CN113265424B - Recombinant adeno-associated virus vector for treating atherosclerosis, genome composition and application - Google Patents

Abstract

The invention provides a recombinant adeno-associated virus vector for treating atherosclerosis, a genome composition and application of the genome composition. The recombinant adeno-associated virus vector is obtained by inserting lectin-like oxidized low-density lipoprotein receptor 1(LOX-1) into the adeno-associated virus vector, and the lectin-like oxidized low-density lipoprotein receptor 1 and a TBG promoter are combined into a genome composition to be used for preparing a gene therapy injection for treating atherosclerosis. Wherein the sequence of the LOX-1 gene is shown as SEQ ID NO. 1 in a sequence table. The invention can efficiently introduce a drug effect element into a body by intravenous injection by utilizing an adeno-associated viral vector to realize ectopic high-efficiency expression of a drug effect element expression product therapeutic action protein LOX-1, successfully express an LOX-1 receptor which is not expressed in liver on liver cells by a TBG promoter, and phagocytose and remove overloaded OX-LDL in circulation of an atherosclerosis patient by means of strong lipid metabolism capability and metabolic pathway of the liver, thereby inhibiting the progression of atherosclerosis.

Description

Recombinant adeno-associated virus vector for treating atherosclerosis, genome composition and application

Technical Field

The invention relates to the field of biotechnology, in particular to a recombinant adeno-associated virus vector for treating atherosclerosis, a genome composition and application, and specifically relates to an AAV (adeno-associated virus) vector system for mediating LOX-1 specificity ectopic expression in liver, wherein LOX-1 receptor which is not expressed in liver is successfully expressed in liver cells, and overloaded OX-LDL in circulation of a patient with atherosclerosis is phagocytized and cleared by means of strong lipid metabolism capability and metabolic pathway of liver, so that the progression of atherosclerosis is inhibited.

Background

Atherosclerosis (atheroclerosis) is a systemic pathological change caused by abnormal deposition of cholesterol-rich lipoproteins in blood vessel walls, which causes atherosclerotic cardiovascular disease to account for the first loss of death and global disease burden, and has continued to rise by 21.1% in the last decade. In centuries of atherosclerosis research and practice, modern clinical practice has made it possible to treat Low Density Lipoprotein (LDL) PCSK9i by enhancing hepatic uptake through the use of statin drugs that inhibit key enzymes of hepatic cholesterol synthesis, ezetimibe that inhibits the major receptors for intestinal cholesterol absorption, and increases the recycling of hepatic cholesterol low density lipoprotein receptor circulation, inhibition from two entrances to cholesterol and enhancement from one exit has achieved the ability to reduce LDL cholesterol to very low states and has achieved tremendous success in inhibiting atherosclerosis. However, the complexity of pathogenesis of the first fatal disease in the world is still not completely analyzed, a way of simply reducing LDL is still insufficient, and the intervention measures of atherosclerosis still need to be explored in many ways to search for a plurality of effective treatment means.

Research has shown that OX-LDL penetrates through the atherosclerotic pathogenic process, and the harm is much higher than LDL. Whether the vascular endothelial barrier is destroyed in the early stage or the atherosclerotic plaque is ruptured in the late stage, the plaque is inseparable from OX-LDL. Thus, removal of OX-LDL is even more urgent than removal of LDL. However, according to the present studies, it has been revealed that the clearance of OX-LDL is closely related to the action of scavenger receptors, among which LOX-1 is the most important clearance receptor, and in physiological cases LOX-1 is mainly expressed in endothelial cells, macrophages, platelet cells, etc., and exerts a series of adverse reactions by OX-LDL in phagocytosis cycle, and it is the fundamental reason why although it phagocytoses OX-LDL, it lacks a pathway for clearance of OX-LDL, thus triggering a series of injury reactions.

Adeno-associated virus (AAV) is a replication-defective parvovirus that requires the assistance of adenovirus or herpes virus for its proliferative replication. The AAV Helper-Free System (AAV Helper-Free System) can produce recombinant adeno-associated virus without Helper virus. Such systems utilize adenovirus gene products that have been defined and regulated for AAV replication and expression, and these gene products are introduced into the host cell by transfection. In the AAV Helper-Free System, the adenovirus gene products (e.g., E2A, E4 and VA RNA genes) required for the production of infectious AAV viral particles are mostly provided by pHelper plasmids co-transfected into cells by other plasmids, and the remaining adenovirus gene products are provided by AAV-293 host cells stably expressing adenovirus E1 genes. AAV-293 Shenmen cell is an aurora Longshen cell line derived by winkle improvement of HEK293 adeno-associated virus production capacity. As a result of no longer requiring live Helper virus, AAV Helper-Free System provides a safer and more convenient gene delivery System for retroviral and adenoviral replacement

Currently, there is a well established set of techniques for the treatment of atherosclerotic diseases, i.e. for the clearance of low density lipoprotein LDL to a large extent. However, the administration of this measure still does not prevent the occurrence of serious cardiovascular diseases to a great extent, especially in the middle and advanced stage of atherosclerosis patients, and therefore it is imperative to find new directions for treating atherosclerosis.

Disclosure of Invention

The invention provides a recombinant adeno-associated virus vector (adeno-associated virus vector) for mediating the specific ectopic expression of LOX-1 in liver to treat atherosclerosis, a gene composition and application thereof, wherein the adeno-associated virus vector is used for efficiently introducing a drug effect element into the body through intravenous injection to realize the ectopic efficient expression of a drug effect element expression product therapeutic action protein LOX-1, and an LOX-1 receptor which is not expressed in liver is successfully expressed in liver cells through a TBG promoter, so that the overloaded OX-LDL in the circulation of an atherosclerotic patient is phagocytized and removed by virtue of the strong lipid metabolism capability and metabolic pathway of the liver, and the progression of atherosclerosis is further inhibited.

