Application of Ago2 in preparation of medicine for treating fatty liver or hyperlipemia and product of Ago2

Abstract

The invention relates to application of Ago2 in preparation of medicines for treating fatty liver or hyperlipemia and a product of the Ago2, belonging to the field of biological medicines. The invention provides an application of Ago2 in preparation of a medicine for treating fatty liver, and provides a medicine for treating fatty liver or hyperlipidemia and a preparation method thereof based on the new application of Ago2, and a protein, a gene, a recombinant expression vector and a transformant with the effect of treating fatty liver or hyperlipidemia. Animal experiments prove that the Ago2-based medicine for resisting fatty liver or hyperlipemia can remarkably reduce the liver lipid and blood fat content of animals with fatty liver caused by high fat, and the effective effect of resisting fatty liver or hyperlipemia is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to the use of Ago2 in the preparation of drugs for treating fatty liver or hyperlipidemia and its products. Background Art

[0002] Non-alcoholic fatty liver is one of the major diseases threatening human health, and the global prevalence of fatty liver in adults is 22.10% to 28.65%. The prevalence of fatty liver in China is approximately 15% - 20%, of which 25% and 14% are males and females respectively. The onset of fatty liver is related to factors such as obesity, diabetes, hypercholesterolemia, and alcohol intake. Fatty liver is an important cause of chronic liver diseases (cirrhosis and liver cancer), and NAFLD is becoming the most common cause of liver diseases and liver-related deaths globally. Clinically, there is currently no effective treatment for fatty liver. More seriously, commonly used lipid-lowering drugs even have the risk of increasing fatty liver. Therefore, new treatment strategies for fatty liver need to be explored.

[0003] Currently, adenoviruses and lentiviruses are mostly used as expression vectors for gene therapy. However, lentiviruses are mostly modified from leukemia viruses or HIV, and adenovirus vectors have a short expression time and are immunogenic to the body, both of which are not suitable for future clinical applications. The recombinant adeno-associated virus vector (rAAV) overcomes the disadvantages that are difficult to overcome by other gene expression vectors, has no immunogenicity, and can drive the long-term expression of the target gene in vivo, thus becoming the most promising vector for gene therapy.

[0004] MicroRNA (miRNAs) is a newly discovered class of non-coding small RNAs with important regulatory roles in gene expression in recent years. Mature miRNAs generally bind completely or incompletely to the 3'-untranslated region (3'UTR) sequence of their target mRNAs through the base complementary pairing principle. Some studies have found that miRNAs are involved in regulating the occurrence and development of fatty liver. In recent years, it has been found that drugs designed targeting miR-320 can reduce blood lipids and improve fatty liver. However, miRNAs are widely expressed, and systemic administration is likely to affect the functions of multiple organs and tissues, and non-specific confounding effects may occur. Therefore, key regulatory factors for the function of miRNAs that play a more important role in fatty liver need to be found. Ago2 has been reported as the core protein for miRNAs to exercise their regulatory functions, and Ago2 is distributed in different subcellular components such as mitochondria, nucleus, and cytoplasm (Science. 2014; 346(6209): 608 - 13; Cell. 2014; 158(3): 607). The functions of Ago2 in different subcellular components are completely different. However, so far, there has been no report on the association between subcellular component Ago2 and fatty liver, and the report on the therapeutic effect of subcellular component Ago2 in fatty liver and the use of related therapeutic drugs is even blank. Summary of the Invention

[0005] In view of the above problems and deficiencies objectively existing in the prior art in this field, the present invention provides the use of Ago2 in the preparation of drugs for treating fatty liver or hyperlipidemia and its products.

[0006] The above technical object of the present invention is achieved through the following technical solutions:

[0007] Use of Ago2 in the preparation of drugs for treating fatty liver or hyperlipidemia.

[0008] The action target of the drug for treating fatty liver or hyperlipidemia is Ago2;

[0009] Preferably, the Ago2 is mitochondrially targeted Ago2;

[0010] Preferably, the mitochondrially targeted Ago2 refers to Ago2 linked with a mitochondrial leader peptide;

[0011] Preferably, the mitochondrial leader peptide is selected from: Cox8, Su9, Sod2;

[0012] Preferably, the Ago2 is selected from: human-derived Ago2, rat-derived Ago2, mouse-derived Ago2.

[0013] A gene for treating fatty liver or hyperlipidemia, which comprises: a mitochondrial leader peptide gene sequence and an Ago2 gene sequence.

[0014] The mitochondrial leader peptide gene sequence is as shown in SEQ ID NO.1, and the Ago2 gene sequence is as shown in SEQ ID NO.2.

[0015] A recombinant expression vector for treating fatty liver or hyperlipidemia, which is an expression vector linked with the gene for treating fatty liver or hyperlipidemia.

[0016] The expression vector is selected from: adeno-associated virus expression vector pAAV-D(+).

[0017] A transformant for treating fatty liver or hyperlipidemia, which is a host cell transformed with the recombinant expression vector for treating fatty liver or hyperlipidemia.

