A shRNA for targeted knockout of Eif2ak2 gene and its application

Abstract

The present invention discloses a shRNA for targeted knockout of the Eif2ak2 gene and its application, belonging to the field of genetic engineering technology. The shRNA comprises a sense chain and an antisense chain that is reverse complementary to the sense chain, and the nucleotide sequence of the sense chain is shown in SEQ ID NO.5. The present invention first discovered 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, 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 promote the contractile function of mouse vascular smooth muscle cells. It can be used for 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.

Description

Technical Field

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

[0002] Abdominal aortic aneurysm (AAA) is a serious cardiovascular disease that threatens human life and health. Currently, no drug therapy can effectively limit the growth or rupture of AAA. In recent years, adeno-associated viral vectors (AAVs) have become a key component of gene therapy. Due to their high safety, low immunogenicity, strong tissue specificity and affinity, and sustained and stable expression in vivo, AAV vectors have become a candidate for gene therapy of cardiovascular diseases. Therefore, exploring drugs related to AAA gene therapy using AAV 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 a shRNA and application for targeted knockout of the Eif2ak2 gene to solve the problems existing in the above-mentioned prior art. The recombinant AAV9 virus is used to specifically knock down the expression of Eif2ak2 in smooth muscle cells, significantly increasing 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 object, the present invention provides the following solutions:

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

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

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

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

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

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

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

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

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

[0014] The present invention, for the first time, discovered that a recombinant adeno-associated virus that inhibits Eif2ak2 gene expression can successfully suppress the phenotype of abdominal aortic aneurysms in mice, significantly reduce the incidence and severity of abdominal aortic aneurysms in mice, reduce vascular diameter and the ratio of vascular weight to body weight, and significantly promote the contractile function of vascular smooth muscle cells in mice. This invention can be used to effectively treat abdominal aortic aneurysms. This invention provides new ideas and strategies for the treatment of abdominal aortic aneurysms and has broad clinical application prospects. Furthermore, this invention provides a reference for gene therapy research for other cardiovascular diseases, promoting the development of the gene therapy field. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 The results of bioinformatics analysis of transcriptome data from patients with abdominal aortic aneurysm and normal subjects;

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

[0018] Figure 3 Flowchart for the construction of abdominal aortic aneurysm mouse model;

[0019] Figure 4 Western blot to verify the knockdown efficiency of AAV9-SM22a-shEif2ak2;

[0020] Figure 5 This is a phenotypic diagram showing the effect of AAV9-SM22a-shEif2ak2 injection on vascular diameter;

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

[0022] Figure 7 This is the echocardiogram of mice after injection of AAV9-SM22a-shEif2ak2;

[0023] Figure 8 The effect of AAV9-SM22a-shEif2ak2 injection on the vascular diameter of mice;

[0024] Figure 9 Effects of AAV9-SM22a-shEif2ak2 injection on the ratio of vascular weight to body weight in mice;

[0025] Figure 10 This is a staining image of mouse vascular tissue after injection of AAV9-SM22a-shEif2ak2;

[0026] Figure 11 The effect of AAV9-SM22a-shEif2ak2 injection on the degree of vascular elastic fiber rupture in mice;

[0027] Figure 12 The effect of AAV9-SM22a-shEif2ak2 injection on the protein expression level of vascular smooth muscle contractile genes;

[0028] Figure 13 The data are statistical results of the effect of AAV9-SM22a-shEif2ak2 injection on the protein expression level of vascular smooth muscle contractile genes. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may 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 associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

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

[0035] The gene and protein sequences involved in the present invention are shown below.

[0036] 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 published transcriptome data of abdominal aortic aneurysm patients and normal subjects (GSE47472). The results showed that the expression of Eif2ak2 in abdominal aortic aneurysm tissue was significantly higher than that in normal vascular tissue (see Figure 1 ).