In order to achieve the above technical objects, the present invention provides a recombinant adeno-associated virus vector for treating atherosclerosis, comprising: the recombinant adeno-associated virus vector is obtained by inserting a Lectin-like Oxidized Low Density Lipoprotein Receptor 1(LOX-1) gene into an adeno-associated virus vector, wherein the sequence of the LOX-1 gene is shown as SEQ ID NO:1 in a sequence table.

The invention has the following excellent technical scheme: the adeno-associated virus vector is AAV8 virus.

The further technical scheme of the invention is as follows: the recombinant adeno-associated virus vector is characterized in that a LOX-1 gene is cloned into an AAV8 vector, and enzyme cutting sites selected are HindIII and BamHI.

The invention also provides a genome composition for treating atherosclerosis, which is characterized in that: the genome composition comprises the recombinant adeno-associated virus vector for treating atherosclerosis according to claim 1 and a TBG promoter; the gene sequence of the TBG promoter is shown as SEQ ID NO:2 in the sequence table.

The invention has the following excellent technical scheme: AAV8 virus is adopted as the adeno-associated virus vector in the recombinant adeno-associated virus vector; the gene sequence of the gene composition is shown as SEQ ID NO. 3 in the sequence table.

The invention also provides application of the genome composition for treating atherosclerosis, and particularly relates to application of the genome composition in preparing a gene therapy injection for treating atherosclerosis.

The recombinant adeno-associated virus vector can efficiently introduce a drug effect element into a human body by utilizing the adeno-associated virus vector (adeno-associated virus vector) through intravenous injection, and realizes the ectopic efficient expression of a drug effect element expression product therapeutic action protein LOX-1. In order to realize liver ectopic specificity high-efficiency expression of LOX-1, AAV8 viral vectors are preferably selected according to the transduction characteristics of different serotypes of AAV in the invention. The invention also provides a composition comprising the recombinant adeno-associated virus vector, wherein the composition selects a TBG promoter, and the TBG promoter not only has strong capability of promoting gene expression, but also can be expressed only in liver, namely the TBG promoter is a promoter sequence which is specifically promoted only in liver; immediately after the TBG promoter is the sequence of the LOX-1 gene of interest, the LOX-1 protein can be expressed, and the combination of the two and the application of the hepatotropic AAV8 vector can successfully transfect the LOX-1 sequence into the liver cells and successfully express the LOX-1 sequence only on the liver cells.

The target Gene is obtained by searching LOX-1 Gene sequence through a Gene database in NCBI, selecting a proper plasmid vector to construct and recombine to obtain a recombinant plasmid after obtaining a corresponding Gene sequence, wherein the selected enzyme cutting sites are HindIII and BamHI; the recombinant plasmid is packaged by adopting the existing adeno-associated virus packaging process, and then is subjected to virus collection, AAV virus concentration, AAV purification and ultrafiltration desalination in sequence, glycerol is added into virus concentrated solution to enable the final concentration to be 5%, and the virus concentrated solution is stored at-80 ℃ after subpackaging. And detecting the genome copy number of the AAV vector by adopting a quantitative PCR method to determine the virus particle number of the AAV. The accuracy and reliability of the standard curve absolute quantitative PCR detection GC titer are the most critical elements of AAV quality control, and the result influences the accuracy of downstream experiments. Therefore, in our core quality control step, GC titration of AAV was well designed to ensure its accuracy and stability.

The construction process of the recombinant plasmid of the invention is as follows:

(1) the LOX-1 Gene is found from a Gene database in NCBI to be mainly expressed in human umbilical vein endothelial cells, RNA is extracted by the cells (Trizol extraction), and after reverse transcription (reverse transcription kit), PCR (high fidelity enzyme is used for PCR to avoid mismatching) is carried out by using a synthesized primer with a protective base and a restriction enzyme site (the design method of the primer with the restriction enzyme site is shown in the end of a document). Adjusting and optimizing PCR conditions as required, then carrying out agarose electrophoresis (1%) on the PCR product, cutting off a gel block corresponding to the target gene fragment, carrying out gel recovery (using a gel recovery kit), and measuring the DNA concentration after the gel recovery, thereby obtaining a gene fragment with an enzyme cutting site;

(2) carrying out double enzyme digestion on the LOX-1 gene fragment with the enzyme digestion site and the vector plasmid respectively to obtain a viscous tail end; the conventional NEB enzyme digestion system has high efficiency and is completed within 5-15 minutes generally. If the enzyme digestion is not complete, the digestion time can be prolonged appropriately (the digestion reaction can be carried out in an EP tube or a pcr tube, using a 37 ℃ metal bath). After the enzyme digestion is finished, agarose electrophoresis and gel recovery are carried out on the LOX-1 and the plasmid, the concentration of gel recovery products is determined, and the LOX-1 and the vector plasmid at the moment are provided with double enzyme digestion site cohesive ends.

(3) The operation of ligation of LOX-1 gene and plasmid was carried out by T4 ligase, and the reaction system, ligation time and temperature were referred to the purchased T4 ligase instruction manual. After ligation was completed, 5. mu.l of the product was taken for transformation (transformation refers to a process of introducing the objective plasmid into competent cells).

