Application of DDX6 inhibitor in preparation of medicine for treating diseases related to rising of cholesterol

Abstract

According to the application of the DDX6 inhibitor in preparing the medicine for treating the diseases related to the rising of the cholesterol, the function of the DDX6 gene in parenchymal hepatic cells is inhibited by means of gene knockout, the content of the cholesterol in mouse blood can be effectively reduced, and the damage to liver tissue is avoided. Furthermore, the influence of DDX6 on the liver is analyzed through transcriptome sequencing, and it is found that DDX6 knockout is likely to play a role by inhibiting synthesis of cholesterol in parenchymal hepatic cells.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and more particularly to the use of a DDX6 inhibitor in the preparation of a medicament for treating diseases associated with elevated cholesterol. Background Technology

[0002] Cholesterol is an essential small molecule in the human body, and its metabolic regulation includes four main processes: endogenous synthesis, exogenous uptake, esterification and storage, and efflux. Although cholesterol is essential for the body, excessive cholesterol synthesis or uptake can induce hypercholesterolemia and a series of related metabolic diseases, including cardiovascular disease, obesity, fatty liver, and diabetes. After decades of research, scientists have gained a deep understanding of the molecular mechanisms of cholesterol metabolism regulation, which has led to the development of two important classes of cholesterol-lowering drugs: (1) Due to the key role of HMG-CoA reductase HMGCR in cholesterol synthesis, its inhibitors (statins) have revolutionized the treatment strategy for hypercholesterolemia; (2) Since PCSK9 can inhibit the clearance of cholesterol from the blood, monoclonal antibody drugs targeting PCSK9 have been used to treat patients with familial hypercholesterolemia and patients who are intolerant to statins. Although statins and PCSK9 inhibitors have achieved clinical success, many patients still cannot tolerate the side effects of existing drugs or are not sensitive to existing therapies, and there is an urgent need to develop new treatment methods to control cholesterol levels.

[0003] Although cholesterol homeostasis is regulated by multiple processes, and theoretically, regulation of any one of these processes could potentially be used to treat hypercholesterolemia, the clinical treatments available to lower cholesterol levels are still very limited. Summary of the Invention

[0004] Based on the aforementioned technical problems in the existing technology, the inventors of this application have discovered a new target that can reduce cholesterol synthesis by knocking out the DDX6 gene or inhibiting the expression of the DDX6 gene.

[0005] The specific technical solution of this application is as follows:

[0006] 1. Use of DDX6 inhibitors in the preparation of medicaments for the treatment and / or prevention of diseases associated with elevated cholesterol.

[0007] 2. Use of DDX6 inhibitors in medicines for the treatment and / or prevention of diseases associated with elevated cholesterol.

[0008] 3. The use according to item 1 or 2, wherein the disease associated with elevated cholesterol is selected from familial hypercholesterolemia, nonfamilial hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, peripheral artery disease, hyperlipidemia, and dyslipidemia.

[0009] 4. Use of DDX6 inhibitors in the preparation of medicines for the treatment and / or prevention of diseases selected from the following:

[0010] Familial hypercholesterolemia, nonfamilial hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, peripheral artery disease, hyperlipidemia, and dyslipidemia.

[0011] 5. Use of DDX6 inhibitors in the treatment and / or prevention of the following diseases:

[0012] Familial hypercholesterolemia, nonfamilial hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, peripheral artery disease, hyperlipidemia, and dyslipidemia.

[0013] 6. The use according to any one of items 1-5, wherein the DDX6 inhibitor is a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule comprising hybridization with a DDX6 nucleic acid molecule, a gene editor, or a mutated DDX6 protein.

[0014] 7. A pharmaceutical composition for treating and / or preventing diseases associated with elevated cholesterol, comprising a DDX6 inhibitor.

[0015] 8. The pharmaceutical composition according to claim 7, wherein the DDX6 inhibitor is a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule comprising hybridization with a DDX6 nucleic acid molecule, a gene editor, or a mutated DDX6 protein.

[0016] 9. The pharmaceutical composition according to item 7 or 8, further comprising a carrier for delivering the DDX6 inhibitor.

[0017] 10. The composition according to claim 7, wherein the carrier is a viral carrier or lipid nanoparticles.

[0018] The effects of the invention

[0019] This application utilizes gene knockout to inhibit the function of the DDX6 gene in hepatocytes, effectively reducing cholesterol levels in mouse blood without damaging liver tissue. Furthermore, transcriptome sequencing analysis of the effects of DDX6 on the liver revealed that DDX6 knockout likely exerts its effect by inhibiting cholesterol synthesis in hepatocytes.

[0020] Furthermore, a mouse model of metabolic disease was established using a high-fat diet, and the function of DDX6 was analyzed. The results showed that a high-fat diet could induce pathological phenotypes such as hypercholesterolemia, obesity, fatty liver, and diabetes. Inhibiting the function of the DDX6 gene in hepatocytes through gene knockout could improve these pathological phenotypes in mice. Therefore, we believe that DDX6 can be a therapeutic target for hypercholesterolemia and related metabolic diseases.