[0018] The host cell includes: 293 cells, DH5α competent cells.

[0019] A drug for treating fatty liver or hyperlipidemia, comprising: a pharmaceutically active ingredient; characterized in that the pharmaceutically active ingredient comprises: the gene for treating fatty liver or hyperlipidemia, the recombinant expression vector for treating fatty liver or hyperlipidemia, and the transformant for treating fatty liver or hyperlipidemia.

[0020] Use of the gene for treating fatty liver or hyperlipidemia, and / or the recombinant expression vector for treating fatty liver or hyperlipidemia, and / or the transformant for treating fatty liver or hyperlipidemia in the preparation of a medicament for treating fatty liver or hyperlipidemia.

[0021] On the premise that the patent laws of some countries or regions permit, the present invention also claims to protect a substance with high expression of mitochondrially targeted Ago2, or a fusion protein composed of a leading peptide sequence capable of localizing to mitochondria and an Ago2 sequence, or a fusion protein composed of Cox8 and Ago2, or a recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2 connecting a Cox8 gene sequence and an Ago2 gene sequence, or a recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2, and its application in the treatment of fatty liver or hyperlipidemia.

[0022] The present invention provides a medicament for treating fatty liver or hyperlipidemia, characterized in that the active ingredient of the medicament comprises a substance that takes mitochondrially targeted Ago2 as a drug target and can increase the expression of mitochondrially targeted Ago2.

[0023] The active ingredient of the medicament comprises a substance with high expression of mitochondrially targeted Ago2.

[0024] The substance of mitochondrially targeted Ago2 comprises a mitochondrial targeting signal sequence fragment and an Ago2 sequence fragment, and the sequence fragment of cox8-Ago2 alone is shown in SEQ ID NO.1 and SEQ ID NO.2 for example.

[0025] The medicament further comprises a pharmaceutically acceptable excipient, and / or a reagent for buffering, synthesizing, and / or purifying the sequence fragment.

[0026] A preparation method of a medicament for treating fatty liver or hyperlipidemia, characterized by comprising: using the substance capable of highly expressing the mitochondrially targeted Ago2 as the active ingredient of the medicament for treating fatty liver or hyperlipidemia.

[0027] Insert the sequence fragment capable of expressing Cox8-Ago2 into an expression vector, thereby preparing a recombinant plasmid capable of stably expressing mitochondrially targeted Ago2. The sequence fragment of Cox8-Ago2 is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0028] An object of the present invention is to provide a drug for treating fatty liver, and the active ingredient of the drug includes targeting Ago2 to mitochondria as the drug target, and the substance that highly expresses the mitochondrially targeted Ago2 has the efficacy of treating fatty liver or hyperlipidemia.

[0029] High expression: That is, by various vectors, Ago2 containing a mitochondrial localization sequence is specifically highly expressed in mitochondria. Ago2 can be specifically highly expressed in mitochondria through leading signals such as mitochondrial localization sequences like Cox8, Su9, Sod2, etc.; or Ago2 is delivered into mitochondria through mitochondrially targeted nanoparticles;

[0030] Any one of the above methods can play a role in increasing the expression level of mitochondrially targeted Ago2. Any act of commercially packaging the above substances in a commodity package marked with the use for treating fatty liver or hyperlipidemia falls within the scope of protection claimed in the present invention.

[0031] Furthermore, the active ingredient of the drug includes a substance that highly expresses mitochondrially targeted Ago2.

[0032] Furthermore, the substance that highly expresses mitochondrially targeted Ago2 includes a leading peptide sequence fragment of mitochondrial localization cox8 and a sequence fragment of Ago2 as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0033] Furthermore, the drug also includes pharmaceutically acceptable excipients, and / or reagents for buffering, synthesizing, and / or purifying the sequence fragments. Those skilled in the art can, according to objective needs, add various pharmaceutically acceptable adjuvants / excipients to the anti-fatty liver or hyperlipidemia drug of the present invention to make various dosage forms for easy sales or promotion.

[0034] Another object of the present invention is to provide a preparation method of a drug for treating fatty liver or hyperlipidemia, including: using the substance that highly expresses mitochondrially targeted Ago2 as the active ingredient of the drug for treating fatty liver or hyperlipidemia.

[0035] In a further embodiment, the preparation method includes: inserting a leading peptide sequence of mitochondrial localization cox8 and a sequence fragment of Ago2 into an expression vector, thereby preparing a recombinant plasmid that can stably express mitochondrially targeted Ago2.

[0036] In a specific embodiment, the expression vector containing the leading peptide sequence of mitochondrial localization cox8 and the sequence fragment of Ago2 is an adeno-associated virus expression vector pAAV-D(+).