[0049] Example 2

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

[0051] 1.1 RNAi target design and double-stranded DNA oligo preparation

[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) RNA interference target design

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

[0055] (3) DNA oligo sequence synthesis

[0056] Design the shRNA interference sequence based on the selected target sequence (sense and antisense strands; the sense strand is shown in SEQ ID NO. 5 in Table 1; the antisense strand is the reverse complement of SEQ ID NO. 5). Add appropriate restriction endonuclease sites at both ends to complete the vector construction. Additionally, add a TTTTT termination signal to the 3' end of the sense strand, and a sequence complementary to the termination signal to the 5' end of the antisense strand. After design, send the design to JERUI for single-stranded DNA oligo synthesis.

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

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

[0059] 1.2 Enzyme digestion

[0060] The mouse Eif2ak2 gene miR30-shRNA was excised using SfaAI / MluI, and the pAV-SM22a-GFP vector was simultaneously digested. The gene digestion system is shown in the table below.

[0061] Table 1

[0062]

[0063] After adding the sample and mixing well, incubate at 37°C for 1 hour for enzyme digestion. After the reaction is completed, use 1% agarose gel electrophoresis to detect the size of the enzyme-digested target band, and use a gel recovery kit to recover the target fragment.

[0064] The enzyme digestion system of the vector pAV-SM22a-GFP is shown in the table below, and the vector is then recovered by gel.

[0065] Table 2

[0066]

[0067] The mouse Eif2ak2 gene miR30-shRNA was ligated to the vector using T4 ligase. The ligation system is shown in the table below.

[0068] Table 3

[0069]

[0070] After mixing, centrifuge and connect at 22℃ for 1h.

[0071] 1.3 Conversion

[0072] The ligation product was transformed into E. coli DH5α competent cells and plated on LB plates with corresponding resistance for screening. The correct vector was selected for sequencing. The correct plasmid pAV-SM22a-GFP-miR30-shRNA (see the map) Figure 2 ) to remove endotoxins and then package the virus.

[0073] Specific steps of conversion:

[0074] (1) Take out the prepared DH5a competent cell from -80℃ and place it in an ice bath.

[0075] (2) After the DH5a competent cells have melted, take 5 μL of the ligation product and add it to 20 μL of DH5a competent cells. Mix thoroughly and let it stand in an 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 move it to an 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 in a shaker at 37°C, 220 rpm, and shake for 1 hour. The purpose is to express the relevant resistance marker gene on the plasmid and allow the bacteria to recover.

[0078] (5) Spread onto a solid culture medium plate with corresponding resistance.

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

[0080] 1.4 Adeno-associated virus packaging

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

[0082] 1.4.2 Packaging Viruses

[0083] (1) One to two hours before transfection, change the cell culture medium to serum-free DMEM (1% HEPES and 1% penicillin-streptomycin).

[0084] (2) Transfection: Prepare the transfection reagent: packaging plasmid: vector plasmid: helper plasmid at a ratio of 15:2:2:1. Incubate at room temperature for 30 min.

[0085] (3) Add the above-mentioned liquid after standing to the HEK293T cells and mark them. Shake and mix well.

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

[0087] 1.4.3 Collecting Poison

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

[0089] (2) Transfer the culture supernatant to a new tube and precipitate PGE8000 overnight (add 2.33 g NaCl + 8.5 g PEG8000 per 100 mL). Centrifuge at 3500 g and 4°C for 30 min the next day. Remove the supernatant and resuspend in PBS + 0.001% PF68.

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

[0091] (4) Mix the resuspension of 2) with the resuspension of 3), shake and mix thoroughly, and then sonicate until it is no longer viscous. During sonication, the probe should be cleaned with 84 solution, 75% alcohol, and water in sequence between different samples. The probe should be kept for 5 to 10 seconds after the sample is sonicated for more than 30 seconds. Depending on the viscosity of the sample, sonicate 3 to 4 times at an AMPL value of 30%, and once at an 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 iodixanol density gradient centrifugation.

