhibitor described herein and a second lipid-lowering agent for use in reducing triglyceride levels in the liver.

In some embodiments, the present invention provides a method of treating nonalcoholic fatty liver disease (NAFLD), the method comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor described herein and a therapeutically effective amount of a second inhibitor fat medicine.

Accordingly, in some embodiments, the present invention provides the use of the combination of the ASGR1 inhibitor described herein and a second lipid-lowering drug in the preparation of a medicament for treating non-alcoholic fatty liver disease (NAFLD).

Accordingly, another aspect of the present invention provides a combination of an ASGR1 inhibitor as described herein and a second lipid-lowering agent for use in the treatment of non-alcoholic fatty liver disease (NAFLD).

In some embodiments, the subject has NAFL, NASH, NAFLD with liver fibrosis, NAFLD with cirrhosis, or NAFLD with hepatocellular carcinoma.

In some embodiments, the invention provides a method of lowering total cholesterol levels in blood, the method comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor described herein and a therapeutically effective amount of a second lipid-lowering agent.

Accordingly, in some embodiments, the present invention provides the use of the combination of the ASGR1 inhibitor described herein and a second lipid-lowering drug in the preparation of a drug for lowering the total cholesterol level in blood.

Accordingly, in some embodiments, the present invention provides a combination of an ASGR1 inhibitor described herein and a second lipid-lowering agent for use in lowering total cholesterol levels in blood.

In some embodiments, the invention provides a method of reducing triglyceride levels in blood comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor described herein and a therapeutically effective amount of a second lipid-lowering agent.

Accordingly, in some embodiments, the present invention provides the use of the combination of the ASGR1 inhibitor described herein and a second lipid-lowering drug in the preparation of a drug for lowering blood triglyceride levels.

Accordingly, in some embodiments, the present invention provides a combination of an ASGR1 inhibitor described herein and a second lipid-lowering agent for use in reducing triglyceride levels in blood.

In some embodiments, the invention provides a method of preventing a cardiovascular disease such as atherosclerosis, myocardial infarction or coronary artery disease, the method comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor described herein and a therapeutically effective amount The second lipid-lowering drug.

Accordingly, in some embodiments, the present invention provides the use of the combination of the ASGR1 inhibitor described herein and a second lipid-lowering drug in the preparation of a drug for preventing cardiovascular disease.

Accordingly, in some embodiments, the present invention provides a combination of an ASGR1 inhibitor described herein and a second lipid-lowering agent for use in the prevention of cardiovascular disease.

In any of the above embodiments, the second lipid-lowering drug is preferably an HMGCR inhibitor, an NPC1L1 inhibitor and/or a PCSK9 inhibitor.

In any of the above embodiments, the second lipid-lowering drug and the ASGR1 inhibitor can be administered simultaneously or at intervals. For example, the second lipid-lowering drug can be administered to the subject prior to administration of the ASGR1 inhibitor, or the second lipid-lowering drug can be administered to the subject after administration of the ASGR1 inhibitor.

In some embodiments, the HMGCR inhibitor is a statin. In some embodiments, the statin is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin, and pravastatin. In some embodiments, the statin is atorvastatin.

In some embodiments, the NPC1L1 inhibitor is ezetimibe.

In some embodiments, the PCSK9 inhibitor is a PCSK9 antibody or a nucleic acid (eg, siRNA or shRNA) targeting a gene encoding PCSK9. In some embodiments, the PCSK9 antibody is Evolocumab, Alirocumab, or Inclisiran. Other exemplary PCSK9 inhibitors include those described in WO2017220701A1 , WO2012088313A1 , WO2009026558A1 , WO2009102427A2 or WO2017035340A1 , the entire disclosures of which are incorporated herein by reference.

Suitable routes of administration include parenteral (eg intramuscular, intravenous or subcutaneous) and oral administration. Other conventional administration methods include administration through tracheal intubation, oral intake, inhalation, topical application or dermal, subcutaneous, intraperitoneal, intraarterial injection.

Appropriate dosages are determined by the clinician based on parameters or factors known or suspected in the art to affect therapy or expected to affect therapy. Generally, dosages will be started slightly lower than optimum and thereafter increased by small amounts until the desired or optimal effect relative to any adverse side effects is achieved. Important monitoring indicators include measuring, for example, inflammatory symptoms or the levels of inflammatory cytokines produced.

pharmaceutical composition

One aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an ASGR1 inhibitor described herein and a pharmaceutically acceptable carrier.

Another aspect of the present invention provides a pharmaceutical composition, which includes a therapeutically effective amount of the ASGR1 inhibitor described herein, a therapeutically effective amount of a second lipid-lowering drug, and a pharmaceutically acceptable carrier.

In any of the above aspects, the pharmaceutical composition is used to promote cholesterol efflux. In some embodiments, the promotion of cholesterol efflux includes promoting the efflux of total cholesterol in the liver into bile and feces.

In any of the above aspects, the pharmaceutical composition is used to treat NAFLD. In some embodiments, the NAFLD is NAFL, NASH, NAFLD with liver fibrosis, NAFLD with cirrhosis, or NAFLD with hepatocellular carcinoma.

In any of the above aspects, the pharmaceutical composition is used for lowering the total cholesterol level in blood and/or liver. In some embodiments, the pharmaceutical composition is used to lower triglyceride levels in blood and/or liver. In some embodiments, the pharmaceutical composition is used to prevent cardiovascular disease, such as atherosclerosis, myocardial infarction, or coronary artery disease.

Another aspect of the present invention provides a kit comprising a first pharmaceutical composition and a second pharmaceutical composition, the first pharmaceutical composition comprising a therapeutically effective amount of the ASGR1 inhibitor described herein and a pharmaceutically acceptable The carrier, the second pharmaceutical composition includes a therapeutically effective amount of the second lipid-lowering drug and a pharmaceutically acceptable carrier.

In any aspect above, the second lipid-lowering drug is preferably an HMGCR inhibitor, an NPC1L1 inhibitor and/or a PCSK9 inhibitor. In some embodiments, the HMGCR inhibitor is a statin. In some embodiments, the statin is selected from lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin, and pravastatin. In some embodiments, the statin is atorvastatin. In some embodiments, the NPC1L1 inhibitor is ezetimibe. In some embodiments, the PCSK9 inhibitor is an anti-PCSK9 antibody or a nucleic acid (eg, siRNA or shRNA) targeting a gene encoding PCSK9. In some embodiments, the PCSK9 antibody is Evolocumab, Alirocumab, or Inclisiran.