The invention has the beneficial effects that: the invention has definite specificity, only transfects the liver, plays a role through the specific ectopic expression of the liver, has definite safety problem, and has proved that the in vivo safety is ensured through experimental detection. The invention not only combines the advanced direction of gene therapy, but also jumps out of the conventional main trend of the current atherosclerosis treatment, searches a new treatment direction, can be successfully transformed and applied to clinical treatment, and provides a new treatment direction and means for clinicians to treat atherosclerosis on the basis of reducing LDL.

Drawings

FIG. 1 is a schematic diagram of the construction sequence of the virus of the present invention, wherein the promoter is selected from the liver-specific promoter, thyroid-associated globulin (TBG) promoter, and the target gene is LOX-1;

FIG. 2 is a schematic diagram of the structure of the GV599 vector in example;

FIG. 3 is a comparison graph of results of observing the expression of green fluorescent protein eGFP carried in AAV8-TBG-LOX-1 viral vector by using DAPI stained nuclei of frozen sections of heart, liver, spleen, lung and kidney of mice in the virus group and the control group in the test group after two weeks of injection under a confocal laser microscope with the same photographing parameters;

FIG. 4(A) is a comparison of LOX-1 expression in the liver of mice in the control group and the virus group in the Westernblot experiment of test two after 1-4 weeks of AAV8 transfection;

FIG. 4(B) is a comparison of the results of statistical analysis of LOX-1 expression levels in control and virus mice in test two after 1-4 weeks of virus transfection;

FIG. 4(C) is a graph showing comparison of LOX-1 expression in livers of mice in the control group and the virus group detected by immunohistochemistry in test two;

FIG. 4(D) is a comparison graph of LOX-1 expression in livers of mice in the control group and the virus group detected by immunofluorescence in experiment two, with a magnification of 100 ×;

FIG. 5 is a photograph showing the Control, AAV8-TBG-LXO-1(2w) and AAV8-TBG-LXO-1(4w) three groups of tissue sections in the third experiment, which are used for observing the phagocytosis of Ox-LDL by LOX-1 in the liver under a laser confocal microscope; observing at a magnification of 100 x;

FIG. 6(A) is a graph comparing the curves of OX-LDL in circulation in mice injected with control and virus mice for 2-8 weeks in the four experiments;

FIG. 6(B) is a bar graph showing statistical comparison of OX-LDL levels in circulation in mice injected for 2-8 weeks in control and virus mice in the four trials;

FIG. 7(A) is a histological structure of liver of a control mouse and a virus mouse in five experimental groups observed under a light microscope after HE staining of liver sections;

FIG. 7(B-G) is a comparison graph of the liver and kidney function-related markers ALT, AST, TBIL, ALB, Cr, and BUN 4 weeks after injection of control mice and virus mice in the fifth experiment;

FIG. 8 is a graph comparing the degree of progression of atherosclerotic plaques in the six groups observed by oil red O staining;

FIG. 9 is a statistical histogram of the area fraction of atherosclerotic plaques in the aortic annulus for the two groups of six experiments.

Detailed Description

The present invention will be further described with reference to the following examples, and the present invention will be further described with reference to the accompanying drawings and examples. The drawings are for purposes of illustrating embodiments of the invention only and for purposes of clarity and conciseness. The following claims presented in the drawings are specific to embodiments of the invention and are not intended to limit the scope of the claimed invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment provides a recombinant adeno-associated virus vector for treating atherosclerosis, which is obtained by inserting LOX-1 gene into AAV8 virus, wherein the sequence of the LOX-1 gene is shown as SEQ ID NO:1 in the sequence table, and the selected enzyme cutting sites are HindIII and BamHI. The genome composition comprises the recombinant adeno-associated virus vector and a TBG promoter; the gene sequence of the TBG promoter is shown as SEQ ID NO. 2 in the sequence table, and the gene sequence of the gene composition is shown as SEQ ID NO. 3 in the sequence table. When genome synthesis is carried out, the TBG promoter can be inserted into an adeno-associated virus vector, and then a target gene is inserted; the vector can also be packaged by directly using an adeno-associated virus vector into which a TBG promoter has been inserted, and then inserting a target gene.

The insertion, transfection, packaging and application of the LOX-1 gene are further described below using AAV8 virus with a TBG promoter inserted directly as a vector. In the following example I, the AAV8 viral vector is selected from the group consisting of the vectors of GV599, a model of Shanghai Jikai GeneSciensis medical science and technology Co., Ltd, and the schematic structure thereof is shown in FIG. 2, and the present inventors used the vectors to directly insert and combine the gene LOX-1 of interest into the AAV8-TBG-LOX-1 adeno-associated viral vector.

EXAMPLE I AAV8-TBG-LOX-1 adeno-associated virus vector preparation and packaging process is detailed as follows:

(1) obtaining a target gene: the LOX-1 Gene is found from a Gene database in NCBI to be mainly expressed in human umbilical vein endothelial cells, RNA is extracted by the cells (Trizol extraction), and after reverse transcription (reverse transcription kit), PCR (high fidelity enzyme is used for PCR to avoid mismatching) is carried out by using a synthesized primer with a protective base and a restriction enzyme site (the design method of the primer with the restriction enzyme site is shown in the end of a document). PCR conditions were adjusted and optimized as needed. The PCR product was then subjected to agarose electrophoresis (1%), and the gel corresponding to the target gene fragment was excised and recovered (using a gel recovery kit), and the DNA concentration was measured after gel recovery. At this time, we obtained a gene fragment with a cleavage site.

