ShRNA (short hairpin ribonucleic acid) for targeted knockout of Eif2ak2 gene and application

Abstract

The invention discloses shRNA (short hairpin Ribonucleic Acid) for targeted knockout of an Eif2ak2 gene and application, and belongs to the technical field of gene engineering. The shRNA comprises a positive-sense strand and an antisense strand which is reversely complementary with the positive-sense strand, and the nucleotide sequence of the positive-sense strand is shown as SEQ ID NO. 5. It is found for the first time that the recombinant adeno-associated virus capable of inhibiting Eif2ak2 gene expression can successfully inhibit the phenotype of the abdominal aortic aneurysm of a mouse, obviously reduce the morbidity of the abdominal aortic aneurysm of the mouse, improve the severity of the abdominal aortic aneurysm of the mouse, reduce the inner diameter of a blood vessel and the ratio of the weight to the body weight of the blood vessel, and promote the contraction function of vascular smooth muscle cells of the mouse; the method can be used for effectively treating the abdominal aortic aneurysm, provides a new thought and strategy for treating the abdominal aortic aneurysm, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to an shRNA for targeted knockout of the Eif2ak2 gene and its application. Background Art

[0002] Abdominal aortic aneurysm (AAA) is a type of cardiovascular disease that seriously threatens human life and health. Currently, there is no drug therapy that can effectively limit the growth or rupture of AAA. In recent years, adeno-associated viral vector (AAV) has become a key component of gene therapy as a vector. Given its characteristics of high safety, low immunogenicity, strong tissue specificity and affinity, and the ability to stably express in vivo, the AAV vector has become a candidate vector for gene therapy of cardiovascular diseases. Therefore, exploring drugs related to AAA gene therapy using adeno-associated virus as a delivery vector is of great significance for the clinical treatment of AAA. Summary of the Invention

[0003] The purpose of the present invention is to provide an shRNA for targeted knockout of the Eif2ak2 gene and its application, so as to solve the problems existing in the above-mentioned prior art. Using recombinant AAV9 virus to specifically knockdown the expression of Eif2ak2 in smooth muscle cells can significantly increase the contractile function of vascular smooth muscle, thereby maintaining the contractile phenotype of vascular smooth muscle cells and inhibiting the occurrence and development of AAA.

[0004] To achieve the above purpose, the present invention provides the following solutions:

[0005] The present invention provides an shRNA for targeted knockout of the Eif2ak2 gene. The shRNA includes a sense strand and an antisense strand that is reverse complementary to the sense strand. The nucleotide sequence of the sense strand is as shown in SEQ ID NO.5.

[0006] The present invention also provides a recombinant adeno-associated viral vector containing the shRNA.

[0007] The present invention also provides the application of the shRNA or the recombinant adeno-associated viral vector in the preparation of a drug for treating abdominal aortic aneurysm.

[0008] The present invention also provides the application of the shRNA or the recombinant adeno-associated viral vector in the preparation of a drug for reducing the incidence of abdominal aortic aneurysm.

[0009] The present invention also provides the application of the shRNA or the recombinant adeno-associated viral vector in the preparation of a drug for improving the severity of abdominal aortic aneurysm.

[0010] The present invention also provides the use of the shRNA or the recombinant adeno-associated virus vector in the preparation of a drug for reducing the inner diameter of blood vessels and / or the ratio of blood vessel weight to body weight.

[0011] The present invention also provides the use of the shRNA or the recombinant adeno-associated virus vector in the preparation of a drug for promoting the contractile function of vascular smooth muscle cells.

[0012] The present invention also provides a drug for treating abdominal aortic aneurysm, and the drug comprises the shRNA or the recombinant adeno-associated virus vector.