To prepare pharmaceutical or sterile compositions, the drug is mixed with a pharmaceutically acceptable carrier or excipient. Formulations in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions can be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers. Pharmaceutically acceptable carriers are well known in the art. It is known in the art how to prepare aqueous compositions containing as active ingredients. Typically, these compositions are prepared as injections or sprays, such as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, prior to injection or spraying can also be prepared.

sequence listing

Example

Materials and Methods

mouse

CRISPR/Cas9-mediated Asgr1 whole-body knockout mice were constructed by Chengdu Jizui Yaokang Co., Ltd. The sgRNA-mediated cas9 endonuclease is located in the 2nd and 3rd introns and the 8th and 9th introns cut between. By homologous recombination, part of exon 3 to exon 8 was deleted to obtain whole-body Asgr1 knockout mice.

In the experiments, wild-type littermates served as controls. All mice were kept in SPF (specific pathogen free) level animal room, and kept light for 12 hours/dark for 12 hours, and 8-week-old male or female mice were fed with high-fat and high-cholesterol cholate according to the time required in the experiment Feed (Research Diets, D12109C) was processed, and in antibody neutralization or antibody and statin drugs or ezetimibe, mice were given intraperitoneal injection or intragastric administration of antibody according to the specified amount. All animals were starved for 4 hours before killing. All animal experiments strictly abide by the relevant regulations of the state and Wuhan University on the welfare and protection of experimental animals.

Materials and Plasmids

Lovastatin (purity≥98%, HPLC) was purchased from Shanghai Pharm Vally. Sodium mevalonate(#4667), anti-FLAG M2beads(#A2220), anti-MYC beads(E6654), Fetuin A(SRP6217), D-Galactose(G5388), N-acetyl-D-galactosamine(A-2795), Phenylmethanesulfonyl fluoride (PMSF, #P7626), Protease inhibitor cocktail (#P8340) and β-mercaptoethanol (#M3148) were purchased from Sigma. Dil(1,1-dioctadecyl-3,3,3,3-tetramethyl-indocarbocyanine perchlorate)-LDL (#20614ES76) was purchased from Shanghai Yisheng. Lipofectamine RNAiMAX (#13778150) was purchased from Theromo Fisher. MG132 (#I-130) was purchased from Boston Biochem. Puromycin (#BS111) was purchased from Biosharp. G418 (#345810), Pepstatin A (#516481) and ALLN (N-acetyl-leu-leu-norleucinal, #208719) were purchased from Calbiochem. Ni-NTA Agarose (#30230) was purchased from Qiagen. LPEI (Linear polyethyleneimine, #23966-1) was purchased from Polysciences. FuGENE HD (#E2311) and M-MLV RTase (#M1701) were purchased from Promega. Leupeptin (#11034626001) was purchased from Roche. DTT (DL-Dithiothreitol, #A100281) and NP-40 (A100109) were purchased from Shanghai Sangong. Phosphatase inhibitor (P1082) was purchased from Biyuntian. The medium used for culturing cells, fetal bovine serum (Fetal Bovine Serum, FBS) was purchased from Life Technology. Taq enzyme was purchased from Tiangen. KOD Hot Start DNA polymerase (#KOD-401; TOYOBO) was purchased from Takara; RNA duplex was synthesized by Guangzhou Ruibo Company, and Q-PCR2×MIX was purchased from Mona. Total cholesterol (TC) kits, total triglycerides (TG) kits, and bile acid kits were purchased from Nanjing Kehua Biological Company. Both NEFA kit (294-63601) and Phospholipid kit (292-63901) were purchased from WAKO. ALT, AST, and AKP kits were purchased from Nanjing Jiancheng Biology Co., Ltd. manage. Blood glucose test strips and blood glucose meters were purchased from Wenhao. Lipoprotein-deficient serum (d>1.215g/mL), that is, LPPS is prepared from newborn calf serum by ultracentrifugation.

The following plasmids were constructed by standard molecular cloning techniques:

Human-derived and mouse-derived Asgr1, Asgr2, Lxrα, and Lxrβ gene fragments were derived from the cDNA formed by reverse transcription of RNA from Huh7 and mouse liver tissue, and the human-derived BARD1 gene fragment was amplified from Huh7 and cloned into p3× Flag-CMV14, pEGFP-C1 and pcDNA3-C-5×Myc vectors. pDEST-FRT/T0-GFP-BRCA1 (#71116) was purchased from Addgene. Various truncations and point mutations of ASGR1 were constructed by point mutation method.

Huh7 and HEK293T cells were grown in monolayer at 37°C and 5% _CO2 . The cells were maintained in medium A (DMEM containing 100 units/mL penicillin and 100 mg/mL streptomycin sulfate) supplemented with 10% fetal bovine serum (FBS), and cholesterol-deficient medium B was supplemented by 5% of medium A Lipoprotein-free serum (LPPS), 1 μM lovastatin and 10 μM mevalonate were obtained. Primary mouse hepatocyte culture medium D (M199) supplemented with 5% FBS, 100units/mL penicillin and 100mg/mL streptomycin sulfate.

western blot

The collected cells or tissues are lysed in RIPA lysis buffer supplemented with protease inhibitors and phosphatase inhibitors. RIPA lysis buffer contained 50 mM Tris-HCl (pH=8.0), 150 mM NaCl, 2 mM MgCl ₂ , 1.5% NP-40, 0.1% SDS and 0.5% sodium deoxycholate. Protease inhibitors included 10 μM MG-132, 10 μg/ml Leupeptin, 1 mM PMSF, 5 μg/ml pepstatin, 25 μg/ml ALLN, 1 mM DTT. The protein concentration of the lysates was determined using the BCA method (Thermo Fisher Scientific). Protein samples were mixed with membrane lysis buffer (62.5mM Tris-HCl (pH=6.8), 15% SDS, 8M urea, 10% glycerol and 100mM DTT) and 4× loading buffer (150mM Tris-HCl (pH=6.8 ), 12% SDS, 30% glycerol, 6% 2-mercaptoethanol and 0.02% bromophenol blue) were incubated at 37°C for 30 minutes. The protein samples were separated by SDS-PAGE gel and transferred to PVDF membrane, and blocked with TBS containing 0.075% Tween 20 and 5% skimmed milk (3% BSA for phosphorylation experiment), that is, TBST for 1 hour. And incubated overnight at 4°C with the indicated primary antibodies, followed by washing 3 times with TBST. Finally, detection was performed with Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific).