[0021] Instruction manual illustrations

[0022] Figures 1A to 1B This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on cholesterol homeostasis in mice. Figure 1A The effect of DDX6 knockout in hepatocytes on total cholesterol levels in mouse serum. Figure 1B This is a schematic diagram showing the relationship between DDX6 knockout in hepatocytes and the expression of genes related to cholesterol synthesis.

[0023] Figures 2A to 2B This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on the physiological state of mice. Figure 2A This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on the body weight of mice fed a low-fat diet. Figure 2B This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on the fat and lean meat quality of low-fat fed mice.

[0024] Figures 3A to 3B This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on the liver of mice. Figure 3A This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on liver quality in low-fat fed mice. Figure 3B This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on liver damage.

[0025] Figures 4A to 4B This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on serum cholesterol levels in obese mice. Figure 4A This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on serum cholesterol levels in mice fed a high-fat diet. Figure 4B This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on the levels of HDL-c and LDL-c in the serum of mice fed a high-fat diet.

[0026] Figures 5A-5C This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on the physiological condition of obese mice. Figure 5A This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on obesity induced by a high-fat diet. Figure 5B This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on the quality of white adipose tissue induced by a high-fat diet in mice. Figure 5CThis is a schematic diagram illustrating the effects of DDX6 knockout in hepatocytes on fat accumulation and lean meat quality caused by a high-fat diet.

[0027] Figures 6A to 6D This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on obesity-related diseases in obese mice. Figure 6A This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on non-alcoholic fatty liver disease. Figure 6B This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on liver quality. Figure 6C This is a schematic diagram illustrating the effect of DDX6 knockout on ALT and AST in hepatocytes. Figure 6D This is a schematic diagram illustrating the effect of DDX6 knockout in hepatocytes on blood glucose.

[0028] Figure 7 This is a schematic diagram illustrating the effect of AAV-mediated DDX6 knockout in hepatocytes on mouse body weight.

[0029] Figures 8A to 8B This is a schematic diagram of blood biochemical analysis showing the inhibition of DDX6 expression in hepatocytes by shRNA. Figure 8A This is a schematic diagram showing the serum total cholesterol content of mice in each group. Figure 8B This is a schematic diagram of ALT and AST activity in each group of mice.

[0030] Figures 9A to 9B This is a schematic diagram of blood biochemical analysis of mice in each group after 4 weeks of high-fat feeding. Figure 9A This is a schematic diagram showing the serum total cholesterol content of mice in each group. Figure 9B This is a schematic diagram of ALT and AST activity in each group of mice.

[0031] Figures 10A to 10C This is a schematic diagram showing paraffin sections stained with HE and Oil Red O, liver weight, and glucose tolerance test results of mice in each group after week 4 of high-fat feeding. Figure 10A These are schematic diagrams of paraffin sections stained with HE and Oil Red O from each group of mice. Figure 10B This is a diagram showing the liver weight of mice in each group. Figure 10C This is a schematic diagram of the glucose tolerance test for each group of mice.

[0032] Figures 11A to 11D This is a schematic diagram showing the blood biochemical analysis, HE and Oil Red O stained paraffin sections, and liver weight of mice in each group after 7 weeks of high-fat feeding. Figure 11A This is a schematic diagram showing the serum total cholesterol content of mice in each group. Figure 11B This is a schematic diagram of ALT and AST activity in each group of mice. Figure 11C These are schematic diagrams of paraffin sections stained with HE and Oil Red O from each group of mice. Figure 11D This is a schematic diagram showing the liver weight of mice in each group.

[0033] Figures 12A to 12B This is a schematic diagram of the blood biochemical analysis of mice in each group during the LNP-siDdx6 knockdown experiment. Figure 12A This is a schematic diagram showing the serum total cholesterol content of mice in each group. Figure 12B This is a schematic diagram of ALT and AST activity in each group of mice.

[0034] Figures 13A to 13B This is a schematic diagram of the blood biochemical analysis of mice in each group during the GalNac-siDdx6 knockdown experiment. Figure 13A This is a schematic diagram showing the serum total cholesterol content of mice in each group. Figure 13B This is a schematic diagram of ALT and AST activity in each group of mice. Detailed Implementation

[0035] The present application will now be described in detail. While specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0036] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0037] The inventors of this application have discovered that by inhibiting the function of the DDX6 gene, the cholesterol content in the blood of mice can be effectively reduced without causing damage to liver tissue. Based on this, this application provides the use of DDX6 inhibitors in the preparation of drugs for treating and / or preventing diseases related to elevated cholesterol.

[0038] In this application, DDX6 is an RNA helicase involved in regulating RNA translation and degradation.

[0039] This application also provides for the use of DDX6 inhibitors in medicaments for the treatment and / or prevention of diseases associated with elevated cholesterol.

[0040] In this application, the DDX6 inhibitor can inhibit the expression, content, or activity of the DDX6 gene or RNA or the protein it encodes.

[0041] The DDX6 inhibitor described in this application inhibits the expression of the DDX6 gene or DDX6 protein, which can effectively reduce the cholesterol content in the blood of mice without causing damage to liver tissue. Furthermore, a high-fat diet can induce pathological phenotypes such as hypercholesterolemia, obesity, fatty liver, and diabetes. By using the DDX6 inhibitor to inhibit the function of the DDX6 gene in liver parenchymal cells, these pathological phenotypes in mice can be improved. Therefore, this can be a therapeutic target for the treatment of hypercholesterolemia and related metabolic diseases.