(2) Carrying out double enzyme digestion on the LOX-1 gene segment with enzyme digestion sites and the GV599 vector plasmid respectively to obtain cohesive ends, wherein the efficiency of a common NEB enzyme digestion system is high and is generally finished within 5-15 minutes; in order to ensure incomplete enzyme digestion, the enzyme digestion time can be properly prolonged (the enzyme digestion reaction can be carried out in an EP tube or a pcr tube, and a 37-degree metal bath is adopted); after enzyme digestion is finished, agarose electrophoresis and gel recovery are carried out on the LOX-1 and the plasmid, the concentration of a gel recovery product is determined, and the LOX-1 and the vector plasmid at the moment are provided with double enzyme digestion site cohesive ends; connecting LOX-1 gene and plasmid by T4 ligase to obtain AAV8-TBG-LOX-1 recombinant expression plasmid, the gene sequence of which is shown as SEQ ID NO. 3 in the sequence table; the reaction system, ligation time and temperature were determined according to the purchased T4 ligase manual, and after ligation, 5. mu.l of AAV8-TBG-LOX-1 recombinant expression plasmid was transformed (transformation refers to the process of introducing the objective plasmid into competent cells).

(3) Packaging of AAV: co-transfecting the AAV8-TBG-LOX-1 recombinant expression plasmid in the step (2) with pHelper (carrying adenovirus-derived genes) and pAAV-RC (carrying AAV replication and capsid genes) into AAV-293 cells (providing trans-acting factors required for AAV replication and packaging), and assembling the AAV8-TBG-LOX-1 recombinant expression plasmid in packaging cells after 2 to 3 days of transfection; the method comprises the following specific steps:

a. preparing AAV-293 cells: 3X 106 AAV-293 cells were added to 10ml DMEM growth medium per 100-mm tissue culture plate for transfection 48 hours later; AAV-293 cells are transfected by calcium phosphate transfection.

b. Two days prior to transfection, host cells passaged should reach 70-80% confluence; taking out AAV8-TBG-LOX-1 recombinant expression plasmid to be cotransfected from a refrigerator of-20 ℃, and adjusting the concentration of the plasmid to 1mg/ml by using a TE buffer solution with pH 7.5;

c. calculating the required transfection system and plasmid dosage according to the number of packing discs, if one disc is packed, sucking 10u (10ug each) of each of the three plasmids into a 1.5ml EP centrifuge tube, then adding 1ml of 0.3M CaCl2, and gently mixing to obtain a DNA/CaCl2 mixed solution;

d. sucking 1ml of 2X HBS solution into another 15ml conical bottom tube, dropwise adding 1.03ml of DNA/CaCl2 mixed solution obtained in the step c (the previous step), and performing turnover or repeated blowing and beating and uniformly mixing to obtain DNA/CaCl2/HBS solution;

e. immediately dripping the mixed DNA/CaCl2/HBS solution onto a cell culture disc, slightly shaking the cell culture disc while adding the mixed DNA/CaCl2/HBS solution to ensure that the solution is uniformly distributed in a culture medium as much as possible, and returning the cell culture disc to a 37-degree culture box to be placed for 6 hours for transfection;

h. after transfection was complete, the medium in the plates was replaced with 10ml of fresh medium and the plates were returned to the incubator for an additional 66-72 hours.

After AAV packaging is completed, the AAV particle packaging progress is judged by observing the morphological change of AAV-293 cells. For convenience, it is necessary to make a negative control for packaging a baculovirus (e.g., transfection group without DNA). The most obvious sign of the success of viral packaging is the color of the medium changing from red to orange to yellow of withdrawal (no negative control). As the virus came out, some of the blasts rounded up and fell off the disc, and they were seen to float in the medium.

(4) And (3) toxin collection: preparing a dry ice ethanol bath (ethanol is poured into a foam box filled with dry ice, or liquid nitrogen is used for replacing the dry ice ethanol bath) and a water bath at 37 ℃, collecting the virus-producing cells and the culture medium into a 15ml centrifuge tube, and when the cells are collected, inclining the culture tray by a certain angle to scrape the cells into the culture medium; centrifuging at 200g for 3 min under centrifugation conditions to separate cells and supernatant, storing the supernatant separately, and resuspending the cells with 1ml of PBS; repeatedly transferring the cell suspension in a dry ice ethanol bath and a water bath at 37 ℃, freezing and thawing for four times, and slightly shaking after each thawing; cell debris was then removed by centrifugation at 10000g and the centrifuged supernatant was transferred to a fresh centrifuge tube.

(5) Concentration of AAV virus: adding 40% PEG8000 to the supernatant collected in the step (4) until the final concentration is 8%, placing on ice for 2 hours (mixing uniformly every 15 minutes), centrifuging at 2500g for 30 minutes, removing the supernatant, resuspending the precipitate with PBS, and mixing with the cell lysis supernatant; then centrifuging at 3000g for 30 min, transferring the supernatant to another clean tube, where the supernatant should not have visible cell debris, and centrifuging again if some debris remains; finally, the residual plasmid DNA (final concentration of 50U/ml) was removed by digestion with Benzonase nuclease, the tube cap was closed, inverted several times to mix well, incubated at 37 ℃ for 30 minutes, filtered through a 0.45 μm filter and the filtrate was taken.

(6) Purification of AAV virus: adding solid CsCl into the virus concentrated solution until the density is 1.41g/ml (the refractive index is 1.372), adding 6.5g CsCl into about 10ml virus solution, and shaking to dissolve the CsCl, wherein the dissolved CsCl absorbs heat and cools; the sample was then added to an ultracentrifuge tube, the remaining space of the tube was filled with a pre-prepared 1.41g/ml CsCl solution, and centrifuged at 175,000g for 24 hours to form a density gradient; collecting samples with different densities step by step in sequence, sampling for titer determination, and collecting the components enriched with AAV particles; the above process is repeated once.