The primary antibody used in the experiment

Both Anti-β-actin (#A5441) and anti-FLAG (#F3165) were purchased from Sigma. anti-AMPK(10929-2-AP), anti-ACC(67373-I-Ig), anti-FASN(10624-2-AP), anti-GAPDH(60004-1-Ig), anti-ASGR1(11739- 1-AP) was purchased from ProteinTech. Anti-CYP7A1 (sc-518007), anti-SREBP1 (sc-13551) and anti-BRCA1 (sc-6954) were purchased from Santa Cruz. Anti-c-Myc and anti-HMGCR were prepared and purified from hybridoma cell lines (ATCC) 9E10 and A9, respectively. anti-LDLR, anti-H2 (HMGCR) by selecting soluble segments, immunized rabbits to obtain antiserum and affinity purified antibodies. Anti-EGFP is obtained by expressing and purifying EGFP protein in Escherichia coli and immunizing rabbits. anti-LXRα (ab41902) was purchased from Abcam. anti-LXRβ (NB100-74457), anti-ABCG8 (NBP1-71706F), anti-ABCA1 (NB400-105) were purchased from Novus. anti-BARD1 (A300-263A) was purchased from Bethy. Anti-phos ACC (118187) and anti phos-AMPK (#2535) were purchased from Cell signaling Tech. Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories.

Ubiquitination experiments

Cells were washed twice with pre-cooled 1xPBS buffer, followed by pre-cooled lysis buffer (adding 0.5% digitonin, 5mM EGTA, 5mM EDTA, protease inhibitors (except DTT and PMSF) and phosphatase Inhibitor in 1xPBS) to lyse the cells, centrifuge at 13400rpm at 4°C for 10 minutes, discard the precipitate, and take 60 μl of the supernatant as the whole cell lysate control (Input component), Mix the remaining supernatant with 30 μl anti-Myc magnetic beads, incubate with rotation at 4°C for 4 hours, centrifuge at 1000 g for 3 minutes, take 60 μl of the supernatant as the supernatant control (Supernatant component), then discard the supernatant and use the lysis buffer The solution was washed 3 times for 10 minutes each time and vortexed continuously to mix. Finally, collect the precipitate, add 100 μl membrane dissolution buffer and incubate in a 37°C metal bath for 30 minutes, then centrifuge at 13,400 rpm for 2 minutes, absorb 90 μl of the supernatant after centrifugation, and remove it with 2× loading buffer (4× loading buffer components) 2-mercaptoethanol and SDS lysis buffer according to 1:1 mixing) 1:1 mixing. Labeled as a pellet component. Then each component was detected by Western blot.

Blood and liver chemistry analysis

Serum was obtained from eyeball blood sampling to detect total cholesterol and triglycerides. The liver was homogenized, and the supernatant was collected for lipid extraction to obtain liver total cholesterol, and liver triglyceride and total cholesterol (Kehua, China) levels were determined according to the instructions. Phospholipid levels were determined with a kit (Phospholipid, WAKO, Japan). Kits made in Nanjing (China) were used to detect the levels of ALT and AST in serum.

results and analysis

Example 1: LXRα is increased after ASGR1 deletion, promotes cholesterol efflux, and reduces lipid levels in the liver and blood.

In order to explore how the loss of ASGR1 affects lipid metabolism, we used small interfering RNA to specifically mediate ASGR1 knockdown in human liver cancer cell Huh7, extracted RNA for eukaryotic transcriptome sequencing, and then enriched with gene set Analysis (Gene set enrichment analysis, GSEA) for analysis. The results showed that the LXR pathway was significantly activated (Fig. 1a), which was also confirmed by real-time quantitative PCR (Fig. 1b). Regardless of whether it was siRNA-mediated ASGR1 knockdown cells (Figure 1c) or ASGR1 knockdown cells (Figure 1d). LXR protein levels were significantly up-regulated. However, in cells with stable expression of ASGR1, the protein levels of LXR and the target genes downstream of LXR were significantly reduced (Fig. 1e, f). Furthermore, we also found that in Huh7 cells, neither siRNA-specific knockdown of ASGR1 nor plasmids co-expressing ASGR1 and LDLR affected the protein stability of LDLR (Fig. 2m–n), and Nor did it affect the endocytosis of DiL-tagged LDL (Fig. 2o-p), nor antagonize PCSK9-induced LDLR degradation (Fig. 2q).

In order to gain a deeper understanding of whether ASGR1 has a regulatory effect on blood lipids, we first detected the expression level of ASGR1 in each tissue of the mouse by extracting various tissues of the mouse (Figure 2a), and found that ASGR1 is mainly in the liver of the mouse in the expression. Based on this tissue expression profile, we constructed ASGR1 systemic knockout mice by CRISPR/Cas9 technology (Fig. 2b-d). Eight-week-old littermate mice were randomly assigned to 3 groups according to their genotypes, fed with high-fat and high-cholesterol cholate diet for 4 weeks, and the metabolic phenotypes of the mice were detected. The results showed that body weight, daily food intake, liver-to-body weight ratio, blood glucose, and aspartate aminotransferase and alanine aminotransferase were not affected in either male or female Asgr1 knockout mice (Fig. 2e-j, Fig. 5c-d and Fig. 5i-j), while significantly reducing total cholesterol and triglycerides in blood (Fig. 1g-h, Fig. 5e-f); total cholesterol and triglycerides in liver also decreased significantly (Fig. 1i-j , Fig. 5g–h), hematoxylin-eosin staining (left) and oil red O staining (right) of liver sections also showed that lipid accumulation in the liver was greatly relieved (Fig. 1k). In addition, we also found that in Asgr1 knockout mice, the gallbladder volume increased significantly (Figure 1l, Figure 5k), and the concentration and total amount of total cholesterol in bile increased significantly (Figure 1m, 1o, Figure 5l-5m), The concentration and total amount of total bile acids in bile also significantly increased (Fig. 1n, 1p, Fig. 5n–5o), phospholipids in bile increased (Fig. 2k), and the ratio of bile cholesterol to phospholipids did not change (Fig. 2l). After liver tissue was homogenized, western blot analysis was performed, and it was found to be consistent with previous results: the protein level of LXR increased significantly, and proteins related to cholesterol efflux, such as ABCG8, ABCA1, and CYP7A1, increased significantly, while SREBP1, FASN, etc. Proteins related to lipid synthesis were significantly reduced, while LDLR, a protein related to cholesterol absorption, was unchanged (Figure 1r, Figure 3a, Figure 5a), and real-time quantitative PCR also showed results consistent with changes in protein levels (Figure 1s, Figure 3b, Figure 5b). In summary, ASGR1 Deletion protects against metabolic disturbances in mice induced by high-fat, high-cholesterol cholate diet.