[0042] In this application, hepatocytes are the main functional cells in the liver, accounting for about 70% of the total number of liver cells, and are in a state of slow replacement and renewal.

[0043] In some embodiments, the diseases associated with elevated cholesterol are selected from familial hypercholesterolemia, nonfamilial hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, peripheral artery disease, hyperlipidemia, and dyslipidemia.

[0044] This application provides for the use of DDX6 inhibitors in the preparation of medicaments for the treatment and / or prevention of diseases selected from the following:

[0045] Familial hypercholesterolemia, nonfamilial hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, peripheral artery disease, hyperlipidemia, and dyslipidemia.

[0046] This application provides for the use of DDX6 inhibitors in medicines for the treatment and / or prevention of diseases selected from the following:

[0047] Familial hypercholesterolemia, nonfamilial hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, peripheral artery disease, hyperlipidemia, and dyslipidemia.

[0048] In some embodiments, the DDX6 inhibitor is a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule comprising hybridization with a DDX6 nucleic acid molecule, a gene editor, or a mutated DDX6 protein.

[0049] In this application, examples of the nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNA (siRNA), and short hairpin RNA (shRNA). Such inhibitory nucleic acid molecules can be designed to target any region of the DDX6 nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence within the DDX6 nucleic acid molecule or mRNA molecule and reduces the expression of the DDX6 protein in the subject's cells. In some embodiments, the DDX6 inhibitor comprises an antisense molecule that hybridizes to the DDX6 nucleic acid molecule or mRNA molecule and reduces the expression of the DDX6 protein in the subject's cells. In some embodiments, the DDX6 inhibitor comprises siRNA that hybridizes to the DDX6 nucleic acid molecule or mRNA molecule and reduces the expression of the DDX6 protein in the subject's cells. In some embodiments, the DDX6 inhibitor comprises shRNA that hybridizes to the DDX6 nucleic acid molecule or mRNA molecule and reduces the expression of the DDX6 protein in the subject's cells.

[0050] In this application, the repressive nucleic acid molecule may comprise RNA or DNA, or both. The repressive nucleic acid molecule may also be linked or fused with a heterologous nucleic acid sequence (such as a heterologous nucleic acid sequence in a vector) or a heterologous label. For example, the repressive nucleic acid molecule may be within a vector containing the repressive nucleic acid molecule and the heterologous nucleic acid sequence, or as an exogenous donor sequence containing the repressive nucleic acid molecule and the heterologous nucleic acid sequence. The repressive nucleic acid molecule may also be linked or fused with a heterologous label. The label may be directly detectable (e.g., a fluorophore) or indirectly detectable (e.g., a hapten, enzyme, or fluorophore quencher). Such labels can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label may also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme. The term "label" may also refer to a "tag" or hapten that selectively binds to a conjugated molecule such that the conjugated molecule is used to generate a detectable signal when subsequently added with a substrate. Exemplary tags that can be used to facilitate purification include, but are not limited to, myc, HA, FLAG or 3XFLAG, 6XHis or polyhistidine, glutathione S-transferase (GST), maltose-binding protein, epitope tags, or the Fc portion of immunoglobulins. Many tags include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent, and chemiluminescent substrates, and other tags.

[0051] In this application, the gene editor comprises a DNA gene editor and an RNA gene editor. The gene editor comprises a gene-editing protein and optionally gRNA.

[0052] Preferably, the gene-editing protein is a Cas protein. In this application, suitable Cas proteins include, for example, wild-type Cas9 and wild-type Cpf1 proteins (e.g., like FnCpf1). The Cas protein may have full cleavage activity to produce double-strand breaks in the DDX6 genomic nucleic acid molecule, or it may be a cleavage enzyme that produces single-strand breaks in the DDX6 genomic nucleic acid molecule. Other examples of Cas proteins include, but are not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), C se3(CasE), Cse4(CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4 and Cu1966, as well as their homologs or modified forms.

[0053] In this application, the mutated DDX6 protein refers to a protein that can inhibit the function of endogenous DDX6 protein. For example, the mutated DDX6 protein can be a DDX6 protein carrying one or more amino acid sequence mutations (such as Glu247Gln, His372Arg, Arg373Gln, Cys390Arg, Thr391Ile, Thr391Pro), or a DDX6 protein lacking certain amino acid sequences.

[0054] In this application, no restrictions are placed on the method of mutation, and those skilled in the art can make conventional choices according to actual needs.

[0055] This application provides a pharmaceutical composition for treating and / or preventing diseases associated with elevated cholesterol, comprising a DDX6 inhibitor.

[0056] In some embodiments, the DDX6 inhibitor is a small molecule compound or an inhibitory nucleic acid molecule that hybridizes with a DDX6 nucleic acid molecule.

[0057] In some embodiments, it also includes a carrier for delivering the DDX6 inhibitor.

[0058] In some embodiments, the carrier is a viral carrier or lipid nanoparticles.

[0059] In this application, the viral vector may be, for example, an adeno-associated virus (AAV) vector, a lentiviral vector, an adenovirus vector, a retroviral vector, a herpesvirus, an SV40 vector, a poxvirus vector, etc.