[0013] The present invention discloses the following technical effects:

[0014] The present invention discovers for the first time that a recombinant adeno-associated virus that inhibits the expression of the Eif2ak2 gene can successfully inhibit the phenotype of abdominal aortic aneurysm in mice, significantly reduce the incidence of abdominal aortic aneurysm in mice, significantly improve the severity of abdominal aortic aneurysm in mice, reduce the inner diameter of blood vessels, the ratio of blood vessel weight to body weight, and significantly promote the contractile function of vascular smooth muscle cells in mice. It can be used in the effective treatment of abdominal aortic aneurysm. The present invention provides new ideas and strategies for the treatment of abdominal aortic aneurysm and has broad clinical application prospects. In addition, the present invention provides a reference for the gene therapy research of other cardiovascular diseases and promotes the development of the gene therapy field. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0016] Figure 1 It is the bioinformatics analysis result of the transcriptome data of abdominal aortic aneurysm patients and normal people;

[0017] Figure 2 It is the map of the recombinant lentiviral vector pAV-SM22a-GFP-miR30-shRNA;

[0018] Figure 3 It is the construction flow chart of the abdominal aortic aneurysm mouse model;

[0019] Figure 4 It is the Western Blot diagram for verifying the knockdown efficiency of AAV9-SM22a-shEif2ak2;

[0020] Figure 5 It is the phenotype diagram of the effect of injecting AAV9-SM22a-shEif2ak2 on the inner diameter of blood vessels;

[0021] Figure 6 To investigate the effect of injecting AAV9-SM22a-shEif2ak2 on the incidence of abdominal aortic aneurysm in mice;

[0022] Figure 7 Echocardiogram of mice after injecting AAV9-SM22a-shEif2ak2;

[0023] Figure 8 To investigate the effect of injecting AAV9-SM22a-shEif2ak2 on the inner diameter of blood vessels in mice;

[0024] Figure 9 To investigate the effect of injecting AAV9-SM22a-shEif2ak2 on the ratio of blood vessel weight to body weight in mice;

[0025] Figure 10 Stained image of mouse blood vessel tissue after injecting AAV9-SM22a-shEif2ak2;

[0026] Figure 11 To investigate the effect of injecting AAV9-SM22a-shEif2ak2 on the degree of elastic fiber breakage in blood vessels of mice;

[0027] Figure 12 To investigate the effect of injecting AAV9-SM22a-shEif2ak2 on the protein expression level of vascular smooth muscle contraction genes;

[0028] Figure 13 Statistical results of the effect of injecting AAV9-SM22a-shEif2ak2 on the protein expression level of vascular smooth muscle contraction genes. Detailed implementation manners

[0029] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0033] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0034] Explanation of technical terms of the present invention: Eif2ak2: The coding gene of the PKR protein; MYH11: Myosin heavy chain 11; MYL9: Myosin light chain 9; CNN1: Calponin 1; α-SMA: α-smooth muscle actin.

[0035] The genes and protein sequences related to the present invention are shown as follows.

[0036] The amino acid sequence of Eif2ak2 (SEQ ID NO.1):

[0037] MASDTPGFYMDKLNKYRQMHGVAITYKELSTSGPPHDRRFTFQVLIDEKEFPEAKGRSKQEARNAAAKLAVDILDNENKVDCHTSASEQGLFVGNYIGLVNSFAQKKKLSVNYEQCEPNSELPQRFICKCKIGQTMYGTGSGVTKQEAKQLAAKEAYQKLLKSPPKTAGTSSSVVTSTFSGFSSSSSMTSNGVSQSAPGSFSSENVFTNGLGENKRKSGVKVSPDDVQRNKYTLDARFNSDFEDIEEIGLGGFGQVFKAKHRIDGKRYAIKRVKYNTEKAEHEVQALAELNHVNIVQYHSCWEGVDYDPEHSMSDTSRYKTRCLFIQMEFCDKGTLEQWMRNRNQSKVDKALILDLYEQIVTGVEYIHSKGLIHRDLKPGNIFLVDERHIKIGDFGLATALENDGKSRTRRTGTLQYMSPEQLFLKHYGKEVDIFALGLILAELLHTCFTESEKIKFFESLRKGDFSNDIFDNKEKSLLKKLLSEKPKDRPETSEILKTLAEWRNISEKKKRNTC。

[0038] The nucleotide sequence of Eif2ak2 (SEQ ID NO.2):

[0039]

[0040] Primer sequences for amplifying Eif2ak2:

[0041] FP (SEQ ID NO.3): 5'-TCGTGACCGGAGTGGAGTAT-3';

[0042] RP (SEQ ID NO.4): 5'-GGCCCAAAGCAAAGATGTCC-3'.

[0043] shRNA sequence (SEQ ID NO.5):

[0044] AGGAGTAGCCATTACGTATAAATAGTGAAGCCACAGATGTATTTATACGTAATGGCTACTCCG.