[0037] The present invention relates to a drug for treating fatty liver or hyperlipidemia. The anti-fatty liver and hyperlipidemia drug relates to a construction and preparation method of a recombinant adeno-associated virus recombinant (rAAV-cox8-Ago2) that can specifically target Ago2 of mitochondria, and exerts the drug effect of treating fatty liver or hyperlipidemia by highly expressing the mitochondria-targeted rAAV-Ago2. The present invention constructs a pAAV-D(+)-cox8-Ago2 expression plasmid by chemical synthesis method, and packages and prepares a recombinant adeno-associated virus containing the target fragment by calcium phosphate co-transfection method of three plasmids and purifies it. Animal experiments confirm that the anti-fatty liver and hyperlipidemia drug provided by the present invention can significantly improve the liver lipid deposition and blood lipid level of fatty liver mice, and play an effective role in anti-fatty liver and hyperlipidemia.

[0038] The present invention accidentally discovered the relationship between mitochondrial Ago2 and the pathogenesis of fatty liver, and further carried out molecular experiments to develop an anti-fatty liver drug targeting mitochondrial Ago2 as a drug target. At the same time, animal experiments were carried out to verify its effect, and it was found that highly expressing mitochondria-targeted Ago2 can significantly improve liver lipid deposition and reduce blood lipid level in mice, and treat fatty liver or hyperlipidemia.

[0039] Based on the cox8 leading base sequence and Ago2 base sequence targeting mitochondria, the present invention designed and synthesized a sequence of mitochondria-targeted Ago2, and successfully inserted it into the eukaryotic expression vector pAAV-D(+) to form a recombinant plasmid pAAV-D(+)-cox8-Ago2. Then, the following three plasmids: 1) pXX9 plasmid, 2) phelper plasmid, 3) pAAV-D(+)-cox8-Ago2 plasmid, were transfected into 293 cells by calcium phosphate co-transfection method to package and prepare a recombinant adeno-associated virus (rAAV9) that can express mitochondria-targeted cox8-Ago2. After purification, the titer was measured by real-time PCR method. Next, the packaged recombinant adeno-associated virus (rAAV-cox8-Ago2) of the same serotype was injected into fatty liver model mice induced by high-fat diet (HFD) via the tail vein, and it was found that the long-term expression of mitochondrial Ago2 in the mouse liver changed significantly, indicating that the recombinant adeno-associated virus can mediate the long-term and effective expression of mitochondrial Ago2. The results of Oil Red and HE showed that the expression of mitochondria-targeted Ago2 mediated by the recombinant adeno-associated virus can significantly improve the liver lipid deposition of fatty liver mice, and the results of plasma biochemical detection showed that the expression of mitochondria-targeted Ago2 can significantly reduce the blood lipid level of fatty liver mice, further supporting the anti-fatty liver and hyperlipidemia effects of mitochondria-targeted Ago2. Description of the Drawings

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0041] Figure 1 It is a schematic diagram of the plasmid composition of pAAV-D(+)-cox8-Ago2 in Experimental Example 1; where cox8 represents the mitochondrial leader sequence, m-Ago2 represents mouse-derived Ago2, promoter(CMV) represents promoter CMV, ITR represents the inverted repeat sequence, and Amp represents the ampicillin resistance gene sequence.

[0042] Figure 2 It is an analysis chart of the content of Ago2 located in the liver mitochondria of fatty liver mice in Experimental Example 2; where A is the western blot result chart of the content of Ago2 in the liver mitochondria of normal diet mice and high-fat diet mice; B is the bar chart of the content of Ago2 in normal diet mice and high-fat diet mice; the meanings of the markings in the figure are as follows: VDAC1 represents the internal reference protein of mitochondria; CHOW represents normal diet control group mice; HFD represents high-fat-induced fatty liver model group mice.

[0043] Figure 3 It is the detection of the expression of Ago2 in the liver of HFD mice treated with different treatments by Western blotting; the meanings of each marking in the figure are listed as follows: rAAV-GFP represents high-fat-induced fatty liver model group mice (HFD mice) injected with a recombinant adeno-associated virus vector of control GFP, rAAV-cox8-Ago2 represents high-fat-induced fatty liver model group mice (HFD mice) injected with a recombinant adeno-associated virus vector with high expression of Ago2 in mitochondria, and VDAC1 represents the internal reference protein of mitochondria; the detection results show that rAAV-cox8-Ago2 can significantly increase the expression of Ago2 in the liver mitochondria of HFD mice.

[0044] Figure 4 It is the detection of the effect of rAAV-cox8-Ago2 treatment on liver lipid deposition in HFD mice by Oil Red and HE; where A is the representative figure and statistical chart of Oil Red, B is the morphological detection of HE, C is the bar chart of plasma triglyceride, and D is the bar chart of total cholesterol in plasma; the results show that rAAV-cox8-Ago2 can significantly reduce liver lipid deposition and blood lipid levels in HFD mice.