[0060] In this application, the DDX6-floxed transgenic mouse refers to a mouse in which loxp sequences are inserted flanking exons 5 and 6 of the DDX6 gene. These two loxp sequences recombine under the action of Cre recombinase, thereby knocking out exons 5 and 6, resulting in a frameshift mutation and achieving gene knockout.

[0061] In this application, the Alb-Cre transgenic mouse refers to a mouse in which the open reading frame of the Cre recombinase is inserted after the promoter of the Albumin gene. Since the promoter of the Albumin gene is active only in hepatocytes, the Cre recombinase is expressed only in hepatocytes, thereby achieving specific knockout of the DDX6 gene in hepatocytes.

[0062] In the sequence listing, nucleic acid sequences can represent either DNA or RNA sequences. When representing RNA sequences, the T in the sequence represents uridine.

[0063] Example

[0064] This application provides a general and / or specific description of the materials and experimental methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. All reagents or instruments used, unless otherwise specified, are commercially available conventional products. The method for constructing knockout mice is as follows:

[0065] DDX6-floxed transgenic mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were crossed with Alb-Cre transgenic mice (purchased from Jackson Laboratory) to obtain hepatocyte DDX6 knockout mice (L-DDX6- / -). The method for identifying the obtained hepatocyte-specific DDX6 knockout mice (L-DDX6- / -) is as follows: To confirm the acquisition of hepatocyte-specific DDX6 knockout mice, genotyping was performed on the offspring mice one week after birth. DNA was extracted from the mouse toes and PCR amplification was performed using specific primers. The PCR products were separated by agarose gel electrophoresis, and the corresponding bands were detected by a UV imaging system. Mice with PCR results showing Alb-Cre positivity and DDX6 f / f double positivity were identified as L-DDX6- / - mice. All mice were housed in pathogen-free cages in a mouse room with 24°C and a light exposure time (7:00-19:00).

[0066] The method for constructing an obesity model is as follows:

[0067] The mice used to construct the obesity model were eight-week-old DDX6 floxed transgenic mice. Mice expressing Alb-Cre (i.e., DDX6 knockout mice in hepatocytes (L-DDX6- / -)) were the experimental group, while those not expressing Alb-Cre were the control group (DDX6 floxed transgenic mice). Mice were normally fed until eight weeks of age, and then the experimental and control groups, with similar body weights, were evenly distributed in their respective cages. Subsequently, the high-fat diet group was replaced with a high-fat diet (purchased from Detz Biotechnology Co., Ltd., containing casein, L-cysteine, gluten, maltodextrin, lard, soybean oil (containing TBHQ), cellulose, complex minerals, calcium carbonate, calcium bicarbonate, potassium citrate monohydrate, multivitamins, choline tartrate, and blue pigment, with fat providing 60% of the calories and protein and carbohydrates each providing 20%). The normal-feeding group, without dietary intervention, continued to receive a low-fat diet (purchased from Keao Xieli (Tianjin) Feed Co., Ltd., a standard feed used in mouse growth and reproduction, typically used by non-specially fed mice; ingredients include corn, soybean meal, fish meal, flour, yeast, vegetable oil, salt, multivitamins, and minerals). The dietary intervention lasted for 20 weeks, during which the mice were weighed and observed weekly.

[0068] The RNA-seq assay method is as follows:

[0069] Mice from both the experimental and control groups were euthanized immediately, and liver tissue was collected. Liver tissue samples were rapidly frozen in liquid nitrogen at -196°C for 5 minutes and stored at -80°C. RNA was extracted using TRIzol reagent. The total RNA concentration was measured using NanoDrop One, and libraries were constructed using the VAHTS Stranded mRNA-seq Library Prep Kit for Illumina V2. Sequencing was performed on the Illumina NovaSeq 6000 platform.

[0070] Blood biochemistry testing methods:

[0071] Mice were anesthetized with isoflurane, and plasma was collected from the heart while the mice were under anesthesia, then collected in 1.5 ml centrifuge tubes. The centrifuge tubes were then centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected to obtain serum. An appropriate amount of serum was collected in a 1.5 ml centrifuge tube and then placed into a fully automated biochemical analyzer for the detection of relevant indicators. In this application, the blood biochemical indicators include total cholesterol (TCH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST).

[0072] Glucose tolerance test method:

[0073] Before the test, the mice were fasted for 16 hours (with normal water intake). At the start of the experiment, glucose was injected into the mice intraperitoneally at a dose of 2 grams of glucose per kilogram of body weight. Subsequently, blood samples were collected from the tail tips of the mice at 0, 15, 30, 60, 90, and 120 minutes after injection, and blood glucose levels were measured using a glucometer.

[0074] Body composition analysis methods:

[0075] Body composition analysis in mice primarily involved detecting the content of fat and lean meat using a low-field nuclear magnetic resonance (NMR) analyzer. This method is based on the differences in relaxation time among fat, lean meat, and water in live animals, and distinguishes and quantifies the content of fat and lean meat by detecting the NMR signals.