[0045] Interference target sequence (SEQ ID NO.6):

[0046] GGAGTAGCCATTACGTATAAA.

[0047] Example 1 Biological analysis

[0048] Bioinformatics analysis was performed using the publicly available transcriptome data of patients with abdominal aortic aneurysm and normal individuals (GSE47472). The results showed that the expression of Eif2ak2 in abdominal aortic aneurysm tissues was significantly higher than that in normal vascular tissues (see Figure 1 ).

[0049] Example 2

[0050] 1. Construction of lentiviral vector for RNA interference of Eif2ak2 gene

[0051] 1.1 Design of RNA interference target and preparation of double-stranded DNA oligo

[0052] (1) The amino acid sequence of the protein encoded by the Eif2ak2 gene is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0053] (2) Design of RNA interference target

[0054] According to the design principle of RNA interference sequence, multiple 19-21nt RNA interference target sequences were designed using the Eif2ak2 gene as a template. After evaluation and determination by design software, the SEQ ID NO.6 sequence shown in Table 1 was selected as the interference target.

[0055] (3) Synthesis of DNA oligo sequence

[0056] Design the shRNA interference sequence (sense strand and antisense strand) according to the selected target sequence (the sense strand is shown as SEQ ID NO.5 in Table 1, and the antisense strand sequence is reverse complementary to the sequence shown in SEQ ID NO.5), and add appropriate restriction endonuclease cleavage sites at both ends to complete the vector construction. In addition, add a TTTTT termination signal at the 3' end of the sense strand, and add a termination signal complementary sequence at the 5' end of the antisense strand. After the design is completed, send it to GenScript for the synthesis of single-stranded DNA oligo.

[0057] (4)Preparation of double-stranded DNA oligo

[0058] Dissolve the synthesized single-stranded DNA oligo dry powder in annealing buffer (final concentration 20 μM), and incubate in a 90°C water bath for 15 min. After natural cooling to room temperature, double-stranded DNA with sticky ends is formed.

[0059] 1.2 Enzyme digestion

[0060] Cut the miR30-shRNA of the mouse Eif2ak2 gene with SfaAI / MluI, and at the same time digest the pAV-SM22a-GFP vector. The gene digestion system is shown in the following table.

[0061] Table 1

[0062]

[0063] After adding the samples and mixing well, incubate at 37°C for 1 h for enzyme digestion. After the reaction is completed, detect the size of the target band of enzyme digestion by 1% agarose gel electrophoresis, and recover the target fragment using a gel extraction kit.

[0064] The enzyme digestion system of the vector pAV-SM22a-GFP is as follows in the table, and then recover the vector by gel extraction.

[0065] Table 2

[0066]

[0067] Ligate the miR30-shRNA of the mouse Eif2ak2 gene with the vector using T4 ligase. The ligation system is shown in the following table.

[0068] Table 3

[0069]

[0070] After mixing, centrifuge and ligate at 22°C for 1 h.

[0071] 1.3 Transformation

[0072] The ligation product was transformed into Escherichia coli DH5α competent cells, which were then spread on LB plates with corresponding resistance for screening. A vector with correct digestion was selected for sequencing. The correct plasmid pAV-SM22a-GFP-miR30-shRNA (map shown in Figure 2 ) was extracted to remove endotoxin for subsequent virus packaging.

[0073] Specific steps for transformation:

[0074] (1) Take out the pre-prepared DH5α competent cells from -80 °C and place them in an ice bath.

[0075] (2) After the DH5α competent cells are melted, take 5 μL of the ligation product and add it to 20 μL of DH5α competent cells. Mix well and let it stand in the ice bath for 15 minutes.

[0076] (3) Place the centrifuge tube in a 42 °C water bath for 40 seconds (do not shake the centrifuge tube during this period), then quickly transfer it to the ice bath and let it stand for 2 minutes.

[0077] (4) Add 200 μL of sterile LB medium (without antibiotics) to the centrifuge tube, mix well, and place it on a shaker at 37 °C and 220 rpm for 1 hour. The purpose is to express the relevant resistance marker genes on the plasmid and resuscitate the bacteria.

[0078] (5) Spread it on solid medium plates with corresponding resistance.

[0079] (6) Incubate overnight in a 37 °C incubator.