Example 2: ASGR1 knockout improves high-fat and high-cholesterol diet-induced metabolic syndrome is dependent on LXR.

In the previous experimental results, we detected that in the case of ASGR1 knockout, the protein level of LXR was significantly increased, its downstream target genes were significantly up-regulated, and it could significantly improve the metabolic phenotype induced by high fat and high cholesterol. But is the improvement effect of ASGR1 on metabolic syndrome achieved through LXR? To address this question, we constructed Asgr1 ^-/- Lxrα ^-/- mice by mating Asgr1 ^-/- with Lxrα ^-/- . Select 8W-old Asgr1 ^-/- (Asgr1 KO) mice and littermate wild-type Asgr1 ^+/+ (WT), Lxrα ^- ^/- mice (Lxrα KO) and Asgr1 ^-/- Lxrα ^-/- (DKO) Six mice in each group were fed a high-fat and high-cholesterol cholate diet (60% fat + 1.25% cholesterol + 0.5% cholate, HF/HC/BS diet) for 4 weeks, and the tissues of the mice were taken to detect various indicators. The experimental results showed that: in the context of Lxrα deletion, the effect of Asgr1 deletion on the increase of downstream ABCG8, ABCA1, CYP7A1 and the reduction effect on SREBP1c and FASN no longer existed (Figure 4a), and real-time quantitative PCR also correlated with Western blot The results of the analysis were consistent, and the difference between the Lxrα KO group and the DKO group disappeared (Fig. 4b).

The analysis of biochemical indicators showed that there was no significant difference in the body weight, daily food intake, blood glucose, ALT, AST, liver and body weight ratio of the mice (Fig. 4c, m-q). Compared with the control group (Lxrα KO), total cholesterol and triglycerides in the blood of DKO mice (Fig. 4e,f) did not show the effect similar to that seen in the Asgr1 KO group vs WT group; and by FPLC The distribution of lipoproteins found that compared with the WT group, the Asgr1 KO group showed significantly less VLDL and LDL, while HDL increased significantly. However, on this basis, Lxrα was deleted, that is, comparing the DKO group and the LxrαKO group, it was found that although the Lxrα deletion would lead to a very high level of cholesterol in the blood, the ASGR1 deletion had almost no effect on the distribution of lipoproteins in the blood (Fig. 4i- j) ; Asgr1 alone KO has no relief effect on liver lipid accumulation, and H&E staining and Oil Red O staining of liver tissue sections confirm this conclusion (Fig. 4d). On the other hand, the total cholesterol in bile showed a significant increase in the Asgr1 KO group, and on this basis, if Lxrα was knocked out, the effect of the increase disappeared (Fig. 4k). In addition, we also detected the cholesterol in the feces. Looking at the WT group and the Asgr1 KO group alone, we found that the total cholesterol in the feces increased significantly, suggesting that part of the reduced cholesterol was excreted into the feces. However, in the context of LxrαKO and then knocking out Asgr1, that is, DKO mice, these effects disappeared (Fig. 4l).

To sum up, the loss of ASGR1 does improve the metabolic syndrome induced by high fat and high cholesterol through LXRα. Therefore, once LXR is lost, the regulatory effect of ASGR1 on blood lipids completely disappears.

Example 3: Asialoglycoprotein-ASGR1-AMPK axis regulates LXRa and SREBP.

The previous results confirmed that ASGR1 can regulate the protein level of LXRα through ubiquitination (Figure 6a-c), so is there an E3 ubiquitin ligase that mediates the degradation of LXRα protein by ASGR1? According to reports, BARD1/BRCA1 is currently found to be the only E3 ubiquitin ligase mediating LXRα degradation. BRCA1 (Breast and ovarian cancer susceptibility 1), a known tumor suppressor gene, is related to DNA damage repair and forms a stable heterodimer with BARD1 (BRCA1-associated RING domain 1) to regulate the response of cells to DNA damage , it has also been reported that the dimer plays an important role in transcriptional regulation, cell cycle and meiosis of repressive chromosomes. BRCA1 and BARD1 contain a RING domain (which can be used to mediate DNA-protein binding or protein-protein binding), which contains the nuclear export signal (NES) at the N-terminus and two tandem BRCA1 carboxy-terminal (BRCT, also with protein-protein combined with the relevant) area. The unique RING region at the N-terminus of BRCA1 and BARD1 allows each of them to play a weak role as E3, and once the two form a heterodimer through the RING domain, the protein stability of the two increases, and the corresponding E3 activity Significantly enhanced, which may be due to their instability alone, and the enhanced E3 activity will be weakened after the 61st and 64th cysteine (C61G, C64G) mutations of BRCA1, and the cancer suppression function will also be weakened, But it does not affect the combination of the two.