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

[0095] Example 3 Animal Experiment

[0096] The flowchart of animal model construction is shown in Figure 3 The specific operations are as follows:

[0097] Angiotensin II (Ang II) was used to induce abdominal aortic aneurysm in mice. The experiment used a MINI-OSMOTIC PUMP (Alzet Model 2004-28) and Human Ang II (Solebol, A9290). The Ang II dose used in this experiment was 1.44 mg / kg / day, and stimulation lasted for 28 days. Before the experiment, mice were weighed to calculate the amount of Ang II required. Ang II powder was dissolved in saline to prepare a 10 mg / mL stock solution. Based on the desired Ang II dose for each mouse, the appropriate volume of Ang II stock solution was transferred to a 1.5 mL EP tube and filled with saline to 250 μL to prepare an Ang II working solution tailored to the mouse's weight. The Ang II working solution was then injected into the MINI-OSMOTIC PUMP using a 1 mL syringe. Avoid injecting or retaining bubbles, as this will impair proper pumping of the Ang II working solution. Place the prepared MINI-OSMOTIC PUMP in a 5 mL sterile centrifuge tube filled with normal saline, label it, and incubate at 37°C for at least 36 hours to simulate the in vivo mouse environment and allow the working solution to be pumped out at a constant rate. In the control group, normal saline was used instead of Ang II.

[0098] To implant the pump, first anesthetize the mouse with an intraperitoneal injection of saturated tribromoethanol (50 mg / kg body weight). Once fully anesthetized, place the mouse in the left lateral decubitus position on a clean bench. Wipe the back of the neck with an ethanol-soaked cotton ball. Cut the skin on the back of the neck to the superficial fascia, approximately 0.5 cm wide. Lift the skin at the lower edge of the incision with straight forceps. Insert the hemostat deep into the incision and bluntly separate a pocket-like space to the mouse's tail (approximately 2.5 cm long and 1.5 cm wide). Be careful to operate gently to avoid stretching the wound. Gently push the MINI-OSMOTIC PUMP from the incision into the band-like space on the mouse's back, with the outlet facing the mouse's tail. Carefully suture the incision.

[0099] Four weeks before surgery, mice were injected with AAV9-ctrl / AAV9-SM22a-shEif2ak2 (2×10 11 vg, 100 μL). Specifically, 8-week-old Apoe knockout mice were divided into two groups and injected with AAV9-ctrl (empty virus without target gene, AAV9-SM22a-GFP) and AAV9-SM22a-shEif2ak2 (2×10 11vg, 100 μL). Four weeks after injection, mice were pumped. Four weeks later, ultrasound was performed and tissue samples (aorta, liver, brain, kidney, heart, and spleen) were collected. Western blot analysis verified the knockdown efficiency of AAV9-SM22a-shEif2ak2 in different tissues.

[0100] The results are as follows Figure 4 As shown, the protein expression level of Eif2ak2 in vascular tissue 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 methods

[0103] Echocardiography in mice: Four weeks after the sustained-release pump was implanted, mice underwent echocardiography using a Visual Sonics Vevo770 echocardiometer. The mice's chest hair was removed with depilatory cream and then wiped clean with toilet paper. After anesthesia with 3% isoflurane, ultrasound gel was applied to the mouse's chest. Measurements were taken along the abdominal aorta using a probe. Data were then collected and the average of five measurements was used as the final result. According to diagnostic guidelines, a vessel diameter exceeding 50% of the normal diameter is considered an aneurysm, and a diameter exceeding 30% is defined as vasodilation.

[0104] 2. Experimental results

[0105] The results are as follows Figure 5-Figure 8 As shown, after injection of the adeno-associated virus vector, the inner diameter of the blood vessels was significantly reduced, the incidence of abdominal aortic aneurysm in mice dropped from 60% to 12.5%, the vascular dilation rate dropped from 80% to 37.5%, and the mortality rate caused by vascular rupture dropped from 17% to 0. These aspects showed significant improvements, 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 Aneurysms in Mice

[0107] 1. Experimental methods

[0108] After ultrasound, the mouse body weight was measured (the pump weight needed to be subtracted), and vascular tissue was obtained to measure the vascular weight and fixed for staining to detect pathological indicators.