[0045] Figures 1 - 4The meanings of each label are listed as follows: rAAV-GFP represents HFD mice injected with a recombinant adeno-associated virus vector carrying control GFP, and rAAV-cox8-Ago2 represents HFD mice injected with a recombinant adeno-associated virus vector highly expressing Ago2 in mitochondria; CHOW represents mice in the normal diet control group, and HFD represents mice in the high-fat-induced fatty liver model group. Detailed implementation manners

[0046] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0047] I. Instruments, reagents and consumables

[0048] (I) Instruments

[0049] ND-1000 nucleic acid analyzer, ABI 9700 PCR instrument, ABI 7900HT fluorescence real-time quantitative PCR instrument, Beckman X-15R low-temperature high-speed centrifuge;

[0050] (II) Reagents and consumables

[0051] Oil Red O dry powder and HE dye (purchased from Hubei Bioscience Co., Ltd.), blood lipid detection kit (purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.), TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (agarose gel DNA recovery kit, purchased from TaKaRa), E.Z.N.A. Endo-Free PlasmidMaxi Kit (plasmid extraction kit, purchased from OMEGA), EasyPure Plasmid MiniPrep Kit (plasmid extraction kit, purchased from TransGen Biotech Co., Ltd.); the eukaryotic expression vector pAAV-D(+) was constructed and gifted by Professor Xiao Xiao in cooperation, and the cox8-Ago2 DNA sequence fragment was synthesized by Wuhan Coristar Biotech Co., Ltd.

[0052] II. Sources of biological materials

[0053] (I) The 293T cells used in Experimental Example 1 of the present invention were purchased from the American Type Culture Collection (ATCC), and the DH5α competent cells were purchased from TransGen Biotech Co., Ltd.

[0054] (2) The C57 background mice used in Experimental Example 2 of the present invention were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0055] Group 1 Examples, Pharmaceutical Use of Ago2

[0056] This group of examples provides the use of Ago2 in the preparation of drugs for treating fatty liver or hyperlipidemia.

[0057] In a specific example, the target of the drug for treating fatty liver or hyperlipidemia is Ago2;

[0058] Preferably, the Ago2 is mitochondrially targeted Ago2;

[0059] Preferably, the mitochondrially targeted Ago2 refers to Ago2 linked with a mitochondrial leader peptide;

[0060] Preferably, the mitochondrial leader peptide is selected from: Cox8, Su9, Sod2;

[0061] Those skilled in the art are familiar that there are three commonly used mitochondrial leader peptides, namely Cox8, Su9, and Sod2. These are known mitochondrial leader peptides that have been reported and all have similar mitochondrial targeting effects.

[0062] In a specific example, the mitochondrial leader peptide Cox8 has the conventional technical meaning well-known to those skilled in the art. For example, it can be the Cox8 described in the article "Doubledeletion of PINK1 and Parkin impairs hepatic mitophagyand exacerbates acetaminophen-induced liver injury in mice";

[0063] The mitochondrial leader peptide Su9 has the conventional technical meaning well-known to those skilled in the art. For example, it can be the Su9 described in the article "MicroRNA directly enhances mitochondrial translation duringmuscledifferentiation";

[0064] The mitochondrial leader peptide Sod2 has the conventional technical meaning well-known to those skilled in the art. For example, it can be the Sod2 described in the article "Specific elimination of mutant mitochondrial genomes in patient-derivedcells by mitoTALENs".

[0065] Preferably, the Ago2 is selected from: human Ago2, rat Ago2, and mouse Ago2.

[0066] As is well known to those skilled in the art, since the sequence of Ago2 is conserved among human / rat / mouse, it can be speculated that Ago2 from other species will also have a similar effect.

[0067] Group 2 Examples, Genes for Treating Fatty Liver or Hyperlipidemia in the Present Invention

[0068] This group of examples provides a gene for treating fatty liver or hyperlipidemia. All examples in this group have the following common characteristics: The gene for treating fatty liver or hyperlipidemia includes: a mitochondrial targeting peptide gene sequence and an Ago2 gene sequence.

[0069] In a specific example, the mitochondrial targeting peptide gene sequence is as shown in SEQ ID NO.1, and the Ago2 gene sequence is as shown in SEQ ID NO.2.

[0070] Group 3 Examples, Recombinant Expression Vectors for Treating Fatty Liver or Hyperlipidemia in the Present Invention

[0071] This group of examples provides a recombinant expression vector for treating fatty liver or hyperlipidemia. All examples in this group have the following common characteristics: The recombinant expression vector for treating fatty liver or hyperlipidemia is an expression vector ligated with the gene for treating fatty liver or hyperlipidemia according to any one of the examples in Group 2.

[0072] In a specific example, the expression vector is selected from: the adeno-associated virus expression vector pAAV-D(+).

[0073] Group 4 Examples, Transformants for Treating Fatty Liver or Hyperlipidemia in the Present Invention

[0074] This group of examples provides a transformant for treating fatty liver or hyperlipidemia. All examples in this group have the following common characteristics: The transformant for treating fatty liver or hyperlipidemia is a host cell transformed with the recombinant expression vector for treating fatty liver or hyperlipidemia according to any one of the examples in Group 3.

[0075] In a specific example, the host cell includes: 293 cells, DH5α competent cells.