So, does BRCA1/BARD1 play a role as an E3 complex in the regulation of LXRα protein degradation by ASGR1? Therefore, in Figure 7a, we designed siRNA specifically targeting BRCA1 and BARD1 to observe whether ASGR1 still has ubiquitination on LXRα under the condition of knocking down the complex. As shown in Figure 7a, under normal conditions, ASGR1 can significantly increase the ubiquitination of LXR (lanes 1-3), while when BRCA1 and BARD1 were knocked down, the ubiquitination of LXRα was significantly reduced (lanes 4-6) . Consistently, compared with the control group, the degradative effect of ASGR1 on LXR almost disappeared after BRCA1 and BARD1 deletion (Fig. 7b). Next, we wondered, what is the connection between ASGR1 and BRCA1 and BARD1? We first examined whether ASGR1 deletion affected the transcript and protein levels of BRCA1 and BARD1. The experimental results showed that whether it was siRNA-mediated knockdown of ASGR1 (Figure 6d) or CRISPR/Cas9-mediated ASGR1 KO (Figure 7c), the protein levels of BRCA1 and BARD1 were significantly reduced compared with the control group. On the contrary, ASGR1, BRCA1 and BARD1 were co-expressed in Huh7 cells, and ASGR1 was also found to stabilize BRCA1 and BARD1 proteins (Fig. 7d).

The structure of mouse ASGR1 is shown in Fig. 6e. Among them, Asp at position 241, Asp at position 265, Asn at position 264, Glu at position 252, Gln at position 239 and Trp at position 243 participate in the formation of the active site for ligand binding.

In order to further understand whether the function of ASGR1 binding to ligand is involved in the regulation of ASGR1 on LXRα. Through analysis, we found that there are a large number of ASGR1 ligands in fetal bovine serum (FBS), so we designed the experiment shown in Figure 6f. , ASGR1 can promote the degradation of LXRα, and once the ligand disappears, the degradation of LXRα by ASGR1 disappears. The ligands of ASGR1, whether asialylated fetuin A (Asialofetuin A, AF) or asialylated acidic protein (Asialoorosomocoid, ASOR), can mediate the degradation of LXRα by ASGR1 (Figure 7e, Figure 6g). Furthermore, studies have reported that the 241st, 265th Asp, 264th Asn, 252th Glu, 239th Gln and 243rd Trp of ASGR1 participate in the formation of the active site for ligand binding. Therefore, we mutated these 6 amino acids into alanine (Alanine, A), that is, the ASGR1 (6A) mentioned in the article, and then tested whether it can respond to the ligand-induced degradation of LXRα. As shown in Figure 6h: after the ligand binding site of ASGR1 was mutated (ASGR1(6A)), its degradation effect on LXRα was almost disappeared compared with wild-type ASGR1. Similarly, in Figure 6i, when there is only ligand and no ASGR1, there is almost no degradation of LXRα, and when wild-type ASGR1 and ligand exist at the same time, ASGR1 has a dose-dependent effect on the degradation of LXRα, while even In the presence of ligand, ASGR1 cannot perform the degradation function after the ligand binding site of ASGR1 is mutated. And desialylated fetuin A can significantly promote the ubiquitination of LXRα by ASGR1 (Fig. 7f).

In the previous experimental results, we confirmed that BRCA1 and BARD1 mediate the degradation of LXRα by ASGR1 (Fig. 7a,b), and we further explored whether BRCA1 and BARD1 played a key role in the ligand-induced degradation of LXRα by ASGR1. As shown in Figure 7g, the binding of ligands to ASGR1 stabilized the protein levels of BRCA1 and BARD1, thereby inducing the degradation of LXRα. With BRCA1 and BARD1 knockdown, LXRα was no longer degraded even in the presence of ligand. It shows that BRCA1 and BARD1 are crucial in the degradation of LXRα by ASGR1 induced by ligand.

After ASGR1 binds to its ligand, asialoglycoprotein, through clathrin-mediated endocytosis, it finally enters the lysosome for degradation. Therefore, we speculated whether blocking endocytosis affects the degradation of LXRα by ASGR1. Using designated small interfering RNA and plasmids to transfect Huh7 cells, we found that after specifically knocking down CHC, the degradation effect of ASGR1 on LXRα almost disappeared (Fig. 6j). And after CHC was knocked down, the downstream protein changes caused by the binding of ASGR1 to the ligand almost disappeared (Fig. 7h).

According to existing literature reports, the nutrients released by lysosome degradation protein include sugar and amino acid to activate mTORC1 and inhibit AMPK. mTORC1 activates SREBP to increase lipid synthesis, whereas AMPK Can directly phosphorylate SREBP1 and inhibit its splicing into the nucleus. And AMPK activation can significantly reduce the expression of BRCA1. So is AMPK involved in the degradation of LXRα by ASGR1? To verify this conjecture, we first used bafilomycin A1 to inhibit the degradation of proteins in lysosomes, and found that after bafilomycin A1 treatment, compared with the control group, the degradation effect of ASGR1 on LXRα almost disappeared, The stabilizing effect of ASGR1 deletion on LXRα was also lost (Fig. 6k, Fig. 7i). After further treatment with AMPK agonist A-769662, compared with the control group, the degradation effect of ASGR1 on LXRα disappeared (Fig. 6l). Moreover, the experimental results showed that wild-type Huh7 cells were treated with A-769662 in a dose-dependent manner, and the protein levels of LXRα and p-ACC were significantly increased, while p-S6K, BRCA1, BARD1, and SREBP1/2 were significantly reduced ( Figure 7j). Conversely, after wild-type Huh7 cells were treated with Dorsomophin (AMPK inhibitor), the results were opposite to those of the agonist (Fig. 7k). In ASGR1-specific knockout cells (ASGR1KO-A,B), the protein levels of LXRα, p-ACC, p-S6K, BRCA1, BARD1, and SREBP1/2 were significantly reduced by ASGR1 deletion. Then it disappeared (Figure 7l).

In summary, the above results show that after ASGR1 binds to asialoglycoprotein, it enters into lysosome through CHC-mediated endocytosis, releases nutrients under the action of acid enzyme in lysosome, activates mTORC1 inhibits AMPK; on the one hand, it activates SREBP1 to increase lipid synthesis, and on the other hand, the increased protein expression of BRCA1/BARD1 promotes the degradation of LXRα, thereby reducing cholesterol efflux. On the contrary, after ASGR1 is mutated or inhibited, the endocytosis and degradation of ligands are blocked, mTORC1 is inhibited, AMPK is activated, and AMPK phosphorylation inhibits the splicing of SREBP1c into the nucleus, reducing the protein stability of BRCA1/BARD1, This increased the protein level of LXRα, which promoted cholesterol efflux by activating ABCA1, ABCG5/8, and CYP7A1 without increasing lipid synthesis (Fig. 7m).