(7) And (3) ultrafiltration desalination: 4ml of deionized water is added into an Amicon-15 ultrafiltration device; adding the recombined virus obtained by density gradient centrifugation in the step (6) into an ultrafiltration device, adding PBS (phosphate buffer solution) to the total volume of 4ml, and covering a cover; centrifugation at 1500g for about 5 to 10 minutes, checking the remaining volume every 5 minutes until the final volume is 200-; the above procedure was repeated 3 times, and the ultrafiltration tube was centrifuged to give a final volume of about 0.5ml of AAV8-TBG-LOX-1 recombinant virus concentrate.

For the determination of virus titer in the examples, the viral particle number of AAV was determined by detecting the genome copy number of the recombinant AAV vector by quantitative PCR method. The accuracy and reliability of GC titer detection by standard curve absolute quantitative PCR are the most key elements of AAV quality control, and the result influences the accuracy of downstream experiments. Therefore, in our core quality control step, GC titration of AAV was well designed to ensure its accuracy and stability.

Example two: diluting the AAV8-TBG-LOX-1 adeno-associated virus vector with the virus titer of 2x 1013vg (virus genes)/ml into 2x 1011 vg/cell/100 mul by using a sterile PBS buffer solution, taking 10ul virus stock solution and 90ul sterile PBS, configuring in a super clean bench, mixing, blowing and uniformly mixing by using a pipette, placing in an ice box for low-temperature storage, and preparing on site to avoid the influence of repeated freeze thawing on the virus titer; then 100ul of the virus dilution was drawn up with an insulin needle and injected into mice via tail vein, waiting for observation of transfection effect. Normally, the virus can be stored in a-80 refrigerator, and after injection, the virus can be stably expressed at about 2 weeks after injection, and the duration of the stable expression can be at least 2 months.

The expression of AAV8-TBG-LOX-1 adeno-associated virus vector in liver, transfection effect and treatment effect of atherosclerosis were studied in combination with different experiments, and the experimental process was carried out by using mice injected with the same amount of physiological saline as a Control group (Control) for comparative study.

The transfection effect of a pair of viruses in mice is tested: observing the expression of the self-carried eGFP protein (enhanced green fluorescent protein) on the adeno-associated virus vector under a laser confocal microscope by DAPI staining; the specific operation process comprises the following steps:

1. 7 mice injected with AAV8-TBG-LOX-1 virus diluent in the second implementation group are taken as the virus group, and 7 mice injected with the same amount of physiological saline are taken as the control group; mice were sacrificed 2 weeks after AAV8 injection.

2. The heart of the mouse is exposed, and the heart perfusion is carried out by using pre-cooled physiological saline, and the samples of aorta, heart, liver, spleen, lung, kidney and the like are reserved.

3. Freezing: the microtome is precooled to-20 ℃ in advance, and the tissue support is placed in the microtome for freezing by using the embedding medium coating in advance.

4. Slicing: the thickness of the liver slices is 10 mu m, and 6-8 slices are reserved for one liver specimen; the frozen sections of the aortic root tissue are slightly different, and the thickness of the sections is 6 mu m; the frozen section method of tissue specimens of spleen, kidney, lung and the like is the same as that of liver;

5. and (4) observation: after the section is finished, the Hoechst staining solution is added into the section gently to stain the nucleus, the section is placed under a laser confocal microscope to observe the expression condition of the virus self-carried fluorescent eGFP (green fluorescence), and the same parameters are fixed during each shooting; the shooting result is shown in fig. 3, and only eGFP green fluorescent protein in the liver-specific expression virus sequence of the frozen section of the heart, liver, spleen, lung and kidney of the mouse is observed through laser confocal observation; showing that the virus sequence is specifically ectopically transfected and expressed in the liver of the mouse after the virus injection; wherein the picture in figure 3 is magnified at 200 x.

And (2) test II: selecting live test mice, injecting 100ul AAV8-TBG-LOX-1 virus diluent (10ul virus stock solution +90ul sterile PBS) prepared in example II into the virus group through tail vein; the Control group (Control) is injected with an equal amount of normal saline through tail vein, mice of the virus group and the Control group are killed in batches 1-4 weeks after injection, and heart, liver, spleen, lung and kidney specimens of the mice are obtained through heart perfusion for corresponding detection.

a. Taking a control group mouse and 0.1g of mouse liver after 1-4 weeks of virus transfection, using 200ul RIPA lysate to crack liver tissues to obtain liver total protein supernatant, using BCA method to measure the total protein concentration of the supernatant, configuring a system with 50mg liver protein amount per hole, preparing 10% SDS-Page gel according to the instruction, setting the first hole as a control group, setting two holes per week of virus group, carrying out sample application, gel running and membrane transfer, then using an anti-LOX-1 antibody and an anti-GAPDH antibody to respectively incubate a PVDF membrane, and after the incubation is finished, carrying out luminescence in a chemiluminescence instrument to obtain a Wensterblot strip. As a result, as shown in FIG. 4(A), the virus group successfully expressed LOX-1 after virus transfection, and the expression amount gradually increased with the passage of time until the maximum was reached at week 4.

b. The mouse intervention mode was as described in a, the LOX-1 and GAPDH bands were measured using Image Lab software, the ratio of the two bands was analyzed statistically, and the corresponding statistical graph is shown in FIG. 4(B), the weekly ratio of the virus group to the control group was subjected to t-test between the two groups,: p < 0.01.

c. Livers of virus group week 4 mice and control group week 4 mice were packaged into paraffin blocks by paraffin embedding and then sectioned (6um) at 1: the anti-LOX-1 antibody was diluted at a dilution ratio of 200 to perform immunohistochemical staining, in which dark brown color was an expression region of LOX-1 and blue color was nuclei, and the results are shown in FIG. 4(C), which shows that the liver of the virus group at week 4 extensively expressed LOX-1 in large amounts, and the expression was significantly different from that of the control group.

d. Livers of the virus group week 4 mice and the control group week 4 mice were packaged into tissue wax blocks by paraffin embedding method, and then sectioned (6um) at 1: the anti-LOX-1 antibody was diluted at a dilution ratio of 100 for immunofluorescence staining, in which red is the LOX-1 expression region and blue is the nucleus, and the comparison results are shown in FIG. 4(D), where the liver of the virus group extensively expressed LOX-1 in large amounts at week 4, and the expression difference from the control group was significant.