[0076] Group 5 Examples, Drugs for Treating Fatty Liver or Hyperlipidemia in the Present Invention

[0077] This group of embodiments provides a drug for treating fatty liver or hyperlipidemia. All embodiments in this group have the following common features: The drug for treating fatty liver or hyperlipidemia includes: a pharmacodynamic active ingredient; The pharmacodynamic active ingredient includes: a gene for treating fatty liver or hyperlipidemia according to any one of the embodiments in Group 2, a recombinant expression vector for treating fatty liver or hyperlipidemia according to any one of the embodiments in Group 3, and a transformant for treating fatty liver or hyperlipidemia according to any one of the embodiments in Group 4.

[0078] This group of embodiments provides a drug for treating fatty liver or hyperlipidemia. The pharmacodynamic component of the drug for treating fatty liver or hyperlipidemia targets mitochondrial Ago2, and exerts the pharmacodynamic effect of treating fatty liver or hyperlipidemia by highly expressing the mitochondrially targeted Ago2. Through some experiments, the present invention found that the content of Ago2 located in the liver mitochondria of fatty liver mice increased significantly ( Figure 1 ), which indicates that Ago2 in mitochondria may play a regulatory role in the pathophysiological processes of fatty liver and hyperlipidemia. Further mouse experiments were carried out. By increasing the content of mitochondrial Ago2, the liver lipid deposition in fatty liver mice can be significantly improved and the blood lipid can be reduced ( Figure 4 ), thereby achieving the therapeutic effect on fatty liver and hyperlipidemia.

[0079] The present invention found through experiments that the level of mitochondrial Ago2 in HFD fatty liver mice was significantly high, and overexpressing mitochondrial Ago2 in HFD fatty liver mice could resist fatty liver. Based on the inventor's many years of experience in clinical research and development, it is speculated that the logical relationship between the two may be as follows: In fatty liver, the increase in mitochondrial Ago2 protein indicates that mitochondrial Ago2 may play an important role in fatty liver or hyperlipidemia. However, whether this increase is a compensatory protective effect or a destructive effect requires subsequent functional studies. In the functional study, the present invention found that overexpressing mitochondrial Ago2 can treat fatty liver and hyperlipidemia, indicating that the increased mitochondrial Ago2 in fatty liver plays a compensatory protective role, but the strength is not large enough, and a larger amount of overexpression is needed to amplify the protective effect.

[0080] Furthermore, the pharmacodynamic component of the drug includes a substance that can increase mitochondrially targeted Ago2. Those skilled in the art are familiar that a mitochondrial localization signal can be added before the Ago2 sequence to specifically increase the expression of mitochondrial Ago2 and thus play a therapeutic role. Other means can also be used, such as certain mitochondrially targeted nanoparticles to directly deliver Ago2 to mitochondria, which can also achieve the purpose of increasing the mitochondrial level of Ago2.

[0081] Preferably, the active ingredient of the drug comprises a mitochondrial-targeted Ago2 sequence fragment. The most common method in molecular biology is to add a mitochondrial localization signal sequence in front of the target sequence fragment to transport Ago2 to the mitochondria for therapeutic purposes. The Cox8 mitochondrial signal peptide is a commonly used mitochondrial localization signal, and the sequences of co8-Ago2 are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0082] In some alternative embodiments of the present invention, some other mitochondrial localization signal sequences, such as the leading sequences of Su9, Sod2, etc., can also specifically deliver Ago2 to the mitochondria. The recombinant plasmids of Ago2 containing these leading sequences have anti-fatty liver or hyperlipidemia effects similar to or equivalent to those in Experimental Example 2 below. Due to limited space, they will not be elaborated one by one herein.

[0083] In some alternative embodiments of the present invention, some Ago2 sequence fragments of other species, such as human Ago2, rat Ago2, etc., are highly homologous to the mouse Ago2 gene sequence. The recombinant plasmids of Ago2 containing these species have anti-fatty liver or hyperlipidemia effects similar to or equivalent to those in Experimental Example 2 below. Due to limited space, they will not be elaborated one by one herein.

[0084] In a most specific embodiment of the present invention, the active ingredient of the drug is the recombinant adeno-associated virus plasmid pAAV-D(+)-cox8-Ago2 expressing mitochondrial-targeted mouse Ago2. The recombinant adeno-associated virus vector (rAAV) adopted in the present invention overcomes the disadvantages that are difficult to overcome by other gene expression vectors. It can carry the target gene to transfect both dividing and non-dividing cells (i.e., has a wide range of transgenic scope), has no side effects (no immunogenicity), has a high infection efficiency, can drive the target gene to express in vivo for a long time, and successfully solves the problem of large-scale replication in vitro without adenovirus contamination, thus becoming the most promising vector for gene therapy.

[0085] More specifically, the cox8 mitochondrial guiding sequence and the mus-Ago2 sequence are inserted into the adenovirus expression vector pAAV-D(+) as shown in SEQ ID NO.1 and SEQ ID NO.2 to construct the pAAV-D(+)-cox-Ago2 plasmid for expression. The above double-stranded nucleotide sequence was synthesized by Wuhan Keresite Biotechnology Co., Ltd. and inserted into the vector pAAV-D(+).