[0076] Liver histological analysis methods:

[0077] After euthanizing mice and dissecting their livers, the liver tissue was preserved in 4% paraformaldehyde solution, then dehydrated and embedded in paraffin. The paraffin-embedded liver tissue blocks were then sliced ​​into thin sections using a microtome to prepare tissue sections. These sections were then stained with hematoxylin and eosin. For oil red staining, the liver tissue was dehydrated, embedded in an OCT chamber, cut into 5-10 μm frozen sections, stained with oil red O staining solution, and then counterstained with hematoxylin. Finally, images were acquired using a microscope.

[0078] Preparation and injection of adenovirus 1

[0079] The construction, packaging, and titer determination of the delivery vectors for the virus AAV2 / 8-hTBG-ICre-WPRE-pA (used for liver-specific expression of Cre recombinase) and the virus AAV2 / 8-hTBG-EGFP-WPRE-pA (used as a control) were all performed by Shanghai Taiting Biotechnology Co., Ltd. In the AAV induction experiment, DDX6 floxed transgenic mice of similar weight were evenly divided into different cages and subjected to a high-fat diet intervention starting at eight weeks of age. At week 7 of high-fat feeding, each cage of mice was randomly divided into two groups: (1) the AAV2 / 8-hTBG-ICre-WPRE-pA group; and (2) the AAV2 / 8-hTBG-EGFP-WPRE-pA group. Subsequently, these mice were injected with 5*10 [units of something] via tail vein injection. 11 The virus expresses Cre recombinase or fluorescent protein GFP, which are specifically expressed in hepatocytes.

[0080] Preparation of adenovirus 2

[0081] The construction, viral packaging, and titer determination of the viral delivery vector AAV2 / 8-hTBG-MasterRNAI155(mDdx6)-EGFP-WPRE-pA (AAV-shDDX6, 5'-ACAAGATTGCGGCATAAGCCA-3'SEQ ID NO:7) for liver-specific knockdown of DDX6 and the viral delivery vector AAV2 / 8-hTBG-MasterRNAI155(NC)-EGFP-WPRE-pA (AAV-GFP) for use as a control were all performed by Shanghai Taiertu Biotechnology Co., Ltd.

[0082] Preparation of GalNac-siRNA

[0083] The preparation and purification of GalNac-siRNA were performed by Shanghai Gemma Pharmaceutical Technology Co., Ltd. The sequence of GalNac-siDdx6 is as follows:

[0084] Justice chain sequence: 5'-T·G·GCTTaTgccGCAATCTTGT-GalNac3-3' (SEQ ID NO:1);

[0085] Antisense sequence: 5'-A·c·AAGaTTgCGGCaTaAGCCA·T·T-3' (SEQ ID NO:2); the uppercase letters above indicate 2′-O-methyl modification of adenosine (A), cytidine (C), guanosine (G) and uridine (T); the lowercase letters indicate 2′-fluorine modification of adenosine (A), cytidine (C), guanosine (G) and uridine (T); GalNAc3 indicates tribranched GalNac; · indicates thiomodification of phosphodiester bond.

[0086] Preparation of lipid nanoparticles

[0087] The components of the packaged lipid nanoparticles include ionizable lipids DLin-MC3-DMA (MC3), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and dimyristicoglycerol-polyethylene glycol 2000 (C14-PEG2000). During preparation, the inorganic phase was mixed in ethanol at a molar ratio of 50:10:38.5:1.5 (MC3 / DSPC / cholesterol / C14-PEG2000), while the aqueous phase was prepared in 10mM citrate buffer (pH 3). siRNA was added, and the siRNA sequences of Ddx6 from the knockdown tissue used were: sense strand sequence: 5'-GGCTTATGCCGCAATCTTGdTdT-3' (SEQ ID NO:3); antisense strand sequence: 5'-CAAGATTGCGGCATAAGCCdTdT-3' (SEQ ID NO:4); the siScramble sequences used as controls were: sense strand sequence: 5'-GTCGGCATACGCGTTATCTdTdT-3' (SEQ ID NO:5); antisense strand sequence: 5'-AGATAACGCGTATGCCGACdTdT-3' (SEQ ID NO:6). The aqueous and organic phases were mixed at a mass ratio of ionizable lipids to RNA of 10:1 and a flow rate ratio of 3:1. Ethanol was then removed by dialysis. The resulting formulation was stored at 2–8°C until use, wherein, in SEQ ID NO:3-6, dT represents deoxyribonucleic acid.

[0088] Analysis method:

[0089] In all experiments, each group contained at least three mice. All statistical data are expressed as mean ± standard error (mean ± sem) or mean ± standard deviation (mean ± SD). All statistical analyses were performed using GraphPad Prism 9 (GraphPad Software). All parameters were analyzed using unpaired two-tailed Student's t-test, one-way ANOVA, or two-way ANOVA. A p-value less than 0.05 was considered statistically significant.

[0090] Example 1

[0091] Control and knockout mice were housed in the same cage for 28 weeks, during which time they were fed a normal low-fat diet.

[0092] (1) Total cholesterol (TCH) was measured in mice, and the results are as follows: Figure 1A As shown.

[0093] from Figure 1A It can be seen that the serum total cholesterol content of mice with DDX6 knockout in hepatocytes is significantly reduced.

[0094] (2) RNA analysis of liver tissue from knockout mice

[0095] Liver tissue samples from control and knockout mice were extracted using conventional methods in the art, and RNA-seq analysis was performed. The results are as follows: Figure 1B As shown.