In the second test, the successful expression of the LOX-1 protein in the liver of the mouse is proved by Western Blot and immunohistochemistry; immunofluorescence demonstrated successful expression of LOX-1 in mouse liver.

And (3) testing three: selecting live test mice, injecting 100ul AAV8-TBG-LOX-1 virus diluent (10ul virus stock solution +90ul sterile PBS) prepared in example II into the virus group through tail vein; the control group was injected with 100ul of physiological saline through the tail vein; at 2 weeks and 4 weeks after injection into mice, mice in virus group and experimental group were respectively injected with OX-LDL 100ul (10ul Dil +90ul PBS) containing red fluorescent probe Dil via tail vein, mice were sacrificed 8h after injection, liver specimens were obtained, 10um sections were obtained by freezing sections, Control, AAV8-TBG-LXO-1(2w), AAV8-TBG-LXO-1(4w) tissue sections were observed under the same parameters by laser confocal microscope, and the observation results were shown in FIG. 5 at a magnification of 100 x. Wherein the red fluorescence represents Dil-Ox-LDL, and the blue is cell nucleus stained by DAPI, the result shows that the liver of the control group does not phagocytose the Dil-Ox-LDL basically, but the virus group phagocytoses obviously, and the phagocytosis effect is more obvious from the time of virus transfection to the 4 th week. These data indicate that our genomic composition is not only successfully expressed in mouse liver, but is also able to successfully exert its function, phagocytosis removes circulating OX-LDL.

And (4) testing four: after 2-8 weeks of virus injection in example two, we obtained blood samples from mice injected with virus by means of orbital and eye-picking (fasting for 12h before blood collection), and after standing for 1h, centrifuged at 3500rpm/min for 15min to obtain supernatant, and stored at-80 ℃. After all blood samples are sampled, detecting the content of OX-LDL by using a 96-hole ELISA kit, and performing statistical analysis on the detection result; in the same time, control mice injected with 100ul of physiological saline through tail vein were compared, and the OX-LDL change trend of both mice was shown in FIG. 6(A) and bar chart 6 (B); the test result shows that the OX-LDL in the circulation is continuously phagocytosed by the liver 2-8 weeks after the injection of the virus, and the content of the OX-LDL in the circulation is reduced. The statistical method uses t test: p <0.05, P < 0.01.

And (5) testing: the group of mice was injected caudally with 100ul of the AAV8-TBG-LOX-1 dilution prepared in example two (10ul of stock virus plus 90ul of sterile PBS); control mice were injected with 100ul of saline through the tail vein; and (3) performing paraffin embedding on the livers of the control group mouse and the virogroup mouse, performing section acquisition on a 6um paraffin section, and performing HE (high-intensity eosin) staining to observe whether the tissue structure of the livers is changed under a light microscope. As shown in FIG. 7(A), the cells in liver of AAV8-TBG-LOX-1 group were structurally intact, morphology was not changed at all, and the magnification was 200X in the virus group compared with the control group.

The inventor adopts the way of orbit blood collection and eyeball-picking blood collection to obtain the blood sample of mice in the 4 th week after virus intervention and a control group (fasting is 12h before blood collection), stands for 1h and then centrifuges at 3500rpm/min for 15min to obtain supernatant, an automatic biochemical detector is used for detecting liver and kidney function related indexes ALT, AST, TBIL, ALB, Cr and BUN to obtain the content of the corresponding indexes, then Prism software is used for carrying out statistical analysis to obtain a histogram such as a graph shown in figure 7(B-G), and the result shows that the liver and kidney function indexes in the detection cycle show that the virus group is not different from the control group, thereby proving that the injection and gene treatment of the virus do not cause other damages. The statistical method uses t test: p <0.05, P < 0.01.

And (6) test six: selecting live test mice, injecting 100ul AAV8-TBG-LOX-1 virus diluent (10ul virus stock solution +90ul sterile PBS) prepared in example II into the virus group through tail vein; the control group was injected with 100ul of physiological saline through the tail vein; in 4 weeks after virus injection, heart specimens of mice of a Control group and a virus group are obtained, frozen sections are obtained to obtain frozen sections (6um) of heart aortic valve annulus positions, then oil red O staining is carried out, the degree of progression of atherosclerotic plaques of the two groups is observed through the oil red O staining, and the result is shown in figure 8, it is found that the Control group not only has rapid atherosclerosis progression and obvious cholesterol necrotic core, but also has the progression of the AAV8-TBG-LOX-1 group, but has obvious slow progression and no necrotic core formation compared with the Control group, and the amplification factor is 100x (left) and 200x (right); the ratio of the area of the oil red O staining positive (dark red zone) to the area of the whole aortic annulus was obtained and statistical analysis was performed using Prism software to obtain a histogram as shown in fig. 9, the statistical method selected t test with P < 0.0001. As can be seen by comparing FIG. 8 and FIG. 9, the clearance of circulating OX-LDL by the liver significantly inhibited the progression of atherosclerosis.