[0086] Furthermore, the drug also includes pharmaceutically acceptable excipients, and / or reagents for buffering, culturing, and / or propagating the recombinant adeno-associated virus plasmid pAAV-D(+)-cox8-Ago2; those skilled in the art can, according to objective needs, add various pharmaceutically acceptable adjuvants / excipients to the anti-fatty liver or hyperlipidemia drug of the present invention to prepare various dosage forms for easy sales or promotion.

[0087] Group 6 Examples, Pharmaceutical Uses of the Genes, Recombinant Expression Vectors, and Transformants of the Present Invention

[0088] This group of examples provides the use of a gene for treating fatty liver or hyperlipidemia according to any one of the examples in Group 2, and / or a recombinant expression vector for treating fatty liver or hyperlipidemia according to any one of the examples in Group 3, and / or a transformant for treating fatty liver or hyperlipidemia according to any one of the examples in Group 4 in the preparation of drugs for treating fatty liver or hyperlipidemia.

[0089] Group 7 Examples, Preparation Methods of the Drugs of the Present Invention

[0090] This group of examples provides a preparation method of a drug for treating fatty liver or hyperlipidemia. All examples in this group have the following common characteristics: expressing a fusion protein composed of a mitochondrial-targeting leader peptide and Ago2.

[0091] In some examples, the gene sequence of the mitochondrial-targeting leader peptide and the gene sequence of Ago2 are ligated to an expression vector to obtain a recombinant expression vector;

[0092] Preferably, the recombinant expression vector is transformed into a host cell to obtain a transformant;

[0093] Preferably, the recombinant expression vector is transformed into a host cell by the calcium phosphate co-transfection method to obtain a transformant;

[0094] Preferably, the Ago2 is selected from: human Ago2, rat Ago2, mouse Ago2;

[0095] Preferably, the expression vector is the adeno-associated virus expression vector pAAV-D(+);

[0096] Preferably, the mitochondrial-targeting leader peptide sequence is selected from: Cox8, Su9, Sod2;

[0097] Preferably, the recombinant expression vector is selected from: the recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2 ligated with the Cox8 gene sequence and the Ago2 gene sequence;

[0098] Preferably, the transformant is selected from: recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2;

[0099] Preferably, the Cox8 gene sequence is as shown in SEQ ID NO.1;

[0100] Preferably, the Ago2 gene sequence is as shown in SEQ ID NO.2.

[0101] In some specific embodiments of this group, the recombinant adeno-associated virus plasmid pAAV-D(+)-cox8-Ago2 containing the sequence fragment of Ago2 with mitochondrial targeting ability - containing the cox8 mitochondrial localization signal is used as the active ingredient of the anti-fatty liver or hyperlipidemia drug.

[0102] Furthermore, the full gene synthesis technology is adopted to synthesize the mus-cox8-Ago2 double-stranded nucleotide and clone it on the pAAV-D(+) vector, thereby preparing a recombinant plasmid capable of stably expressing mitochondrially targeted Ago2. In a more specific scheme, the cox8-Ago2 sequence fragment capable of expressing mitochondrial targeting is as shown in SEQ ID NO.1 and SEQ ID NO.2; the expression vector is the adeno-associated virus expression vector pAAV-D(+).

[0103] The specific experimental operation steps of this group of embodiments can be referred to in Experimental Example 1.

[0104] The technical effects of the present invention will be further verified and illustrated through the following experimental examples.

[0105] Experimental Example 1: Construction of Recombinant Adeno-Associated Virus

[0106] 1. Construction of pAAV-D(+) Vector

[0107] According to the base sequences of the mitochondrial leader sequence Cox8 and mus-Ago2 (Ago2 derived from mice), the full gene synthesis technology is adopted to synthesize the cox8-Ago2 double-stranded nucleotide and clone it on the pAAV-D(+) vector ( Figure 1 ), and the double-stranded nucleotide is synthesized by Wuhan Keruister Biotechnology Co., Ltd. The cox8 and Ago2 sequences are as shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0108] 2. Plasmid Transformation

[0109] Add the pAAV-D(+)-cox8-Ago2 plasmid to 100 μl of DH5α competent cells and place on ice for 30 min; heat at 42 °C for 45 sec, then place on ice for 1 min; add 500 μl of antibiotic-free LB medium and incubate with shaking at 37 °C and 100 rpm for 60 min; culture on an Amp+ LB plate medium and select white monoclonal colonies for identification.