[0096] from Figure 1B It can be seen that DDX6 knockout in hepatocytes can inhibit the expression of genes involved in cholesterol synthesis.

[0097] (3) Measurement of mouse weight and analysis of body composition

[0098] A. The control and knockout mice were weighed and observed every Friday, and the results were as follows: Figure 2A As shown, from Figure 2A It can be seen that under normal low-fat feeding conditions, DDX6 knockout of hepatocytes had no significant effect on the body weight of mice, and there was no significant difference in the physiological state of the mice.

[0099] B. Body composition analysis

[0100] The results of body component analysis of control and knockout mice are as follows: Figure 2B As shown. From Figure 2B It can be seen that DDX6 knockout of hepatocytes had no significant effect on the quality of fat and muscle tissue in mice.

[0101] (3) Impact on liver health

[0102] First, after euthanizing the mice, they were immediately dissected, and liver fingers were collected. The livers of the knockout mice and control mice were weighed, and the results are as follows: Figure 3A As shown, from Figure 3A It can be seen that DDX6 knockout in hepatocytes does not affect liver weight in mice. Furthermore, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum were measured, and the results are as follows: Figure 3B As shown. From Figure 3B It can be seen that the activities of ALT and AST in the serum of knockout mice were not significantly different from those in control mice, indicating that DDX6 knockout in hepatocytes does not cause liver damage in mice.

[0103] In summary, DDX6 knockout in hepatocytes significantly reduced total cholesterol in mouse serum, but had no significant effect on mouse body weight and did not cause liver damage.

[0104] (4) Effects on obese mice

[0105] Given the close relationship between cholesterol and obesity and related metabolic diseases, the effects of DDX6 knockout in hepatocytes on obese mice were investigated through a high-fat diet intervention.

[0106] First, an obese mouse model (high-fat model group) was constructed according to the above method, and the serum of the mice was subjected to blood biochemistry tests. The results are as follows: Figure 4A As shown, from Figure 4A As can be seen, similar to the results of normally fed mice, the total cholesterol content in the serum of mice with DDX6 knockout of hepatocytes was significantly reduced.

[0107] Furthermore, the cholesterol levels of high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) in mouse serum were detected using a fully automated biochemical analyzer. The results are as follows: Figure 4B As shown.

[0108] from Figure 4B It can be seen that the levels of HDL-c and LDL-c were significantly decreased in mice with DDX6 knockout of hepatocytes, while the HDL / LDL ratio was significantly increased, which has positive significance for the prevention of cardiovascular diseases.

[0109] Secondly, the body weight of control mice and mice in the high-fat model group was monitored, and the results were as follows: Figure 5A As shown, from Figure 5AIt can be seen that the body weight of DDX6 knockout mice in hepatocytes was significantly lower than that of control mice, indicating that the weight gain of knockout mice was significantly reduced under high-fat feeding conditions. Simultaneously, body composition analysis was also performed on the high-fat model group mice, and the results are as follows... Figure 5B and Figure 5C As shown, from Figure 5B and Figure 5C It can be seen that, compared with control mice, DDX6 knockout of hepatocytes can significantly alleviate the increase in white fat mass caused by high fat intake and give mice a higher lean meat mass.

[0110] (5) Impact on obesity-related diseases

[0111] Obesity is often accompanied by many metabolic-related diseases, such as non-alcoholic fatty liver disease and insulin resistance. To address this, paraffin sections of mouse liver tissue were analyzed using HE and Oil Red O staining, and the livers were weighed. The results are as follows: Figure 6A and Figure 6B As shown.

[0112] from Figure 6A and Figure 6B It can be seen that control mice fed a high-fat diet exhibited characteristics of non-alcoholic fatty liver disease, such as steatosis and the appearance of ballooning cells. However, the livers of DDX6 knockout mice in hepatocytes did not show these characteristics, and the liver mass of DDX6 knockout mice fed a high-fat diet was significantly lower than that of control mice. This indicates that DDX6 knockout of hepatocytes can prevent non-alcoholic fatty liver disease induced by a high-fat diet.

[0113] Simultaneously, serum biochemical tests and glucose tolerance tests were performed on the serum of control mice and DDX6 knockout mice in hepatocytes. The results are as follows: Figure 6C and Figure 6D As shown.

[0114] from Figure 6C It can be seen that the serum ALT activity of DDX6 knockout mice was significantly reduced, indicating that DDX6 knockout in hepatocytes can significantly alleviate liver damage associated with obesity. Figure 6D It can be seen that, compared with control mice, DDX6 knockout mice in hepatocytes responded to the rise in blood glucose more quickly and recovered blood glucose to normal levels within 2 hours. In contrast, obese control mice could not recover to normal blood glucose levels within 2 hours, and thus showed symptoms of insulin resistance. This indicates that knocking out DDX6 in hepatocytes can prevent the development of insulin resistance symptoms associated with obesity.

[0115] (6) To test the therapeutic effect of this application, we used AAV as a vector to achieve time-space gene knockout by injecting Cre specifically expressed in hepatocytes via tail vein injection. Using this method, we injected AAV carrying either Cre or GFP specifically expressed in hepatocytes into the seventh week of feeding a high-fat diet, thus obtaining experimental and control groups. The high-fat diet was maintained, and weekly weight monitoring was conducted. The results are as follows: Figure 7 As shown.