[0080] 1.4 Adeno-associated virus packaging

[0081] 1.4.1 Prepare HEK 293T cells. When packaging, the cell density is 85% - 90% and the cells are evenly distributed and in good condition.

[0082] 1.4.2 Package the virus

[0083] (1) Replace the cell medium with serum-free DMEM medium (1% HEPES and 1% penicillin-streptomycin) one to two hours before transfection.

[0084] (2) Transfection: Prepare according to the ratio of transfection reagent: packaging plasmid: vector plasmid: helper plasmid = 15:2:2:1. Let it stand at room temperature for 30 min.

[0085] (3) Add the above-mentioned standing liquid to HEK293T cells and make marks. After adding, shake well to mix.

[0086] (4) Incubate the cells in a 37 °C, 5% CO2 incubator for 72 h and then collect the virus.

[0087] 1.4.3 Virus collection

[0088] (1)Resuspend the cells and collect them together with the culture medium into a 50 mL centrifuge tube. Centrifuge to separate the cell pellet and the supernatant.

[0089] (2)Transfer the culture medium supernatant to a new tube. Precipitate PGE8000 overnight (add 2.33 g NaCl + 8.5 g PEG8000 per 100 mL). The next day, centrifuge at 3500 g for 30 min at 4 °C, discard the supernatant, and resuspend with PBS + 0.001% PF68.

[0090] (3)Resuspend the cell pellet with PBS + 0.001% PF68. After one freeze-thaw cycle, add 5 M NaCl and vortex to mix evenly.

[0091] (4)Mix the resuspended solution in step (2) with the resuspended solution in step (3). After shaking and mixing evenly, sonicate until not viscous. When sonicating, the probe should be sequentially cleaned with 84 solution, 75% alcohol, and water between different samples. Sonicate for 30 s and stop for 5 - 10 s. Depending on the viscosity of the sample, sonicate 3 - 4 times under the condition of AMPL value of 30% and once under the condition of AMPL value of 20%.

[0092] (5)Centrifuge the sonicated liquid at 3500 g for 30 min and collect the supernatant.

[0093] 1.4.4 Purify the virus using the iodixanol density gradient centrifugation method.

[0094] 1.4.5 Collect the virus and place it in an ultrafiltration tube to concentrate the virus, obtaining AAV9-SM22a-shEif2ak2.

[0095] Example 3 Animal experiment

[0096] The flow chart for constructing the animal model is shown in Figure 3 . The specific operations are as follows:

[0097] Angiotensin II (Ang II)-induced abdominal aortic aneurysm model in mice. The experiment used MINI-OSMOTIC PUMP (Alzet Model 2004-28) and Human Ang II (Solarbio, A9290). The dose of Ang II used in this experiment was 1.44 mg / kg / d for 28 days of stimulation. Before the experiment, the body weight of the mice was weighed to calculate the amount of Ang II required during the experiment. The Ang II powder was dissolved in physiological saline to make a stock solution of 10 mg / mL. According to the dose of Ang II required for each mouse experiment, the corresponding volume of the Ang II stock solution was taken into a 1.5 mL EP tube, and physiological saline was added to make up to 250 μL to prepare an Ang II working solution matching the body weight of the mice. Then, the Ang II working solution was injected into the MINI-OSMOTIC PUMP using a 1 mL syringe, avoiding injecting or leaving air bubbles, otherwise it would affect the normal pumping of the Ang II working solution. The prepared MINI-OSMOTIC PUMP was placed in a 5 mL sterile centrifuge tube filled with physiological saline, marked, and placed at 37°C for more than 36 h to simulate the in vivo environment of the mice so that the working solution could be pumped out evenly. In the control group, physiological saline was used to replace Ang II.

[0098] When implanting the pump, the mice were first anesthetized by intraperitoneal injection of saturated tribromoethanol (50 mg / kg body weight). After the mice were completely anesthetized, they were placed on their left side in a laminar flow hood. The neck and back of the mice were wiped with a cotton ball soaked in ethanol, and the skin of the neck and back of the mice was cut open to the superficial fascia, about 0.5 cm wide. The skin at the lower edge of the incision was lifted with straight forceps, and a hemostat was inserted into the incision to bluntly separate a pocket space to the tail of the mice (about 2.5 cm long and about 1.5 cm wide), noting that the operation should be gentle to avoid enlarging the wound of the mice. The MINI-OSMOTIC PUMP was gently pushed into the pocket space on the back of the mice from the incision, with the liquid outlet facing the tail end of the mice, and the incision was carefully sutured.