Example 4: AAV-shRNA-mediated knockdown of ASGR1 promotes cholesterol efflux and reduces lipid levels in liver and blood.

RNAi is an effective therapeutic approach. Since ASGR1 is selectively expressed in the liver, we used adeno-associated virus (AAV) serotype 2/8 to specifically knock down Asgr1 in the liver. The experimental results showed that after AAV-shAsgr1 specifically knocked down Asgr1, the results were consistent with Asgr1 knockout mice, and the proteins related to cholesterol efflux increased significantly, such as LXRα, LXRβ, ABCG8, ABCA1, CYP7A1, while lipid synthesis The protein FASN decreased significantly, while the cholesterol synthesis or absorption proteins HMGCR and LDLR had no significant changes (Fig. 8a). Real-time quantitative PCR was also consistent with the protein expression results (Fig. 8b). Biochemical indicators showed that total cholesterol and triglycerides in serum and total cholesterol and triglycerides in liver decreased significantly (Fig. It was shown that the lipid accumulation in the liver was alleviated to a great extent (Fig. 8g-h). While the total amount of total cholesterol in bile and the total amount of total bile acid in bile were also significantly increased (Fig. ).

Example 5: ASGR1 neutralizing antibody 4B9 promotes the efflux of cholesterol into bile and feces, showing a lipid-lowering therapeutic effect.

ASGR1 can bind to a large number of desialylated glycoproteins. In order to better simulate the improvement effect of Asgr1 knockout on high-fat and high-cholesterol-induced metabolic syndrome, we purified ASGR1 protein from HEK293T cells and immunized rabbits to obtain the inhibitory effect. Neutralizing antibody against ASGR1 function. The specific process is shown in Figure 9a: steps for preparing neutralized ASGR1 monoclonal clones. The purified ASGR1 protein was used as an antigen to immunize rabbits. After the first round of screening, 4 B cell monoclonal strains were obtained as candidates. Next, the coding region of the variable region of the antibody is sequenced, and it is constructed on an antibody expression vector, and transfected into mammalian cells to purify the protein. The effect was confirmed by western blotting and real-time quantitative PCR, and finally the rabbit-derived Fc fragment was replaced with the mouse-derived Fc fragment. Finally, the neutralizing antibody 4B9 with the best effect was selected for large-scale production and used in subsequent experiments. The experimental results verified that 4B9 has a potential blood lipid-lowering effect. 4B9 and its His three ASGR1 monoclonal neutralizing antibodies could significantly increase the protein stability of ASGR1 (Figure 9c, 9d), and the genes involved in cholesterol efflux were also significantly up-regulated (Figure 9e). In a more detailed way, we transfected different ASGR1 truncated plasmids, detected the segment recognized by 4B9, and found that 4B9 mainly recognized the sequence between 182-274aa (Fig. 9f).

Further, we tested whether 4B9 can completely mimic the improvement of high fat and high cholesterol caused by Asgr1 deletion. Eight-week-old Asgr1 knockout mice and littermate wild-type mice were randomly divided into 4 groups as shown in Figure 10 according to genotype, with 6 mice in each group. Free drinking water, high fat, high cholesterol and cholate (HF/HC/BS) diet (60% fat, 1.25% cholesterol and 0.5% cholate) were fed. At the same time, the mice were intraperitoneally injected with the control antibody or ASGR1 neutralizing antibody 4B9 at a dose of 10 mg/kg/day every other day. After 14 days, the mice were uniformly starved for 4 hours before killing. As a result of the experiment, wild-type mice injected with 4B9 antibody showed results consistent with Asgr1 deletion, that is, proteins related to cholesterol efflux, such as LXR, ABCG8, ABCA1, and CYP7A1, were significantly increased, and p-ACC was also significantly up-regulated, while Lipid synthesis-related proteins SREBP1 and FASN were significantly reduced, while LDLR was not affected. BARD1 was significantly decreased; and there was no effect of injection or no injection of 4B9 in Asgr1 knockout mice, indicating that 4B9 specifically targets ASGR1 ( FIG. 10 a ). Real-time quantitative PCR also showed results consistent with changes in protein levels (Fig. 10b). Biochemical indicators showed that compared with the control group, the total cholesterol and triglycerides in the serum of mice injected with 4B9 antibodies were significantly decreased (Figure 10c, d), and the total cholesterol and triglycerides in the liver were significantly decreased (Figure 10e , f), the volume of gallbladder increased significantly (Figure 10g, j), the concentration and total amount of total cholesterol in bile increased significantly (Figure 10h, i, j), the total cholesterol in feces increased significantly (Figure 10k), and the total cholesterol in bile increased significantly (Figure 10k). The concentration and total amount of bile acids increased significantly (Fig. 10l,m). There were no significant changes in body weight, daily food intake, ratio of liver to body weight, blood sugar, alanine aminotransferase and aspartate aminotransferase (Fig. 10n-s). Based on the above results, ASGR1 neutralizing antibody 4B9 showed a very significant lipid-lowering effect, which can greatly alleviate the metabolic syndrome induced by high-fat and high-cholesterol diet.

Example 6: The combination of antibody 4B9 and atorvastatin shows a synergistic lipid-lowering effect.

Statins, commonly used to reduce LDL-c, are the first-line drugs used to lower cholesterol. Currently, the statin drugs approved by the FDA mainly include lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin and pravastatin, among which atorvastatin and Rosuvastatin is the most widely used. However, some related studies have found that long-term use of statins can lead to statin resistance in patients, resulting in liver toxicity, and statin intolerance in some patients, resulting in side effects such as myasthenia, rhabdomyolysis, diabetes, etc.; in addition, the dissolution of statins in water The performance is poor, and the utilization rate of the body is not high, which also limits its use to a certain extent. 4B9 was proved to be able to significantly improve the metabolic syndrome phenotype induced by high-fat and high-cholesterol diet in the previous experimental results (Fig. 10). Further, we wanted to test whether there is a synergistic lipid-lowering effect of 4B9 combined with atorvastatin. The 8-week-old Asgr1 knockout mice and wild-type littermates were randomly divided into 8 groups as shown in Figure 11 according to genotype, with 6 mice in each group. Free drinking water, high fat high cholesterol and cholate (HF/HC/BS) diet (60% fat, 1.25% cholesterol and 0.5% cholate) feeding.