[0116] from Figure 7 It can be seen that, in cases where obesity has already occurred, knocking out the DDX6 gene in liver parenchymal cells can alleviate weight gain in mice caused by a high-fat diet.

[0117] Example 2

[0118] I. Injection of adenovirus 2

[0119] The mice used to establish the obesity model were eight-week-old wild-type c57BL / 6 mice. After normal feeding until eight weeks of age, mice of similar weight in the experimental and control groups were evenly distributed in each cage. Subsequently, the diet of the high-fat feeding group was replaced with a high-fat diet containing 60% fat calories. During the high-fat feeding period, the mice were weighed and observed weekly.

[0120] In the normal feeding experiment, wild-type C57BL / 6 mice of similar weight were evenly distributed into different cages. At the eighth week of age, each cage of mice was randomly divided into two groups: (1) control group-AAV-GFP group; (2) AAV-shDdx6 group; and then 1*10 mg / L of each mouse was injected into the tail vein of both groups. 11 The corresponding virus was collected 9 days later.

[0121] In the high-fat feeding experiment, wild-type C57BL / 6 mice of similar weight were evenly distributed into different cages and began a high-fat diet at eight weeks of age. Two experimental protocols were used in this high-fat feeding experiment:

[0122] A. At week 7 of high-fat feeding, mice in each cage were randomly divided into two groups: (1) high-fat feeding group - AAV-GFP group; (2) high-fat feeding group - AAV-shDdx6 group; and then 1*10 mg of each mouse was injected into the tail vein of both groups. 11 The corresponding virus was collected 10 weeks later.

[0123] B. During week 4 of high-fat feeding, each cage of mice was randomly divided into two groups: (1) high-fat feeding group - AAV-GFP group; (2) high-fat feeding group - AAV-shDdx6 group; subsequently, 1*10 mg of high-fat feed was injected into each group of mice via the tail vein. 11 The corresponding virus was collected 6 weeks later.

[0124] II. Injection of lipid nanoparticles

[0125] In the formal experiment, wild-type C57BL / 6 mice with similar weights were evenly divided into different cages. At the eighth week of age, each cage of mice was randomly divided into two groups: (1) knockdown group - LNP-siDdx6 group; (2) control group - LNP-siScramble group. Subsequently, 50 μg of the corresponding LNP-siRNA was injected into the tail vein of the two groups of mice (LNP-siDdx6 was injected into the knockdown group and LNP-siScramble was injected into the control group). Samples were collected 9 days later.

[0126] III. Injection of GalNac-siRNA

[0127] In the formal experiment, wild-type C57BL / 6 mice with similar body weights were evenly divided into different cages. At the eighth week of age, each cage of mice was randomly divided into two groups: (1) knockdown group - GalNac-siDdx6 group; (2) control group. The knockdown group was subcutaneously administered 10 μg of GalNac-siRNA per gram of body weight, while the control group mice were given an equal volume of phosphate-buffered saline (PBS). Samples were collected 9 days later.

[0128] IV. Experimental Results

[0129] Based on the experimental results, hepatocyte-specific knockout of Ddx6 can significantly reduce the total cholesterol content in mouse serum and significantly alleviate obesity, fatty liver, and insulin resistance caused by a high-fat diet. In order to further verify the therapeutic potential of this gene at different expression levels, a variety of knockdown methods were used and its effects on different physiological indicators were evaluated.

[0130] First, using AAV as a vector, the shRNA was injected into mice via tail vein injection, thereby concentrating its expression in hepatocytes and inhibiting Ddx6 expression in hepatocytes. By separating mouse serum and performing blood biochemical monitoring, it was found that Ddx6 knockdown in mouse hepatocytes significantly reduced serum total cholesterol levels. Figure 8A Meanwhile, because serum ALT and AST activities showed no significant change compared to wild-type mice, it indicates that AAV-shDdx6-mediated Ddx6 knockdown in hepatocytes does not cause liver damage in mice. Figure 8B ).

[0131] Similarly, given the close relationship between cholesterol and obesity and related metabolic diseases, we investigated the effects of Ddx6 knockdown in hepatocytes on obese mice by subjecting them to a high-fat diet at 8 weeks of age.

[0132] In a high-fat diet-induced experiment, the therapeutic effect of knocking down Ddx6 in hepatocytes was investigated at different stages of obesity development by knocking down interventions at different time points. Therefore, in this experiment, knockdown interventions were performed at weeks 4 and 7 of a high-fat diet.

[0133] In the experiment of knocking down Ddx6 in hepatocytes during week 4 of a high-fat diet, firstly, similar to the results of normally fed mice, blood biochemistry tests were performed on the mice's serum, and the results were as follows: Figure 9A As shown, mice with Ddx6 knockdown in hepatocytes had significantly lower serum total cholesterol levels. Figure 9A Furthermore, by detecting ALT and AST activities in mouse serum, the results were as follows: Figure 9B As shown in the results, the levels of both were not significantly different from the control group. In conclusion, the experimental results of Ddx6 knockdown intervention in hepatocytes during the 4th week of high-fat feeding indicate that Ddx6 knockdown in hepatocytes can significantly reduce total cholesterol in mouse serum without significantly affecting liver health.