[0099] Four weeks before the operation, AAV9-ctrl / AAV9-SM22a-shEif2ak2 (2×10 11 v.g, 100 μL) was injected via the tail vein of the mice. Specifically, 8-week-old Apoe knockout mice were divided into two groups and injected with AAV9-ctrl (empty virus without the target gene, AAV9-SM22a-GFP) and AAV9-SM22a-shEif2ak2 (2×10 11v.g., 100 μL), and 4 weeks after injection, a pump was implanted in the mice. After another 4 weeks, ultrasound was performed and samples were taken (aorta, liver, brain, kidney, heart, spleen). Western Blot experiments were conducted to verify the knockdown efficiency of AAV9-SM22a-shEif2ak2 in different tissues.

[0100] The results are as Figure 4 shown, and the protein expression level of Eif2ak2 in vascular tissues was significantly reduced.

[0101] Example 4 Effect of AAV9-SM22a-shEif2ak2 Adeno-Associated Virus Vector on the Incidence of Abdominal Aortic Aneurysm in Mice

[0102] 1. Experimental Method

[0103] Echocardiography in Mice: Four weeks after the implantation of the slow-release pump, echocardiography was performed on the mice. The echocardiography instrument for mice was the Vevo770 type echocardiograph of Visual Sonics. The chest hair of the mice was removed with depilatory cream and wiped clean with toilet paper. After anesthesia with 3% isoflurane, ultrasonic gel was applied to the chest of the mice, and the probe was used to measure along the abdominal aorta. Subsequently, data statistics were performed, and the average value of five measurement results was taken as the final result. According to the diagnostic guidelines, an aneurysm was defined as a vascular diameter exceeding 50% of the normal diameter, and a vascular dilation was defined as exceeding 30%.

[0104] 2. Experimental Results

[0105] The results are as Figures 5 - 8 shown. After injection of the adeno-associated virus vector, the internal diameter of the blood vessels was significantly reduced. The incidence of abdominal aortic aneurysm in mice decreased from 60% to 12.5%, the vascular dilation rate decreased from 80% to 37.5%, and the mortality rate caused by vascular rupture decreased from 17% to 0. All these aspects showed significant improvement, indicating the effectiveness of the adeno-associated virus vector in inhibiting abdominal aortic aneurysm.

[0106] Example 5 Effect of AAV9-SM22a-shEif2ak2 Adeno-Associated Virus Vector on the Severity of Abdominal Aortic Aneurysm in Mice

[0107] 1. Experimental Method

[0108] After ultrasound of the mice, the body weight of the mice was measured (the weight of the pump needed to be removed). The vascular tissues were taken to measure the vascular weight and fixed for staining to detect pathological indexes.

[0109] The weighed blood vessels were fixed in 4% paraformaldehyde to denature and coagulate the proteins of tissues and cells. Ethanol with a low concentration (70%) to a high concentration (100%) was used as the dehydrating agent to gradually remove the water in the blood vessels. Then the blood vessels were made transparent in xylene to replace the alcohol in the blood vessels with xylene for paraffin embedding. The transparent blood vessels were put into the melted paraffin by heating. After the paraffin was completely immersed in the blood vessels, embedding was carried out. The embedded wax blocks were fixed on the microtome and cut into thin slices, generally 5 - 8 μm thick, pasted onto the glass slides, and dried in an incubator at 65°C for standby for section baking. The paraffin sections were soaked in xylene for 20 min and repeated 3 times. Then they were hydrated through alcohol from high concentration to low concentration and finally rinsed with distilled water for staining.

[0110] Hematoxylin - eosin staining: The sections were treated with a high - definition constant - staining pretreatment solution for 1 min. The sections were stained with hematoxylin solution for 3 - 5 min, washed with tap water, differentiated with the differentiating solution, washed with tap water, blued with the bluing solution, and rinsed with running water. The sections were dehydrated in 95% alcohol for 1 min and stained with eosin solution for 15 s. The sections were successively placed in absolute ethanol I for 2 min - absolute ethanol II for 2 min - absolute ethanol III for 2 min - n - butanol I for 2 min - n - butanol II for 2 min - xylene I for 2 min - xylene II for 2 min for transparency, and sealed with neutral gum. Microscopic examination was carried out and image acquisition and analysis were performed.