At the same time, the mice were intraperitoneally injected with the control antibody or ASGR1 neutralizing antibody 4B9 at a dose of 10 mg/kg/day every other day, and atorvastatin was administered orally at a dose of 30 mg/kg/day every day. After 14 days, mice were uniformly starved for 4 hours before killing. The experimental results show that when 4B9 is used alone, it still increases the levels of genes and proteins related to cholesterol efflux, inhibits the levels of genes and proteins related to lipid synthesis, and does not affect the levels of genes or proteins related to cholesterol synthesis or absorption. However, the combination of atorvastatin and 4B9 does not affect the expression effect of 4B9 on promoting cholesterol efflux-related genes and proteins such as LXRs, ABCG8, ABCA1, and CYP7A1, but can more significantly inhibit the expression of lipid synthesis-related genes and proteins, such as SREBP1, FASN (Fig. 11a, 11o-p). When 4B9 or atorvastatin is used alone, it can effectively reduce the total cholesterol level and triglyceride level in serum and liver, and when 4B9 and atorvastatin are used in combination, it shows a more significant lipid-lowering effect ( Figure 11b-e, Figure 11q-r), 4B9 can be used alone increased the total amount of total cholesterol and total bile acids in bile, whereas atorvastatin administration had little effect on total cholesterol and total bile acids in bile (Fig. 11f,g). 4B9 alone significantly increased fecal total cholesterol efflux, whereas atorvastatin had no effect on fecal cholesterol (Fig. 11h). Whether 4B9 or atorvastatin was used alone or in combination, there were no significant effects on the body weight, daily food intake, ratio of liver to body weight, blood glucose, alanine aminotransferase and aspartate aminotransferase of the mice among the 8 groups (Fig. 11i -n). In summary, the combination of 4B9 and atorvastatin can achieve better lipid-lowering effect than the two alone.

Example 7: The combined use of antibody 4B9 and ezetimibe showed a synergistic lipid-lowering effect.

Ezetimibe (EZ) is a lipid-lowering drug whose main function is to inhibit NPC1L1-mediated intestinal brush border absorption of cholesterol from diet and bile, but does not affect the absorption of other fat-soluble nutrients, and is mainly used for treatment Familial hypercholesterolemia. EZ treatment reduces the accumulation of lipids in the liver, reduces total cholesterol, triglycerides and LDL-c in the blood, and increases HDL-c at the same time. There was no significant effect on fat-soluble vitamins such as vitamin A, vitamin D and vitamin E. 4B9 has been proven to have a certain lipid-lowering effect in mice (Figure 10). Therefore, will the combination of 4B9 and EZ increase the lipid-lowering effect to a greater extent? Therefore, we designed an experiment as shown in Figure 12 to verify the conjecture. The mice were randomly divided into 8 groups (n=6) according to their genotypes. They were fed with HF/HC/BS feed, EZ was administered orally at 10 mg/kg every day, and 4B9 (10 mg/kg) was intraperitoneally injected once every other day. mouse, mouse tissue was taken. The results showed that when 4B9 was used alone, it still showed an increase in cholesterol efflux-related genes and their protein levels, such as LXRs, ABCG8, ABCA1, and CYP7A1, and inhibited lipid synthesis-related genes and their protein levels, such as SREBP1 and FASN, without affecting cholesterol Synthesis or absorption-related genes or their protein levels, and when ezetimibe was used alone or in combination with 4B9, the genes and proteins related to cholesterol efflux, and the genes and proteins related to lipid synthesis decreased significantly (Fig. 12a ,12n,o). When 4B9 or ezetimibe was used alone, it could effectively reduce the total cholesterol level and triglyceride level in serum and liver, and when 4B9 and ezetimibe were used in combination, it showed a more significant lipid-lowering effect ( Figure 12b-e, Figure 12p-q), 4B9 alone can increase the total amount of total cholesterol and total bile acid in bile, and whether ezetimibe alone or in combination with 4B9 can significantly inhibit the amount of total cholesterol and total bile acid in bile Total amount of total bile acids (Fig. 12f,g). Whether 4B9 or ezetimibe was used alone or in combination, there were no significant effects on the body weight, daily food intake, ratio of liver to body weight, blood glucose, alanine aminotransferase and aspartate aminotransferase of the mice in the 8 groups (Fig. 12h -m). In summary, the combined use of 4B9 and ezetimibe can achieve a better lipid-lowering effect than the use of the two alone.

Example 8: Asgr1 knockdown ameliorates atherosclerosis.

Coronary artery disease (CAD) is one of the most important cardiovascular diseases (CVD). The occurrence of CAD is mainly due to the closure of coronary arteries caused by atherosclerosis. When the function of endothelial cells in the arterial wall is damaged, a large number of lipoprotein particles accumulate in the intima of arteries. High concentrations of LDL can penetrate damaged endothelial cells to form oxidized low-density lipoprotein (ox-LDL), and ox-LDL will attract a large number of white blood cells to the intima of coronary arteries, and macrophages will then play a role in clearing Foam cells are formed, a large number of foam cells replicate, and finally damage is formed, leading to the appearance of fatty streaks. The earliest damage is regarded as atherosclerosis. The emergence of a large number of injuries releases a "help" signal, recruiting smooth muscle cells (SMCs) to the area where fat streaks appear for "rescue", SMCs begin to proliferate rapidly, produce extracellular matrix (mainly collagen and proteoglycan), and atherosclerosis Plaques begin to form and accumulate large amounts of extracellular matrix produced by SMCs, resulting in the eventual transformation of the lesion into a fibrotic plaque. Fibrotic plaques continue to accumulate in the coronary vessels, gradually narrowing the vessels, and eventually calcifying the plaques.

When using adeno-associated virus (AAV) serotype 2/8-mediated knockdown of Asgr1, it was able to significantly reduce the content of cholesterol and triglyceride in blood and the accumulation of lipid in liver (Fig. 13a-c, 13i) , body weight, the ratio of liver weight to body weight, and blood glucose had no significant changes (Fig. 13d-f), while the results of aortic chromogenicity proved that the deletion of Asgr1 could significantly reduce the formation of atherosclerotic plaques (Fig. 13g-h). In summary, inhibiting the expression of Asgr1 can significantly reduce the accumulation of atherosclerotic plaques.