[0134] Obesity is often accompanied by many metabolic-related diseases, such as non-alcoholic fatty liver disease and insulin resistance. To address this, paraffin sections of mouse liver tissue were analyzed using HE and Oil Red O staining. The results are as follows: Figure 10A and Figure 10B As shown, wild-type mice were found to exhibit characteristics of non-alcoholic fatty liver disease when fed a high-fat diet. Figure 10A Symptoms such as fatty degeneration and the appearance of ballooning cells were observed in the livers of Ddx6 knockdown mice. However, these symptoms were significantly alleviated in the livers of Ddx6 knockdown mice fed a high-fat diet, and the liver quality of Ddx6 knockdown mice was significantly lower than that of wild-type mice. Figure 10B This indicates that Ddx6 knockdown in hepatocytes can improve non-alcoholic fatty liver disease induced by a high-fat diet. Furthermore, glucose tolerance tests showed that, compared to wild-type mice, Ddx6 knockdown mice responded to elevated blood glucose levels much faster. Figure 10C This indicates that knocking down Ddx6 in hepatocytes can alleviate the symptoms of insulin resistance associated with obesity.

[0135] Similar to the intervention in week 4, in the experiment of knocking down Ddx6 in hepatocytes during week 7 of high-fat feeding, serum biochemistry was performed on mice, and the results were as follows: Figures 11A to 11B As shown, the total cholesterol content in the serum of mice with Ddx6 knockdown of hepatocytes was also significantly reduced. Figure 11A Meanwhile, the levels of ALT and AST did not change significantly. Figure 11BMeanwhile, paraffin sections of mouse liver tissue were analyzed using HE and Oil Red O staining, and the results are as follows: Figure 11C As shown, wild-type mice also exhibited characteristics of non-alcoholic fatty liver disease when fed a high-fat diet. Figure 11C Ddx6 knockdown of hepatocytes significantly alleviated these symptoms and reduced liver weight in mice. Figure 11D ).

[0136] Furthermore, to further validate the AAV-shDdx6 knockdown effect and explore gene intervention strategies with greater clinical translation potential, two methods, GalNAc-siRNA and LNP-siRNA, were employed. The GalNAc-siRNA method, through optimized liver-targeted delivery, can specifically target hepatocytes, achieving more efficient gene knockdown; while LNP-siRNA utilizes a lipid nanoparticle delivery system, improving the stability of siRNA.

[0137] In the LNP-siDdx6 knockdown experiment, eight-week-old wild-type mice were administered the drug via tail vein injection. Serum was collected nine days after injection for blood biochemical analysis. The results are as follows: Figures 12A to 12B As shown. Experimental results showed that, similar to the results of knockout and AAV-shDdx6 experiments, LNP-siDdx6-mediated Ddx6 knockdown in hepatocytes also significantly reduced the total cholesterol content in mouse serum. Figure 12A It can significantly reduce ALT activity, thus protecting liver health. Figure 12B ).

[0138] In the GalNac-siDdx6 knockdown experiment, eight-week-old wild-type mice were administered the drug subcutaneously, and serum was collected nine days later for blood biochemical analysis. The results are as follows: Figures 13A to 13B As shown. Experimental results show that GalNac-siDdx6-mediated Ddx6 knockdown in hepatocytes also significantly reduced the total cholesterol content in mouse serum ( ). Figure 13A It does not have a significant impact on the liver. Figure 13B ).

[0139] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. Use of DDX6 inhibitors in the preparation of medicaments for the treatment and / or prevention of diseases associated with elevated cholesterol.

2. Use of DDX6 inhibitors in medicines for the treatment and / or prevention of diseases associated with elevated cholesterol.

3. The use according to claim 1 or 2, wherein, The diseases associated with elevated cholesterol are selected from familial hypercholesterolemia, nonfamilial hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, peripheral artery disease, hyperlipidemia, and dyslipidemia.

4. Use of DDX6 inhibitors in the preparation of medicines for the treatment and / or prevention of diseases selected from the following: Familial hypercholesterolemia, nonfamilial hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, peripheral artery disease, hyperlipidemia, and dyslipidemia.

5. Use of DDX6 inhibitors in the treatment and / or prevention of the following diseases: Familial hypercholesterolemia, nonfamilial hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, peripheral artery disease, hyperlipidemia, and dyslipidemia.

6. The use according to any one of claims 1-5, wherein, The DDX6 inhibitor is a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule containing a hybridization with a DDX6 nucleic acid molecule, a gene editor, or a mutated DDX6 protein.

7. A pharmaceutical composition for treating and / or preventing diseases associated with elevated cholesterol, comprising a DDX6 inhibitor.

8. The pharmaceutical composition according to claim 7, wherein the DDX6 inhibitor is a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule comprising hybridization with a DDX6 nucleic acid molecule, a gene editor, or a mutated DDX6 protein.

9. The pharmaceutical composition according to claim 7 or 8, further comprising a carrier for delivering the DDX6 inhibitor.

10. The composition according to claim 7, wherein the carrier is a viral carrier or lipid nanoparticles.

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