Sequence listing

<110> affiliated cooperation hospital of college of Tongji medical college of Huazhong university of science and technology

<120> recombinant adeno-associated virus vector for treating atherosclerosis, genome composition and application

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3581

<212> DNA

<213> mouse (Mus musculus)

<400> 1

ggcagttggc tgaggtcctc gactgtttca gtttttcact cttagcagga atttggagat 60

gacttttgat gacaagatga agcctgcgaa tgacgagcct gatcagaagt catgtggcaa 120

gaagcctaaa ggtctgcatt tgctttcttc cccatggtgg ttccctgctg ctatgactct 180

ggtcatcctc tgcctggtgt tgtcagtgac ccttattgta cagtggacac aattacgcca 240

ggtatctgac ctcttaaaac aataccaagc gaaccttact cagcaggatc gtatcctgga 300

agggcagatg ttagcccagc agaaggcaga aaacacttca caggaatcaa agaaggaact 360

gaaaggaaag atagacaccc tcacccagaa gctgaacgag aaatccaaag agcaggagga 420

gcttctacag aagaatcaga acctccaaga agccctgcaa agagctgcaa actcttcaga 480

ggagtcccag agagaactca agggaaagat agacaccatc acccggaagc tggacgagaa 540

atccaaagag caggaggagc ttctgcagat gattcagaac ctccaagaag ccctgcagag 600

agctgcaaac tcttcagagg agtcccagag agaactcaag ggaaagatag acaccctcac 660

cttgaagctg aacgagaaat ccaaagagca ggaggagctt ctacagaaga atcagaacct 720

ccaagaagcc ctgcaaagag ctgcaaactt ttcaggtcct tgtccacaag actggctctg 780

gcataaagaa aactgttacc tcttccatgg gccctttagc tgggaaaaaa accggcagac 840

ctgccaatct ttgggtggcc agttactaca aattaatggt gcagatgatc tgacattcat 900

cttacaagca atttcccata ccacctcccc gttctggatt ggattgcatc ggaagaagcc 960

tggccaacca tggctatggg agaatggaac tcctttgaat tttcaattct ttaagaccag 1020

gggcgtttct ttacagctat attcatcagg caactgtgca taccttcaag acggagctgt 1080

gttcgctgaa aactgcattc taattgcatt cagcatatgt cagaagaaga caaatcattt 1140

gcaaatttag tgaatctaaa gattctggag aagaccatga gaagactttt gactgtcgct 1200

ctgaaattta agctattctt tattacctgc atgtaaagca tgttggcctt gacatctgtc 1260

agttacctga tagctacagt tcacctcaac aaagacaagg accagaagca aatactggtg 1320

gatccagatg tttgaaatct ttgtatcaaa acgtgtgagt tcaattgttt atccatatac 1380

actggccttg cccctccaaa gtctcccaac caacctgcaa tccttcttcc ccttcgtgtt 1440

ttaaatgatg cttcctgcct gacctggcca tgctttgtac tcagtctcct ctacctcagt 1500

atgcctcctg ttgccgcatg aaagacagga tgtagaaatc ctcctcaagt gcaggcagag 1560

ggctcaaagg caaagctcgt ccgagaaata acatgcaaag aaacagaact ggaaaaacta 1620

cactgcaaac aggaactcat gatctctaaa aagccatggc ttgatcttta tgacaggagt 1680

ccatctccag actgcacttt tacacacata ttttttattt tccttttatt gtaagtttat 1740

ggatagtttg cctacttgaa ttctgtgtac cacatgagtg cctggtgcca ctgaagatca 1800

gaagaggaca tcagattctc tggatctgga attgcagatg gttgtaagct gctatgtaga 1860

tgtgaagaat tgaattcgtg tcccctggaa gaacagccag tactctttgc catgagctat 1920

ctctcaagct cctgtgatca attcttgtat cagttatgtc tccatttttg ctctaccaaa 1980

gaacagtgtt ataactttaa aacagtaagt attttcatta ttctaggggt attatagcag 2040

atatatagat atagatatag atgatataga tatagatata gatatagata tagatataga 2100

tatagatata gatatagata tagatataga tatagatata gatatagagt gtgtgtgtgt 2160

gtgtgtgtgt gtgtgtgtgt gtgtttctat gctagataca tccttgagaa gatgagacag 2220

ttttgtgtga aatgagtttg taataatcca aatttaaaaa taaattcgat gattacctgt 2280

agtggtcata ttaccacagc taagatgatg aacatacctg tcacttctgc ccctttccaa 2340

agcctccccc ctaaaacaaa caccaatctg ctttcagttc gcattttata gagcttatca 2400

ttttgttttt aagacagaat ctcattatat agttctggct ggcctggaac tcactacaca 2460

gagcaggctg gcctccacct tctagagctc ctcctgccag actcccaaac cttaagactg 2520

aaggagtgcc ctgccatgtg tgactcaaac accttaatgt gaatggaata gcataagatg 2580

tccaggtttt ttcagtccag cttcttccac ttggtacaat ttttaatttt tgtgttcata 2640

catctccacc acagtgtttg tatcagttca tcattctttt caaatgttga gccttcccct 2700

gtggatctat agtgtcattt gttatctgtg tatttgttga tgcgatttgg gttgttttta 2760

tttggggtca cctacaaata aagctgctat ggatgtccat ggacaaggct aatatcttag 2820

gtaagcacct acgagtaaga tgcttgggtc attcagtgtg ggaatatatg gttggctatt 2880

ttaaccattc ctgtttgaaa acattaattt ttttattttt gaaatcaatt ttttaaaaaa 2940

ttagtctatt ttacatctca accccagttt ctcttcctcc tctcctctca accttctccc 3000

accttctctc cctgacccca tccacccctc ctccctttct ctccagaaga ggggaggcct 3060