[0110] 3. Small-scale plasmid extraction

[0111] Pick a monoclonal colony and add it to 3 ml of Amp+ LB liquid medium. Incubate with shaking at 37 °C and 280 rpm overnight. Extract the plasmid using the EasyPure Plasmid MiniPrep Kit from Beijing Quanshijin Company. The specific operation steps are as follows: 1. Centrifuge 1.5 ml of overnight cultured bacteria at 10,000 g for 1 min and aspirate the supernatant as completely as possible; 2. Add 250 μl of colorless solution RB (containing RNase A) and vortex to suspend the bacterial pellet; 3. Add 250 μl of blue solution LB and gently invert the mixture up and down 4 - 6 times to fully lyse the bacteria and form a blue and transparent solution; 4. Add 350 μl of yellow solution NB and gently mix 5 - 6 times until a firm yellow clot is formed, and let stand at room temperature for 2 min; 5. Centrifuge at 15,000 g for 5 min and carefully aspirate the supernatant and add it to the adsorption column; 6. Centrifuge at 15,000 g for 1 min and discard the effluent; 7. Add 650 μl of solution WB, centrifuge at 15,000 g for 1 min and discard the effluent; 8. Centrifuge at 15,000 g for 2 min to completely remove the residual WB; 9. Place the adsorption column in a new Ep tube, add 20 μl of EB preheated to 70 °C in the center of the column, and let stand at room temperature for 1 min; 10. Centrifuge at 10,000 g for 1 min to elute the DNA, and store the eluted DNA at -20 °C.

[0112] 4. Large-scale plasmid extraction

[0113] Prepare a 1 L sterile conical flask, add 300 ml of sterile LB medium, and add ampicillin solution to a final concentration of 100 μg / ml. Add 50 μl of the required plasmids (pXX9, phelper, pAAV-D(+)-GFP, pAAV-D(+)-cox8-Ago2) respectively, and incubate with shaking at 280 rpm and 37 °C overnight. Operate according to the instructions of the Endo-Free Plasmid Maxi Kit from OMEGA Company to extract the plasmid. The specific steps are as follows: 1. Centrifuge the bacteria at 5000 g for 10 min at room temperature to collect; 2. Discard the medium, add 10 ml of Solution I / RNase A mixture, and vortex to completely resuspend; 3. Add 10 ml of Solution II to the resuspended mixture, gently invert and mix 10 - 15 times, and then let stand at room temperature.

[0114] 5. rAAV-Mediated Virus Packaging

[0115] When the 293T cells grow to 90%, 1 - 2 hours before calcium phosphate transfection, change the fresh medium (containing serum) in each culture dish to 12 - 15 ml. First add calcium chloride (CaCl2) to a 50 ml centrifuge tube, then add the plasmid to form a Ca-DNA mixture, mix well. Slowly add 2X HEBS BUFFER to the Ca-DNA mixture to form a Ca-DNA-P mixture. While adding 2X HEBS, shake the centrifuge tube to mix well to form calcium phosphate particles. After 8 - 12 hours, change to 18 - 20 ml of serum-free medium. After 72 hours, aspirate the medium, wash 3 times with PBS. Add 1 ml of Tris + NaCl (pH 8.5) to each culture dish, scrape the cells with a spatula, collect them in a clean centrifuge tube, and store at -80 °C.

[0116] 6. Virus Purification

[0117] Take out the cells frozen at -80 °C, thaw and dissolve them at 37 °C, freeze-thaw 4 times repeatedly, centrifuge at 8,000 g for 15 min, transfer the supernatant to a clean centrifuge tube, and discard the cell pellet. Mix anhydrous ethanol pre-cooled at -20 °C with rAAV in a volume ratio of 3:1, place in a -20 °C refrigerator for 2 hours, then centrifuge at 4 °C and 13,000 rpm for 15 min, discard the supernatant; after the ethanol evaporates, add the corresponding volume of Tris + NaCl (pH 8.5) to dissolve the precipitate. Filter with a Millipore small filter (0.22 μm).

[0118] 7. Virus Titer Determination

[0119] Sample Treatment:

[0120] 40 μl of rAAV virus solution

[0121] 5 μl of Proteinase K (20 mg / ml)

[0122] React at 55 °C for 1 hour;

[0123] 45 μl of Phenol:Chloroform:Isoamyl Alcohol

[0124] Centrifuge at 4 °C and 12,000 g for 5 min to recover the aqueous phase;

[0125] 45 μl of Chloroform

[0126] Centrifuge at 4 °C and 12,000 g for 5 min to recover the aqueous phase.

[0127] Real-time PCR:

[0128] 0.4 μl of Primer 1 (10 μm)

[0129] Primer 2 (10 μm) 0.4 μl

[0130] SYBR Green I Mix 10 μl

[0131] ddH2O 8.2 μl

[0132] Template 1 μl

[0133] 95°C 30 sec --- (95°C 5 sec --- 60°C 5 sec --- 72°C 20 sec) × 40 cycles --- Melting Curve Experiment Example 2. Taking the recombinant adeno-associated virus expressing cox8-Ago2 of rAAV9 type as an example, its therapeutic effect on fatty liver or hyperlipidemia was detected

[0134] 1. Detection of mitochondrial Ago2 expression in HFD mice:

[0135] 8-week-old C57 mice were used: fed a high-fat diet (High Fat Diet, HFD, fatty liver model group caused by high fat) and a normal control diet (CHOW, normal diet control group). At the end of the experiment (after 12 weeks), liver mitochondria were extracted using the Beyotime tissue mitochondrial extraction kit, and Western blot was used to detect the expression of mitochondrial Ago2 in the liver of HFD mice. The results showed that the content of mitochondrial Ago2 in the liver of HFD mice increased significantly ( Figure 2 ).