[0109] The weighed blood vessels are fixed in 4% paraformaldehyde to denature and coagulate the proteins in the tissues and cells. Low (70%) to high (100%) concentrations of ethanol are used as dehydrating agents to gradually remove the water from the blood vessels. The blood vessels are then placed in xylene to make them transparent, replacing the alcohol in the blood vessels with xylene for paraffin embedding. The transparent blood vessels are placed in heated and dissolved paraffin and embedded after the paraffin is completely immersed in the blood vessels. The embedded wax block is fixed on a microtome and cut into thin slices, generally 5-8 μm thick. The slices are attached to a glass slide and placed in a 65°C constant temperature oven to dry. The slices are then baked for later use. The paraffin sections are soaked in xylene for 20 minutes, repeated three times. The sections are then hydrated using high to low concentrations of alcohol and finally rinsed with distilled water before staining.

[0110] Hematoxylin-eosin staining: Sections were treated in high-definition constant stain pretreatment solution for 1 minute. Sections were stained in hematoxylin solution for 3-5 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with bluing solution, and rinsed with running water. Sections were dehydrated in 95% alcohol for 1 minute and stained in eosin solution for 15 seconds. Sections were then placed in anhydrous ethanol I for 2 minutes, anhydrous ethanol II for 2 minutes, anhydrous ethanol III for 2 minutes, n-butanol I for 2 minutes, n-butanol II for 2 minutes, xylene I for 2 minutes, and xylene II for 2 minutes to clear the sections. Sections were then mounted with neutral gum. Microscopic examination and image acquisition and analysis were performed.

[0111] EVG staining: Dewax paraffin sections to water: Soak paraffin sections in xylene, changing the xylene every 20 minutes for three times (high-wax, low-wax, pure xylene). Then, rehydrate the sections in a gradient of ethanol: 100%, 100%, 95%, 85%, and 70%, soaking for 2 minutes in each concentration. Transfer to PBS, slowly shake at room temperature, and wash three times for 5 minutes each. Dry the sections, apply potassium permanganate dropwise to the tissue, stain for 5 minutes, and rinse with distilled water.

[0112] Dry the sections, add oxalic acid dropwise to the tissue, bleach for 5 minutes, and rinse thoroughly with distilled water. After a brief rinse with 95% ethanol, add Elastin stain dropwise to cover the entire tissue and incubate overnight at 4°C (place in a humidified chamber and add water to maintain humidity to minimize evaporation). Wash away the stain with 95% ethanol and rinse with distilled water. Dry the sections and counterstain with VG stain for 1 minute. Rapidly differentiate with 95% ethanol and place in anhydrous ethanol. Transparent with xylene and mount the sections.

[0113] 2. Experimental results

[0114] The results are as follows Figures 9-11 As shown, after injection of the adeno-associated virus vector, the ratio of blood vessel weight to body weight was significantly reduced, and the degree of vascular elastic fiber rupture was significantly improved.

[0115] Example 6 Effect of AAV9-SM22a-shEif2ak2 Adeno-Associated Virus Vector on Contractile Gene Protein Expression in Mouse Vascular Smooth Muscle Cells

[0116] 1. Experimental methods

[0117] Vascular tissue proteins were extracted according to the instructions of the vascular tissue total protein extraction kit, and then the expression levels of vascular smooth muscle contractile gene proteins (PKR, MYH11, MYL9, CNN1, αSMA) were detected by western blot. The internal reference protein for relative quantification was GAPDH. The protein grayscale value was calculated by comparison, and the protein expression between different groups was statistically analyzed.

[0118] 2. Experimental results

[0119] like Figure 12-13 As shown, after injection of the adeno-associated virus vector, the protein expression level of vascular smooth muscle contractile genes in mice increased, indicating that vascular function was significantly improved.

[0120] According to the experimental results of the above examples, it can be seen that the use of recombinant AAV9 virus to specifically knock down 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 embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of shRNA or a recombinant adeno-associated virus vector containing the shRNA in the preparation of a drug for treating abdominal aortic aneurysm, characterized in that: 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 shown in SEQ ID NO.

5.

2. Use of shRNA or a recombinant adeno-associated virus vector containing the shRNA in the preparation of a drug for reducing the incidence of abdominal aortic aneurysm, characterized in that: 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 shown in SEQ ID NO.

5.

3. Use of shRNA or a recombinant adeno-associated virus vector containing the shRNA in the preparation of a drug for improving the severity of abdominal aortic aneurysm, characterized in that: 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 shown in SEQ ID NO.5.

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