cccatggatg ccaaccagcc tcagcatctc aagttacagt aagaataggt ttgtcttctc 3120

ttgtgaaaat cttaattttt agatttatct attatatatg cagtactttg cttgcacata 3180

tgtattggta ccatgtacat gaatggtacc agagaaagtc agaagaggcc attgtatttt 3240

ctgggactgg aattacagac ggttttgaac aatcctatag actctgggaa ctgaacccag 3300

gtcctctgga aaggcaagca gtgctcttaa cccctgagcc atttcttctg gccttttagc 3360

aatttttatt aatatataac tgtgtataat tgcactttta gctcaaagtt cttaagtgtc 3420

aaatagtctt ggatttattt tcatgttatc actgtctgta caatttctgt gatgaaataa 3480

ctgagcatat attttgagaa tttggttttc ttaaatttta aatctgaagg atttacatat 3540

ttgagaataa aaaccagcca gatatgaaaa aaaaaaaaaa a 3581

<210> 2

<211> 561

<212> DNA

<213> mouse (Mus musculus)

<400> 2

agggctggaa gctacctttg acatcatttc ctctgcgaat gcatgtataa tttctacaga 60

acctattaga aaggatcacc cagcctctgc ttttgtacaa ctttccctta aaaaactgcc 120

aattccactg ctgtttggcc caatagtgag aactttttcc tgctgcctct tggtgctttt 180

gcctatggcc cctattctgc ctgctgaaga cactcttgcc agcatggact taaacccctc 240

cagctctgac aatcctcttt ctcttttgtt ttacatgaag ggtctggcag ccaaagcaat 300

cactcaaagt tcaaacctta tcattttttg ctttgttcct cttggccttg gttttgtaca 360

tcagctttga aaataccatc ccagggttaa tgctggggtt aatttataac taagagtgct 420

ctagttttgc aatacaggac atgctataaa aatggaaaga tgttgctttc tgagagacag 480

ctttattgcg gtagtttatc acagttaaat tgctaacgca gtcagtgctt ctgacacaac 540

agtctcgaac ttaagctgca c 561

<210> 3

<211> 1367

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggttaatgct ggggttaatt tataactaag agtgctctag ttttgcaata caggacatgc 60

tataaaaatg gaaagatgaa ttcggaactg gaggtggagg tagtggaatg gatccgccac 120

catgactttt gatgacaaga tgaagcctgc gaatgacgag cctgatcaga agtcatgtgg 180

caagaagcct aaaggtctgc atttgctttc ttccccatgg tggttccctg ctgctatgac 240

tctggtcatc ctctgcctgg tgttgtcagt gacccttatt gtacagtgga cacaattacg 300

ccaggtatct gacctcttaa aacaatacca agcgaacctt actcagcagg atcgtatcct 360

ggaagggcag atgttagccc agcagaaggc agaaaacact tcacaggaat caaagaagga 420

actgaaagga aagatagaca ccctcaccca gaagctgaac gagaaatcca aagagcagga 480

ggagcttcta cagaagaatc agaacctcca agaagccctg caaagagctg caaactcttc 540

agaggagtcc cagagagaac tcaagggaaa gatagacacc atcacccgga agctggacga 600

gaaatccaaa gagcaggagg agcttctgca gatgattcag aacctccaag aagccctgca 660

gagagctgca aactcttcag aggagtccca gagagaactc aagggaaaga tagacaccct 720

caccttgaag ctgaacgaga aatccaaaga gcaggaggag cttctacaga agaatcagaa 780

cctccaagaa gccctgcaaa gagctgcaaa cttttcaggt ccttgtccac aagactggct 840

ctggcataaa gaaaactgtt acctcttcca tgggcccttt agctgggaaa aaaaccggca 900

gacctgccaa tctttgggtg gccagttact acaaattaat ggtgcagatg atctgacatt 960

catcttacaa gcaatttccc ataccacctc cccgttctgg attggattgc atcggaagaa 1020

gcctggccaa ccatggctat gggagaatgg aactcctttg aattttcaat tctttaagac 1080

caggggcgtt tctttacagc tatattcatc aggcaactgt gcataccttc aagacggagc 1140

tgtgttcgct gaaaactgca ttctaattgc attcagcata tgtcagaaga agacaaatca 1200

tttgcaaatt ggctccggag ccacgaactt ctctctgtta aagcaagcgg gagatgtgga 1260

agaaaacccc ggtcccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc 1320

catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcg 1367

Claims (2)

1. A genomic composition for the treatment of atherosclerosis, wherein: the gene composition comprises a recombinant adeno-associated virus vector for treating atherosclerosis and a TBG promoter; the recombinant adeno-associated virus vector for treating atherosclerosis is obtained by cloning LOX-1 gene into AAV8 vector, and the selected enzyme cutting sites are HindIII and BamHI; wherein the sequence of the LOX-1 gene is shown as SEQ ID NO. 1 in a sequence table; the gene sequence of the TBG promoter is shown as SEQ ID NO. 2 in a sequence table; the gene sequence of the gene composition is shown as SEQ ID NO. 3 in the sequence table.

2. Use of the genomic composition for the treatment of atherosclerosis according to claim 1, characterized in that: the use of the genomic composition of claim 1 for the preparation of a gene therapy injection for the treatment of atherosclerosis.

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