[0136] 8-week-old C57 mice were used. rAAV-cox8-Ago2 and control rAAV-GFP viruses were injected into the tail vein respectively. The virus titer was 1×10 11 PFU / each. Fed a high-fat diet (HFD, fatty liver model group caused by high fat) and a normal control diet (normal control diet group). At the end of the experiment (after 12 weeks), liver mitochondria were extracted and Western blot was used to detect the expression of mitochondrial Ago2 in the liver of HFD mice. The results showed that rAAV-cox8-Ago2 treatment could significantly increase the expression of mitochondrial Ago2 in the liver ( Figure 3 ).

[0137] 2. Detection of liver lipids in HFD fatty liver mice: At the end of the experiment, oil red and HE staining were used to detect the liver lipid and blood lipid conditions of HFD mice. The method is as follows:

[0138] Formulation of Oil Red stock solution: 100 mL of 100% isopropanol + 0.5 g of Oil Red O dry powder, fully dissolve, filter, and store in the dark at 4°C. Formulation of Oil Red working solution: Before the experiment, dilute the stock solution with distilled water at a ratio of 3:2 and filter. Frozen sections of mouse liver are stained with the Oil Red working solution for 2 - 5 min, washed with water, counterstained with hematoxylin, differentiated with hydrochloric acid alcohol for 1 - 5 s, blued with running water, and photographed after mounting. For HE staining (hematoxylin-eosin staining), place the sections in an aqueous hematoxylin solution for staining for several minutes, differentiate in acid water and ammonia water for several seconds each, rinse with running water for 1 hour, place in distilled water for a moment, dehydrate in 70% and 90% alcohol for 10 minutes each, stain with alcoholic eosin staining solution for 2 - 3 minutes, dehydrate and clear, and mount and photograph. The contents of blood lipids (triglyceride, total cholesterol) in plasma are detected using a biochemical kit from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. The results showed that rAAV-cox8-Ago2 treatment could significantly reduce the liver lipid content and blood lipid levels in HFD mice ( Figure 4 ).

Claims

1. Use of Ago2 in the preparation of a medicament for treating fatty liver or hyperlipidemia.

2. Use of Ago2 according to claim 1 in the preparation of a medicament for treating fatty liver or hyperlipidemia, characterized in that, The target of the medicament for treating fatty liver or hyperlipidemia is Ago2; and / or, the Ago2 is mitochondrion-targeted Ago2; and / or, the mitochondrion-targeted Ago2 refers to Ago2 linked with a mitochondrial leader peptide; and / or, the mitochondrial leader peptide is selected from: Cox8, Su9, Sod2; and / or, the Ago2 is selected from: human Ago2, rat Ago2, mouse Ago2.

3. A gene for treating fatty liver or hyperlipidemia, characterized in that, It includes: mitochondrial leader peptide gene sequence and Ago2 gene sequence.

4. A gene for preventing fatty liver or hyperlipidemia according to claim 3, characterized in that, The mitochondrial leader peptide gene sequence is as shown in SEQ ID NO.1, and the Ago2 gene sequence is as shown in SEQ ID NO.

2.

5. A recombinant expression vector for treating fatty liver or hyperlipidemia, characterized in that, It is an expression vector linked with a gene for treating fatty liver or hyperlipidemia as claimed in claim 2 or 3.

6. The recombinant expression vector for anti-fatty liver or hyperlipidemia according to claim 5, characterized in that, The expression vector is selected from: adeno-associated virus expression vector pAAV-D(+).

7. A transformant against fatty liver or hyperlipidemia, characterized in that, It is a host cell transformed with a recombinant expression vector for treating fatty liver or hyperlipidemia as claimed in claim 4 or 5.

8. A transformant for treating fatty liver or hyperlipidemia according to claim 7, characterized in that, The host cell includes: 293 cells, DH5α competent cells.

9. A drug for treating fatty liver or hyperlipidemia, comprising: a pharmacodynamic active ingredient; characterized in that, The pharmacologically active ingredient contains: a gene for treating fatty liver or hyperlipidemia as claimed in claim 3 or 4, a recombinant expression vector for treating fatty liver or hyperlipidemia as claimed in claim 5 or 6, a transformant for treating fatty liver or hyperlipidemia as claimed in claim 7 or 8.

10. Use of a gene for treating fatty liver or hyperlipidemia as claimed in claim 3 or 4, and / or a recombinant expression vector for treating fatty liver or hyperlipidemia as claimed in claim 5 or 6, and / or a transformant for treating fatty liver or hyperlipidemia as claimed in claim 7 or 8 in the preparation of a medicament for treating fatty liver or hyperlipidemia.

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