The body maintains the dynamic balance of cholesterol mainly by coordinating the three processes of cholesterol synthesis, absorption and efflux. We specifically targeted ASGR1 through small interfering RNA, and performed eukaryotic transcriptome sequencing. After GSEA analysis, we found that the genes related to the LXR pathway were significantly enriched, and through a series of biochemical experiments and animal experiments. The influence of lipid level is mainly realized through the regulation of LXR protein level, and BRCA1/BARD1 is involved in the degradation of LXR by ASGR1 as a ubiquitinated ligase.

mTORC1 is a nutrient sensor and hub for energy storage. Our study shows that ASGR1 binds to the ligand asialoglycoprotein in the blood and enters into the lysosome for degradation through clathrin-mediated activation of the mTORC1-AMPK signaling pathway to stabilize the protein level of BRCA1/BARD1 , thereby degrading LXR, thereby inhibiting the efflux of cholesterol. After knocking out ASGR1 or inhibiting its function with neutralizing antibodies, this signaling pathway can be blocked, thereby promoting the efflux of cholesterol. Can specifically activate AMPK in the liver. First, due to the increased stability of LXR protein, it can significantly promote the efflux of cholesterol into the gallbladder and feces; second, due to the activation of AMPK, it inhibits the splicing of SREBP1c into the nucleus, thereby inhibiting the activation of lipid synthesis-related genes, thereby Greatly relieved the accumulation of lipids in the liver and relieved fatty liver.

In summary, the present invention activates the mTORC1-AMPK-BRCA1/BARD1 pathway by studying the combination of asialoglycoprotein and ASGR1, and finally regulates the protein stability of LXR, thereby playing an important role in lipid metabolism, and then provides ASGR1 deletion New uses for improving metabolic syndrome such as alleviating NAFLD and reducing blood lipids and liver lipids.

It should be understood that while the invention has been specifically disclosed by way of preferred embodiments and optional features, those skilled in the art may make modifications, improvements and variations to the invention disclosed herein which are considered within the scope of the present invention. The materials, methods and examples provided herein are representative and exemplary of preferred embodiments and are not intended as limitations on the scope of the invention.

Claims

Application of an asialoglycoprotein receptor 1 (ASGR1) inhibitor in the preparation of a drug for promoting cholesterol efflux.
The application according to claim 1, wherein the drug for promoting cholesterol efflux is used for reducing total cholesterol in the liver, reducing triglyceride in the liver or treating non-alcoholic fatty liver disease (NAFLD).
The application according to claim 1, wherein the ASGR1 inhibitor is selected from the group consisting of anti-ASGR1 monoclonal antibodies or antigen-binding fragments thereof, nucleic acids encoding nucleic acids targeting ASGR1, nucleic acid aptamers targeting ASGR1, and combinations thereof.
The application according to claim 3, wherein the anti-ASGR1 monoclonal antibody binds to the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and inhibits or blocks ASGR1 and its natural ligand binding and/or endocytosis of ASGR1.
The application according to claim 3, wherein the epitope bound by the ASGR1 monoclonal antibody comprises Q240, D242, W244, E253, N265, D266, D267, R237, N209, H257, T259 and One or more of Y273.
The application according to claim 3, wherein the epitope to which the ASGR1 monoclonal antibody binds comprises Q240, D242, W244, E253, N265 and D266 in SEQ ID NO:1.
The application according to claim 3, wherein the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region and is selected from any of the following antibodies:

(a) The light chain variable region comprises LCDR1, LCDR2, LCDR3, respectively comprising the sequence shown in SEQ ID NO: 4-6, and the heavy chain variable region comprises HCDR1, HCDR2, HCDR3, respectively comprising SEQ ID NO : the sequence shown in 7-9;

(b) The light chain variable region comprises LCDR1, LCDR2, LCDR3, respectively comprising the sequence shown in SEQ ID NO: 10-12, and the heavy chain variable region comprises HCDR1, HCDR2, HCDR3, respectively comprising SEQ ID NO : the sequence shown in 13-15;

(c) The light chain variable region comprises LCDR1, LCDR2, LCDR3, respectively comprising the sequence shown in SEQ ID NO: 16-18, and the heavy chain variable region comprises HCDR1, HCDR2, HCDR3, respectively comprising SEQ ID NO : the sequence shown in 19-21;

(d) The light chain variable region comprises LCDR1, LCDR2, LCDR3, respectively comprising the sequence shown in SEQ ID NO: 22-24, and the heavy chain variable region comprises HCDR1, HCDR2, HCDR3, respectively comprising SEQ ID NO : the sequence shown in 25-27;

(e) the light chain variable region comprises the sequence shown in SEQ ID NO: 28, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 29;

(f) the light chain variable region comprises the sequence shown in SEQ ID NO: 30, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 31;

(g) the light chain variable region comprises the sequence shown in SEQ ID NO: 32, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 33; and

(h) The light chain variable region comprises the sequence shown in SEQ ID NO: 34, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 35.
The use according to claim 3, wherein the nucleic acid is antisense oligonucleotide (ASO), siRNA, shRNA or gRNA.
The application according to claim 8, wherein the gRNA and the CRISPR/Cas9 system constitute a gene editing system.
The use according to claim 8, wherein the siRNA is delivered by GalNAc, LNP or AAV.
The application according to claim 8, wherein the nucleic acid targets the sequence shown in SEQ ID NO: 36 and inhibits the expression of the coding gene of ASGR1.
The application according to claim 2, wherein the NAFLD is non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), NAFLD with liver fibrosis, NAFLD with liver cirrhosis, or hepatocellular carcinoma. NAFLD.
The use according to claim 1, wherein the ASGR1 inhibitor is used in combination with a second lipid-lowering agent.
The use according to claim 13, wherein the second lipid-lowering agent is an HMGCR inhibitor, an NPC1L1 inhibitor or a PCSK9 inhibitor.
The use according to claim 14, wherein the HMGCR inhibitor is a statin.
The use according to claim 15, wherein the statin drug is atorvastatin.
The use according to claim 14, wherein the NPC1L1 inhibitor is ezetimibe.
The use according to claim 14, wherein the PCSK9 inhibitor is Evolocumab, Alirocumab or Inclisiran.