[0111] EVG staining: Deparaffinization of paraffin sections to water: The paraffin sections were soaked in xylene, and the xylene was changed every 20 minutes for 3 consecutive times (successively for more wax, less wax, pure xylene). Then the sections were hydrated in a gradient of 100%, 100%, 95%, 85%, 70% ethanol, and soaked in each concentration for 2 minutes. They were changed to PBS and slowly shaken at room temperature and washed 3 times, 5 minutes each time. The sections were dried, potassium permanganate was dropped onto the tissue and stained for 5 minutes, and then rinsed thoroughly with distilled water.

[0112] The sections were dried, oxalic acid was dropped onto the tissue and bleached for 5 minutes, and then rinsed thoroughly with distilled water. After a brief wash with 95% ethanol, Elastin staining solution was dropped to cover all the tissues and incubated overnight at 4°C (placed in a wet box, adding water to make the environment humid to reduce evaporation). The stain was removed by differentiation with 95% ethanol and rinsed with distilled water. The sections were dried, counter - stained with VG staining solution for 1 minute. Differentiated rapidly with 95% ethanol and placed in absolute ethanol. Transparent in xylene and sealed.

[0113] 2. Experimental results

[0114] The results were as Figures 9 - 11 shown. After injecting the adeno - associated virus vector, the ratio of blood vessel weight to body weight decreased significantly, and the degree of rupture of blood vessel elastic fibers was significantly improved.

[0115] Effect of AAV9-SM22a-shEif2ak2 Adeno-associated Virus Vector on the Expression of Contractile Genes and Proteins in Mouse Vascular Smooth Muscle Cells

[0116] 1. Experimental Method

[0117] Extract the protein of vascular tissue according to the instruction manual of the total protein extraction kit for vascular tissue, and then detect the expression levels of the proteins (PKR, MYH11, MYL9, CNN1, αSMA) of vascular smooth muscle contraction genes through western blot experiment. The relative quantitative internal reference protein is GAPDH. Calculate the protein gray value by comparison and count the protein expression in different groups.

[0118] 2. Experimental Results

[0119] As Figures 12 - 13 shown, after injecting this adeno-associated virus vector, the protein expression levels of vascular smooth muscle contraction genes in mice increased, indicating that the vascular function was significantly improved.

[0120] According to the experimental results of the above embodiments, it can be seen that using recombinant AAV9 virus to specifically knockdown the expression of Eif2ak2 in smooth muscle cells significantly increases the contractile function of vascular smooth muscle, thereby maintaining the contractile phenotype of vascular smooth muscle cells and inhibiting the occurrence and development of AAA.

[0121] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. shRNA targeting the knockout of the Eif2ak2 gene, characterized in that, The shRNA includes a sense strand and an antisense strand that is reverse complementary to the sense strand, and the nucleotide sequence of the sense strand is as shown in SEQ ID NO.

5.

2. A recombinant adeno-associated virus vector containing the shRNA according to claim 1.

3. Use of the shRNA according to claim 1 or the recombinant adeno-associated virus vector according to claim 2 in the preparation of a medicament for treating abdominal aortic aneurysm.

4. Use of the shRNA according to claim 1 or the recombinant adeno-associated virus vector according to claim 2 in the preparation of a medicament for reducing the incidence of abdominal aortic aneurysm.

5. Use of the shRNA according to claim 1 or the recombinant adeno-associated virus vector according to claim 2 in the preparation of a medicament for improving the severity of abdominal aortic aneurysm.

6. Use of the shRNA according to claim 1 or the recombinant adeno-associated virus vector according to claim 2 in the preparation of a medicament for reducing the inner diameter of blood vessels and / or the ratio of blood vessel weight to body weight.

7. Use of the shRNA according to claim 1 or the recombinant adeno-associated virus vector according to claim 2 in the preparation of a medicament for promoting the contractile function of vascular smooth muscle cells.

8. A drug for treating abdominal aortic aneurysm, characterized in that, The medicament includes the shRNA according to claim 1 or the recombinant adeno-associated virus vector according to claim 2.

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